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Better Cancer Medication

Posted in Acute myelocytic leukemia, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Clinical & Translational, Curation, tagged AML, Cancer - General, Chemotherapy, new classes of drugs on October 9, 2015| Leave a Comment »

Better Cancer Medication

Larry H. Bernstein, MD, FCAP, Curator

LPBI

A Better Class of Cancer Drugs

http://www.technologynetworks.com/medchem/news.aspx?ID=183124

An SDSU chemist has developed a technique to identify potential cancer drugs that are less likely to produce side effects.

A class of therapeutic drugs known as protein kinase inhibitors has in the past decade become a powerful weapon in the fight against various life-threatening diseases, including certain types of leukemia, lung cancer, kidney cancer and squamous cell cancer of the head and neck. One problem with these drugs, however, is that they often inhibit many different targets, which can lead to side effects and complications in therapeutic use. A recent study by San Diego State University chemist Jeffrey Gustafson has identified a new technique for improving the selectivity of these drugs and possibly decreasing unwanted side effects in the future.

Why are protein kinase–inhibiting drugs so unpredictable? The answer lies in their molecular makeup.

Many of these drug candidates possess examples of a phenomenon known as atropisomerism. To understand what this is, it’s helpful to understand a bit of the chemistry at work. Molecules can come in different forms that have exactly the same chemical formula and even the same bonds, just arranged differently. The different arrangements are mirror images of each other, with a left-handed and a right-handed arrangement. The molecules’ “handedness” is referred to as chirality. Atropisomerism is a form of chirality that arises when the spatial arrangement has a rotatable bond called an axis of chirality. Picture two non-identical paper snowflakes tethered together by a rigid stick.

Some axes of chirality are rigid, while others can freely spin about their axis. In the latter case, this means that at any given time, you could have one of two different “versions” of the same molecule.

Watershed treatment

As the name suggests, kinase inhibitors interrupt the function of kinases—a particular type of enzyme—and effectively shut down the activity of proteins that contribute to cancer.

“Kinase inhibition has been a watershed for cancer treatment,” said Gustafson, who attended SDSU as an undergraduate before earning his Ph.D. in organic chemistry from Yale University, then working there as a National Institutes of Health poctdoctoral fellow in chemical biology.

“However, it’s really hard to inhibit a single kinase,” he explained. “The majority of compounds identified inhibit not just one but many kinases, and that can lead to a number of side effects.”

Many kinase inhibitors possess axes of chirality that are freely spinning. The problem is that because you can’t control which “arrangement” of the molecule is present at a given time, the unwanted version could have unintended consequences.

In practice, this means that when medicinal chemists discover a promising kinase inhibitor that exists as two interchanging arrangements, they actually have two different inhibitors. Each one can have quite different biological effects, and it’s difficult to know which version of the molecule actually targets the right protein.

“I think this has really been under-recognized in the field,” Gustafson said. “The field needs strategies to weed out these side effects.”

Applying the brakes

So that’s what Gustafson did in a recently published study. He and his colleagues synthesized atropisomeric compounds known to target a particular family of kinases known as tyrosine kinases. To some of these compounds, the researchers added a single chlorine atom which effectively served as a brake to keep the atropisomer from spinning around, locking the molecule into either a right-handed or a left-handed version.

When the researchers screened both the modified and unmodified versions against their target kinases, they found major differences in which kinases the different versions inhibited. The unmodified compound was like a shotgun blast, inhibiting a broad range of kinases. But the locked-in right-handed and left-handed versions were choosier.

“Just by locking them into one or another atropisomeric configuration, not only were they more selective, but they inhibited different kinases,” Gustafson explained.

If drug makers incorporated this technique into their early drug discovery process, he said, it would help identify which version of an atropisomeric compound actually targets the kinase they want to target, cutting the potential for side effects and helping to usher drugs past strict regulatory hurdles and into the hands of waiting patients.

Inroads Against Leukaemia

http://www.technologynetworks.com/medchem/news.aspx?ID=183594

Potential for halting disease in molecule isolated from sea sponges.

A molecule isolated from sea sponges and later synthesized in the lab can halt the growth of cancerous cells and could open the door to a new treatment for leukemia, according to a team of Harvard researchers and other collaborators led by Matthew Shair, a professor of chemistry and chemical biology.

“Once we learned this molecule, named cortistatin A, was very potent and selective in terms of inhibiting the growth of AML [acute myeloid leukemia] cells, we tested it in mouse models of AML and found that it was as efficacious as any other molecule we had seen, without having deleterious effects,” Shair said. “This suggests we have identified a promising new therapeutic approach.”

It’s one that could be available to test in patients relatively soon.

“We synthesized cortistatin A and we are working to develop novel therapeutics based on it by optimizing its drug-like properties,” Shair said. “Given the dearth of effective treatments for AML, we recognize the importance of advancing it toward clinical trials as quickly as possible.”

The drug-development process generally takes years, but Shair’s lab is very close to having what is known as a development candidate that could be taken into late-stage preclinical development and then clinical trials. An industrial partner will be needed to push the technology along that path and toward regulatory approval. Harvard’s Office of Technology Development (OTD) is engaged in advanced discussions to that end.

The molecule works, Shair explained, by inhibiting a pair of nearly identical kinases, called CDK8 and CDK19, that his research indicates play a key role in the growth of AML cells.

The kinases operate as part of a poorly understood, massive structure in the nucleus of cells called the mediator complex, which acts as a bridge between transcription factors and transcriptional machinery. Inhibiting these two specific kinases, Shair and colleagues found, doesn’t shut down all transcription, but instead has gene-specific effects.

“We treated AML cells with cortistatin A and measured the effects on gene expression,” Shair said. “One of the first surprises was that it’s affecting a very small number of genes — we thought it might be in the thousands, but it’s in the low hundreds.”

When Shair, Henry Pelish, a senior research associate in chemistry and chemical biology, and then-Ph.D. student Brian Liau looked closely at which genes were affected, they discovered many were associated with DNA regulatory elements known as “super-enhancers.”

“Humans have about 220 different types of cells in their body — they all have the same genome, but they have to form things like skin and bone and liver cells,” Shair explained. “In all cells, there are a relatively small number of DNA regulatory elements, called super-enhancers. These super-enhancers drive high expression of genes, many of which dictate cellular identity. A big part of cancer is a situation where that identity is lost, and the cells become poorly differentiated and are stuck in an almost stem-cell-like state.”

While a few potential cancer treatments have attacked the disease by down-regulating such cellular identity genes, Shair and colleagues were surprised to find that their molecule actually turned up the activity of those genes in AML cells.

“Before this paper, the thought was that cancer is ramping these genes up, keeping the cells in a hyper-proliferative state and affecting cell growth in that way,” Shair said. “But our molecule is saying that’s one part of the story, and in addition cancer is keeping the dosage of these genes in a narrow range. If it’s too low, the cells die. If they are pushed too high, as with cortistatin A, they return to their normal identity and stop growing.”

Shair’s lab became interested in the molecule several years ago, shortly after it was first isolated and described by other researchers. Early studies suggested it appeared to inhibit just a handful of kinases.

“We tested approximately 400 kinases, and found that it inhibits only CDK8 and CDK19 in cells, which makes it among the most selective kinase inhibitors identified to date,” Shair said. “Having compounds that precisely hit a specific target, like cortistatin A, can help reduce side effects and increase efficacy. In a way, it shatters a dogma because we thought it wasn’t possible for a molecule to be this selective and bind in a site common to all 500 human kinases, but this molecule does it, and it does it because of its 3-D structure. What’s interesting is that most kinase-inhibitor drugs do not have this type of 3-D structure. Nature is telling us that one way to achieve this level of specificity is to make molecules more like cortistatin A.”

Shair’s team successfully synthesized the molecule, which helped them study how it worked and why it affected the growth of a very specific type of cell. Later on, with funding and drug-development expertise provided by Harvard’s Blavatnik Biomedical Accelerator, Shair’s lab created a range of new molecules that may be better suited to clinical application.

“It’s a complex process to make [cortistatin A] — 32 chemical steps,” said Shair. “But we have been able to find less complex structures that act just like the natural compound, with better drug-like properties, and they can be made on a large scale and in about half as many steps.”

“Over the course of several years, we have watched this research progress from an intriguing discovery to a highly promising development candidate,” said Isaac Kohlberg, senior associate provost and chief technology development officer. “The latest results are a real testament to Matt’s ingenuity and dedication to addressing a very tough disease.”

While there is still much work to be done — in particular, to better understand how CDK8 and CDK19 regulate gene expression — the early results have been dramatic.

“This is the kind of thing you do science for,” Shair said, “the idea that once every 10 or 20 years you might find something this interesting, that sheds new light on important, difficult problems. This gives us an opportunity to generate a new understanding of cancer and also develop new therapeutics to treat it. We’re very excited and curious to see where it goes.”

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Treatment of Acute Leukemias

Posted in Academic Publishing, Acute lymphocytic leukemia, Acute myelocytic leukemia, Anticancer Resistance, Biomarkers & Medical Diagnostics, Bone marrow derived cells, Cancer and Current Therapeutics, Cancer Screening, Cell Biology, Circulating Progenitor Cells, Clinical & Translational, Clinical Diagnostics, Curation, Diagnostics and Lab Tests, Disease Biology, Hematology, Hematopoiesis, Umbilical cord cells, tagged Acute lymphocytic leukemia, Acute myeloid leukemia, Chemotherapy, lymphoblast, lymphoblastic leukemia, Pharmaceuticals on October 5, 2015| Leave a Comment »

Treatment of Acute Leukemias

Author and Curator: Larry H. Bernstein, MD, FCAP

2.4.4 Treatment of Acute Leukemias

Treatment of Acute Lymphoblastic Leukemia

Ching-Hon Pu, and William E. Evans
N Engl J Med Jan 12, 2006; 354:166-178
http://dx.doi.org:/10.1056/NEJMra052603

Although the overall cure rate of acute lymphoblastic leukemia (ALL) in children is about 80 percent, affected adults fare less well. This review considers recent advances in the treatment of ALL, emphasizing issues that need to be addressed if treatment outcome is to improve further.

Acute Lymphoblastic Leukemia

Ching-Hon Pui, Mary V. Relling, and James R. Downing
N Engl J Med Apr 8, 2004; 350:1535-1548
http://dx.doi.org:/10.1056/NEJMra023001

This comprehensive survey emphasizes how recent advances in the knowledge of molecular mechanisms involved in acute lymphoblastic leukemia have influenced diagnosis, prognosis, and treatment.

Gene-Expression Patterns in Drug-Resistant Acute Lymphoblastic Leukemia Cells and Response to Treatment

Amy Holleman, Meyling H. Cheok, Monique L. den Boer, et al.
N Engl J Med 2004; 351:533-42

Childhood acute lymphoblastic leukemia (ALL) is curable with chemotherapy in approximately 80 percent of patients. However, the cause of treatment failure in the remaining 20 percent of patients is largely unknown.

Methods We tested leukemia cells from 173 children for sensitivity in vitro to prednisolone, vincristine, asparaginase, and daunorubicin. The cells were then subjected to an assessment of gene expression with the use of 14,500 probe sets to identify differentially expressed genes in drug-sensitive and drug-resistant ALL. Gene-expression patterns that differed according to sensitivity or resistance to the four drugs were compared with treatment outcome in the original 173 patients and an independent cohort of 98 children treated with the same drugs at another institution.

Results We identified sets of differentially expressed genes in B-lineage ALL that were sensitive or resistant to prednisolone (33 genes), vincristine (40 genes), asparaginase (35 genes), or daunorubicin (20 genes). A combined gene-expression score of resistance to the four drugs, as compared with sensitivity to the four, was significantly and independently related to treatment outcome in a multivariate analysis (hazard ratio for relapse, 3.0; P=0.027). Results were confirmed in an independent population of patients treated with the same medications (hazard ratio for relapse, 11.85; P=0.019). Of the 124 genes identified, 121 have not previously been associated with resistance to the four drugs we tested.

Conclusions Differential expression of a relatively small number of genes is associated with drug resistance and treatment outcome in childhood ALL.

Leukemias Treatment & Management

Author: Lihteh Wu, MD; Chief Editor: Hampton Roy Sr
http://emedicine.medscape.com/article/1201870-treatment

The treatment of leukemia is in constant flux, evolving and changing rapidly over the past few years. Most treatment protocols use systemic chemotherapy with or without radiotherapy. The basic strategy is to eliminate all detectable disease by using cytotoxic agents. To attain this goal, 3 phases are typically used, as follows: remission induction phase, consolidation phase, and maintenance therapy phase.

Chemotherapeutic agents are chosen that interfere with cell division. Tumor cells usually divide more rapidly than host cells, making them more vulnerable to the effects of chemotherapy. Primary treatment will be under the direction of a medical oncologist, radiation oncologist, and primary care physician. Although a general treatment plan will be outlined, the ophthalmologist does not prescribe or manage such treatment.

The initial treatment of ALL uses various combinations of vincristine, prednisone, and L-asparaginase until a complete remission is obtained.
Maintenance therapy with mercaptopurine is continued for 2-3 years following remission.
Use of intrathecal methotrexate with or without cranial irradiation to cover the CNS varies from facility to facility.
Daunorubicin, cytarabine, and thioguanine currently are used to obtain induction and remission of AML.
Maintenance therapy for 8 months may lengthen remission. Once relapse has occurred, AML generally is curable only by bone marrow transplantation.
Presently, treatment of CLL is palliative.
CML is characterized by a leukocytosis greater than 100,000 cells. Emergent treatment with leukopheresis sometimes is necessary when leukostastic complications are present. Otherwise, busulfan or hydroxyurea may control WBC counts. During the chronic phase, treatment is palliative.
When CML converts to the blastic phase, approximately one third of cases behave as ALL and respond to treatment with vincristine and prednisone. The remaining two thirds resemble AML but respond poorly to AML therapy.
Allogeneic bone marrow transplant is the only curative therapy for CML. However, it carries a high early mortality rate.
Leukemic retinopathy usually is not treated directly. As the hematological parameters normalize with systemic treatment, many of the ophthalmic signs resolve. There are reports that leukopheresis for hyperviscosity also may alleviate intraocular manifestations.
When definite intraocular leukemic infiltrates fail to respond to systemic chemotherapy, direct radiation therapy is recommended.
Relapse, manifested by anterior segment involvement, should be treated by radiation. In certain cases, subconjunctival chemotherapeutic agents have been injected.
Optic nerve head infiltration in patients with ALL is an emergency and requires prompt radiation therapy to try to salvage some vision.

Treatments and drugs

http://www.mayoclinic.org/diseases-conditions/leukemia/basics/
treatment/con-20024914

Common treatments used to fight leukemia include:

Chemotherapy. Chemotherapy is the major form of treatment for leukemia. This drug treatment uses chemicals to kill leukemia cells.

Depending on the type of leukemia you have, you may receive a single drug or a combination of drugs. These drugs may come in a pill form, or they may be injected directly into a vein.

Biological therapy. Biological therapy works by using treatments that help your immune system recognize and attack leukemia cells.
Targeted therapy. Targeted therapy uses drugs that attack specific vulnerabilities within your cancer cells.

For example, the drug imatinib (Gleevec) stops the action of a protein within the leukemia cells of people with chronic myelogenous leukemia. This can help control the disease.

Radiation therapy. Radiation therapy uses X-rays or other high-energy beams to damage leukemia cells and stop their growth. During radiation therapy, you lie on a table while a large machine moves around you, directing the radiation to precise points on your body.

You may receive radiation in one specific area of your body where there is a collection of leukemia cells, or you may receive radiation over your whole body. Radiation therapy may be used to prepare for a stem cell transplant.

Stem cell transplant. A stem cell transplant is a procedure to replace your diseased bone marrow with healthy bone marrow.

Before a stem cell transplant, you receive high doses of chemotherapy or radiation therapy to destroy your diseased bone marrow. Then you receive an infusion of blood-forming stem cells that help to rebuild your bone marrow.

You may receive stem cells from a donor, or in some cases you may be able to use your own stem cells. A stem cell transplant is very similar to a bone marrow transplant.

2.4.4.2 Acute Myeloid Leukemia

New treatment approaches in acute myeloid leukemia: review of recent clinical studies.

Norsworthy K¹, Luznik L, Gojo I.
Rev Recent Clin Trials. 2012 Aug; 7(3):224-37.
http://www.ncbi.nlm.nih.gov/pubmed/22540908

Standard chemotherapy can cure only a fraction (30-40%) of younger and very few older patients with acute myeloid leukemia (AML). While conventional allografting can extend the cure rates, its application remains limited mostly to younger patients and those in remission. Limited efficacy of current therapies and improved understanding of the disease biology provided a spur for clinical trials examining novel agents and therapeutic strategies in AML. Clinical studies with novel chemotherapeutics, antibodies, different signal transduction inhibitors, and epigenetic modulators demonstrated their clinical activity; however, it remains unclear how to successfully integrate novel agents either alone or in combination with chemotherapy into the overall therapeutic schema for AML. Further studies are needed to examine their role in relation to standard chemotherapy and their applicability to select patient populations based on recognition of unique disease and patient characteristics, including the development of predictive biomarkers of response. With increasing use of nonmyeloablative or reduced intensity conditioning and alternative graft sources such as haploidentical donors and cord blood transplants, the benefits of allografting may extend to a broader patient population, including older AML patients and those lacking a HLA-matched donor. We will review here recent clinical studies that examined novel pharmacologic and immunologic approaches to AML therapy.

Novel approaches to the treatment of acute myeloid leukemia.

Roboz GJ¹Hematology Am Soc Hematol Educ Program. 2011:43-50.
http://dx.doi.org:/10.1182/asheducation-2011.1.43.

Approximately 12 000 adults are diagnosed with acute myeloid leukemia (AML) in the United States annually, the majority of whom die from their disease. The mainstay of initial treatment, cytosine arabinoside (ara-C) combined with an anthracycline, was developed nearly 40 years ago and remains the worldwide standard of care. Advances in genomics technologies have identified AML as a genetically heterogeneous disease, and many patients can now be categorized into clinicopathologic subgroups on the basis of their underlying molecular genetic defects. It is hoped that enhanced specificity of diagnostic classification will result in more effective application of targeted agents and the ability to create individualized treatment strategies. This review describes the current treatment standards for induction, consolidation, and stem cell transplantation; special considerations in the management of older AML patients; novel agents; emerging data on the detection and management of minimal residual disease (MRD); and strategies to improve the design and implementation of AML clinical trials.

Age ≥ 60 years has consistently been identified as an independent adverse prognostic factor in AML, and there are very few long-term survivors in this age group.5 Poor outcomes in elderly AML patients have been attributed to both host- and disease-related factors, including medical comorbidities, physical frailty, increased incidence of antecedent myelodysplastic syndrome and myeloproliferative disorders, and higher frequency of adverse cytogenetics.28 Older patients with multiple poor-risk factors have a high probability of early death and little chance of long-term disease-free survival with standard chemotherapy. In a retrospective analysis of 998 older patients treated with intensive induction at the M.D. Anderson Cancer Center, multivariate analysis identified age ≥ 75 years, unfavorable karyotype, poor performance status, creatinine > 1.3 mg/dL, duration of antecedent hematologic disorder > 6 months, and treatment outside a laminar airflow room as adverse prognostic indicators.29 Patients with 3 or more of these factors had expected complete remission rates of < 20%, 8-week mortality > 50%, and 1-year survival < 10%. The Medical Research Council (MRC) identified cytogenetics, WBC count at diagnosis, age, and de novo versus secondary disease as critical factors influencing survival in > 2000 older patients with AML, but cautioned in their conclusions that less objective factors, such as clinical assessment of “fitness” for chemotherapy, may be equally important in making treatment decisions in this patient population.30 It is hoped that data from comprehensive geriatric assessments of functional status, cognition, mood, quality of life, and other measures obtained during ongoing cooperative group trials will improve our ability to predict how older patients will tolerate treatment.

Current treatment of acute myeloid leukemia.

Roboz GJ¹.
Curr Opin Oncol. 2012 Nov; 24(6):711-9.
http://dx.doi.org:/10.1097/CCO.0b013e328358f62d.

The objectives of this review are to discuss standard and investigational nontransplant treatment strategies for acute myeloid leukemia (AML), excluding acute promyelocytic leukemia.

RECENT FINDINGS: Most adults with AML die from their disease. The standard treatment paradigm for AML is remission induction chemotherapy with an anthracycline/cytarabine combination, followed by either consolidation chemotherapy or allogeneic stem cell transplantation, depending on the patient’s ability to tolerate intensive treatment and the likelihood of cure with chemotherapy alone. Although this approach has changed little in the last three decades, increased understanding of the pathogenesis of AML and improvements in molecular genomic technologies are leading to novel drug targets and the development of personalized, risk-adapted treatment strategies. Recent findings related to prognostically relevant and potentially ‘druggable’ molecular targets are reviewed.

SUMMARY: At the present time, AML remains a devastating and mostly incurable disease, but the combination of optimized chemotherapeutics and molecularly targeted agents holds significant promise for the future.

Adult Acute Myeloid Leukemia Treatment (PDQ®)
http://www.cancer.gov/cancertopics/pdq/treatment/adultAML/healthprofessional/page9

About This PDQ Summary

This summary is reviewed regularly and updated as necessary by the PDQ Adult Treatment Editorial Board, which is editorially independent of the National Cancer Institute (NCI). The summary reflects an independent review of the literature and does not represent a policy statement of NCI or the National Institutes of Health (NIH).

Board members review recently published articles each month to determine whether an article should:

be discussed at a meeting,
be cited with text, or
replace or update an existing article that is already cited.

Treatment Option Overview for AML

Successful treatment of acute myeloid leukemia (AML) requires the control of bone marrow and systemic disease and specific treatment of central nervous system (CNS) disease, if present. The cornerstone of this strategy includes systemically administered combination chemotherapy. Because only 5% of patients with AML develop CNS disease, prophylactic treatment is not indicated.[1–3]

Treatment is divided into two phases: remission induction (to attain remission) and postremission (to maintain remission). Maintenance therapy for AML was previously administered for several years but is not included in most current treatment clinical trials in the United States, other than for acute promyelocytic leukemia. (Refer to the Adult Acute Myeloid Leukemia in Remission section of this summary for more information.) Other studies have used more intensive postremission therapy administered for a shorter duration of time after which treatment is discontinued.[4] Postremission therapy appears to be effective when given immediately after remission is achieved.[4]

Since myelosuppression is an anticipated consequence of both the leukemia and its treatment with chemotherapy, patients must be closely monitored during therapy. Facilities must be available for hematologic support with multiple blood fractions including platelet transfusions and for the treatment of related infectious complications.[5] Randomized trials have shown similar outcomes for patients who received prophylactic platelet transfusions at a level of 10,000/mm3 rather than 20,000/mm3.[6] The incidence of platelet alloimmunization was similar among groups randomly assigned to receive pooled platelet concentrates from random donors; filtered, pooled platelet concentrates from random donors; ultraviolet B-irradiated, pooled platelet concentrates from random donors; or filtered platelets obtained by apheresis from single random donors.[7] Colony-stimulating factors, for example, granulocyte colony–stimulating factor (G-CSF) and granulocyte-macrophage colony–stimulating factor (GM-CSF), have been studied in an effort to shorten the period of granulocytopenia associated with leukemia treatment.[8] If used, these agents are administered after completion of induction therapy. GM-CSF was shown to improve survival in a randomized trial of AML in patients aged 55 to 70 years (median survival was 10.6 months vs. 4.8 months). In this Eastern Cooperative Oncology Group (ECOG) (EST-1490) trial, patients were randomly assigned to receive GM-CSF or placebo following demonstration of leukemic clearance of the bone marrow;[9] however, GM-CSF did not show benefit in a separate similar randomized trial in patients older than 60 years.[10] In the latter study, clearance of the marrow was not required before initiating cytokine therapy. In a Southwest Oncology Group (NCT00023777) randomized trial of G-CSF given following induction therapy to patients older than 65 years, complete response was higher in patients who received G-CSF because of a decreased incidence of primary leukemic resistance. Growth factor administration did not impact on mortality or on survival.[11,12] Because the majority of randomized clinical trials have not shown an impact of growth factors on survival, their use is not routinely recommended in the remission induction setting.

The administration of GM-CSF or other myeloid growth factors before and during induction therapy, to augment the effects of cytotoxic therapy through the recruitment of leukemic blasts into cell cycle (growth factor priming), has been an area of active clinical research. Evidence from randomized studies of GM-CSF priming have come to opposite conclusions. A randomized study of GM-CSF priming during conventional induction and postremission therapy showed no difference in outcomes between patients who received GM-CSF and those who did not receive growth factor priming.[13,14][Level of evidence: 1iiA] In contrast, a similar randomized placebo-controlled study of GM-CSF priming in patients with AML aged 55 to 75 years showed improved disease-free survival (DFS) in the group receiving GM-CSF (median DFS for patients who achieved complete remission was 23 months vs. 11 months; 2-year DFS was 48% vs. 21%), with a trend towards improvement in overall survival (2-year survival was 39% vs. 27%, P = .082) for patients aged 55 to 64 years.[15][Level of evidence: 1iiDii]

References

Kebriaei P, Champlin R, deLima M, et al.: Management of acute leukemias. In: DeVita VT Jr, Lawrence TS, Rosenberg SA: Cancer: Principles and Practice of Oncology. 9th ed. Philadelphia, Pa: Lippincott Williams & Wilkins, 2011, pp 1928-54.
Wiernik PH: Diagnosis and treatment of acute nonlymphocytic leukemia. In: Wiernik PH, Canellos GP, Dutcher JP, et al., eds.: Neoplastic Diseases of the Blood. 3rd ed. New York, NY: Churchill Livingstone, 1996, pp 283-302.
Morrison FS, Kopecky KJ, Head DR, et al.: Late intensification with POMP chemotherapy prolongs survival in acute myelogenous leukemia–results of a Southwest Oncology Group study of rubidazone versus adriamycin for remission induction, prophylactic intrathecal therapy, late intensification, and levamisole maintenance. Leukemia 6 (7): 708-14, 1992. [PUBMED Abstract]
Cassileth PA, Lynch E, Hines JD, et al.: Varying intensity of postremission therapy in acute myeloid leukemia. Blood 79 (8): 1924-30, 1992. [PUBMED Abstract]
Supportive Care. In: Wiernik PH, Canellos GP, Dutcher JP, et al., eds.: Neoplastic Diseases of the Blood. 3rd ed. New York, NY: Churchill Livingstone, 1996, pp 779-967.
Rebulla P, Finazzi G, Marangoni F, et al.: The threshold for prophylactic platelet transfusions in adults with acute myeloid leukemia. Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto. N Engl J Med 337 (26): 1870-5, 1997. [PUBMED Abstract]
Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. The Trial to Reduce Alloimmunization to Platelets Study Group. N Engl J Med 337 (26): 1861-9, 1997. [PUBMED Abstract]
Geller RB: Use of cytokines in the treatment of acute myelocytic leukemia: a critical review. J Clin Oncol 14 (4): 1371-82, 1996. [PUBMED Abstract]
Rowe JM, Andersen JW, Mazza JJ, et al.: A randomized placebo-controlled phase III study of granulocyte-macrophage colony-stimulating factor in adult patients (> 55 to 70 years of age) with acute myelogenous leukemia: a study of the Eastern Cooperative Oncology Group (E1490). Blood 86 (2): 457-62, 1995. [PUBMED Abstract]
Stone RM, Berg DT, George SL, et al.: Granulocyte-macrophage colony-stimulating factor after initial chemotherapy for elderly patients with primary acute myelogenous leukemia. Cancer and Leukemia Group B. N Engl J Med 332 (25): 1671-7, 1995. [PUBMED Abstract]
Dombret H, Chastang C, Fenaux P, et al.: A controlled study of recombinant human granulocyte colony-stimulating factor in elderly patients after treatment for acute myelogenous leukemia. AML Cooperative Study Group. N Engl J Med 332 (25): 1678-83, 1995. [PUBMED Abstract]
Godwin JE, Kopecky KJ, Head DR, et al.: A double-blind placebo-controlled trial of granulocyte colony-stimulating factor in elderly patients with previously untreated acute myeloid leukemia: a Southwest oncology group study (9031). Blood 91 (10): 3607-15, 1998. [PUBMED Abstract]
Buchner T, Hiddemann W, Wormann B, et al.: GM-CSF multiple course priming and long-term administration in newly diagnosed AML: hematologic and therapeutic effects. [Abstract] Blood 84 (10 Suppl 1): A-95, 27a, 1994.
Löwenberg B, Boogaerts MA, Daenen SM, et al.: Value of different modalities of granulocyte-macrophage colony-stimulating factor applied during or after induction therapy of acute myeloid leukemia. J Clin Oncol 15 (12): 3496-506, 1997. [PUBMED Abstract]
Witz F, Sadoun A, Perrin MC, et al.: A placebo-controlled study of recombinant human granulocyte-macrophage colony-stimulating factor administered during and after induction treatment for de novo acute myelogenous leukemia in elderly patients. Groupe Ouest Est Leucémies Aiguës Myéloblastiques (GOELAM). Blood 91 (8): 2722-30, 1998. [PUBMED Abstract]

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Proceedings of the NYAS

Posted in Biological Networks, CANCER BIOLOGY & Innovations in Cancer Therapy, Curation, Disease Biology, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Hematopoiesis, Innovation in Immunology Diagnostics, tagged Cancer - General, NYAS, P13K signaling on September 25, 2015| Leave a Comment »

Proceedings of the NYAS

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Harnessing Cell Signaling to Treat Cancer

The 2015 Ross Prize in Molecular Medicine

Speakers: José Baselga (Memorial Sloan-Kettering Cancer Center) and Lewis C. Cantley (Weill Cornell Medical College)Presented by the Feinstein Institute for Medical Research, Molecular Medicine, and the New York Academy of Sciences

Reported by Hema Bashyam | Posted September 10, 2015

http://www.nyas.org/Publications/EBriefings/Detail.aspx?cid=9f87d38e-7295-4205-a46e-6d5839c8a471

The Ross Prize in Molecular Medicine was established in conjunction with the Feinstein Institute for Medical Research and Molecular Medicine. The Prize recognizes biomedical scientists whose discoveries have changed the way medicine is practiced. It is awarded to scientists who have made a significant impact in the understanding of human disease pathogenesis and/or treatment and who hold significant promise for making even greater contributions to the general field of molecular medicine.

The 2015 Ross Prize in Molecular Medicine was awarded to Dr. Lewis C. Cantley for his critical discoveries regarding signaling pathways in cancer cells. Dr. Cantley discovered the phosphoinositide 3-kinases (PI3K), which are critical for cell growth, proliferation, and survival. Following this discovery, he demonstrated that activity-enhancing mutations in PI3K were present in several types of human cancer, and he continued to investigate small molecule inhibitors of PI3K that are now approved for use in the treatment of cancer.

PI3K.Akt signaling pathway

PI3K signaling pathway inhibitors in solid tumors and hematological malignancies

On June 8, 2015, the Feinstein Institute for Medical Research and its journal Molecular Medicine presented the 2015 Ross Prize at the New York Academy of Sciences. The Feinstein Institute’s focus is on advancing science to prevent disease and cure patients. Established in 2013, the prize has gone to scientists who have made seminal scientific observations and translated their findings into clinical applications. The symposium, titled Harnessing Cell Signaling Pathways to Treat Cancer, honored Lewis C. Cantley, the Meyer Director of the Sandra and Edward Meyer Cancer Center at Weill Cornell Medical College and New York-Presbyterian Hospital.

Cantley’s work on cellular responses to growth factors and hormones and his discovery of the enzyme phosphoinositide 3-kinase (PI3K), its signaling pathway, and the PI3K activity-enhancing mutations in several cancers furthered the current understanding of cell growth, malignant transformation, and the relationship between metabolism and cancer. The PI3K enzyme, which produces a cancer-driving lipid, is the target of several inhibitor molecules now in clinical trials for different types of cancer. Idelalisib, the first PI3K inhibitor to win FDA approval, has been a second-line treatment for chronic lymphocytic leukemia since mid-2014.

Harnessing Cell Signaling to Treat Cancer: The 2015 Ross Prize in Molecular Medicine. Academy eBriefings. 2015. Available at: www.nyas.org/RossPrize2015-eB

Advances in Immunomodulation

The 2014 Ross Prize in Molecular Medicine

Speakers: James P. Allison (University of Texas MD Anderson Cancer Center), Charles A. Dinarello (University of Colorado–Denver), and John J. O’Shea (National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH)

Presented by the Feinstein Institute for Medical Research, Molecular Medicine, and the New York Academy of Sciences

Reported by Hema Bashyam | Posted August 12, 2014

On June 9, 2014, the Feinstein Institute for Medical Research and Molecular Medicine presented the 2014 Ross Prize in Molecular Medicine at New York Academy of Sciences. The symposium, titled Advances in Immunomodulation, honored this year’s awardee, John J. O’Shea, scientific director of the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) at the National Institutes of Health (NIH). The award ceremony was followed by presentations by O’Shea and other immunologists, who described discoveries that have enabled therapeutic targeting of cytokine signaling molecules in autoimmune and inflammatory diseases and of immune checkpoint modulators such as CTLA4 in cancer.

https://youtu.be/3dtKDonbraA

The 2014 Ross Prize in Molecular Medicine was awarded to Dr. John J. O’Shea for his discoveries in immunology and cytokine biology.

Cancer Cell Metabolism

Unique Features Inform New Therapeutic Opportunities

Organizers: Lydia Finley (Memorial Sloan-Kettering Cancer Center), Steven S. Gross (Weill Cornell Medical College), Costas A. Lyssiotis (Weill Cornell Medical College), and Sonya Dougal (The New York Academy of Sciences)
Keynote Speaker: Craig B. Thompson (Memorial Sloan-Kettering Cancer Center)Presented by Hot Topics in Life Sciences

Reported by Paul Riccio | Posted August 14, 2015

Cancer cells become lethal when they form large tumors, metastasize, and colonize diverse tissue types. These functions depend on different metabolic pathways from those active in non-transformed cells. Rapidly growing and proliferating cells require constant biosynthesis, in addition to energy in the form of ATP that all cells need for normal functions. The glucose and glutamine that are exclusively catabolized to water and carbon dioxide in quiescent cells are partly diverted to macromolecule production in dividing cells. Cell metabolism has long been a focus of molecular biology; for the cancer field its study represents a return to original lines of inquiry after years of focus on the genetics of cell transformation and the oncogenes involved in signal transduction pathways. At the May 28, 2015, Hot Topics in Life Sciences symposium Cancer Cell Metabolism: Unique Features Inform New Therapeutic Opportunities, the speakers expressed hope that a synthesis of these two approaches will yield progress in cancer research.

Craig B. Thompson of the Memorial Sloan-Kettering Cancer Center reviewed the basics of cell metabolism. After the initial step in glucose metabolism—glycolysis, conversion of glucose to two molecules of pyruvate—mitochondrial oxidative phosphorylation usually proceeds to yield ATP. But even in the presence of oxygen, many cancer cells divert pyruvate to fermentation, producing lactate. This less rewarding mode of ATP production demands a relatively high rate of glycolysis. Otto Warburg described this shift toward “aerobic glycolysis” in cancer cells in 1924. The molecular and genetic basis of the Warburg effect, however, has only recently come to light. Contrary to Warburg’s hypothesis that mitochondrial defects necessitate this shift, most cancer cells maintain the ability to execute oxidative phosphorylation and do fully catabolize a small amount of glucose.

Cancer cells are genetically differentiated from normal cells, but it is now clear that the metabolic shifts they exhibit are also partly required for division of normal cells. In a quiescent cell, maximum ATP production yields enough energy for cellular machinery, and at least 50% of free energy is used for ion transport across the membrane. When a cell divides, glycolytic intermediates are diverted from the tricarboxylic acid (TCA/Krebs) cycle to reserve carbon and nitrogen for fatty acid synthesis and for production of nonessential amino acids. DNA replication demands de novo nucleotide synthesis, beyond the supply garnered from recycling pathways in a non-dividing cell. Ribose, serine, and glycine (byproducts of glucose metabolism), as well as glutamine for pyrimidine production, are needed for nucleotide synthesis.

cancer cells consume glucose and glutamine

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Cancer cells consume glucose and glutamine at higher levels than normal cells; many oncogenes implicated in signaling cascades also regulate metabolism of these nutrients. Research in cell signaling and metabolism may produce more effective combination therapies to treat cancer. (Image courtesy of Craig B. Thompson)

Glutamine carbon is required for hypoxic cell viability

glutamine-supplies-the-succinate-pool-through-oxidative-and-reductive-metabolism

Thompson noted that previously overlooked clues from cell biology research are fundamental to current work to understand the unique metabolism of cancer cells. Harvey Eagle, who is largely credited with establishing a protocol and eponymous growth medium for culturing mammalian cells, did much of his foundational work with the HeLa cell line, derived from a pancreatic tumor. He observed a key feature of proliferating cells’ metabolism when he found that these cells need to be cultured in a buffered solution supplemented with glucose and, to his surprise, high levels of the nonessential amino acid glutamine. In an anabolic cell where Kreb’s intermediates are diverted to biosynthesis, glutamine metabolism provides a means of anaplerosis. Indeed, some tumors are said to be “glutamine addicted” and cannot survive without exogenous glutamine. The pharmaceutical company Calithera seeks to exploit this property with a new drug, CB-839, that inhibits glutaminase. It is in phase I clinical trials for treating cancer.

Christian Metallo of the University of California, San Diego, has used metabolic tracing to show that the glutamine contribution to lipogenesis shifts in mammalian cells in an oxygen-dependent manner. Cells cultured under hypoxic conditions, which might mimic those experienced by cancer cells in a poorly vascularized tumor, shift to reductive carboxylation of α-ketoglutarate. This glucose and glutamine derivative is also critical in stem cells.Lydia Finley of the Memorial Sloan-Kettering Cancer Center linked α-ketoglutarate levels in embryonic stem cells to the maintenance of demethylation and pluripotency.

Thompson argued that metabolic shifts that define cancer cells are not a secondary consequence of transformation, as has long been thought. The most common genetic mutations that drive cancers are in proto-oncogenes and tumor suppressor genes, which regulate pathways that control cell division. The constitutive activation of the cell cycle and the loss of checkpoints, however, are not enough to drive unmitigated growth; the cell must also undergo metabolic transformation to meet the energetic and synthetic demands of growth. This hypothesis, although perhaps intuitive, has only gained prominence with observations linking metabolic genes to control by signal transduction pathways.

The most commonly mutated gene in cancers is KRAS. The KRAS protein, a GTPase, normally functions as a molecular switch, relaying signals received by receptor tyrosine kinases and other receptors of extracellular signals. Two of its main targets include the MAPK and PI3K signal transduction cascades. But many indirect targets of KRAS are involved in cellular metabolism, including glucose transporters that are positively regulated by the PI3K/Akt pathway. Glutamine-addicted tumors are often characterized by the oncogenic expression of Myc, a transcription factor that promotes the expression of glutamine transporters as well as metabolic enzymes needed for biosynthesis. Constitutively activated KRAS thus primes a cell to undergo aerobic glycolysis by ensuring a steady influx of glucose. Selina Chen-Kiang of the Weill Cornell Medical College showed that therapies targeting the cell cycle indirectly reduce PI3K activation in cancer cells. In collaboration with Pfizer, her research group has tested the Cdk4/6 inhibitor palbociclib in early-phase clinical trials in Mantle cell lymphoma (MCL) patients. Genetic and biochemical analysis of tumors from palbociclib-responsive MCL patients exhibited reduced glucose transporter expression. Palbociclib is currently being tested as a cancer therapy in five separate clinical trials.

Jon Blenis of the Weill Cornell Medical Center identified another common genetic hallmark of cancer linked to metabolism, activated mTORC1 (mTOR Complex 1) signaling. Pathways that activate this complex integrate metabolism by sensing levels of nutrients, such as amino acids leucine and glutamine. The complex then regulates glucose and glutamine metabolism, amino acid production, and lipid biosynthesis. Blenis’s group has screened for targets in the pathways to identify combination therapies for mTORC1-activated cancers. One combines the glutamine metabolism inhibitor BAPTES with the HSP90 inhibitor 17-AAG to reduce transformed cells’ ability to carry out aerobic glycolysis and confront oxidative stress. A novel downstream target of mTORC1, the kinase SPRK2, is another promising lead.

Each tumor arises from a unique sequence of genetic lesions. Does each also have a unique metabolic signature? Most conclusions on this topic are drawn from studying isolated cancer cell lines, but tumor cells may execute metabolism differently in vivo. Matthew G. Vander Heiden of the Massachusetts Institute of Technology described rodent models of non-small cell lung carcinoma and pancreatic cancer, both driven by KRAS-activation and p53-knockout mutations. The first model had tumors characterized by increased glucose uptake; surprisingly, these tumors had elevated glucose catabolism through oxidative phosphorylation, in addition to elevated glycolytic metabolism. Glutamine tracing, however, showed almost no glutamine anaplerosis in vivo. But when explanted to culture conditions, cells from these tumors acquired glutamine dependence. In the pancreatic cancer model, no single nutrient was clearly favored as a metabolite. Albumin labeling experiments, however, showed that most of the pancreatic cancer cell biomass was derived from this extracellular protein.

It has long been assumed that extracellular protein is not a major fuel for most cells, but as Thompson observed, there is evidence that mammalian cells can draw on this energy source. Most cell culture media are supplemented with serum containing albumin, and the 0.74 mM concentration of albumin found in human plasma is equivalent to a 400 mM source of amino acids. The finding that cancer cells and cultured normal cells ingest extracellular protein through micropinocytosis has invigorated the field. The normal pathway to nutrient acquisition in slime molds, this process involves forming a vesicle around the extracellular contents and ingesting whatever is present in the surrounding media. The tumor cells in Vander Heiden’s pancreatic cancer model fed biosynthesis and anaplerosis through this unusual process of macropinocytosis and protein catabolism.

branched-chain amino acids are significant contributors to fatty acid synthesis in proliferating adipocytes

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Using stable isotope tracers and mass spectrometry to quantify label incorporation, Metallo made the surprising observation that branched-chain amino acids are significant contributors to fatty acid synthesis in proliferating adipocytes. Cancer cells also catabolize protein through a lysosomal pathway to fuel biosynthesis. Inhibiting autophagy and protein catabolism in tumor cells could thus be new strategies for cancer therapies. (Image courtesy of Christian Metallo)

Thompson’s data show that mouse embryonic fibroblasts grown in leucine-depleted media cease proliferating. But their growth is rescued by the addition of albumin in excess of 3%. The addition of chloroquine to inhibit lysosomal proteolysis confirmed that albumin is metabolized via a macropinocytic mechanism; blocking the cell’s ability to form lysosomal vesicles abrogated the albumin rescue. Alec C. Kimmelman of Harvard Medical School explained that autophagy, another lysosome-dependent pathway, could be a therapeutic target in cancers such as pancreatic adenocarcinoma. Activated KRAS–driven pancreatic cancers have historically been among the most difficult to treat, with a 5-year survival rate of 6%. Kimmelman’s data show that genetic or pharmacologic inhibition of autophagy slows growth of pancreatic cancer cell lines and of tumors in genetically engineered mouse models. This work also demonstrates that autophagy has key roles in the metabolism of these tumors.

Altered metabolism facilitates the rapid proliferation of transformed cells; it is also implicated in metastasis and in the maintenance of pluripotency. Elena Piskounova of the University of Texas Southwestern Medical Center explained that successful metastasis, which is associated with worse cancer outcomes, is a relatively rare event; for every thousand cells that dissociate from a tumor, only one or two will successfully colonize a new site. Using a xenograft model with human melanoma cells introduced to immunocompromised mice, Piskounova showed that the limiting step in metastasis was cell survival in the circulatory system. Metabolic profiling revealed that these cells have high levels of oxidized glutathione and reactive oxygen species. Treatment of the xenografted mice with antioxidants increased the rate of metastasis, suggesting that, to survive, circulating tumor cells (CTCs) must overcome oxidative stress. Piskounova explained that cancer cells’ need for NADPH, a reducing molecule that helps cells combat oxidative stress, might be met through folate metabolism.

If it survives oxidative stress, a CTC must adapt metabolically to the target tissue it colonizes. Sohail Tavazoie of the Rockefeller University studies how colon cancer–derived cells colonize the liver. A screen for microRNAs that suppress colon-to-liver metastasis identified mir-483 and mir-551a, which targeted the brain-type creatine kinase (CKB). Further study revealed that this kinase is secreted from cells and then converts creatine to phosphocreatine in an ATP-dependent reaction. Transport of the phosphocreatine back into the cells provides a catabolic substrate to fuel cellular energy and biosynthetic needs. Drugs that promote oxidative stress or block folate metabolism, NADPH production, or creatine utilization hold promise as therapies to prevent metastasis.

The New York Academy of Sciences. Cancer Cell Metabolism: Unique Features Inform New Therapeutic Opportunities. Academy eBriefings. 2015. Available at: www.nyas.org/TumorMetabolism-eB

Click Chemistry in Biology and Medicine

Organizers: Peng Wu (Albert Einstein College of Medicine) and Jennifer Henry (formerly at The New York Academy of Sciences)
Keynote Speakers: Jim Paulson (Scripps Research Institute) and K. Barry Sharpless (Scripps Research Institute)Presented by the Chemical Biology Discussion Group

Reported by Megan Stephan | Posted November 21, 2014

click chemistry

http://www.nyas.org/image.axd?id=b9daf7c7-763f-4fe7-8b85-959be16e67e0&t=635513990247670000

Click chemistry is a new approach to synthetic organic chemistry that uses simple chemical building blocks and mild aqueous conditions to produce high-specificity ligands and labeling molecules for the investigation of biological systems. These reactions mimic those found in nature for building large complex molecules such as proteins, DNA, and RNA. Since its inception by K. Barry Sharpless, click chemistry has yielded several novel reactions that have been used to produce experimental tools and therapeutic agents, some of which are now in preclinical testing. The products of these reactions are particularly useful for investigating difficult-to-synthesize biological products such as glycans, and challenging, highly transient signaling activities such as those depending on redox reactions. On September 15, 2014, pioneers and young investigators in the field presented their recent work, providing a glimpse into the expanding possibilities of this synthetic methodology. The symposium, Click Chemistry in Biology and Medicine: New Developments and Strategies, was presented by the Academy’s Chemical Biology Discussion Group.

Lung Cancer

Advances in Current Treatment Modalities and Patient Classification

Organizers: Magdalena Alonso-Galicia (Forest Research Institute), Shashidhar S. Jatiani (Forest Research Institute), Huiping Jiang (Boehringer Ingelheim Pharmaceuticals), George Zavoico (HC Wainwright), and Jennifer Henry (The New York Academy of Sciences)
Speakers: Rolf Brekken (UT Southwestern Medical Center), Jessica S. Donington (NYU Langone Medical Center), Balazs Halmos (Columbia University Medical Center), Roy S. Herbst (Yale School of Medicine), and Suresh S. Ramalingam (Emory University)Presented by the Biochemical Pharmacology Discussion Group

Reported by Paul Riccio | Posted May 28, 2014

Like other cancers of the internal organs not often diagnosed until late stages of disease, lung cancer is among the deadliest. In the U.S., the 5-year survival rate after diagnosis is lower than 15%. Lung cancer is also among the most common cancers, in part because of the continued prevalence of tobacco smoking. The American Cancer Association estimates that in 2014, 224 210 people will be newly diagnosed with lung cancer and 159 260 deaths will be associated with the disease. On March 25, 2014, the Biochemical Pharmacology Discussion Group convened physicians and scientists for the Lung Cancer: Advances in Current Treatment Modalities and Patient Classification symposium, to discuss advances in treatment as well as new information about genetic heterogeneity that may inform future trials. Despite poor outcomes for current treatments, the speakers were optimistic that therapeutic innovations will gradually improve prognoses for this seemingly intractable disease.

From refinements in surgical techniques to drugs targeting specific oncoproteins, the range of precision therapeutic tools to combat lung cancer is growing. Several speakers cautioned, however, that technological innovation alone will not dramatically transform lung cancer treatment. Rather, physicians must tailor treatments for each patient and deploy tools discriminately, biopsying and sequencing each tumor, for example, and choosing the most appropriate drugs among many. Roy Herbst and Balazs Halmos both related adaptive treatments that resulted in several rounds of remission in patients. However, successes like these have been achieved in large medical centers where clinicians had access to drug trials. Adapting protocols for personalized medicine in a wide range of clinical settings remains challenging.

In his introduction to the symposium, Shashidhar S. Jatiani of the Forest Research Institute reviewed the sub-classification of lung cancer into small cell and non-small cell types. The former is a grim diagnosis: patients almost always present with advanced disease because metastasis occurs early. Chemotherapy is the standard treatment. More common, however, is a diagnosis of non-small cell lung cancer (NSCLC), which accounts for approximately 85% of new cases. The speakers focused on the treatment of NSCLCs, which usually arise in the epithelium of the branched lung, most commonly as an adenocarcinoma.

When NSCLC is identified as a pre-metastatic tumor, the most effective treatment is surgery. Jessica S. Doningtonof NYU Langone Medical Center explained that a balance must be achieved between complete removal of cancerous cells and preservation of lung function. The standard of care is removal of the lobe containing the tumor, one of five lobes in the lungs. A complete lobectomy is an invasive procedure, requiring the surgeon to break several ribs to access the thoracic cavity. The average age of patients at diagnosis is 70 years old, and many patients require substantial care to recover from such a surgery. But it has become possible to perform more precise surgery: rather than remove the lobe entirely, many surgeons now resect a minimal amount of tissue, directed by the branched morphology of the lung, in a procedure called a segmentectomy. In addition, the use of laparoscopic cameras in a technique known as video-assisted thoracic surgery (VATS) now limits the size of the incision to the circumference of the tumor itself. An increasing number of hospitals now support robot-assisted surgery using the da Vinci Surgical System, which can also be used to achieve less-invasive segmentectomies. Both techniques decreased morbidity and mortality when compared to open thoracic surgery. Although purchasing and maintaining a da Vinci system is a substantial investment, significantly less money is required for postoperative intensive care and respiratory therapy when VATS and robot-assisted surgery are used compared to open surgery.

New technologies in the operating room, such as the robotic da Vinci Surgical System pictured above, allow surgeons to remove malignant tissue more precisely. Laparoscopic and robot-assisted surgeries yield faster patient recoveries and fewer post-operative expenses. (Image courtesy of Jessica S. Donington)

http://www.nyas.org/image.axd?id=9c706d8a-56c0-4a41-a38c-1070e94d8301&t=635358375925400000

Inoperable tumors that arise close to the main airways, or in patients too frail to undergo surgery, are treated with radiation therapy. In an adaptation of a radiotherapy technique originally applied to brain tumors, lung tumors are now targeted more precisely and with higher doses of radiation using hypo-fractionated stereotactic body radiation therapy (SBRT). In the Netherlands, where practitioners have aggressively phased out older modes of radiation therapy in favor of SBRT, a greater than 36% increase in 8-month survival has been observed concurrent with the change.

Underlying the success of VATS, robot-assisted surgery, and SBRT is the theme that “less is more,” Donington said. Less healthy tissue is destroyed, less time and resources are required for patient recovery, and patients are ideally better prepared to undergo adjuvant therapy.

When the primary tumor has metastasized, as in more advanced cases, chemotherapy is often prescribed in addition to surgical or radiological interventions. Unfortunately, in most patients traditional chemotherapy regimens afford delayed progression at best and are rarely curative. No progress has been achieved in developing more potent chemotherapies or combinations of cytotoxic drugs for lung cancer in over ten years. In response, many physician-scientists, including symposium speakers Roy S. Herbst, Balazs Halmos, and Suresh S. Ramalingam, have begun studying the efficacy of new drugs targeting specific oncogenic pathways. Through whole exome sequencing of lung tumors, a portrait of the most common genetic aberrations has emerged. Mutations in KRAS have been known to occur in lung adenocarcinomas for at least ten years, and comprehensive sequencing efforts have additionally implicated oncogenes, including the receptor tyrosine kinases EGFR, MET, RET, NTRK1, ROS1, and HER2; the serine threonine kinase BRAF; and the atypical receptor tyrosine kinase ALK. In fact, only 37% of examined lung adenocarcinomas do not harbor mutations in any of these genes.

Molecular testing of biopsied lung tumors

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Molecular testing of biopsied lung tumors is necessary to match patients with the best therapeutic options. Through clinical trials and collaborative sequencing efforts, a comprehensive list of the mutations that drive lung cancer is emerging. As shown above in data reported by the Lung Cancer Mutation Consortium, these mutations are frequently in oncogenes, particularly receptor tyrosine kinases. (Image courtesy of Suresh S. Ramalingam)

Providing some historical perspective, Roy S. Herbst of Yale School of Medicine told participants that the first-generation receptor tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib entered the clinic in 1997, delivering modest response rates of less than 10%. Ten years later, with the emergence of deeper tumor exome sequencing, it became clear that responders were much more likely to harbor specific EGFR mutations, such as deletion of exons 19 or 21, which encode parts of the catalytic domain. Tumors with EGFR exon 19 deletion, in particular, exhibit susceptibility to erlotinib, which functions as a competitive inhibitor at the ATP binding site. Balazs Halmosof Columbia University Medical Center observed that based on the specificity of the TKIs, the conscientious clinician will need to become something of a “mutation aficionado,” to be able to match the most efficacious TKI to each patient. At present, such choices are largely confined to clinical trials because erlotinib, gefitinib, imatinib, and afatinib are the only TKIs approved for lung cancer. Physician training will be needed as more of these drugs achieve FDA approval. To illustrate the complexity of genetics-based drug choice, Herbst mentioned the BATTLE-2 clinical trial, which he oversees. It compares several TKIs and employs a full-time statistician to conduct a Bayesian learning method to help oncologists to make the best drug/tumor match.

Receptor tyrosine kinase (RTK) inhibitors may target the mutated pathways that manifest in many lung adenocarcinomas, but a considerable proportion of tumors do not exhibit obvious mutations. From a therapeutic perspective, therefore, drugs must be developed to target more general molecular pathways that are active in tumor cells. Suresh S. Ramalingam of Emory University reviewed one such strategy, which targets the heat-shock protein HSP90. Heat-shock proteins bind and stabilize client proteins, including oncoproteins, thus preventing their degradation through the proteasome. Drugs that inhibit HSPs, such as the second-generation HSP90 inhibitor ganetespib, should theoretically facilitate the indirect degradation of oncoproteins. Moreover, such drugs will only target cancer cells, because HSP90 remains at very low levels in healthy tissue. Ramalingam outlined an active phase III study of ganetespib, the GALAXY-2 trial.

The TKIs are indeed revolutionary tools in the field of molecular oncology, but frustratingly, these novel agents only delay disease progression and do not increase survival rates among lung cancer patients. Selective pressures in the tumor microenvironment favor either cells that acquire subsequent mutations or the activation of downstream effectors that thwart the loss of the RTK. As Halmos explained, these mutations often occur in the RTKs themselves. In the case of epidermal growth factor receptor (EGFR), insertions into exon 20, or a threonine to methionine substitution in a part of the protein encoded by exon 20, render the mutant receptor resistant to TKIs. The speakers speculated that combinations of drugs targeting several oncoproteins may circumvent the problem of tumor resistance, but available agents are limited and there are no obvious putative combination therapies.

Rolf Brekken of UT Southwestern Medical Center outlined the rationale for using immunotherapy to combat lung cancer. A hallmark of tumor cells is their evasion of immune detection, achieved in part by the presentation of phosphatidylserine (PS) in the outer cell membrane. In healthy cells, this phospholipid is confined to the inner leaflet of the cell membrane. When PS becomes externalized, as in normal apoptosis, PS receptors in macrophages that detect the dying cell trigger the release of immunosuppressive cytokines. Brekken hypothesized that if this process can be inhibited, an adaptive immune response might ensue. The monoclonal antibody bavituximab, developed by Peregrine Pharmaceuticals, targets glycoprotein-bound PS. Preclinical data using bavituximab to combat a rodent tumor model is encouraging, with tumors completely regressing in some rats. Brekken’s lab has observed several lines of evidence suggesting that an innate immune response occurred in bavituximab-treated rats, leading to the development of anti-tumor T cells. As several speakers noted, the best test of this therapy would be to reintroduce tumor cells to the rats and observe whether the cells are targeted and destroyed by an immune response. Such a result would be astonishing. Indeed, the potential of immunotherapy in cancer treatment has recently received considerable attention from the popular media. Several antibodies have entered clinical trials, including bavituximab, which is in a phase III NSCLC trial called SUNRISE.

From new developments in surgery and radiation therapy to a rapidly expanding collection of drugs in the field of molecular oncology, we are poised to improve the prognosis for many lung cancer patients. Key to success will be a continued emphasis on personalized medicine, augmenting the standard diagnostic regimen to include molecular testing to match each patient with a targeted therapeutic approach.

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Steroids, Inflammation, and CAR-T Therapy

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, FDA, FDA Regulatory Affairs, Immuno-Oncology & Genomics, Innovation in Immunology Diagnostics, Lymphoma, Personal Health Applications: Tech Innovations serves HealhCare, Personalized and Precision Medicine & Genomic Research, tagged acute and chronic leukemias, b cell lymphoma, Cancer - General, Cancer immunotherapy, CAR-T, chemotherapeutic toxicity, Conditions and Diseases, FDA, Food and Drug Administration, Hodgkin's lymphoma, medicine, Non-Hodgkin lymphoma, Personalized medicine, research, toxicity on September 14, 2015| Leave a Comment »

Steroids, Inflammation, and CAR-T Therapy [6.3.8] (UPDATED)

Reporter: Stephen J. Williams, Ph.D.

UPDATED: 08/31/2020 (CRISPR edited CAR-T clinical trials)

Word Cloud by Daniel Menzin

Corticosteroids have been used as anticancer agents since the 1940s, with activity reported in a wide variety of solid tumors, including breast and prostate cancer, and the lymphoid hematologic malignancies. They are commonly found in regimens for acute lymphocytic leukemia, Hodgkin’s and non-Hodgkin’s lymphoma, myeloma, and chronic lymphocytic leukemia.

A great review on the mechanism of action of prednisone’s antitumoral activity is seen in

Corticosteroids in the Treatment of Neoplasms Lorraine I. McKay, PhD and John A. Cidlowski, PhD. in Holland-Frei Cancer Medicine. 6th edition.

It was first discovered that cortisone caused tumor regression in a transplantable mouse lymphosarcoma,⁸¹ a finding soon extended to a wide variety of murine lymphatic tumors. The effects of corticosteroids were also evaluated on many nonendocrine and nonlymphoid transplantable rodent tumors. Pharmacologic doses of steroid inhibited growth of various tumor systems.⁸² Tissue culture studies confirmed that lymphoid cells were the most sensitive to glucocorticoids, and responded to treatment with decreases in DNA, ribonucleic acid (RNA), and protein synthesis.⁸³ Studies of proliferating human leukemic lymphoblasts supported the hypothesis that glucocorticoids have preferential lymphocytolytic effects. The mechanism of action was initially thought to be caused by impaired energy use via decreased glucose transport and/or phosphorylation; it was later discovered that glucocorticoids induce apoptosis, or programmed cell death, in certain lymphoid cell populations.^84,⁸⁵

–For review on corticosteroids in cancer therapy see more at: http://www.cancernetwork.com/review-article/corticosteroids-advanced-cancer#sthash.IwHbekuI.dpuf

However, as Dana Farber’s Dr. George Canellos, M.D. ponders in Can MOPP be replaced in the treatment of advanced Hodgkin’s disease? Semin Oncol. Canellos GP¹. 1990 Feb;17(1 Suppl 2):2-6., many dose-limiting toxicities occur with MOPP (mechlorethamine, vincristine, procarbazine, prednisone) therapy used in advanced Hodgkin’s disease. Although, at the time, he generally was looking to establish combination therapies with less side effect, the advent of more personalized therapies as well as immunotherapies may indeed replace the older regimens for B-cell malignancies and Hodgkin’s disease, and their panels of toxicities.

Short-term side effects of prednisone (Cancer.gov prednisone description with side effects) as with all glucocorticoids, include high blood glucose levels (especially in patients with diabetes mellitus or on other medications that increase blood glucose, such as tacrolimus) and mineralocorticoid effects such as fluid retention.^[10] The mineralocorticoid effects of prednisone are minor, which is why it is not used in the management of adrenal insufficiency, unless a more potent mineralocorticoid is administered concomitantly.

Long-term side effects include Cushing’s syndrome, steroid dementia syndrome, truncal weight gain, osteoporosis, glaucoma and cataracts, type II diabetes mellitus, and depression upon dose reduction or cessation.

Therefore the oncology world has been moving toward therapies which are more selective with less dose-limiting toxicities (e.g. Rituximab), and are looking to CAR-T therapies as a possible replacement for standard chemotherapeutic regimens. However, as with prednisone, there have been serious adverse events in some CAR-T clinical trials. Luckily clinicians, as discussed below, have found supportive therapies to alleviate the most severe side effects to CAR-T.

This section will be refer to supportive therapies as those adjuvant therapy given to alleviate patient discomfort, reduce toxicities and adverse event, or support patient well-being during their course of chemotherapy, not adjuvant therapy to enhance antitumoral effect.

For more background information of CAR-T therapies and related issues please see my previous post

NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee

The following is a brief re-post of some of the important points for reference to this new posting.

1. Evolution of Chimeric Antigen Receptors

Early evidence had suggested that adoptive transfer of tumor-infiltrating lymphocytes, after depletion of circulating lymphocytes, could result in a clinical response in some tumor patients however developments showed autologous T-cells (obtained from same patient) could be engineered to express tumor-associated antigens (TAA) and replace the TILS in the clinical setting.

A brief history of construction of 2^nd and 3^rd generation CAR-T cells given by cancer.gov:

http://www.cancer.gov/cancertopics/research-updates/2013/CAR-T-Cells

Differences between second- and third-generation chimeric antigen receptor T cells. (Adapted by permission from the American Association for Cancer Research: Lee, DW et al. The Future Is Now: Chimeric Antigen Receptors as New Targeted Therapies for Childhood Cancer. Clin Cancer Res; 2012;18(10); 2780–90. doi:10.1158/1078-0432.CCR-11-1920)

Constructing a CAR T Cell (from cancer.gov)

The first efforts to engineer T cells to be used as a cancer treatment began in the early 1990s. Since then, researchers have learned how to produce T cells that express chimeric antigen receptors (CARs) that recognize specific targets on cancer cells.

The T cells are genetically modified to produce these receptors. To do this, researchers use viral vectors that are stripped of their ability to cause illness but that retain the capacity to integrate into cells’ DNA to deliver the genetic material needed to produce the T-cell receptors.

The second- and third-generation CARs typically consist of a piece of monoclonal antibody, called a single-chain variable fragment (scFv), that resides on the outside of the T-cell membrane and is linked to stimulatory molecules (Co-stim 1 and Co-stim 2) inside the T cell. The scFv portion guides the cell to its target antigen. Once the T cell binds to its target antigen, the stimulatory molecules provide the necessary signals for the T cell to become fully active. In this fully active state, the T cells can more effectively proliferate and attack cancer cells.

2. Consideration for Design of Trials and Mitigating Toxicities

Early Toxic effects– Cytokine Release Syndrome– The effectiveness of CART therapy has been manifested by release of high levels of cytokines resulting in fever and inflammatory sequelae. One such cytokine, interleukin 6, has been attributed to this side effect and investigators have successfully used an IL6 receptor antagonist, tocilizumab (Acterma™), to alleviate symptoms of cytokine release syndrome (see review Adoptive T-cell therapy: adverse events and safety switches by Siok-Keen Tey).

Early Toxic effects – Over-activation of CAR T-cells; mitigation by dose escalation strategy (as authors in reference [3] proposed). Most trials give billions of genetically modified cells to a patient.
Late Toxic Effects – long-term depletion of B-cells . For example CART directing against CD19 or CD20 on B cells may deplete the normal population of CD19 or CD20 B-cells over time; possibly managed by IgG supplementation

References

Ertl HC, Zaia J, Rosenberg SA, June CH, Dotti G, Kahn J, Cooper LJ, Corrigan-Curay J, Strome SE: Considerations for the clinical application of chimeric antigen receptor T cells: observations from a recombinant DNA Advisory Committee Symposium held June 15, 2010. Cancer research 2011, 71(9):3175-3181.
Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA: Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Molecular therapy : the journal of the American Society of Gene Therapy 2010, 18(4):843-851.
Kandalaft LE, Powell DJ, Jr., Coukos G: A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer. Journal of translational medicine 2012, 10:157.

3. Case Reports of Adverse Events and Their Amelioration During CAR-T Therapy

CAR-T Therapy have Had reports of Serious Adverse Events

From FierceBiotech UPDATED: Two deaths force MSK to hit the brakes on engineered T cell cancer study

April 6, 2014 | By John Carroll

Safety concerns forced investigators at Memorial Sloan-Kettering Cancer Center to suspend patient recruitment for an early-stage study of a closely watched approach to reengineering the immune system to fight cancer. Several days ago MSK updated a site on clinicaltrials.gov to note that it was halting recruitment for a small study using T cells reengineered with chimeric antigen receptors (CARs) against CD19-positive B cells for aggressive non-Hodgkin lymphoma, triggering concerns about the potential fallout at Juno Therapeutics, the biotech formed to commercialize the effort. And Sunday evening representatives for MSK revealed at the meeting of the American Association for Cancer Research in San Diego that the deaths of two patients spurred investigators to rethink the trial protocol on recruitment, revamping the patient profile to account for the threat of comorbidities while adjusting the dose “based on the extent of disease at the time of treatment.”

For more on this story please see

Source: http://www.fiercebiotech.com/story/memorial-sloan-kettering-hits-brakes-engineered-t-cell-cancer-study/2014-04-06

Keynote presentation by Carl H. June, recipient of The Richard V. Smalley MD 2013 Award

As reported in 2013 in Highlights and summary of the 28th annual meeting of the Society for Immunotherapy of Cancer by Paolo A Ascierto^1†, David H Munn^2†, Anna K Palucka^34† and Paul M Sondel in Journal of ImmunoTherapy of Cancer

“

Since 2005, SITC honors a luminary in the field who has significantly contributed to the advancement of cancer immunotherapy research by presenting the annual Richard V. Smalley MD Memorial Award, which is associated with the Smalley keynote lecture at the Annual SITC meeting. The awardee this year Carl H. June of the University of Pennsylvania, has led innovative translational research for over 25 years, with the most recent focus being the development of the Chimeric Antigen Receptor modified T-cell (CART) approach. Carl June summarized how the past 15 years of progress have expanded upon the original concept presented by Zelig Eshhar [4], in which variable regions of tumor-reactive monoclonal antibodies (mAbs) (V_H and V_L) are linked to transmembrane and signaling domains of T cell activating molecules to create membrane based receptors with specificity for the tumor antigen recognized by the original mAb [4]. These receptors can be transfected into T cells, for example with lentiviruses. Pre-clinical work demonstrated how CD3-ζ and 41BB signaling components enhanced proliferation and survival of T cells in hypoxic conditions. The initial clinical work has been done with CART reactive to CD-19 on malignant B cells, with progress particularly in chronic lymphocytic leukemia (CLL) in adults and acute lymphoblastic leukemia (ALL) in children [5,6]. As of the SITC meeting, CarlJune’s team had treated 35 patients with CLL and 20 with ALL. Of the 20 with ALL, ½ had relapsed after allogeneic BMT. Of these 20 children, 17 were in complete remission, and with persistent B cell aplasia; documenting the persistent effects of the CART cells. Toxicities included the persistent B cell aplasia and profound tumor lysis and cytokine storm, seen 1–2 weeks into the treatment for ALL. This cytokine storm has been ameliorated by using anti-IL6 mAb. The B cell aplasia, while undesired, is acceptable, as patients can receive passive replacement of IgG, thus making their B cells “expendable”. These CART cells can traffic into the CNS. In ALL patients, it appears that each individual CART cell (or its progeny) can destroy 1000 tumor cells. Ongoing efforts in CarlJune’s program, and at other centers, are now moving into analyses of CART reactive with other tumor targets, by using mAbs that recognize antigens expressed on other tumors. Among these are EGFR on glioblastoma, PSMA on prostate cancer, mesothelin on ovarian cancer, HER2 on breast (and other) cancers, and several other targets. Because some of these targets are also expressed on normal tissues that are “not expendable”, novel approaches are being developed to decrease the potency or longevity of the CART effect, to decrease potential toxicity. This includes generating “short lived” CART cells by inducing CAR expression with short-lived RNA, rather than transfecting with a DNA construct that remains permanently.

“

In T-Cell Immunotherapy: Looking Forward Molecular Therapy (2014); 22 9, 1564–1574. doi:10.1038/mt.2014.148 many of the leading CAR-T clinicians and investigators reported on some of the adverse events related o CAR-T therapy including

40 severe adverse events (SAE) had been reported from 2010 to 2013.
B-cell aplasia
Systemic inflammatory release syndrome (CRS) {the most sever toxicity seen}
Tumor lysis syndrome
CNS toxicity
Macrophage activation syndrome

According to the investigators the systemic inflammatory release syndrome (CRS) is the most severe toxicity seen

“

The most commonly reported adverse event is CRS,⁴⁹ with about three-quarters of the patients with CRS requiring admission to an intensive care unit. In the case of CAR therapy, the onset of CRS is related to the particular signaling domain in the CAR, with early-onset CRS in the first several days after infusion related to CARs that encode a CD28 signaling domain.^4,16 By contrast, CARs encoding a 4-1BB signaling domain tend to have delayed-onset CRS (range, 7 to 50 days) after CAR T-cell infusion.⁶ CRS has also been reported after the infusion of TCR-modified T cells, with onset typically five to seven days after infusion. The development of CRS is often, but not invariably, associated with clinically beneficial tumor regression. Several cytokines have been reported to be elevated in the serum—most commonly, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-6. Management of CRS has included supportive care, corticosteroids, etanercept, tocilizumab, and alemtuzumab. The role of suicide genes in the management of CRS remains unknown.⁵⁰

“

This supportive therapy have now been included in all protocols now and sites are engaged in developing pharmacovigilance protocol development for CAR-T therapy.

UPDATED 08/31/2020

The following articles discuss the use of the CRISPR Cas9 system to improve the efficacy and reduce immune tolerance and rejection of engineered CAR-T therapies and the evolution of the CAR-T therapy in clinical trials for lung cancer and leukemias.

CRISPR-engineered immune cells reach the bedside

Bence György Science Translational Medicine 13 May 2020: Vol. 12, Issue 543, eabb7095 DOI: 10.1126/scitranslmed.abb7095

Abstract

In a clinical trial, CRISPR editing of T cells to fight lung cancer was feasible and safe.

Lung cancer is the most prevalent and fatal malignancy worldwide. Whereas the overall five-year cancer survival rates in the United States increased by 16.7% over the past five decades, lung cancer five-year survival rates only increased by 5.9%. Immune checkpoint inhibitors, such as antibodies against programmed cell death protein 1 (PD-1), are front-line anticancer treatments because PD-1 ligand on tumor cells efficiently inhibits immune responses.

Lu et al. published the results of a clinical trial, which aimed at knocking out PD-1 on T cells to confer antitumor activity against metastatic non–small cell lung cancer. The T cells were expanded ex vivo, then reinfused into 12 patients. The primary aim of the trial was to test feasibility and safety, and the secondary aims included efficiency and tracking of edited T cells. Overall, the treatment was well tolerated, with only mild to moderate adverse effects and no dose-limiting toxicities. Considerable care was taken to analyze clustered regularly interspaced short palindromic repeats (CRISPR)–induced off-target effects, and the study showed that median mutation frequency was 0.05% in off-target sites. Edited T cells were detected in the peripheral blood of 11 out of 12 patients and in tumor biopsy specimens from one patient up to 52 weeks after infusion. After infusion, two patients had stable disease, and it lasted up to 76 weeks in one patient.

This trial was performed cautiously to avoid major issues with off-target effects, and the researchers used T cells with relatively low editing rates (median of 5.8%). Nevertheless, the authors observed signs of biological activity in patients with metastatic disease who had failed several other lines of treatment. To improve biological efficiency, it will be critical to increase editing rates without increasing off-target effects. Although the off-target effects were close to background level in this study, genome-wide unbiased screens will be necessary, particularly when editing rates are boosted. A separate problem to address before broader clinical translation is T cell expansion, which failed in five patients in this study.

These early data suggest that CRISPR editing of T cells in humans is feasible and safe. Several other trials are ongoing and should help assess the potential benefit of ex vivo edited immune cells. With CRISPR-edited immune cells, it may be possible to get a better grip on metastatic lung cancer and other malignancies.

Source: Science at https://stm.sciencemag.org/content/12/543/eabb7095?_ga=2.977329.1688000087.1598874689-944049349.1598112445

Highlighted Article

Lu, J. Xue, T. Deng, X. Zhou, K. Yu, L. Deng, M. Huang, X. Yi, M. Liang, Y. Wang, H. Shen, R. Tong, W. Wang, L. Li, J. Song, J. Li, X. Su, Z. Ding, Y. Gong, J. Zhu, Y. Wang, B. Zou, Y. Zhang, Y. Li, L. Zhou, Y. Liu, M. Yu, Y. Wang, X. Zhang, L. Yin, X. Xia, Y. Zeng, Q. Zhou, B. Ying, C. Chen, Y. Wei, W. Li, T. Mok, Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat. Med. 26, 732–740 (2020).

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 editing of immune checkpoint genes could improve the efficacy of T cell therapy, but the first necessary undertaking is to understand the safety and feasibility. Here, we report results from a first-in-human phase I clinical trial of CRISPR–Cas9 PD-1-edited T cells in patients with advanced non-small-cell lung cancer (ClinicalTrials.gov NCT02793856). Primary endpoints were safety and feasibility, and the secondary endpoint was efficacy. The exploratory objectives included tracking of edited T cells. All prespecified endpoints were met. PD-1-edited T cells were manufactured ex vivo by cotransfection using electroporation of Cas9 and single guide RNA plasmids. A total of 22 patients were enrolled; 17 had sufficient edited T cells for infusion, and 12 were able to receive treatment. All treatment-related adverse events were grade 1/2. Edited T cells were detectable in peripheral blood after infusion. The median progression-free survival was 7.7 weeks (95% confidence interval, 6.9 to 8.5 weeks) and median overall survival was 42.6 weeks (95% confidence interval, 10.3–74.9 weeks). The median mutation frequency of off-target events was 0.05% (range, 0–0.25%) at 18 candidate sites by next generation sequencing. We conclude that clinical application of CRISPR–Cas9 gene-edited T cells is generally safe and feasible. Future trials should use superior gene editing approaches to improve therapeutic efficacy.

CRISPR-engineered T cells in patients with refractory cancer

BY EDWARD A. STADTMAUER, JOSEPH A. FRAIETTA, MEGAN M. DAVIS, ADAM D. COHEN, KRISTY L. WEBER, ERIC LANCASTER, PATRICIA A. MANGAN, IRINA KULIKOVSKAYA, MINNAL GUPTA, FANG CHEN, LIFENG TIAN, VANESSA E. GONZALEZ, JUN XU, IN-YOUNG JUNG, J. JOSEPH MELENHORST, GABRIELA PLESA, JOANNE SHEA, TINA MATLAWSKI, AMANDA CERVINI, AVERY L. GAYMON, STEPHANIE DESJARDINS, ANNE LAMONTAGNE, JANUARY SALAS-MCKEE, ANDREW FESNAK, DONALD L. SIEGEL, BRUCE L. LEVINE, JULIE K. JADLOWSKY, REGINA M. YOUNG, ANNE CHEW, WEI-TING HWANG, ELIZABETH O. HEXNER, BEATRIZ M. CARRENO, CHRISTOPHER L. NOBLES, FREDERIC D. BUSHMAN, KEVIN R. PARKER, YANYAN QI, ANSUMAN T. SATPATHY, HOWARD Y. CHANG, YANGBING ZHAO, SIMON F. LACEY, CARL H. JUNE

SCIENCE 28 FEB 2020

CRISPR takes first steps in humans

CRISPR-Cas9 is a revolutionary gene-editing technology that offers the potential to treat diseases such as cancer, but the effects of CRISPR in patients are currently unknown. Stadtmauer et al. report a phase 1 clinical trial to assess the safety and feasibility of CRISPR-Cas9 gene editing in three patients with advanced cancer (see the Perspective by Hamilton and Doudna). They removed immune cells called T lymphocytes from patients and used CRISPR-Cas9 to disrupt three genes (TRAC, TRBC, and PDCD1) with the goal of improving antitumor immunity. A cancer-targeting transgene, NY-ESO-1, was also introduced to recognize tumors. The engineered cells were administered to patients and were well tolerated, with durable engraftment observed for the study duration. These encouraging observations pave the way for future trials to study CRISPR-engineered cancer immunotherapies.

Structured Abstract

INTRODUCTION

Most cancers are recognized and attacked by the immune system but can progress owing to tumor-mediated immunosuppression and immune evasion mechanisms. The infusion of ex vivo engineered T cells, termed adoptive T cell therapy, can increase the natural antitumor immune response of the patient. Gene therapy to redirect immune specificity combined with genome editing has the potential to improve the efficacy and increase the safety of engineered T cells. CRISPR coupled with CRISPR-associated protein 9 (Cas9) endonuclease is a powerful gene-editing technology that potentially allows the ability to target multiple genes in T cells to improve cancer immunotherapy.

RATIONALE

Our first-in-human, phase 1 clinical trial (clinicaltrials.gov; trial NCT03399448) was designed to test the safety and feasibility of multiplex CRISPR-Cas9 gene editing of T cells from patients with advanced, refractory cancer. A limitation of adoptively transferred T cell efficacy has been the induction of T cell dysfunction or exhaustion. We hypothesized that removing the endogenous T cell receptor (TCR) and the immune checkpoint molecule programmed cell death protein 1 (PD-1) would improve the function and persistence of engineered T cells. In addition, the removal of PD-1 has the potential to improve safety and reduce toxicity that can be caused by autoimmunity. A synthetic, cancer-specific TCR transgene (NY-ESO-1) was also introduced to recognize tumor cells. In vivo tracking and persistence of the engineered T cells were monitored to determine if the cells could persist after CRISPR-Cas9 modifications.

RESULTS

Four cell products were manufactured at clinical scale, and three patients (two with advanced refractory myeloma and one with metastatic sarcoma) were infused. The editing efficiency was consistent in all four products and varied as a function of the single guide RNA (sgRNA), with highest efficiency observed for the TCR α chain gene (TRAC) and lowest efficiency for the TCR β chain gene (TRBC). The mutations induced by CRISPR-Cas9 were highly specific for the targeted loci; however, rare off-target edits were observed. Single-cell RNA sequencing of the infused CRISPR-engineered T cells revealed that ~30% of cells had no detectable mutations, whereas ~40% had a single mutation and ~20 and ~10% of the engineered T cells were double mutated and triple mutated, respectively, at the target sequences. The edited T cells engrafted in all three patients at stable levels for at least 9 months. The persistence of the T cells expressing the engineered TCR was much more durable than in three previous clinical trials during which T cells were infused that retained expression of the endogenous TCR and endogenous PD-1. There were no clinical toxicities associated with the engineered T cells. Chromosomal translocations were observed in vitro during cell manufacturing, and these decreased over time after infusion into patients. Biopsies of bone marrow and tumor showed trafficking of T cells to the sites of tumor in all three patients. Although tumor biopsies revealed residual tumor, in both patients with myeloma, there was a reduction in the target antigens NY-ESO-1 and/or LAGE-1. This result is consistent with an on-target effect of the engineered T cells, resulting in tumor evasion.

CONCLUSION

Preliminary results from this pilot trial demonstrate that multiplex human genome engineering is safe and feasible using CRISPR-Cas9. The extended persistence of the engineered T cells indicates that preexisting immune responses to Cas9 do not appear to present a barrier to the implementation of this promising technology.

Crispr takes on human trials from UPenn trial Crispr edited T cells

CRISPR-Cas9 engineering of T cells in cancer patients. T cells were isolated from the blood of a patient with cancer. CRISPR-Cas9 rebonuclear protein complexes loaded with three sgRNAs were electroporated into the normal T cells, resulting in gene editing of the TRAC, TRBC1, TRBC2, and PDCD1 (encoding PD-1) loci. The cells were then transduced with a lentiviral vector to express a TCR specific for the cancer-testis antigens NY-ESO-1 and LAGE-1 (right). The engineered T cells were then returned to the patient by intravenous infusion, and patients monitored for safety and efficacy. Taken from Science 28 Feb 2020:Vol. 367, Issue 6481, eaba7365 DOI: 10.1126/science.aba7365

UPDATED 10/04/2021 (Advances in CAR-T therapy for solid tumors)

The following contains a curation of the latest advances on CAR-T therapy for solid tumors, which has been a challenge for the field.

NK cells expressing a chimeric activating receptor eliminate MDSCs and rescue impaired CAR-T cell activity against solid tumors

From: Parihar, R., Rivas, C., Huynh, M., Omer, B., Lapteva, N., Metelitsa, L. S., Gottschalk, S. M., & Rooney, C. M. (2019). NK Cells Expressing a Chimeric Activating Receptor Eliminate MDSCs and Rescue Impaired CAR-T Cell Activity against Solid Tumors. Cancer immunology research, 7(3), 363–375. https://doi.org/10.1158/2326-6066.CIR-18-0572

Abstract

Solid tumors are refractory to cellular immunotherapies in part because they contain suppressive immune effectors such as myeloid-derived suppressor cells (MDSCs) that inhibit cytotoxic lymphocytes. Strategies to reverse the suppressive tumor microenvironment (TME) should also attract and activate immune effectors with antitumor activity. To address this need, we developed gene-modified natural killer (NK) cells bearing a chimeric receptor in which the activating receptor NKG2D is fused to the cytotoxic ζ-chain of the T-cell receptor (NKG2D.ζ). NKG2D.ζ–NK cells target MDSCs, which overexpress NKG2D ligands within the TME. We examined the ability of NKG2D.ζ–NK cells to eliminate MDSCs in a xenograft TME model and improve the antitumor function of tumor-directed chimeric antigen receptor (CAR)-modified T cells. We show that NKG2D.ζ–NK cells are cytotoxic against MDSCs, but spare NKG2D ligand-expressing normal tissues. NKG2D.ζ–NK cells, but not unmodified NK cells, secrete pro-inflammatory cytokines and chemokines in response to MDSCs at the tumor site and improve infiltration and antitumor activity of subsequently infused CAR-T cells, even in tumors for which an immunosuppressive TME is an impediment to treatment. Unlike endogenous NKG2D, NKG2D.ζ is not susceptible to TME-mediated down-modulation and thus maintains its function even within suppressive microenvironments. As clinical confirmation, NKG2D.ζ–NK cells generated from patients with neuroblastoma killed autologous intra-tumoral MDSCs capable of suppressing CAR-T function. A combination therapy for solid tumors that includes both NKG2D.ζ–NK cells and CAR-T cells may improve responses over therapies based on CAR-T cells alone.

INTRODUCTION

T lymphocytes can be engineered to target tumor-associated antigens by forced expression of CARs (1). Although successful when directed against leukemia-associated antigens such as CD19 (2, 3), CAR-T cell therapy for solid tumors has been less effective, with best responses in patients with minimal disease (4, 5). Solid tumors recruit inhibitory cells such as myeloid-derived suppressor cells (MDSCs) (6). These immature myeloid cells are a component of innate immunity and strengthen the suppressive TME (7, 8). The frequency of circulating or intra-tumoral MDSCs correlates with cancer stage, disease progression, and resistance to standard chemo- and radio-therapies (9). Hence, MDSCs are worth targeting in the quest to enhance CAR-T cell efficacy against solid tumors.

Natural killer (NK) cells, a lymphoid component of the innate immune system, produce MHC-unrestricted cytotoxicity and secrete pro-inflammatory cytokines and chemokines (10). NK cells also modulate the activity of antigen-presenting myeloid cells within lymphoid organs, and recruit and activate effector T cells at sites of inflammation (11, 12). NK cells express NKG2D, a cytotoxicity receptor that is activated by non-classical MHC molecules expressed on cells stressed by events such as DNA damage, hypoxia, or viral infection (13). NKG2D ligands are overexpressed on several solid tumors and on tumor-infiltrating MDSCs (14). NK cells, therefore, could alter the TME in favor of an antitumor response by eliminating suppressive elements such as MDSCs. However, the NKG2D cytotoxic adapter molecule, DAP10, is downregulated by suppressive molecules of the TME, such as TGFβ (15), limiting the antitumor functions of NK cells.

To overcome the repressive effect of the solid TME on NKG2D function, we used a retroviral vector to modify NK cells with a chimeric NKG2D receptor (NKG2D.ζ) comprising the extracellular domain of the native NKG2D molecule fused to the intracellular cytotoxic ζ-chain of the T-cell receptor (16). We hypothesized that primary human NK cells expressing NKG2D.ζ (NKG2D.ζ–NK cells) would maintain NKG2D.ζ expression within the suppressive TME, kill NKG2D ligand-expressing MDSCs, secrete pro-inflammatory cytokines and chemokines, and recruit and activate effector cells, including CAR-T cells, derived from the adaptive immune system. These benefits are not attainable from NK cells expressing the native NKG2D receptor as its functions are down-modulated in the TME. Here we show that when NK cells express NKG2D.ζ, immune suppression is sufficiently countered to enable tumor-specific CAR-T cells to persist within the TME and eradicate otherwise resistant tumors.

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MATERIALS AND METHODS

Cytokines, cell lines, and antibodies.

Recombinant human interleukin (IL)2 was obtained from National Cancer Institute Biologic Resources Branch (Frederick, MD). Recombinant human IL6, GM-CSF, IL7, and IL15 were purchased from Peprotech (Rocky Hill, NJ, USA). The human neuroblastoma cell line LAN-1 was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM culture medium supplemented with 2 mM L-glutamine (Gibco-BRL) and 10% FBS (Hyclone, Waltham, MA, USA). The human CML cell line K562 was purchased from American Type Culture Collection and cultured in complete-RPMI culture medium composed of RPMI-1640 medium (Hyclone) supplemented with 2 mM L-glutamine and 10% FBS. A modified version of parental K562 cells, genetically modified to express a membrane-bound version of IL15 and 41BB-ligand, K562-mb15–41BB-L, was kindly provided by Dr. Dario Campana (National University of Singapore). All cell lines were verified by either genetic or flow cytometry-based methods (LAN-1 and K562 authenticated by ATCC in 2009) and tested for mycoplasma contamination monthly via MycoAlert (Lonza) mycoplasma enzyme detection kit (last mycoplastma testing of LAN-1, K562 parental line, and K562-mb15–41BB-L on November 2, 2018; all negative). All cell lines were used within one month of thawing from early-passage (< 3 passages of original vial) lots.

CAR-encoding retroviral vectors.

The construction of the SFG-retroviral vector encoding GD2-CAR.41BB.ζ, as shown in Supplementary Fig. S1A, was previously described (17). The SFG-retroviral vector encoding NKG2D.ζ, an internal ribosomal entry site (IRES), and truncated CD19 (tCD19), was generated by sub-cloning NKG2D.ζ from a retroviral vector (18) kindly provided by Dr. Charles L. Sentman (Dartmouth Geisel School of Medicine, Hanover, NH, USA) into pSFG.IRES.tCD19 (19). RD114-speudotyped viral particles were produced by transient transfection in 293T cells, as previously described (20).

Expansion and retroviral transduction of human NK and T cells.

Human NK cells were activated, transduced with retroviral constructs (Fig. 1A) and expanded as previously described by our laboratory (21). Briefly, peripheral blood mononuclear cells (PBMCs) obtained from healthy donors under Baylor College of Medicine IRB-approved protocols, were cocultured with irradiated (100 Gy) K562-mb15–41BB-L at a 1:10 (NK cell:irradiated tumor cell) ratio in G-Rex^® cell culture devices (Wilson Wolf, St. Paul, MN, USA) for 4 days in Stem Cell Growth Medium (CellGenix) supplemented with 10% FBS and 500 IU/mL IL2. Cell suspensions on day 4 (containing 50–70% expanded/activated NK cells) were transduced with SFG-based retroviral vectors, as previously described (22). The transduced cell population was then subjected to secondary expansion to generate adequate cell numbers for experiments in G-Rex^® devices at the same NK cell:irradiated tumor cell ratio with 100 IU/mL IL2. This 17-day human gene-modified NK cell protocol resulted in > 97% pure CD56⁺/CD3⁻ NK cell population with avg. 77.4% ± 18.2% (n = 25) of NK cells transduced with the construct of interest. For most experiments, transduced NK cells were purified to > 95% by magnetic column selection of truncated CD19 selection marker-positive cells.

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Figure 1.

NKG2D.ζ–NK cells expand and kill ligand-expressing targets.

(A) Schematic of SFG-based retroviral vector constructs for transduction of human NK cells. (B) Human NK cells were expanded as described in Methods and percentage of CD56⁺/CD3⁻ NK cells at time of retroviral transduction (day 4) is shown. Expanded NK cells (red circle in A) purified via depletion of CD3⁺ cells were transduced with NKG2D.ζ retroviral vector or empty vector control (referred to as “unmodified”), and transduction efficiencies are shown inset. (C) NKG2D expression on NK cells (MFI, inset) was assessed with isotype antibody as control. Non-transduced NK cells exhibited similar NKG2D expression to empty vector-transduced NK cells. * p = 0.003 vs. unmodified condition. (D) Expression of NKG2D (absolute MFI on y-axis) on NK cells from each donor (n = 25) transduced with either empty vector or NKG2D.ζ construct was determined by flow cytometry. Each pair of data points connected by a line represent cells from a single donor, to confirm surface expression of our chimeric molecule after transduction. Black line with grey block next to each group are mean MFI ± SEM. (E) NKG2D.ζ–NK cell cytotoxicity against K562 and LAN-1 tumor targets in a 4-hour ⁵¹Cr-release assay. Given that K562 and LAN-1 are both NK-sensitive targets, low E:T ratios were utilized to observe differences. Experiment is representative of at least three separate determinations from n = 10 donors. * p < 0.01 vs. unmodified NK cells at same E:T ratio. (F) NKG2D.ζ–NK cells were expanded after transduction culture (as shown in schema), and fold-expansion and cytotoxicity both pre- (day 7) and post- (day 17) secondary expansion were determined.

For production of GD2.CAR-T cells (autologous to MDSCs and NK cells), PBMCs from healthy donors were suspended in T-cell medium (TCM) consisting of RPMI-1640 supplemented with 45% Click’s Medium (Gibco-BRL), 10% FBS, and 2 mM L-glutamine, and cultured in wells pre-coated with CD3 (OKT3, CRL-8001, American Type Culture Collection) and CD28 (Clone CD28.2, BD Biosciences) antibodies for activation. Human IL15 and IL7 were added on day +1, and cells underwent retroviral transduction on day +2, as previously described (22). T-cells were used for experiments between days +9 to +14 post-transduction, with phenotype as shown in Supplementary Figs. S1B–C.

Induction and enrichment of human MDSCs.

Our method for ex vivo generation of human PBMC-derived MDSCs was derived from published reports (23), with slight modifications. Briefly, PBMCs were sequentially depleted of CD25^hi-expressing cells and CD3-expressing cells by magnetic column separation (Miltenyi Biotec). Resultant CD25^lo/−, CD3⁻ PBMCs were plated at 4×10⁶ cells/mL in complete-RPMI medium with human IL6 and GM-CSF (both at 20 ng/mL) onto 12-well culture plates (Sigma Corning) at 1 mL/well. Plates were incubated for 7 days with medium and cytokines being replenished on days 3 and 5. Resultant cells were harvested by gentle scraping and MDSCs were purified by magnetic selection using CD33 magnetic microbeads (Miltenyi Biotec). Cells were analyzed by multi-color flow cytometry for CD33, CD14, CD15, HLA-DR, CD11b, CD83, and CD163 (BD Biosciences). MDSCs were defined as either monocytic (M-MDSCs; CD14⁺, HLA-DR^low/−), PMN-MDSCs (CD14⁻, CD15⁺, CD11b⁺), or early-stage MDSCs (lineage⁻, HLA-DR^low/−, CD33⁺), as per published guidelines (24). In addition to the above markers, MDSCs were stained for PD-L1, PD-L2, and NKG2D ligands via an NKG2D-Fc chimera (BD Biosciences) followed by FITC-labeled anti-Fc. This pan-ligand staining approach was determined to be the most efficient way to assess NKG2D ligand expression on human MDSCs because (1.) NKG2D ligand expression had not previously been reported for human MDSCs and thus simultaneous evaluation of the eight different NKG2D ligands would have been required, and (2.) we found poor reproducibility in staining patterns using individual commercially-available ligand antibodies, even within the same donor.

In vitro T-cell suppression assay.

T-cell proliferation was assessed using Cell Trace Violet (Thermo Fisher) dye dilution analysis, as per manufacturer’s recommendations. Briefly, 1×10⁵ Cell Trace Violet-labeled T cells (isolated at the time of MDSC generation) were plated onto 96-well plates in the presence of plate-bound 1 μg/mL CD3 and 1 μg/mL CD28 antibodies with 50 IU/mL IL2 in the absence or presence of autologous MDSCs or peripheral blood monocytes (as a myeloid cell control) at 1:1, 4:1 and 8:1 T cell:MDSC ratios. In some experiments, only the 4:1 ratio is shown as this was determined as optimal for assessment of suppression. After 4 days of coculture, T cells were labeled with CD3 antibody and assessed for cell division using Cell Trace Violet dye dilution by flow cytometry. Percent suppression was calculated as follows: [(% proliferating T cells in the absence of MDSCs – % proliferating T cells in presence of MDSCs)/% proliferating T cells in the absence of MDSCs] x 100. Proliferation was defined as % T cells undergoing active division as represented by Cell Trace Violet dilution peaks, as previously described (25).

In vitro CAR-T chemotaxis assay.

Transwell 5-μm pore inserts (Corning, Somerset, NJ) for migration experiments were prepared by coating with 0.01% gelatin at 37 °C overnight, followed by 3 μg of human fibronectin (Life Technologies, Grand Island, NY) at 37 °C for 3 hours to mimic endothelial and extracellular matrix components, as previously described (26). Briefly, 2×10⁵ purified GD2.CAR-T cells were placed in 100 μL of TCM in the upper chambers of the pre-coated Transwell inserts that were then transferred into wells of a 24-well plate. Culture supernatants (400 μL) from NKG2D.ζ or unmodified NK cells cultured with autologous MDSCs or monocytes, were placed in the lower chambers of the wells. Plain medium or medium supplemented with 1 μg/mL of the T-cell recruiting chemokine, MIG, served as negative and positive controls, respectively. The plates were then incubated for 4 hours at 37 °C with 5% CO₂, followed by a 10-minute incubation at 4 °C to loosen any cells adhering to the undersides of the insert membranes. The fluid in the lower chambers was collected separately and migrated cells were counted using trypan blue exclusion. The cells were analyzed for CAR expression by flow cytometry to confirm phenotype of migrated T cells.

In vivo tumor microenvironment model.

12–16 week old female NSG mice were implanted subcutaneously in the dorsal right flank with 1×10⁶ Firefly luciferase(FfLuc)-expressing LAN-1 neuroblastoma cells admixed with 3×10⁵ ex vivo-generated MDSCs, suspended in 100 μL of basement membrane Matrigel (Corning). Matrigel basement membrane was important in keeping tumor and MDSCs confined so as to establish a localized solid TME. 10–14 days later, when tumors measured at least 100 mm³ by caliper measurement, mice were injected intravenously with 5×10⁶ GD2.CAR-T cells. Tumor growth was measured twice weekly by live bioluminescence imaging using the IVIS^® system (IVIS, Xenogen Corporation) 10 minutes after 150 mg/kg D-luciferin (Xenogen)/mouse was injected intraperitoneally. In experiments examining the ability of NKG2D.ζ–NK cells to reduce intra-tumoral MDSCs, 1×10⁷ unmodified or NKG2D.ζ–NK cells were injected intravenously when tumors measured at least 100 mm³. At end of experiment, tumors were harvested en bloc, digested ex vivo, and intra-tumoral human MDSCs (CD33⁺, HLA-DR^low cells) were enumerated by flow cytometry. The absolute number of human MDSCs within a tumor digest was enumerated per mouse (n = 5 mice/group), compared to pre-treatment MDSC numbers, and presented as mean % MDSCs remaining per treatment group. In experiments examining the effects of NKG2D.ζ–NK cells on GD2.CAR-T cell antitumor activity, 5×10⁶ (cell dose chosen to mitigate direct antitumor effects of NK cells) unmodified or NKG2D.ζ–NK cells were injected intravenously 3 days prior to GD2.CAR-T injection. In GD2.CAR-T cell homing experiments, CAR-T were transduced with GFP-luciferase retroviral construct prior to injection into mice bearing unmodified tumor cells (27). Mice received 5000 IU human IL2 intraperitoneally three times per week for 3 weeks following NK cell injection to promote NK cell survival in NSG mice (28). Tumor size was measured twice weekly with calipers and the mice were imaged for bioluminescence signal from T cells at the same time. Mice were euthanized for excessive tumor burden, as per protocol guidelines. The animal studies protocol was approved by Baylor College of Medicine Institutional Animal Care and Use Committee and mice were treated in strict accordance with the institutional guidelines for animal care.

Immunohistochemistry of neuroblastoma xenografts.

On day 32 of in vivo experiments, animals were sacrificed, tumors were harvested and sectioned bluntly ex vivo to separate tumor periphery (outer 1/3 of tumor volume) vs. core (non-necrotic inner 2/3 of tumor volume), and n = 5 sections/tumor sample were analyzed for presence of GD2.CAR-T and NKG2D.ζ–NK cells by H&E and human CD3 and CD57 immunostaining performed by the Human Tissue Acquisition and Pathology Core of Baylor College of Medicine. Lack of CD57 expression on infused GD2.CAR-T was confirmed by flow cytometry prior to administration. CD57 was chosen as the marker for NK cells in tumor tissue in our study because LAN-1 tumors naturally express the prototypical NK marker CD56, truncated CD19 expression was inadequate for in situ staining, and CD57 had previously been used as a marker for tissue-localized activated NK cells (29). The number of human CD3+ and CD57+ cells in representative sections of tumors from periphery vs. core of the treatment groups indicated were enumerated per high-powered field (HPF) at 40x magnification and percent of the total number of cells enumerated within tumors found in the periphery vs. core in each treatment group indicated from tumors with and without MDSCs is shown as mean ± SEM of n = 5 sections/periphery or core, n = 5 tumors/group.

Analysis of intra-tumoral MDSCs from patients with neuroblastoma.

Tumor tissue and matched peripheral blood of neuroblastoma patients obtained in the context of a specimen/laboratory study after patient identification had been removed were thawed and analyzed for MDSC subsets by flow cytometry or utilized in in vitro assays, as described in legends or Results. The tissue acquisition protocol was performed after review and approval by the Baylor College of Medicine Institutional Review Board. Briefly, subjects with a diagnosis of high-risk or intermediate-risk neuroblastoma were eligible to participate. Written informed consent, or appropriate assent for participation, in accordance with the Declaration of Helsinki was obtained from each subject or subject’s guardian for procurement of patient blood and tumor tissue and for subsequent analyses of stored patient materials.

Statistics.

Data are presented as mean ± SEM of either experimental replicates or number of donors, as indicated. Paired two-tailed t-test was used to determine significance of differences between means with p < 0.05 indicating a significant difference. For in vivo bioluminescence, changes in tumor radiance from baseline at each time point were calculated and compared between groups using two-sample t-test. Multiple group comparisons were conducted via ANOVA via GraphPad Prism v7 software. Survival determined from the time of tumor cell injection was analyzed by Kaplan-Meier and differences in survival between groups were compared by the log-rank test.

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RESULTS

NKG2D.ζ NK cells expand and have cytotoxicity against target cells.

To increase killing of NKG2D ligand-expressing MDSCs, we generated primary human NK cells stably expressing NKG2D.ζ and a truncated CD19 (tCD19) marker from a retroviral vector (Fig. 1A). NK cells were expanded from PBMCs obtained from normal donors, transduced with retroviral construct expressing chimeric NKG2D, then cultured for 3 additional days. Transduction efficiency, as measured by the expression of tCD19 on CD56⁺CD3⁻ NK cells after the additional 3 days, was 71.3 ± 16% (n = 25 normal donors) and produced a 5.4 ± 1.1-fold increase in NKG2D expression on the NK cell surface (Fig. 1B–D). NKG2D.ζ–NK cells showed greater cytotoxicity (79.2 ± 5.6%, n = 10 normal donors) against wild-type K562, a highly NK cell-sensitive tumor cell line that naturally expresses NKG2D ligands, than mock vector-transduced (hereafter referred to as, unmodified) NK cells (40.5 ± 2.1%) at 2:1 E:T ratio in a 4-hr cytotoxicity assay (Fig. 1E). In contrast, transgenic NKG2D.ζ expression did not increase NK cell killing of LAN-1 neuroblastoma cells that are marginally NK-sensitive, but lack NKG2D ligands. To determine if in vitro expansion affected the cytotoxic function of NKG2D.ζ–NK cells, we secondarily expanded NKG2D.ζ–NK cells for an additional 10-days (Fig. 1F schema). As seen in Fig. 1F, NKG2D.ζ–NK cells expanded (120 ± 7.3-fold by day 17 of culture; n = 10 donors) similarly to unmodified and non-transduced NK cells and maintained stable cytotoxic function between days 7 and 17 of expansion. Thus, we generated and expanded high numbers of primary human NKG2D.ζ-expressing NK cells capable of cytotoxicity against ligand-expressing targets, even after prolonged culture.

Transgenic NKG2D.ζ is unaffected by TGFβ or soluble NKG2D ligands.

Expression of the native NKG2D receptor on NK cells is down-modulated by tumor-derived TGFβ and soluble NKG2D ligands, both of which are abundant in the TME (15, 30) and likely impair NK cell function in solid tumors. To determine the effect of TGFβ and soluble NKG2D ligands on NKG2D.ζ receptor expression and function, we cultured NKG2D.ζ–NK cells in the presence of TGFβ or the soluble NKG2D ligands, MICA and MICB, and examined NKG2D expression and NK cytotoxicity after 24-, 48-, and 72-hours. After exposure to TGFβ or soluble MICA/B, unmodified NK cells significantly down-regulated NKG2D (MFI of 25 vs. 95 in non-exposed NK cells at 48 hours) and were less cytotoxic (20 ± 5.1% killing vs. 40 ± 3.7% killing by non-exposed NK cells at 48 hours) to NKG2D ligand-expressing K562 targets (Fig. 2A, ,B).B). In contrast, NKG2D.ζ–NK cells maintained NKG2D expression and cytotoxicity after exposure to the same concentrations of TGFβ and soluble MICA/B (Fig. 2C, ,D).D). This lack of sensitivity to down-regulation by these tumor-associated components should benefit the function of NKG2D.ζ–NK cells within the TME.

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Figure 2.

Transgenic NKG2D.ζ is unaffected by TGFβ or soluble NKG2D ligands.

NKG2D.ζ or unmodified NK cells (n = 5 donors) were cultured in the presence of TGFβ (5 ng/mL) (A, B) or the soluble NKG2D ligands MICA and MICB (C, D) for 24-, 48-, and 72-hours. NKG2D receptor expression was determined by flow cytometry and NK cytotoxicity against K562 targets was assessed in a 4-hr Cr-release assay at an 5:1 E:T ratio using 48-hr exposed NK cells. Viability of transduced NK cells after exposure to TGFβ for 24, 48, and 72 hours, as assessed by 7-AAD vital staining, was > 90%. * p = 0.001 vs. non-TGFβ/MICA-treated NK groups at same time-points.

Human MDSCs express NKG2D ligands and are killed by NKG2D.ζ–NK cells.

To study the effects of human NK cells on autologous MDSCs, we generated human MDSCs by culture of CD3-/CD25^lo PBMC with IL6 plus GM-CSF for 7 days, followed by CD33⁺ selection, as described in the Methods. The phenotypic characterization of these MDSCs and confirmation of their suppressive capacity is shown in Supplementary Fig. S2. Routinely, our ex vivo-generated MDSCs contained monocytic (M)-MDSC and early(e)-MDSC subsets, with few (avg. < 1%) polymorphonuclear (PMN)-MDSCs (Supplementary Fig. S2A), roughly reflecting the subset composition reported in patients with solid tumors (9, 31). The MDSCs expressed the suppressive factors TGFβ, IL6, IL10, and PDL-1 in amounts often greater than tumor cells (Supplementary Figs. S2B–C), and suppressed proliferation and cytokine secretion by autologous T cells stimulated with plate-bound CD3/CD28 antibodies (Supplementary Figs. S2D–E) and by 2^nd generation GD2.CAR-T cells encoding 4–1BB and CD3-ζ endodomains stimulated with the GD2⁺ tumor line LAN-1 (Supplementary Figs. S2F–G). As seen in Fig. 3A, MDSCs expressed as much or more NKG2D ligand than the positive control tumor line, K562 (ligand MFI of 78.2 vs. 29.7, respectively). Freshly isolated peripheral blood T cells did not express NKG2D ligands, whereas immature and mature dendritic cells expressed little, consistent with previous data (13). The neuroblastoma cell line, LAN-1, subsequently used in our in vivo TME model, did not express NKG2D ligands.

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Figure 3.

Human MDSCs express ligands for NKG2D and are killed by NKG2D.ζ–NK cells.

(A) NKG2D ligand expression on human MDSCs by flow cytometry. Immature dendritic cells (iDC) and mature DCs (mDC) were used as myeloid controls. T cells activated with CD3 and CD28 mAbs plus 100 IU/mL IL2 for 24 hours were used as lymphocyte control. LAN-1 and K562 cells were used as negative and positive controls, respectively. MFI of NKG2D ligand expression in parenthesis. Representative data from single donor (of n = 25 normal donors). Isotype control for NKG2D staining routinely fell within the 1^st log. (B) NKG2D.ζ–NK cell cytotoxicity against autologous MDSCs as targets in a 4-hour ⁵¹Cr-release assay. In some wells of the cytotoxicity assay, a blocking mAb to NKG2D was added. Representative data from triplicate samples per data point from a single donor (of n = 25 normal donors) is shown. * p < 0.01 vs. unmodified NK cells at same E:T ratio. (C) In the same experiment as (B), the same batch of NKG2D.ζ–NK cells were analyzed for cytotoxicity against autologous B cells, monocytes, monocyte-derived iDC and mDC, and activated T cells (n = 10 donors examined). (D) M-MDSC frequency by flow cytometry from neuroblastoma tumor samples obtained from high-risk patients, as described in Methods. (E) Cytotoxicity by NKG2D.ζ–NK cells derived from patient PBMC (harvested and frozen at time of tumor sampling) against autologous tumor-derived MDSCs in a 4-hour ⁵¹Cr-release assay. Data shown are from triplicate samples per data point at a 10:1 E:T ratio. * p < 0.001 vs. unmodified NK cells from same donor. (F) NKG2D.ζ–NK cells were cocultured with autologous MDSCs at 1:1 ratio plus low-dose 50 IU/mL IL2 to maintain NK survival, and fold change in the number of each cell type from the start of coculture was determined by flow cytometry at indicated time-points. * p < 0.001 vs. NK/MDSC fold-change in unmodified NK cell cocultures. (G) Cell-free supernatants were harvested from cocultures at day 3 and analyzed for IFNγ, TNFα, IL6, and IL10 by ELISA. # p < 0.01 vs. corresponding cytokine in cocultures with unmodified NK cells. (H) NKG2D ligand expression was determined for activated T cells (ATCs) expressing NKG2D.ζ and NKG2D.ζ–NK cells. Expression of NKG2D ligands on non-transduced ATCs as control for T-cell activation. (I) NKG2D.ζ–NK cells or NKG2D.ζ T cells were cocultured with autologous ATCs at 1:1 ratio and fold change in the number of each cell type from the start of coculture was determined by flow cytometry at indicated time-points. * p < 0.001 vs. ATC fold-change at days 0 and 3 cocultures.

To evaluate MDSC susceptibility to killing by NKG2D.ζ–NK cells, we performed both short- and long-term killing assays. Fig. 3B shows enhanced killing of MDSCs by autologous NKG2D.ζ–NK cells compared to unmodified NK cells (35 ± 5.5% vs. 8 ± 2.4% cytotoxicity, respectively, at an E:T ratio of 5:1) in a 4-hr chromium-release assay. MDSC killing was dependent on NKG2D, as pre-incubation with an NKG2D blocking Ab reduced the cytotoxicity to levels achieved by unmodified NK cells. NKG2D.ζ–NK cells mediated no cytotoxicity against other autologous immune cells such as freshly-isolated monocytes, monocyte-derived mature dendritic cells, T cells, or B cells (Fig. 3C). Only immature dendritic cells, which expressed little NKG2D ligand (approx. 7% of cells; MFI 11.4), were mildly susceptible to lysis by NKG2D.ζ–NK cells (4.2 ± 1.7 % lysis at an E:T ratio of 20:1). As confirmation of the clinical applicability of our approach, we assessed whether NKG2D.ζ–NK cells generated from patient PBMCs were able to kill highly suppressive MDSCs isolated from the patient’s tumor. Tumor samples obtained from two patients with high-risk neuroblastoma at time of first biopsy/resection contained M-MDSCs (Fig. 3D). NKG2D.ζ–NK cells generated from patient PBMCs (harvested and frozen at time of tumor sampling) mediated significant cytotoxicity in vitro against M-MDSCs purified from patient tumors, whereas unmodified patient NK cells did not (Fig. 3E). These results provide further clinical evidence for the capacity of NKG2D.ζ–NK cells to eliminate MDSCs in patients with suppressive TMEs.

To determine whether NKG2D.ζ–NK cells could control MDSC survival in long-term cultures, we cocultured NKG2D.ζ–NK cells with autologous MDSCs at a 1:1 ratio for 7 days in the presence of low-dose IL2 to maintain NK survival, and quantified each cell type by flow cytometry every two days. As shown in Fig. 3F, NKG2D.ζ–NK cells expanded in cocultures (mean 9.5 ± 0.7-fold increase) with a concomitant reduction in MDSCs (mean 81.3 ± 9.4-fold decrease), whereas unmodified NK cells failed to expand or eliminate MDSCs. NK cells cultured alone or with autologous monocyte controls did not expand (0.8 ± 0.1-fold change). As seen in Fig. 3G, NK cell expansion and MDSC reduction correlated with a shift in the culture cytokine milieu from one that is immune suppressive (more IL6 and IL10; less IFN-γ and TNF-α) in cocultures containing unmodified NK cells, to one that is immune stimulatory and enhances CAR-T antitumor function (less IL6 and IL10; more IFN-γ and TNF-α) in cocultures containing NKG2D.ζ–NK cells. Hence, NKG2D.ζ–NK cells mediate potent cytotoxicity against suppressive MDSCs via their highly expressed NKG2D ligands. In addition, through selective depletion of MDSCs in combination with immune stimulatory cytokine secretion, NKG2D.ζ–NK cells skew the cytokine microenvironment to one that can support CAR-T effector functions (32).

Previous studies have reported that expression of chimeric NKG2D constructs in T lymphocytes can direct these cells to target NKG2D ligand-expressing tumors (16, 33). However, activated T cells (ATCs) themselves upregulate NKG2D ligands (34), with variable ligand expression intensity dependent on the T-cell activation protocol employed, leading to fratricide when the chimeric NKG2D is expressed. To determine if this off-tumor side-effect occurred when the same NKG2D.ζ was expressed in NK cells, we compared the killing of ATCs by autologous NK cells or by autologous T cells expressing our NKG2D.ζ transgene. ATCs and NKG2Dζ.-T cells both upregulated NKG2D ligands during ex vivo expansion with CD3/CD28 antibodies plus IL7 and IL15, whereas NKG2D.ζ-transduced NK cells undergoing expansion in our K562-mb15–41BB-L culture system did not (Fig. 3H). Coculture without additional stimulation of NKG2D.ζ-T cells with autologous ATCs produced fratricide, of both the NKG2D.ζ effector T cells (35 ± 7.2% decrease in cell number) and the non-transduced ATC targets (98 ± 11.5% decrease in cell number) (n = 3). By contrast, ATC numbers were unaffected by coculture with autologous NKG2D.ζ–NK cells (Fig. 3I). These results show that NK cells expressing NKG2D.ζ can kill autologous MDSCs while sparing other NKG2D ligand expressing populations, thus avoiding the fratricide seen with NKG2D.ζ-expressing T cells.

NKG2D.ζ–NK cells eliminate intra-tumoral MDSCs and reduce tumor burden.

To determine if NKG2D.ζ–NK cells could eliminate MDSCs from tumor sites in vivo, we created an MDSC-containing TME in a xenograft model of neuroblastoma. We chose NKG2D ligand-negative LAN-1 tumor for this experiment so that the effects of NKG2D.ζ–NK cells on MDSCs were not confused with their effects on the tumor cells. LAN-1 tumor cells admixed with human MDSCs were inoculated subcutaneously in NSG mice. These animals had increases in the suppressive cytokines IL10 (10-fold vs. tumor alone) and TGFβ (2.6-fold vs. tumor alone) in circulation by day 16 as compared to animals bearing tumors initiated without MDSCs, and the resultant tumors grew more rapidly due to increased neovascularization and tumor-associated stroma (Supplementary Fig. S3A–D), consistent with clinical reports of MDSC-dense tumors (35). As seen in Fig. 4A, in mice bearing NKG2D ligand-negative tumors without MDSCs, a single infusion of 1×10⁷ NKG2D.ζ–NK cells resulted in a small delay in tumor growth but eventual progression, suggesting that the LAN-1 tumor itself (a marginally NK-sensitive target) can be killed at higher NK cell doses independent of NKG2D ligand expression. In mice bearing MDSC-containing tumors, 1×10⁷ NKG2D.ζ–NK cells inhibited tumor growth (Fig. 4B), reduced NKG2D ligand-expressing intra-tumoral MDSCs with only 8.7 ± 3.5% of the input MDSCs remaining (Fig. 4C), and prolonged mouse survival (median survival of 73 days vs. 29 days after unmodified NK cells; Fig. 4D). Since LAN-1 tumor cells do not express NKG2D ligands and are only marginally sensitive to ligand-independent lysis, tumors subsequently regrew in these mice once the NKG2D.ζ–NK cells had disappeared (> day 40). Thus, NKG2D.ζ–NK cells can traffic to tumor sites and reduce intra-tumoral MDSCs but cannot themselves eradicate NKG2D ligand-negative malignant cells in our model.

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Figure 4.

NKG2D.ζ–NK cells eliminate intra-tumoral MDSCs and reduce tumor burden.

LAN-1 tumor cells, either alone (A) or admixed with human MDSCs (B), were injected S.C. in the flanks of NSG mice. When tumors reached a volume of approx. 100 mm³ (day 14, gray block arrow inset), no NK cells (PBS control), 1×10⁷ unmodified or NKG2D.ζ–NK cells were injected I.V. and tumor growth was measured over time via calipers. * p < 0.03 vs. other conditions shown at same time point. (C) On day 26, intra-tumoral human MDSCs (CD33⁺, HLA-DR^low) were enumerated by flow cytometry and are presented as mean % MDSCs remaining per treatment group. ** p < 0.005 vs. unmodified NK treatment. (D) Survival of groups by Kaplan-Meyer analysis. # p = 0.024. Representative experiment of three performed.

NKG2D.ζ–NK cells secrete chemokines that recruit GD2.CAR-T cells.

To determine if NKG2D.ζ–NK cells can recruit T cells modified with a tumor-specific CAR to tumor sites containing MDSCs, we cocultured NKG2D.ζ–NK cells with autologous MDSCs and analyzed culture supernatants for chemokines by multiplex ELISA. Compared to unmodified NK cells, NKG2D.ζ–NK cells produce significantly greater CCL5 (RANTES; 10-fold increase), CCL3 (MIP-1α; 2-fold increase), and CCL22 (MDC; 5-fold increase) in response to autologous MDSCs (Fig. 5A). Large amounts of CXCL8 (IL8) were also produced, but there was no significant difference from the production by unmodified NK cells. Analysis of chemokine receptor expression on 2^nd generation GD2.CAR-T cells revealed CXCR1 (binds CXCL8), CCR2 (binds CCL2), CCR5 (binds CCL3), and CCR4 (binds CCL5) (see Supplementary Fig. S1C). These GD2.CAR-T cells were assayed for chemotaxis to supernatants derived from unmodified or NKG2D.ζ–NK cells cocultured with autologous MDSCs. Supernatants from NKG2D.ζ–NK cell-containing cocultures induced chemotaxis of 41.1 ± 5.5% of GD2.CAR-T cells (Fig. 5B), whereas supernatants from unmodified NK cells induced chemotaxis no greater than produced by medium (14.9 ± 6.4% vs. 17.3 ± 1.9%, respectively). Chemotaxis was not induced by supernatants from unmodified or NKG2D.ζ–NK cells cocultured with monocytes. Thus, following their encounter with MDSCs, NKG2D.ζ–NK cells secrete chemokines that recruit CAR-Ts in vitro.

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Figure 5.

NKG2D.ζ–NK cells secrete chemokines that recruit GD2.CAR-T cells.

(A) NKG2D.ζ or unmodified NK cells were cocultured with autologous MDSCs and cell-free culture supernatants harvested at 48 hours were analyzed for chemokines CXCL8, CCL5, CCL3, and CCL22 by ELISA. Shown are mean chemokine concentration ± SEM for n = 5 cocultures/donor (data from one of five representative donors is shown). * p < 0.005 vs. unmodified NK cocultures. (B) GD2.CAR-T cells were assayed for chemotaxis in Transwells (described in Methods) in response to supernatants derived from unmodified or NKG2D.ζ–NK cells cocultured with autologous MDSCs. Supernatants derived from monocyte (non-suppressive myeloid cell control)-stimulated NK cells were also used. # p < 0.001 vs. Medium, ** p = 0.009 vs. “unmodified NK plus MDSCs” condition. (C) LAN-1 tumor cells, alone or admixed with human MDSCs, were injected S.C. into the flank of NSG mice. When tumors reached a volume ~100 mm³, 5×10⁶ GD2.CAR-T cells were injected I.V. alone on day 13 (GD2.CAR-T), or preceded by 5×10⁶ NKG2D.ζ–NK cells I.V. injected on day 10 (chNK + GD2.CAR-T). GD2.CAR-T signal at tumor site was measured over time via live-animal bioluminescence imaging. (D) Shown is mean ± SEM (n=5 mice/group) bioluminescent signal of GD2.CAR-T cells expressed as radiance. * p = 0.01 vs. all other groups.

NKG2D.ζ NK cells improve GD2.CAR-T cell trafficking to tumor sites.

To determine the effects of the MDSC-induced, NKG2D.ζ–NK cell chemokines on CAR-T cell recruitment in vivo, we used our MDSC-containing TME xenograft model (see Fig. 4). When tumors reached a volume of ~100 mm³ (day 10), 5×10⁶ NKG2D.ζ–NK cells were infused, followed three days later (day 13) by infusion of 5×10⁶ luciferase gene-transduced GD2.CAR-T cells. Tumor localization and expansion of GD2.CAR-T cells was measured over time via live-animal bioluminescence imaging. As seen in Fig. 5C, GD2.CAR-T cells injected alone on day 13 after tumor inoculation (without pre-administration of NKG2D.ζ–NK cells) into mice bearing tumors devoid of MDSCs localized effectively to subcutaneous tumors in the flank (4 of 5 mice showed bioluminescent signal on days 14 and 18; Fig. 5C). There was a 10.5 ± 0.8-fold increase in bioluminescent signal on day 18, with CAR-T cell bioluminescence remaining above baseline levels for the duration of the experiment (Fig. 5D). However, in tumors containing MDSCs, CAR-T cells localized poorly: only 1 of 5 mice exhibited bioluminescent signal (Fig. 5C), with only a 1.02 ± 0.1-fold increase in bioluminescent signal on day 18 and bioluminescence falling below pre-infusion levels within 10 days after injection (Fig. 5D). In contrast, pre-administration of NKG2D.ζ–NK cells on day 10 into mice bearing MDSC-containing tumors allowed subsequently infused GD2.CAR-T cells to localize effectively to tumor sites, with bioluminescence in 5 of 5 mice at the tumor site and a 10.9 ± 0.2-fold increase in bioluminescent signal on day 18, within 5 days of injection (Fig. 5D).

To determine if NKG2D.ζ–NK cells could promote GD2.CAR-T infiltration into the tumor bed, we compared the frequency of human GD2.CAR-T and human NK cells in the tumor periphery and the tumor core by immunohistochemistry (Supplementary Fig. S4A–B). In tumors without MDSCs, 89 ± 11% of the total T cells in the tumor had infiltrated into the tumor core. In contrast, a much smaller fraction (39 ± 16%) infiltrated into the core of tumors containing MDSCs, suggesting TME suppression of CAR-T infiltration. However, pre-treatment of tumors containing MDSCs with NKG2D.ζ–NK cells increased the fraction of intra-tumoral CAR-T cells (70 ± 13%) within the tumor core. Equal numbers of NKG2D.ζ–NK cells were observed within both peripheral and core samples from MDSC-positive and MDSC-negative tumors (Supplementary Fig. S5), suggesting the ability of NK cells to traffic well within tumors despite the presence of MDSCs.

Elimination of MDSCs increases antitumor activity of GD2.CAR-T cells.

To determine if the activities of NKG2D.ζ–NK cells described above enhance the antitumor function of CAR-T cells, we treated mice bearing subcutaneous, luciferase-labeled neuroblastoma containing MDSCs with GD2.CAR-T cells preceded by NKG2D.ζ–NK cells, in a similar set-up to experiments in Fig. 5C. As seen in Fig. 6A–B, a single injection of 5×10⁶ NKG2D.ζ–NK cells (a dose that achieved intra-tumoral MDSC depletion with only 26.8 ± 5.8% of the input MDSCs remaining) resulted in no significant tumor regression or prolongation of survival in mice bearing xenografts containing human MDSCs. A single infusion of 5×10⁶ GD2.CAR-T cells significantly reduced tumor in mice whose xenografts lacked human MDSCs with a median survival of 95 days (Fig. 6C–D). However, the same GD2.CAR-T cells were ineffective against xenografts containing human MDSCs, worsening overall median survival to 39 days (Fig. 6B). In contrast, when the same GD2.CAR-T cell injection was preceded 3 days earlier by a single injection of 5×10⁶ NKG2D.ζ–NK cells (that had no direct antitumor effect by themselves within the other arm of the same experiment, see Fig. 6A–B), the antitumor activity of the GD2.CAR-T cells in mice bearing MDSC-containing tumors was restored to the level observed in mice whose tumors lacked MDSCs (Fig. 6C). NKG2D.ζ–NK cells pre-injection also improved the overall survival of the mice with MDSC-containing tumors to a median 120 days with durable cure in 2 of 5 mice (Fig. 6D). Taken together, our results suggest that NKG2D.ζ–NK cells not only eliminate MDSCs from the TME, but also recruit CAR-T cells to intra-tumoral sites which facilitates antitumor efficacy.

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Figure 6.

Elimination of MDSCs by NKG2D.ζ–NK cells increases antitumor activity of GD2.CAR-T cells.

Luciferase gene-transduced LAN-1 tumor cells, alone or admixed with human MDSCs, were injected S.C. into NSG mice. (A) When tumors reached a volume ~100 mm³, no treatment (No Tx; PBS control) or 5×10⁶ NKG2D.ζ–NK cells alone (chNK) were injected I.V. on day 10 and tumor growth was measured over time via live-animal bioluminescence imaging. Shown is mean ± SEM (n=5 mice/group) bioluminescent signal expressed as radiance. # ns, p = 0.18 vs. No treatment (+MDSC) group. (B) Survival of groups in A was determined by Kaplan-Meyer analysis. # ns, p = 0.059. (C) In other groups of mice within the same experiment, 5×10⁶ GD2.CAR-T cells were injected I.V. alone on day 13 (GD2.CAR-T), or preceded by 5×10⁶ NKG2D.ζ–NK cells injected on day 10 (chNK+GD2.CAR-T). * p = 0.001; # ns, p = 0.59 vs. each other. (D) Survival of groups in C by Kaplan-Meyer analysis. Representative experiment of 5 separate experiments. * p = 0.002; ** p = 0.001.

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DISCUSSION

We have developed a TME-disrupting approach that eliminates MDSCs and rescues MDSC-mediated impairment of tumor-directed CAR-T cells. We show that when co-implanted with a neuroblastoma cell line, human MDSCs both enhance tumor growth and suppress the infiltration, expansion, and antitumor efficacy of tumor-specific CAR T-cells. In this model, NK cells bearing a chimeric version of the activating receptor NKG2D (NKG2D.ζ–NK cells) are directly cytotoxic to autologous MDSCs, thus eliminating MDSCs from tumors. In addition, NKG2D.ζ–NK cells secrete pro-inflammatory cytokines and chemokines in response to MDSCs at the tumor site, improving CAR-T cell infiltration and function, and resulting in tumor regression and prolonged survival compared to treatment with CAR-T cells alone. Our cell therapy approach utilizes an engineered innate immune effector that targets the TME, and shows potential to enhance efficacy of combination immune-based therapies for solid tumors.

NKG2D.ζ–NK cells directly killed highly suppressive MDSCs generated in vitro as well as those from patient tumors. NKG2D.ζ–NK cells also secreted cytokines that favored immune activation in response to MDSCs. Unmodified NK cells were unable to mediate these effects. The ability of NKG2D.ζ–NK cells to eliminate MDSCs from the TME should have several beneficial effects for antitumor immunity. First, as MDSCs express suppressive cytokines such as TGFβ and the checkpoint ligands PDL-1 and PDL-2, elimination of MDSCs should help relieve the suppression of endogenous T cell responses and potentiate the activity of adoptive T cell therapies. Given that high baseline numbers of MDSCs have been reported as a biomarker of poor response in the context of trials with the checkpoint inhibitors ipilimumab and pembrolizumab (36, 37), elimination of MDSCs by NKG2D.ζ–NK cells may also enhance checkpoint inhibition. Second, elimination of MDSCs should also decrease other MDSC-associated effects, including neovascularization via their expression of VEGF, production of immunosuppressive metabolic products such as PGE₂ and adenosine, and establishment of tumor-supportive stroma via their expression of iNOS, FGF, and matrix metalloproteinases (8). In short, the ability of NKG2D.ζ–NK cells to eliminate MDSCs alters the tumor microenvironment in multiple ways that should improve antitumor immunity.

Previous strategies for modulation of MDSCs within the TME have included use of agents that block single functions such as secretion of nitric oxide (38) or expression of checkpoint molecules (39); induce MDSC differentiation such as with all-trans retinoic acid (40); or eliminate MDSCs such as with the cytotoxic agents doxorubicin or cyclophosphamide (41). The MDSC eliminating effects were dependent on continued administration of the agents, with a rapid rebound in MDSCs after discontinuation. Moreover, many of these agents have off-target toxicities that include damage to endogenous tumor-specific T cells. In contrast, NKG2D.ζ–NK cells produce prolonged and specific elimination of MDSCs with the potential to kill MDSCs that are recruited to the tumor from the bone marrow, while continually secreting cytokines and chemokines which respectively alter TME suppression and recruit and activate tumor-specific T cells. Thus, NKG2D.ζ–NK cells exert a prolonged combination of simultaneous immune modulatory effects that enhance antitumor immune function in ways that could not be achieved by previous methods that target MDSCs.

We observed no toxicity against normal hematopoietic cells when NKG2D.ζ was expressed in autologous human NK cells. Previous studies overexpressing an NKG2D.ζ receptor containing co-stimulatory endodomains (e.g., CD28 or 41BB) and DAP10, a signaling adaptor molecule for enhanced surface expression of NKG2D, in T cells showed activity against NKG2D ligand-overexpressing tumors, but at the cost of fratricide in vitro and lethal toxicity in mice (16, 33, 34). Using our standard T-cell activation/expansion protocol (22), we also observed upregulation of NKG2D ligands, leading to fratricide in T cells expressing NKG2D.ζ. When NKG2D.ζ-T cells engage NKG2D ligands expressed on normal tissues, they will not receive the physiologic NK cell-directed inhibitory inputs that would safely regulate this potent and unopposed chimeric receptor activity. By contrast, when NKG2D.ζ is expressed on NK cells, they are able to recognize inhibitory NK cell ligands such as self-MHC expressed on healthy self-tissues, counteracting otherwise unopposed positive signals from NKG2D ligands. Thus, an NK cell platform for NKG2D enhancement may limit toxicity while taking advantage of the cytotoxic and immune modulatory potential of the receptor-ligand system.

Unlike wild-type NKG2D, transgenic NKG2D.ζ expression and activity were not sensitive to down-modulation by TGFβ or soluble NKG2D ligands, allowing improved function in the TME. Native NKG2D relies solely on the intra-cytoplasmic adaptor DAP10 for mediating its cytolytic activity in human NK cells (42). TGFβ1 and soluble NKG2D ligands both decrease DAP10 gene transcription and protein activity, and thus reduce NKG2D function in endogenous NK cells (43, 44). In contrast, transgenic NKG2D.ζ does not rely on DAP10-based signaling for its activity, since signaling occurs through the ζ-chain. Thus, this construct provides a stable cytolytic pathway capable of circumventing TME-mediated down-modulation of native NKG2D activity. A previous study expressing a chimeric NKG2D.ζ molecule that incorporated DAP10 reported enhanced NK cytotoxicity compared to NKG2D.ζ alone in vitro against a variety of human cancer cell lines as well as in a xenograft model of osteosarcoma (45). However, this report did not address the susceptibility of this complex to down-modulation by TGFβ or soluble NKG2D ligands, or whether these NK cells had activity against MDSCs.

NKG2D.ζ–NK cells countered immunosuppression mediated by MDSCs leading to enhanced CAR-T cell tumor infiltration and expansion at tumor sites, CAR-T functions that are impaired in patients with solid tumors (46). Unlike the GD2.CAR-T cells in our model, NKG2D.ζ–NK cells homed effectively to MDSC-engrafted tumors and released an array of chemokines that increased T cell infiltration of tumor. Unlike pharmacologic strategies aimed at enhancing leukocyte trafficking, including administration of lymphotactin or TNFα (47), our approach does not require continuous cytokine administration. In fact, the ability of chimeric NKG2D to augment NK immune function specifically within the immunosuppressive TME provides for the local release of chemotactic factors, reflecting a more homeostatic method by which to increase CAR-T infiltration. Once there, CAR-T cells should meet an environment favorably modified by NKG2D.ζ–NK cell mediated elimination of MDSCs and production of pro-inflammatory cytokines. Indeed, elimination of MDSCs from a GD2⁺ tumor xenograft enhanced the activity of GD2.CAR-T cells in our model, including T-cell survival and intratumoral expansion. Given the suppressive effects of MDSCs in neuroblastoma (48, 49), the model shows how reversal of an MDSC-mediated suppressive microenvironment can improve antitumor functions of effector T cells.

Clinical neuroblastoma contains intense infiltrates of MDSCs (50), which are not included in tumor xenograft models currently used to study human cell therapeutics. Our data suggest that co-inoculation of tumors with suppressive components (such as MDSCs) can model TME-mediated suppression of CAR-T activity against solid tumors, and provides a method by which to understand and counter immunosuppression. Although NSG mice lack a complete immune system in which to examine the effects of multiple endogenous immune components, our ability to engraft exogenous components (e.g., human MDSCs) within our TME model provides the possibility of simulating different immunosuppressive aspects of the solid TME. In fact, further model development utilizing human inhibitory macrophages and regulatory T cells (Tregs) as additional suppressive components of the TME is currently underway in our laboratory.

In summary, we describe an approach to reverse the suppressive TME using engineered human NK cells. We have shown that generation and expansion of our NK cell product is feasible and that NKG2D.ζ–NK cells have antitumor activity within a suppressive solid tumor microenvironment without toxicity to normal NKG2D ligand-expressing tissues. Hence, the elimination of suppressive MDSCs by NKG2D.ζ–NK cells may safely enhance adoptive cellular immunotherapy for neuroblastoma and for many other tumors that are supported and protected by MDSCs.

Combined anti-CTLA4 and anti-PD1 immunotherapy shows promising results against advanced melanoma

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Observing the spleen colonies in mice and proving the existence of stem cells

Posted in Competition in regenerative stem cell science, Hematology, Hematopoiesis, Human Immune System in Health and in Disease, Pharmaceutical Drug Discovery, Scientist: Career considerations, Stem Cells for Regenerative Medicine, tagged stem cells on September 8, 2015| Leave a Comment »

Observing the spleen colonies in mice and proving the existence of stem cells – Till and McCulloch

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Innovation

Series E. 2; 7.2

Till & McCulloch are Doctors James Till and Ernest McCulloch who, while studying the effect of radiation on the bone marrow of mice at the Ontario Cancer Institute, in Toronto, demonstrated the existence of multipotent stem cells in 1961.

Now recognized as the Fathers of Stem Cell Science, Till & McCulloch exemplified the importance of multidisciplinary collaboration in scientific research and have received many awards for their collaborative and ground-breaking research.

They first published their findings of the discovery of stem cells in the journal Radiation Research.[1][2] In later work, joined by graduate student Andy Becker, they cemented their stem cell theory and published the results in the journal Nature in 1963.[3]

After their pioneering discovery, Till & McCulloch continued to help this new field develop; not only by continuing to expand their research activities, but also by mentoring other young scientists. Together, Till & McCulloch spawned successive generations of scientists who continue to deepen the understanding of how the different types of stem cells work and their application to different diseases and medical conditions—many have also become globally recognized leaders in their field.

Dr. Till’s focus shifted increasingly towards the evaluation of cancer therapies and quality of life issues in the 1980s. He has held a wide range of positions in organizations ranging from the Stem Cell Network to Project Open Source to the Canadian Breast Cancer Foundation, and many others.

Dr. McCulloch continued to expand the depth of work in his field with a heavy emphasis on cellular and molecular mechanisms affecting the growth of malignant blast stem cells from the blood of patients with Acute Myeloblastic Leukemia. Unfortunately, Dr. McCulloch died on January 20, 2011, shortly before the 50th anniversary of the publication of the 1961 paper in Radiation Research.

Lifetime Achievement: Drs. James Till and Ernest McCulloch

http://oicr.on.ca/news/portal-news/lifetime-achievement-drs-james-till-and-ernest-mcculloch

In the early 1960s, two Canadian scientists started a series of experiments involving injection of bone marrow cells into irradiated mice.

Dr. James E. Till, a native of Saskatchewan who completed his PhD in biophysics at Yale, and Dr. Ernest McCulloch, a Toronto-born doctor who completed his research training in England, were working together on research related to leukemia at the Ontario Cancer Institute. Their immediate aim was to investigate a controversial new finding by Colorado scientist Theodore Puck, which seemed to show that normal cells are just as susceptible to radiation as cancer cells. At the time, scientists believed radiation “melted” away cancer cells while leaving normal tissue intact. While there was no doubt that radiation is an effective way to kill cancer cells, Puck’s research suggested scientists must be wrong about the way it acts on cells.

Till and McCulloch’s study proved Puck’s finding was correct. But this wasn’t all that their research proved.

In the mouse experiments, they observed nodules in the animals’ spleens when the bone marrow cells were injected. These nodules appeared in proportion to the number of cells injected, leading the two young scientists to speculate that the nodules – which they termed “spleen colonies” – were arising from a single marrow cell. If this were true, the experiment would be a breakthrough, since scientists had not yet proved that it was possible for cells to act in this fashion.

Till and McCulloch conducted further experiments that proved the cells they were observing were indeed stem cells. The rest, as they say, is history.

Still a groundbreaking field

Stem cell research is often discussed in the media as a new, groundbreaking field, but the idea that certain special cells might be responsible for creating many other types of cell goes back quite a bit further than Till and McCulloch’s experiments in the 1960s. The problem of where cells come from is fundamental to biology; for centuries, or perhaps longer, scientists have searched for the origin of the building blocks of life.

Since early in the 1900s, scientists had suspected that there must be some sort of stem cell in the blood forming system. But stem cells proved extraordinarily tricky to observe.

By observing the spleen colonies in mice and proving the existence of stem cells, Till and McCulloch sparked worldwide interest. Once they had established proof that spleen colonies originate from stem cells, there was solid reason to believe that other cells originate from them too – something that has been confirmed through further research.

Developments in technology, biology and research ethics have recently propelled stem cell research to the forefront of public debates on science. Scientists now know that embryonic stem cells can differentiate into all of the specialized embryonic tissues, while adult organisms’ stem cells and progenitor cells can act as a repair system for the body, replenishing specialized cells and maintaining the normal turnover of regenerative organs, such as blood, skin or intestinal tissues.
In the United States, and to a lesser extent in other countries, controversy has erupted as scientists have proposed to explore using human embryonic stem cells – which, by definition, have to be harvested from human embryos – as treatments for disease.

While they tend to garner fewer headlines, there are also many projects exploring the use of adult stem cells in medicine to regenerate parts of the body affected by disease or injury. Research in this area has become very promising since 2006, when Shinya Yamanaka, a researcher at Kyoto University in Japan, showed that adult somatic cells can be “reprogrammed” to act like embryonic stem cells – opening the possibility of using pluripotent stem cells in medicine without harvesting cells from human embryos. The reprogrammed cells, called induced pluripotent stem cells, are an area of intense research activity. In the few years since Yamanaka’s discovery, researchers have already refined and improved techniques for creating induced pluripotent stem cells.

Remarkable careers

In the decades after their discovery, Till and McCulloch continued their research on stem cells, publishing several groundbreaking papers and eventually developing the framework through which stem cells are currently understood. They later moved on to other projects, with McCulloch focusing on cellular and molecular mechanisms affecting the growth of malignant blast stem cells obtained from the blood of patients with acute myeloblastic leukemia, and Till branching out into a number of other health-related fields including evaluation of cancer therapies, quality of life issues and the ethics of Internet research.

Till and McCulloch have received many honours for their research, including the Albert Lasker Award for Basic Medical Research and the Gairdner International Award, Canada’s major award for biomedical research. Both are University Professors Emeritus at the University of Toronto, Officers of the Order of Canada and members of the Order of Ontario and the Canadian Medical Hall of Fame. Till’s research on the impact of the Internet and advocacy for open access to research publications continues to this day. McCulloch is now retired.

Although Till and McCulloch are no longer working in the stem cell field, there are plenty of Ontario scientists who are. The University of Toronto and Ontario Cancer Institute have retained their early lead, developing programs to harness stem cell research for a wide range of applications in medicine. The province rose to international prominence again in the 1990s when Dr. John Dick, a scientist at the Ontario Cancer Institute, proved the existence of cancer stem cells – a subpopulation of cancer cells that are responsible for the growth and spread of cancer.

In the years since, Dick has established a major hub of cancer stem cell research in Ontario. In 2007 the Ontario Institute for Cancer Research appointed Dick as Director of a new Cancer Stem Cell Program to develop and implement a strategy to further understand cancer stem cells and use the concept as the basis for developing new treatments. The program has already recruited rising stars in the cancer stem cell field and has begun working on its ambitious research plan.

“The truly remarkable thing about Drs. Till and McCulloch is that the stem cell discovery was just one part of two very outstanding careers. They also worked tirelessly behind the scenes as builders, teachers and mentors in the decades when Ontario solidified its presence in cancer research,” says Dr. Bob Phillips, Deputy Director of OICR and a former colleague of Till and McCulloch’s at the Ontario Cancer Institute.

“And the remarkable thing about the discovery itself is that we’re just starting to realize the potential of stem cells for medicine. In the 1960s, scientists recognized that Drs. Till and McCulloch’s discovery was important, but I don’t think anyone could have imagined that more than 45 years down the road their work would still be laying the basis for new ideas, new strategies, even new research institutes built around the concept of stem cells.”

A seminal paper published in 1961 by Drs. James Till and Ernest McCulloch was the first academic study to prove the existence of an exciting new type of cell, the stem cell. Since this foundational study, the promise of stem cells and their application to human disease has grown tremendously, their potential only beginning to be realized. This animated video, narrated by Dr. Till, provides a brief explanation of this early study as well as explaining the two basic concepts that define the answer to the question –

“What is a stem cell?”. https://i.vimeocdn.com/portrait/1257429_75x75.webp

Who really discovered stem cells? The history you need to know

Posted on April 11, 2012

Who really discovered stem cells?

Is it even possible that one scientific team all by themselves discovered something so ubiquitous as stem cells?

In theory “yes”, but after much historical research including this great historical article in Cell Stem Cell, I would argue that no one group really discovered stem cells.

Instead I believe the “discovery” of stem cells was an ongoing team effort over a period of many decades and there is much credit to go around.

Who gets the credit now according to most people now for “discovering” stem cells?

Canada rightly takes pride in the work of their scientists Drs. James Till and Ernset McCulloch, who did pioneering studies in hematopoietic stem cell research.

In Canada, Till and McCulloch are unambiguously called the world’s discoverers of stem cells. Period. No ambiguity.

Based on the answers that I found, I believe that Till and McCulloch did not discover stem cells.

But what they did do–publish the first clonal colony formation assay indicating multipotency–deserves great credit.

What information in the history of the stem cell research field makes me come to this conclusion that they did not discover stem cells?

Let’s start with the most widely acclaimed “discoverers” as a reference point.

In 1963, Till and McCulloch published their high impact paper on hematopoietic stem cells in Nature: Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells.

You can read the full paper here (pdf). I found it fascinating.

Ernest McCulloch: Cell Biology – Conducted a series of experiments that would eventually result in the first proof of the existence of stem cells, a discovery that would revolutionize our understanding of human biology and disease.

“I learned enough about myself to settle on a career in medicine: I did not like discipline – therefore I wanted to work for myself – to be my own boss.”

On an ordinary Sunday more than half a century ago, so ordinary a day that its exact date would later be forgotten, a young faculty member at the Ontario Cancer Institute in Toronto went to work to perform a routine check on his experimental animals. Many years later, he only remembered that it was a cold day, perhaps in the autumn. Navigating his way through quiet streets, Dr. Ernest McCulloch arrived at the Institute and entered the building. After donning his lab coat, McCulloch went to the animal quarters and checked his experimental mice. McCulloch followed a routine process for obtaining samples of their blood-forming tissues, a process which he had done many times before. His goal, working with his research partner James Till, was to determine if, by irradiating mouse bone marrow cells before transplanting them into irradiated mice, changes might later be found in the kinds of cells responsible for blood formation. It was a routine collection of samples on an ordinary day, noteworthy only because it was a Sunday.

After the samples were processed McCulloch, ever the sharp-eyed observer, noticed the unexpected presence of several small rounded bumps on the spleens of mice that had received bone marrow cells, and he decided to count them. He found that the number of nodules on each spleen was directly related to the number of bone marrow cells the mouse had received.

Suddenly things got very exciting for this unlikely duo of researchers. McCulloch was short, a medical doctor, raised in affluent downtown Toronto, with a penchant for classical literature, cinema and poetry. Till, on the other hand, was tall and athletic, a straight-shooting biophysicist who grew up on the Canadian Prairies and loved the sport of curling.

Although it had long been postulated that a single type of cell—a so-called stem cell— could give rise to multiple different cell types, no definitive evidence proved that they existed. The potential of such a “stem cell”, if discovered, would be dramatic, because its ability to regenerate different human body tissues could be used to treat all sorts of diseases. Following this cold, ordinary yet ultimately incredibly exciting day, McCulloch and Till went on to perform a series of seminal experiments in the 1960s that proved, for the first time, the existence of stem cells detected by their “spleen colony formation” assays.

The initial discovery of a direct relationship between the number of colonies and the number of transplanted cells suggested that single rare cells were able to initiate these colonies, but the suggestion required further validation. They knew that they were onto something very interesting, because they found that the colonies contained a variety of precursors of mature blood cell types—red cells, white cells and platelets—the normal cellular components of blood. These foundational observations were published in the specialty journal “Radiation Research” in 1961 under the un-dramatic title “A Direct Measurement of Radiation Sensitivity of Normal Bone Marrow Cells”. The paper did not use the words ”stem cell”, because Till and McCulloch, being rigorous scientists, required stronger evidence before making such a bold interpretation of their findings. Hence, their paper went unnoticed by the general biology community.

Their next paper, published in Nature in 1963, changed this and really brought Till and McCulloch to the forefront of hematological biology —the study of blood. Till’s PhD student Andy Becker found a way to trace the source of the cells in the spleen colonies to demonstrate that they originated from individual cells (not clusters of cells) in the bone marrow and could generate three types of progenitors required to make blood. The paper, titled “Cytological Demonstration of the Clonal Nature of Spleen Colonies Derived from Transplanted Mouse Marrow Cells”, still did not use the word “stem cell” as this was not the nature of these exacting scientists, who demanded that any degree of doubt be extinguished before making such claims.

McCulloch and Till went on to publish a number of subsequent papers, which have now been cited thousands of times, unequivocally demonstrating the presence of special cells within the bone marrow. They, with colleague Louis Siminovitch, offered the first biological definition of stem cells, which included two key characteristics: 1) self renewal – to be a stem cell, a cell must be able to give rise to new copies of itself; 2) differentiation – stem cells are able to divide and generate more mature cells that, following subsequent divisions, are eventually able to generate the highly specialized and functional cells essential for complex multi-cellular organisms work. An example of this can be seen in the hematopoietic (e.g. blood forming) stem cells they described, with a single undifferentiated stem cell being able to eventually form all the different types of cells that comprise our blood.

After these breakthroughs in the 1960s, the pair continued to work together in the field of experimental hematology for the next two decades. Although they continued to make more discoveries, it was those first findings that caused a huge impact on biology today by demonstrating the presence of stem cells. The field of stem cell biology has expanded dramatically and is now on the verge of a potential revolution in how we understand health and treat disease.

Born in an affluent neighborhood of Toronto, on Warren Road south of St. Clair Avenue, Ernest “Bun” McCulloch was raised well, with a private school education at Upper Canada College and summers at the cottage in the country. Given the nickname “Bun” by his grandmother, the name stuck with him for his entire life. McCulloch was educated as a medical doctor at the University of Toronto, graduating with an MD in 1948, then going on to the Lister Institute in London, England, where he had his first experience with scientific research.

“Bun” returned to Canada in 1949 where he interned at the Toronto General Hospital, specializing in internal medicine. His medical career began at the Sunnybrook Hospital in Toronto where he became an assistant resident and a research fellow in pathology at the Banting Institute. In 1954, McCulloch joined the University of Toronto as a teacher in the Department of Medicine. His next move, taking on the Head of Hematology in the Biology Division at the Ontario Cancer Institute in 1957, would result in his most famous work. He became part of a team of new promising young cancer researchers in the newly founded Department of Medical Biophysics, McCulloch quickly partnered up with James Till to study the effects of radiation on mouse bone marrow cells. The pair conducted a series of experiments that would eventually result in the first proof of the existence of stem cells, a discovery that would revolutionize our understanding of human biology and disease.

Ernest McCulloch was a man of incredible personality and charm. He was extremely well read and enjoyed discussing a wide variety of poetry, classical literature and theatre with his colleagues. He is known for his long-lasting impact on the Canadian medical research community. A list of the notable scientists mentored by Till and McCulloch is a who’s who of Canadian medical scientists, including (but not limited to): former president of the Canadian Institute for Health Research, Alan Bernstein; the discoverer of the T-cell receptor, Tak Mak, and a world leader in the field of hematopoietic stem cell biology, Connie Eaves.

McCulloch and Till’s work resulted in almost every top honor in science, except for the Noble Prize. Widely expected to be a joint winner of this top prize in science with Jim Till, sadly McCulloch passed away in 2011 preventing him from receiving this distinction. Till and McCulloch’s legacy in Canadian biomedical research cannot be understated, with their foundational work in establishing the presence of stem cells within bone marrow and prolific scientific mentorship. With two recent Nobel prizes, 2007 and 2012, going to stem cell researchers who worked on embryonic stem cells and induced pluripotent stem cells, respectively, it is still expected by many scientists that Till’s seminal experiments on adult stem cells will garner him the Nobel prize in the future.

by Ben Paylor

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Treatment of Lymphomas [2.4.4C]

Posted in Academic Publishing, Automated Cell Processing, Best evidence, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Cancer Screening, Cell Biology, Circulating Progenitor Cells, Clinical & Translational, Clinical Diagnostics, Clinical Genomics, Curation, Discovery process, Disease Biology, Evolutionary cognition, Experimental validation, Explanatory, Gene Regulation, Genetics & Pharmaceutical, Genome Biology, Genomic Expression, Hematology, Hematopoiesis, Historical relevance, Lymphoma, Pharmaceutical Analytics, Pharmaceutical Drug Discovery, Pharmacogenomics, Technology Advance Assessment of, Translational Science, tagged choice of treatment, hematological cancers, lymphomas, treatment of lymphomas on August 11, 2015| Leave a Comment »

Treatment of Lymphomas [2.4.4C]

Larry H. Bernstein, MD, FCAP, Author, Curator, Editor

http://pharmaceuticalinnovation.com/2015/8/11/larryhbern/Treatment-of-Lymphomas-[2.4.4C]

Lymphoma treatment

Overview

http://www.emedicinehealth.com/lymphoma/page8_em.htm#lymphoma_treatment

The most widely used therapies are combinations of chemotherapyand radiation therapy.

Biological therapy, which targets key features of the lymphoma cells, is used in many cases nowadays.

The goal of medical therapy in lymphoma is complete remission. This means that all signs of the disease have disappeared after treatment. Remission is not the same as cure. In remission, one may still have lymphoma cells in the body, but they are undetectable and cause no symptoms.

When in remission, the lymphoma may come back. This is called recurrence.
The duration of remission depends on the type, stage, and grade of the lymphoma. A remission may last a few months, a few years, or may continue throughout one’s life.
Remission that lasts a long time is called durable remission, and this is the goal of therapy.
The duration of remission is a good indicator of the aggressiveness of the lymphoma and of the prognosis. A longer remission generally indicates a better prognosis.

Remission can also be partial. This means that the tumor shrinks after treatment to less than half its size before treatment.

The following terms are used to describe the lymphoma’s response to treatment:

Improvement: The lymphoma shrinks but is still greater than half its original size.
Stable disease: The lymphoma stays the same.
Progression: The lymphoma worsens during treatment.
Refractory disease: The lymphoma is resistant to treatment.

The following terms to refer to therapy:

Induction therapy is designed to induce a remission.
If this treatment does not induce a complete remission, new or different therapy will be initiated. This is usually referred to as salvage therapy.
Once in remission, one may be given yet another treatment to prevent recurrence. This is called maintenance therapy.

Chemotherapy

Many different types of chemotherapy may be used for Hodgkin lymphoma. The most commonly used combination of drugs in the United States is called ABVD. Another combination of drugs, known as BEACOPP, is now widely used in Europe and is being used more often in the United States. There are other combinations that are less commonly used and not listed here. The drugs that make up these two more common combinations of chemotherapy are listed below.

ABVD: Doxorubicin (Adriamycin), bleomycin (Blenoxane), vinblastine (Velban, Velsar), and dacarbazine (DTIC-Dome). ABVD chemotherapy is usually given every two weeks for two to eight months.

BEACOPP: Bleomycin, etoposide (Toposar, VePesid), doxorubicin, cyclophosphamide (Cytoxan, Neosar), vincristine (Vincasar PFS, Oncovin), procarbazine (Matulane), and prednisone (multiple brand names). There are several different treatment schedules, but different drugs are usually given every two weeks.

The type of chemotherapy, number of cycles of chemotherapy, and the additional use of radiation therapy are based on the stage of the Hodgkin lymphoma and the type and number of prognostic factors.

Adult Non-Hodgkin Lymphoma Treatment (PDQ®)

http://www.cancer.gov/cancertopics/pdq/treatment/adult-non-hodgkins/Patient/page1

Key Points for This Section

Adult non-Hodgkin lymphoma is a disease in which malignant (cancer) cells form in the lymph system.

Because lymph tissue is found throughout the body, adult non-Hodgkin lymphoma can begin in almost any part of the body. Cancer can spread to the liver and many other organs and tissues.

Non-Hodgkin lymphoma in pregnant women is the same as the disease in nonpregnant women of childbearing age. However, treatment is different for pregnant women. This summary includes information on the treatment of non-Hodgkin lymphoma during pregnancy

Non-Hodgkin lymphoma can occur in both adults and children. Treatment for children, however, is different than treatment for adults. (See the PDQ summary on Childhood Non-Hodgkin Lymphoma Treatment for more information.)

There are many different types of lymphoma.

Lymphomas are divided into two general types: Hodgkin lymphoma and non-Hodgkin lymphoma. This summary is about the treatment of adult non-Hodgkin lymphoma. For information about other types of lymphoma, see the following PDQ summaries:

Age, gender, and a weakened immune system can affect the risk of adult non-Hodgkin lymphoma.

If cancer is found, the following tests may be done to study the cancer cells:

Immunohistochemistry : A test that uses antibodies to check for certain antigens in a sample of tissue. The antibody is usually linked to a radioactive substance or a dye that causes the tissue to light up under a microscope. This type of test may be used to tell the difference between different types of cancer.
Cytogenetic analysis : A laboratory test in which cells in a sample of tissue are viewed under a microscope to look for certain changes in the chromosomes.
Immunophenotyping : A process used to identify cells, based on the types of antigens ormarkers on the surface of the cell. This process is used to diagnose specific types of leukemia and lymphoma by comparing the cancer cells to normal cells of the immune system.

Certain factors affect prognosis (chance of recovery) and treatment options.

The prognosis (chance of recovery) and treatment options depend on the following:

The stage of the cancer.
The type of non-Hodgkin lymphoma.
The amount of lactate dehydrogenase (LDH) in the blood.
The amount of beta-2-microglobulin in the blood (for Waldenström macroglobulinemia).
The patient’s age and general health.
Whether the lymphoma has just been diagnosed or has recurred (come back).

Stages of adult non-Hodgkin lymphoma may include E and S.

Adult non-Hodgkin lymphoma may be described as follows:

E: “E” stands for extranodal and means the cancer is found in an area or organ other than the lymph nodes or has spread to tissues beyond, but near, the major lymphatic areas.

S: “S” stands for spleen and means the cancer is found in the spleen.

Stage I adult non-Hodgkin lymphoma is divided into stage I and stage IE.

Stage I: Cancer is found in one lymphatic area (lymph node group, tonsils and nearby tissue, thymus, or spleen).
Stage IE: Cancer is found in one organ or area outside the lymph nodes.

Stage II adult non-Hodgkin lymphoma is divided into stage II and stage IIE.

Stage II: Cancer is found in two or more lymph node groups either above or below the diaphragm (the thin muscle below the lungs that helps breathing and separates the chest from the abdomen).
Stage IIE: Cancer is found in one or more lymph node groups either above or below the diaphragm. Cancer is also found outside the lymph nodes in one organ or area on the same side of the diaphragm as the affected lymph nodes.

Stage III adult non-Hodgkin lymphoma is divided into stage III, stage IIIE, stage IIIS, and stage IIIE+S.

Stage III: Cancer is found in lymph node groups above and below the diaphragm (the thin muscle below the lungs that helps breathing and separates the chest from the abdomen).
Stage IIIE: Cancer is found in lymph node groups above and below the diaphragm and outside the lymph nodes in a nearby organ or area.
Stage IIIS: Cancer is found in lymph node groups above and below the diaphragm, and in the spleen.
Stage IIIE+S: Cancer is found in lymph node groups above and below the diaphragm, outside the lymph nodes in a nearby organ or area, and in the spleen.

In stage IV adult non-Hodgkin lymphoma, the cancer:

is found throughout one or more organs that are not part of a lymphatic area (lymph node group, tonsils and nearby tissue, thymus, or spleen), and may be in lymph nodes near those organs; or
is found in one organ that is not part of a lymphatic area and has spread to organs or lymph nodes far away from that organ; or
is found in the liver, bone marrow, cerebrospinal fluid (CSF), or lungs (other than cancer that has spread to the lungs from nearby areas).

Adult non-Hodgkin lymphomas are also described based on how fast they grow and where the affected lymph nodes are in the body. Indolent & aggressive.

The treatment plan depends mainly on the following:

The type of non-Hodgkin’s lymphoma
Its stage (where the lymphoma is found)
How quickly the cancer is growing
The patient’s age
Whether the patient has other health problems
If there are symptoms present such as fever and night sweats (see above)

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Treatment for Chronic Leukemias [2.4.4B]

Posted in Academic Publishing, Automated Cell Processing, BioIT: BioInformatics, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Cancer Screening, Clinical & Translational, Clinical Genomics, Curation, Diagnostics and Lab Tests, Discovery process, Disease Biology, Experimental validation, Explanatory, Gene Regulation, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genomic Expression, Hematopoiesis, Historical relevance, Human Immune System in Health and in Disease, Molecular Genetics & Pharmaceutical, Mutant Gene Expression, Myelodysplasia, Myelofibrosis, Neutrophilia, Pharmaceutical Analytics, Pharmaceutical Drug Discovery, Platelet count disorder, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Technology Advance Assessment of, tagged Chemotherapy, chronic lymphocytic leukemia, Chronic myelogenous leukemia, myelodyspalstic syndromes on August 11, 2015| Leave a Comment »

Treatment for Chronic Leukemias [2.4.4B]

Larry H. Bernstein, MD, FCAP, Author, Curator, Editor

http://pharmaceuticalintelligence.com/2015/8/11/larryhbern/Treatment-for-Chronic-Leukemias-[2.4.4B]

2.4.4B1 Treatment for CML

Chronic Myelogenous Leukemia Treatment (PDQ®)

http://www.cancer.gov/cancertopics/pdq/treatment/CML/Patient/page4

Treatment Option Overview

Key Points for This Section

There are different types of treatment for patients with chronic myelogenous leukemia.

Six types of standard treatment are used:

Targeted therapy
Chemotherapy
Biologic therapy
High-dose chemotherapy with stem cell transplant
Donor lymphocyte infusion (DLI)
Surgery

New types of treatment are being tested in clinical trials.

Patients may want to think about taking part in a clinical trial.

Patients can enter clinical trials before, during, or after starting their cancer treatment.

Follow-up tests may be needed.

There are different types of treatment for patients with chronic myelogenous leukemia.

Different types of treatment are available for patients with chronic myelogenous leukemia (CML). Some treatments are standard (the currently used treatment), and some are being tested in clinical trials. A treatment clinical trial is a research study meant to help improve current treatments or obtain information about new treatments for patients with cancer. When clinical trials show that a new treatment is better than the standard treatment, the new treatment may become the standard treatment. Patients may want to think about taking part in a clinical trial. Some clinical trials are open only to patients who have not started treatment.

Six types of standard treatment are used:

Targeted therapy

Targeted therapy is a type of treatment that uses drugs or other substances to identify and attack specific cancer cells without harming normal cells. Tyrosine kinase inhibitors are targeted therapy drugs used to treat chronic myelogenous leukemia.

Imatinib mesylate, nilotinib, dasatinib, and ponatinib are tyrosine kinase inhibitors that are used to treat CML.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

Chemotherapy

Chemotherapy is a cancer treatment that uses drugs to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. When chemotherapy is taken by mouth or injected into a vein or muscle, the drugs enter the bloodstream and can reach cancer cells throughout the body (systemic chemotherapy). When chemotherapy is placed directly into the cerebrospinal fluid, an organ, or a body cavity such as the abdomen, the drugs mainly affect cancer cells in those areas (regional chemotherapy). The way the chemotherapy is given depends on the type and stage of the cancer being treated.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

Biologic therapy

Biologic therapy is a treatment that uses the patient’s immune system to fight cancer. Substances made by the body or made in a laboratory are used to boost, direct, or restore the body’s natural defenses against cancer. This type of cancer treatment is also called biotherapy or immunotherapy.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

High-dose chemotherapy with stem cell transplant

High-dose chemotherapy with stem cell transplant is a method of giving high doses of chemotherapy and replacing blood-forming cells destroyed by the cancer treatment. Stem cells (immature blood cells) are removed from the blood or bone marrow of the patient or a donor and are frozen and stored. After the chemotherapy is completed, the stored stem cells are thawed and given back to the patient through an infusion. These reinfused stem cells grow into (and restore) the body’s blood cells.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

Donor lymphocyte infusion (DLI)

Donor lymphocyte infusion (DLI) is a cancer treatment that may be used after stem cell transplant.Lymphocytes (a type of white blood cell) from the stem cell transplant donor are removed from the donor’s blood and may be frozen for storage. The donor’s lymphocytes are thawed if they were frozen and then given to the patient through one or more infusions. The lymphocytes see the patient’s cancer cells as not belonging to the body and attack them.

Surgery

Splenectomy

What`s new in chronic myeloid leukemia research and treatment?

http://www.cancer.org/cancer/leukemia-chronicmyeloidcml/detailedguide/leukemia-chronic-myeloid-myelogenous-new-research

Combining the targeted drugs with other treatments

Imatinib and other drugs that target the BCR-ABL protein have proven to be very effective, but by themselves these drugs don’t help everyone. Studies are now in progress to see if combining these drugs with other treatments, such as chemotherapy, interferon, or cancer vaccines (see below) might be better than either one alone. One study showed that giving interferon with imatinib worked better than giving imatinib alone. The 2 drugs together had more side effects, though. It is also not clear if this combination is better than treatment with other tyrosine kinase inhibitors (TKIs), such as dasatinib and nilotinib. A study going on now is looking at combing interferon with nilotinib.

Other studies are looking at combining other drugs, such as cyclosporine or hydroxychloroquine, with a TKI.

New drugs for CML

Because researchers now know the main cause of CML (the BCR-ABL gene and its protein), they have been able to develop many new drugs that might work against it.

In some cases, CML cells develop a change in the BCR-ABL oncogene known as a T315I mutation, which makes them resistant to many of the current targeted therapies (imatinib, dasatinib, and nilotinib). Ponatinib is the only TKI that can work against T315I mutant cells. More drugs aimed at this mutation are now being tested.

Other drugs called farnesyl transferase inhibitors, such as lonafarnib and tipifarnib, seem to have some activity against CML and patients may respond when these drugs are combined with imatinib. These drugs are being studied further.

Other drugs being studied in CML include the histone deacetylase inhibitor panobinostat and the proteasome inhibitor bortezomib (Velcade).

Several vaccines are now being studied for use against CML.

2.4.4.B2 Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia Treatment (PDQ®)

General Information About Chronic Lymphocytic Leukemia

Key Points for This Section

Chronic lymphocytic leukemia is a type of cancer in which the bone marrow makes too many lymphocytes (a type of white blood cell).
Leukemia may affect red blood cells, white blood cells, and platelets.
Older age can affect the risk of developing chronic lymphocytic leukemia.
Signs and symptoms of chronic lymphocytic leukemia include swollen lymph nodes and tiredness.
Tests that examine the blood, bone marrow, and lymph nodes are used to detect (find) and diagnose chronic lymphocytic leukemia.
Certain factors affect treatment options and prognosis (chance of recovery).
Chronic lymphocytic leukemia is a type of cancer in which the bone marrow makes too many lymphocytes (a type of white blood cell).

Chronic lymphocytic leukemia (also called CLL) is a blood and bone marrow disease that usually gets worse slowly. CLL is one of the most common types of leukemia in adults. It often occurs during or after middle age; it rarely occurs in children.

http://www.cancer.gov/images/cdr/live/CDR755927-750.jpg

Anatomy of the bone; drawing shows spongy bone, red marrow, and yellow marrow. A cross section of the bone shows compact bone and blood vessels in the bone marrow. Also shown are red blood cells, white blood cells, platelets, and a blood stem cell.

Anatomy of the bone. The bone is made up of compact bone, spongy bone, and bone marrow. Compact bone makes up the outer layer of the bone. Spongy bone is found mostly at the ends of bones and contains red marrow. Bone marrow is found in the center of most bones and has many blood vessels. There are two types of bone marrow: red and yellow. Red marrow contains blood stem cells that can become red blood cells, white blood cells, or platelets. Yellow marrow is made mostly of fat.

Leukemia may affect red blood cells, white blood cells, and platelets.

Normally, the body makes blood stem cells (immature cells) that become mature blood cells over time. A blood stem cell may become a myeloid stem cell or a lymphoid stem cell.

A myeloid stem cell becomes one of three types of mature blood cells:

Red blood cells that carry oxygen and other substances to all tissues of the body.
White blood cells that fight infection and disease.
Platelets that form blood clots to stop bleeding.

A lymphoid stem cell becomes a lymphoblast cell and then one of three types of lymphocytes (white blood cells):

B lymphocytes that make antibodies to help fight infection.
T lymphocytes that help B lymphocytes make antibodies to fight infection.
Natural killer cells that attack cancer cells and viruses.

Blood cell development. CDR526538-750

http://www.cancer.gov/images/cdr/live/CDR526538-750.jpg

Blood cell development; drawing shows the steps a blood stem cell goes through to become a red blood cell, platelet, or white blood cell. A myeloid stem cell becomes a red blood cell, a platelet, or a myeloblast, which then becomes a granulocyte (the types of granulocytes are eosinophils, basophils, and neutrophils). A lymphoid stem cell becomes a lymphoblast and then becomes a B-lymphocyte, T-lymphocyte, or natural killer cell.

Blood cell development. A blood stem cell goes through several steps to become a red blood cell, platelet, or white blood cell.

In CLL, too many blood stem cells become abnormal lymphocytes and do not become healthy white blood cells. The abnormal lymphocytes may also be called leukemia cells. The lymphocytes are not able to fight infection very well. Also, as the number of lymphocytes increases in the blood and bone marrow, there is less room for healthy white blood cells, red blood cells, and platelets. This may cause infection, anemia, and easy bleeding.

This summary is about chronic lymphocytic leukemia. See the following PDQ summaries for more information about leukemia:

Adult Acute Lymphoblastic Leukemia Treatment.
Childhood Acute Lymphoblastic Leukemia Treatment.
Adult Acute Myeloid Leukemia Treatment.
Childhood Acute Myeloid Leukemia/Other Myeloid Malignancies Treatment.
Chronic Myelogenous Leukemia Treatment.
Hairy Cell Leukemia Treatment

Older age can affect the risk of developing chronic lymphocytic leukemia.

Anything that increases your risk of getting a disease is called a risk factor. Having a risk factor does not mean that you will get cancer; not having risk factors doesn’t mean that you will not get cancer. Talk with your doctor if you think you may be at risk. Risk factors for CLL include the following:

Being middle-aged or older, male, or white.
A family history of CLL or cancer of the lymph system.
Having relatives who are Russian Jews or Eastern European Jews.

Signs and symptoms of chronic lymphocytic leukemia include swollen lymph nodes and tiredness.

Usually CLL does not cause any signs or symptoms and is found during a routine blood test. Signs and symptoms may be caused by CLL or by other conditions. Check with your doctor if you have any of the following:

Painless swelling of the lymph nodes in the neck, underarm, stomach, or groin.
Feeling very tired.
Pain or fullness below the ribs.
Fever and infection.
Weight loss for no known reason.

Tests that examine the blood, bone marrow, and lymph nodes are used to detect (find) and diagnose chronic lymphocytic leukemia.

The following tests and procedures may be used:

Physical exam and history : An exam of the body to check general signs of health, including checking for signs of disease, such as lumps or anything else that seems unusual. A history of the patient’s health habits and past illnesses and treatments will also be taken.

Complete blood count (CBC) with differential : A procedure in which a sample of blood is drawn and checked for the following:

The number of red blood cells and platelets.

The number and type of white blood cells.

The amount of hemoglobin (the protein that carries oxygen) in the red blood cells.

The portion of the blood sample made up of red blood cells.

Results from the Phase 3 Resonate™ Trial

Significantly improved progression free survival (PFS) vs ofatumumab in patients with previously treated CLL

Patients taking IMBRUVICA® had a 78% statistically significant reduction in the risk of disease progression or death compared with patients who received ofatumumab¹
In patients with previously treated del 17p CLL, median PFS was not yet reached with IMBRUVICA® vs 5.8 months with ofatumumab (HR 0.25; 95% CI: 0.14, 0.45)¹

Significantly prolonged overall survival (OS) with IMBRUVICA® vs ofatumumab in patients with previously treated CLL

In patients with previously treated CLL, those taking IMBRUVICA® had a 57% statistically significant reduction in the risk of death compared with those who received ofatumumab (HR 0.43; 95% CI: 0.24, 0.79; P<0.05)¹

Typical treatment of chronic lymphocytic leukemia

http://www.cancer.org/cancer/leukemia-chroniclymphocyticcll/detailedguide/leukemia-chronic-lymphocytic-treating-treatment-by-risk-group

Treatment options for chronic lymphocytic leukemia (CLL) vary greatly, depending on the person’s age, the disease risk group, and the reason for treating (for example, which symptoms it is causing). Many people live a long time with CLL, but in general it is very difficult to cure, and early treatment hasn’t been shown to help people live longer. Because of this and because treatment can cause side effects, doctors often advise waiting until the disease is progressing or bothersome symptoms appear, before starting treatment.

If treatment is needed, factors that should be taken into account include the patient’s age, general health, and prognostic factors such as the presence of chromosome 17 or chromosome 11 deletions or high levels of ZAP-70 and CD38.

Initial treatment

Patients who might not be able to tolerate the side effects of strong chemotherapy (chemo), are often treated with chlorambucil alone or with a monoclonal antibody targeting CD20 like rituximab (Rituxan) or obinutuzumab (Gazyva). Other options include rituximab alone or a corticosteroid like prednisione.

In stronger and healthier patients, there are many options for treatment. Commonly used treatments include:

FCR: fludarabine (Fludara), cyclophosphamide (Cytoxan), and rituximab
Bendamustine (sometimes with rituximab)
FR: fludarabine and rituximab
CVP: cyclophosphamide, vincristine, and prednisone (sometimes with rituximab)
CHOP: cyclophosphamide, doxorubicin, vincristine (Oncovin), and prednisone
Chlorambucil combined with prednisone, rituximab, obinutuzumab, or ofatumumab
PCR: pentostatin (Nipent), cyclophosphamide, and rituximab
Alemtuzumab (Campath)
Fludarabine (alone)

Other drugs or combinations of drugs may also be also used.

If the only problem is an enlarged spleen or swollen lymph nodes in one region of the body, localized treatment with low-dose radiation therapy may be used. Splenectomy (surgery to remove the spleen) is another option if the enlarged spleen is causing symptoms.

Sometimes very high numbers of leukemia cells in the blood cause problems with normal circulation. This is calledleukostasis. Chemo may not lower the number of cells until a few days after the first dose, so before the chemo is given, some of the cells may be removed from the blood with a procedure called leukapheresis. This treatment lowers blood counts right away. The effect lasts only for a short time, but it may help until the chemo has a chance to work. Leukapheresis is also sometimes used before chemo if there are very high numbers of leukemia cells (even when they aren’t causing problems) to prevent tumor lysis syndrome (this was discussed in the chemotherapy section).

Some people who have very high-risk disease (based on prognostic factors) may be referred for possible stem cell transplant (SCT) early in treatment.

Second-line treatment of CLL

If the initial treatment is no longer working or the disease comes back, another type of treatment may help. If the initial response to the treatment lasted a long time (usually at least a few years), the same treatment can often be used again. If the initial response wasn’t long-lasting, using the same treatment again isn’t as likely to be helpful. The options will depend on what the first-line treatment was and how well it worked, as well as the person’s health.

Many of the drugs and combinations listed above may be options as second-line treatments. For many people who have already had fludarabine, alemtuzumab seems to be helpful as second-line treatment, but it carries an increased risk of infections. Other purine analog drugs, such as pentostatin or cladribine (2-CdA), may also be tried. Newer drugs such as ofatumumab, ibrutinib (Imbruvica), and idelalisib (Zydelig) may be other options.

If the leukemia responds, stem cell transplant may be an option for some patients.

Some people may have a good response to first-line treatment (such as fludarabine) but may still have some evidence of a small number of leukemia cells in the blood, bone marrow, or lymph nodes. This is known as minimal residual disease. CLL can’t be cured, so doctors aren’t sure if further treatment right away will be helpful. Some small studies have shown that alemtuzumab can sometimes help get rid of these remaining cells, but it’s not yet clear if this improves survival.

Treating complications of CLL

One of the most serious complications of CLL is a change (transformation) of the leukemia to a high-grade or aggressive type of non-Hodgkin lymphoma called diffuse large cell lymphoma. This happens in about 5% of CLL cases, and is known as Richter syndrome. Treatment is often the same as it would be for lymphoma (see our document called Non-Hodgkin Lymphoma for more information), and may include stem cell transplant, as these cases are often hard to treat.

Less often, CLL may transform to prolymphocytic leukemia. As with Richter syndrome, these cases can be hard to treat. Some studies have suggested that certain drugs such as cladribine (2-CdA) and alemtuzumab may be helpful.

In rare cases, patients with CLL may have their leukemia transform into acute lymphocytic leukemia (ALL). If this happens, treatment is likely to be similar to that used for patients with ALL (see our document called Leukemia: Acute Lymphocytic).

Acute myeloid leukemia (AML) is another rare complication in patients who have been treated for CLL. Drugs such as chlorambucil and cyclophosphamide can damage the DNA of blood-forming cells. These damaged cells may go on to become cancerous, leading to AML, which is very aggressive and often hard to treat (see our document calledLeukemia: Acute Myeloid).

CLL can cause problems with low blood counts and infections. Treatment of these problems were discussed in the section “Supportive care in chronic lymphocytic leukemia.”

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Treatments other than Chemotherapy for Leukemias and Lymphomas [2.5]

Posted in Acute lymphocytic leukemia, Acute myelocytic leukemia, Autologous Cell Therapy, Automated Cell Processing, Biomarkers & Medical Diagnostics, Bone marrow derived cells, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Circulating Progenitor Cells, Clinical Diagnostics, Clinical Genomics, Curation, Diagnostics and Lab Tests, Disease Biology, Evidence-based decision-making, Explanatory, Gene Regulation, Hematology, Hematopoiesis, Human Immune System in Health and in Disease, Imaging-based Cancer Patient Management, Lymphoma, Medical Imaging Technology, Myelodysplasia, Myelofibrosis, Regenerative Biology and Medicine, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Stem Cells for Regenerative Medicine, Technology Advance Assessment of, Translational Science, Umbilical cord cells, tagged allogeneic hemopoietic transplant, Autologous stem cell transplant, blood transfusion, hematological malignancies, nonchemotherapy support, Radiation therapy, stem cell therapy on August 11, 2015| Leave a Comment »

Treatments other than Chemotherapy for Leukemias and Lymphomas

Author, Curator, Editor: Larry H. Bernstein, MD, FCAP

2.5.1 Radiation Therapy

http://www.lls.org/treatment/types-of-treatment/radiation-therapy

Radiation therapy, also called radiotherapy or irradiation, can be used to treat leukemia, lymphoma, myeloma and myelodysplastic syndromes. The type of radiation used for radiotherapy (ionizing radiation) is the same that’s used for diagnostic x-rays. Radiotherapy, however, is given in higher doses.

Radiotherapy works by damaging the genetic material (DNA) within cells, which prevents them from growing and reproducing. Although the radiotherapy is directed at cancer cells, it can also damage nearby healthy cells. However, current methods of radiotherapy have been improved upon, minimizing “scatter” to nearby tissues. Therefore its benefit (destroying the cancer cells) outweighs its risk (harming healthy cells).

When radiotherapy is used for blood cancer treatment, it’s usually part of a treatment plan that includes drug therapy. Radiotherapy can also be used to relieve pain or discomfort caused by an enlarged liver, lymph node(s) or spleen.

Radiotherapy, either alone or with chemotherapy, is sometimes given as conditioning treatment to prepare a patient for a blood or marrow stem cell transplant. The most common types used to treat blood cancer are external beam radiation (see below) and radioimmunotherapy.
External Beam Radiation

External beam radiation is the type of radiotherapy used most often for people with blood cancers. A focused radiation beam is delivered outside the body by a machine called a linear accelerator, or linac for short. The linear accelerator moves around the body to deliver radiation from various angles. Linear accelerators make it possible to decrease or avoid skin reactions and deliver targeted radiation to lessen “scatter” of radiation to nearby tissues.

The dose (total amount) of radiation used during treatment depends on various factors regarding the patient, disease and reason for treatment, and is established by a radiation oncologist. You may receive radiotherapy during a series of visits, spread over several weeks (from two to 10 weeks, on average). This approach, called dose fractionation, lessens side effects. External beam radiation does not make you radioactive.

2.5.2 Bone marrow (BM) transplantation

http://www.nlm.nih.gov/medlineplus/ency/article/003009.htm

There are three kinds of bone marrow transplants:

Autologous bone marrow transplant: The term auto means self. Stem cells are removed from you before you receive high-dose chemotherapy or radiation treatment. The stem cells are stored in a freezer (cryopreservation). After high-dose chemotherapy or radiation treatments, your stems cells are put back in your body to make (regenerate) normal blood cells. This is called a rescue transplant.

Allogeneic bone marrow transplant: The term allo means other. Stem cells are removed from another person, called a donor. Most times, the donor’s genes must at least partly match your genes. Special blood tests are done to see if a donor is a good match for you. A brother or sister is most likely to be a good match. Sometimes parents, children, and other relatives are good matches. Donors who are not related to you may be found through national bone marrow registries.

Umbilical cord blood transplant: This is a type of allogeneic transplant. Stem cells are removed from a newborn baby’s umbilical cord right after birth. The stem cells are frozen and stored until they are needed for a transplant. Umbilical cord blood cells are very immature so there is less of a need for matching. But blood counts take much longer to recover.

Before the transplant, chemotherapy, radiation, or both may be given. This may be done in two ways:

Ablative (myeloablative) treatment: High-dose chemotherapy, radiation, or both are given to kill any cancer cells. This also kills all healthy bone marrow that remains, and allows new stem cells to grow in the bone marrow.

Reduced intensity treatment, also called a mini transplant: Patients receive lower doses of chemotherapy and radiation before a transplant. This allows older patients, and those with other health problems to have a transplant.

A stem cell transplant is usually done after chemotherapy and radiation is complete. The stem cells are delivered into your bloodstream usually through a tube called a central venous catheter. The process is similar to getting a blood transfusion. The stem cells travel through the blood into the bone marrow. Most times, no surgery is needed.

Donor stem cells can be collected in two ways:

Bone marrow harvest. This minor surgery is done under general anesthesia. This means the donor will be asleep and pain-free during the procedure. The bone marrow is removed from the back of both hip bones. The amount of marrow removed depends on the weight of the person who is receiving it.
Leukapheresis. First, the donor is given 5 days of shots to help stem cells move from the bone marrow into the blood. During leukapheresis, blood is removed from the donor through an IV line in a vein. The part of white blood cells that contains stem cells is then separated in a machine and removed to be later given to the recipient. The red blood cells are returned to the donor.

Why the Procedure is Performed

A bone marrow transplant replaces bone marrow that either is not working properly or has been destroyed (ablated) by chemotherapy or radiation. Doctors believe that for many cancers, the donor’s white blood cells can attach to any remaining cancer cells, similar to when white cells attach to bacteria or viruses when fighting an infection.

Your doctor may recommend a bone marrow transplant if you have:

Certain cancers, such as leukemia, lymphoma, and multiple myeloma

A disease that affects the production of bone marrow cells, such as aplastic anemia, congenital neutropenia, severe immunodeficiency syndromes, sickle cell anemia, thalassemia

Had chemotherapy that destroyed your bone

2.5.3 Autologous stem cell transplantation

Phase II trial of ¹³¹I-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphomas

O.W Press, F Appelbaum, P.J Martin, et al.
http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(95)92225-3/abstract

25 patients with relapsed B-cell lymphomas were evaluated with trace-labelled doses (2·5 mg/kg, 185-370 MBq [5-10 mCi]) of 131I-labelled anti-CD20 (B1) antibody in a phase II trial. 22 patients achieved 131I-B1 biodistributions delivering higher doses of radiation to tumor sites than to normal organs and 21 of these were treated with therapeutic infusions of 131I-B1 (12·765-29·045 GBq) followed by autologous hemopoietic stem cell reinfusion. 18 of the 21 treated patients had objective responses, including 16 complete remissions. One patient died of progressive lymphoma and one died of sepsis. Analysis of our phase I and II trials with 131I-labelled B1 reveal a progression-free survival of 62% and an overall survival of 93% with a median follow-up of 2 years. 131I-anti-CD20 (B1) antibody therapy produces complete responses of long duration in most patients with relapsed B-cell lymphomas when given at maximally tolerated doses with autologous stem cell rescue.

Autologous (Self) Transplants

http://www.leukaemia.org.au/treatments/stem-cell-transplants/autologous-self-transplants

An autologous transplant (or rescue) is a type of transplant that uses the person’s own stem cells. These cells are collected in advance and returned at a later stage. They are used to replace stem cells that have been damaged by high doses of chemotherapy, used to treat the person’s underlying disease.

In most cases, stem cells are collected directly from the bloodstream. While stem cells normally live in your marrow, a combination of chemotherapy and a growth factor (a drug that stimulates stem cells) called Granulocyte Colony Stimulating Factor (G-CSF) is used to expand the number of stem cells in the marrow and cause them to spill out into the circulating blood. From here they can be collected from a vein by passing the blood through a special machine called a cell separator, in a process similar to dialysis.

Most of the side effects of an autologous transplant are caused by the conditioning therapy used. Although they can be very unpleasant at times it is important to remember that most of them are temporary and reversible.

Procedure of Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the patient’s own stem cells are used) or allogeneic (the stem cells come from a donor).

Hematopoietic Stem Cell Transplantation

Author: Ajay Perumbeti, MD, FAAP; Chief Editor: Emmanuel C Besa, MD
http://emedicine.medscape.com/article/208954-overview

Hematopoietic stem cell transplantation (HSCT) involves the intravenous (IV) infusion of autologous or allogeneic stem cells to reestablish hematopoietic function in patients whose bone marrow or immune system is damaged or defective.

The image below illustrates an algorithm for typically preferred hematopoietic stem cell transplantation cell source for treatment of malignancy.

An algorithm for typically preferred hematopoietic stem cell transplantation cell source for treatment of malignancy: If a matched sibling donor is not available, then a MUD is selected; if a MUD is not available, then choices include a mismatched unrelated donor, umbilical cord donor(s), and a haploidentical donor.

Supportive Therapies

2.5.4 Blood transfusions – risks and complications of a blood transfusion

Allogeneic transfusion reaction (acute or delayed hemolytic reaction)
Allergic reaction
Viruses Infectious Diseases

The risk of catching a virus from a blood transfusion is very low.

HIV. Your risk of getting HIV from a blood transfusion is lower than your risk of getting killed by lightning. Only about 1 in 2 million donations might carry HIV and transmit HIV if given to a patient.

Hepatitis B and C. The risk of having a donation that carries hepatitis B is about 1 in 205,000. The risk for hepatitis C is 1 in 2 million. If you receive blood during a transfusion that contains hepatitis, you’ll likely develop the virus.

Variant Creutzfeldt-Jakob disease (vCJD). This disease is the human version of Mad Cow Disease. It’s a very rare, yet fatal brain disorder. There is a possible risk of getting vCJD from a blood transfusion, although the risk is very low. Because of this, people who may have been exposed to vCJD aren’t eligible blood donors.

Fever
Iron Overload
Lung Injury
Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) is a condition in which white blood cells in the new blood attack your tissues.

2.5.5 Erythropoietin

Erythropoietin, (/ɨ ˌ r ɪ θ r ɵ ˈ p ɔɪ .ɨ t ɨ n /, UK /ɛ ˌ r ɪ θ r oʊ .p oʊ ˈ iː t ɪ n /) also known as EPO, is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. It is a cytokine (protein signaling molecule) for erythrocyte (red blood cell) precursors in the bone marrow. Human EPO has a molecular weight of 34 kDa.

Also called hematopoietin or hemopoietin, it is produced by interstitial fibroblasts in the kidney in close association with peritubular capillary and proximal convoluted tubule. It is also produced in perisinusoidal cells in the liver. While liver production predominates in the fetal and perinatal period, renal production is predominant during adulthood. In addition to erythropoiesis, erythropoietin also has other known biological functions. For example, it plays an important role in the brain’s response to neuronal injury.^[1] EPO is also involved in the wound healing process.^[2]

Exogenous erythropoietin is produced by recombinant DNA technology in cell culture. Several different pharmaceutical agents are available with a variety ofglycosylation patterns, and are collectively called erythropoiesis-stimulating agents (ESA). The specific details for labelled use vary between the package inserts, but ESAs have been used in the treatment of anemia in chronic kidney disease, anemia in myelodysplasia, and in anemia from cancer chemotherapy. Boxed warnings include a risk of death, myocardial infarction, stroke, venous thromboembolism, and tumor recurrence.^[3]

2.5.6 G-CSF (granulocyte-colony stimulating factor)

Granulocyte-colony stimulating factor (G-CSF or GCSF), also known as colony-stimulating factor 3 (CSF 3), is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream.

There are different types, including

Lenograstim (Granocyte)
Filgrastim (Neupogen, Zarzio, Nivestim, Ratiograstim)
Long acting (pegylated) filgrastim (pegfilgrastim, Neulasta) and lipegfilgrastim (Longquex)

Pegylated G-CSF stays in the body for longer so you have treatment less often than with the other types of G-CSF.

2.5.7 Plasma Exchange (plasmapheresis)

http://emedicine.medscape.com/article/1895577-overview

Plasmapheresis is a term used to refer to a broad range of procedures in which extracorporeal separation of blood components results in a filtered plasma product.^{[1, 2]}The filtering of plasma from whole blood can be accomplished via centrifugation or semipermeable membranes.^[3]Centrifugation takes advantage of the different specific gravities inherent to various blood products such as red cells, white cells, platelets, and plasma.^[4]Membrane plasma separation uses differences in particle size to filter plasma from the cellular components of blood.^[3]

Traditionally, in the United States, most plasmapheresis takes place using automated centrifuge-based technology.^[5]In certain instances, in particular in patients already undergoing hemodialysis, plasmapheresis can be carried out using semipermeable membranes to filter plasma.^[4]

In therapeutic plasma exchange, using an automated centrifuge, filtered plasma is discarded and red blood cells along with replacement colloid such as donor plasma or albumin is returned to the patient. In membrane plasma filtration, secondary membrane plasma fractionation can selectively remove undesired macromolecules, which then allows for return of the processed plasma to the patient instead of donor plasma or albumin. Examples of secondary membrane plasma fractionation include cascade filtration,^[6]thermofiltration, cryofiltration,^[7]and low-density lipoprotein pheresis.

The Apheresis Applications Committee of the American Society for Apheresis periodically evaluates potential indications for apheresis and categorizes them from I to IV based on the available medical literature. The following are some of the indications, and their categorization, from the society’s 2010 guidelines.[2]

The only Category I indication for hemopoietic malignancy is Hyperviscosity in monoclonal gammopathies

2.5.8 Platelet Transfusions

Indications for platelet transfusion in children with acute leukemia

Scott Murphy, Samuel Litwin, Leonard M. Herring, Penelope Koch, et al.
Am J Hematol Jun 1982; 12(4): 347–356
http://onlinelibrary.wiley.com/doi/10.1002/ajh.2830120406/abstract;jsessionid=A6001D9D865EA1EBC667EF98382EF20C.f03t01
http://dx.doi.org:/10.1002/ajh.2830120406

In an attempt to determine the indications for platelet transfusion in thrombocytopenic patients, we randomized 56 children with acute leukemia to one of two regimens of platelet transfusion. The prophylactic group received platelets when the platelet count fell below 20,000 per mm3 irrespective of clinical events. The therapeutic group was transfused only when significant bleeding occurred and not for thrombocytopenia alone. The time to first bleeding episode was significantly longer and the number of bleeding episodes were significantly reduced in the prophylactic group. The survival curves of the two groups could not be distinguished from each other. Prior to the last month of life, the total number of days on which bleeding was present was significantly reduced by prophylactic therapy. However, in the terminal phase (last month of life), the duration of bleeding episodes was significantly longer in the prophylactic group. This may have been due to a higher incidence of immunologic refractoriness to platelet transfusion. Because of this terminal bleeding, comparison of the two groups for total number of days on which bleeding was present did not show a significant difference over the entire study period.

Clinical and Laboratory Aspects of Platelet Transfusion Therapy
Yuan S, Goldfinger D
http://www.uptodate.com/contents/clinical-and-laboratory-aspects-of-platelet-transfusion-therapy

INTRODUCTION — Hemostasis depends on an adequate number of functional platelets, together with an intact coagulation (clotting factor) system. This topic covers the logistics of platelet use and the indications for platelet transfusion in adults. The approach to the bleeding patient, refractoriness to platelet transfusion, and platelet transfusion in neonates are discussed elsewhere.

Pooled Platelets – A single unit of platelets can be isolated from every unit of donated blood, by centrifuging the blood within the closed collection system to separate the platelets from the red blood cells (RBC). The number of platelets per unit varies according to the platelet count of the donor; a yield of 7 x 10¹⁰ platelets is typical [1]. Since this number is inadequate to raise the platelet count in an adult recipient, four to six units are pooled to allow transfusion of 3 to 4 x 10¹¹ platelets per transfusion [2]. These are called whole blood-derived or random donor pooled platelets.

Advantages of pooled platelets include lower cost and ease of collection and processing (a separate donation procedure and pheresis equipment are not required). The major disadvantage is recipient exposure to multiple donors in a single transfusion and logistic issues related to bacterial testing.

Apheresis (single donor) Platelets – Platelets can also be collected from volunteer donors in the blood bank, in a one- to two-hour pheresis procedure. Platelets and some white blood cells are removed, and red blood cells and plasma are returned to the donor. A typical apheresis platelet unit provides the equivalent of six or more units of platelets from whole blood (ie, 3 to 6 x 10¹¹ platelets) [2]. In larger donors with high platelet counts, up to three units can be collected in one session. These are called apheresis or single donor platelets.

Advantages of single donor platelets are exposure of the recipient to a single donor rather than multiple donors, and the ability to match donor and recipient characteristics such as HLA type, cytomegalovirus (CMV) status, and blood type for certain recipients.

Both pooled and apheresis platelets contain some white blood cells (WBC) that were collected along with the platelets. These WBC can cause febrile non-hemolytic transfusion reactions (FNHTR), alloimmunization, and transfusion-associated graft-versus-host disease (ta-GVHD) in some patients.

Platelet products also contain plasma, which can be implicated in adverse reactions including transfusion-related acute lung injury (TRALI) and anaphylaxis. (See ‘Complications of platelet transfusion’ .)

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Treatments for Leukemias and Lymphomas [2.4.4]

Posted in Academic Publishing, Best evidence, Genetics & Pharmaceutical, Genome Biology, Genomic Testing: Methodology for Diagnosis, Hematology, Imaging-based Cancer Patient Management, Immunodiagnostics, Lymphoma, Pharmaceutical Drug Discovery, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Translational Effectiveness, Translational Research, Translational Science, tagged Hodgkin's lymphoma, Non-Hodgkin lymphoma, treatment of lymphomas on August 11, 2015| Leave a Comment »

Treatments for Lymphomas and Leukemias

Curator and Editor: Larry H. Bernstein, MD, FCAP

2.4.4 Treatments for leukemia by type

2.4.4.1 Acute Lymphocytic Leukemias

Treatment of Acute Lymphoblastic Leukemia

Ching-Hon Pu, and William E. Evans
N Engl J Med Jan 12, 2006; 354:166-178
http://dx.doi.org:/10.1056/NEJMra052603

Acute Lymphoblastic Leukemia

Ching-Hon Pui, Mary V. Relling, and James R. Downing
N Engl J Med Apr 8, 2004; 350:1535-1548
http://dx.doi.org:/10.1056/NEJMra023001

This comprehensive survey emphasizes how recent advances in the knowledge of molecular mechanisms involved in acute lymphoblastic leukemia have influenced diagnosis, prognosis, and treatment.

Gene-Expression Patterns in Drug-Resistant Acute Lymphoblastic Leukemia Cells and Response to Treatment

Amy Holleman, Meyling H. Cheok, Monique L. den Boer, et al.
N Engl J Med 2004; 351:533-42

Conclusions Differential expression of a relatively small number of genes is associated with drug resistance and treatment outcome in childhood ALL.

Leukemias Treatment & Management

Author: Lihteh Wu, MD; Chief Editor: Hampton Roy Sr
http://emedicine.medscape.com/article/1201870-treatment

The initial treatment of ALL uses various combinations of vincristine, prednisone, and L-asparaginase until a complete remission is obtained.
Maintenance therapy with mercaptopurine is continued for 2-3 years following remission.
Use of intrathecal methotrexate with or without cranial irradiation to cover the CNS varies from facility to facility.
Daunorubicin, cytarabine, and thioguanine currently are used to obtain induction and remission of AML.
Maintenance therapy for 8 months may lengthen remission. Once relapse has occurred, AML generally is curable only by bone marrow transplantation.
Presently, treatment of CLL is palliative.
CML is characterized by a leukocytosis greater than 100,000 cells. Emergent treatment with leukopheresis sometimes is necessary when leukostastic complications are present. Otherwise, busulfan or hydroxyurea may control WBC counts. During the chronic phase, treatment is palliative.
When CML converts to the blastic phase, approximately one third of cases behave as ALL and respond to treatment with vincristine and prednisone. The remaining two thirds resemble AML but respond poorly to AML therapy.
Allogeneic bone marrow transplant is the only curative therapy for CML. However, it carries a high early mortality rate.
Leukemic retinopathy usually is not treated directly. As the hematological parameters normalize with systemic treatment, many of the ophthalmic signs resolve. There are reports that leukopheresis for hyperviscosity also may alleviate intraocular manifestations.
When definite intraocular leukemic infiltrates fail to respond to systemic chemotherapy, direct radiation therapy is recommended.
Relapse, manifested by anterior segment involvement, should be treated by radiation. In certain cases, subconjunctival chemotherapeutic agents have been injected.
Optic nerve head infiltration in patients with ALL is an emergency and requires prompt radiation therapy to try to salvage some vision.

Treatments and drugs

http://www.mayoclinic.org/diseases-conditions/leukemia/basics/
treatment/con-20024914

Common treatments used to fight leukemia include:

Chemotherapy. Chemotherapy is the major form of treatment for leukemia. This drug treatment uses chemicals to kill leukemia cells.

Depending on the type of leukemia you have, you may receive a single drug or a combination of drugs. These drugs may come in a pill form, or they may be injected directly into a vein.

Biological therapy. Biological therapy works by using treatments that help your immune system recognize and attack leukemia cells.
Targeted therapy. Targeted therapy uses drugs that attack specific vulnerabilities within your cancer cells.

For example, the drug imatinib (Gleevec) stops the action of a protein within the leukemia cells of people with chronic myelogenous leukemia. This can help control the disease.

Radiation therapy. Radiation therapy uses X-rays or other high-energy beams to damage leukemia cells and stop their growth. During radiation therapy, you lie on a table while a large machine moves around you, directing the radiation to precise points on your body.

Stem cell transplant. A stem cell transplant is a procedure to replace your diseased bone marrow with healthy bone marrow.

You may receive stem cells from a donor, or in some cases you may be able to use your own stem cells. A stem cell transplant is very similar to a bone marrow transplant.

2.4.4.2 Acute Myeloid Leukemia

New treatment approaches in acute myeloid leukemia: review of recent clinical studies.

Norsworthy K¹, Luznik L, Gojo I.
Rev Recent Clin Trials. 2012 Aug; 7(3):224-37.
http://www.ncbi.nlm.nih.gov/pubmed/22540908

Novel approaches to the treatment of acute myeloid leukemia.

Roboz GJ¹Hematology Am Soc Hematol Educ Program. 2011:43-50.
http://dx.doi.org:/10.1182/asheducation-2011.1.43.

Current treatment of acute myeloid leukemia.

Roboz GJ¹.
Curr Opin Oncol. 2012 Nov; 24(6):711-9.
http://dx.doi.org:/10.1097/CCO.0b013e328358f62d.

The objectives of this review are to discuss standard and investigational nontransplant treatment strategies for acute myeloid leukemia (AML), excluding acute promyelocytic leukemia.

Adult Acute Myeloid Leukemia Treatment (PDQ®)
http://www.cancer.gov/cancertopics/pdq/treatment/adultAML/healthprofessional/page9

About This PDQ Summary

Board members review recently published articles each month to determine whether an article should:

be discussed at a meeting,
be cited with text, or
replace or update an existing article that is already cited.

Treatment Option Overview for AML

References

Kebriaei P, Champlin R, deLima M, et al.: Management of acute leukemias. In: DeVita VT Jr, Lawrence TS, Rosenberg SA: Cancer: Principles and Practice of Oncology. 9th ed. Philadelphia, Pa: Lippincott Williams & Wilkins, 2011, pp 1928-54.
Wiernik PH: Diagnosis and treatment of acute nonlymphocytic leukemia. In: Wiernik PH, Canellos GP, Dutcher JP, et al., eds.: Neoplastic Diseases of the Blood. 3rd ed. New York, NY: Churchill Livingstone, 1996, pp 283-302.
Morrison FS, Kopecky KJ, Head DR, et al.: Late intensification with POMP chemotherapy prolongs survival in acute myelogenous leukemia–results of a Southwest Oncology Group study of rubidazone versus adriamycin for remission induction, prophylactic intrathecal therapy, late intensification, and levamisole maintenance. Leukemia 6 (7): 708-14, 1992. [PUBMED Abstract]
Cassileth PA, Lynch E, Hines JD, et al.: Varying intensity of postremission therapy in acute myeloid leukemia. Blood 79 (8): 1924-30, 1992. [PUBMED Abstract]
Supportive Care. In: Wiernik PH, Canellos GP, Dutcher JP, et al., eds.: Neoplastic Diseases of the Blood. 3rd ed. New York, NY: Churchill Livingstone, 1996, pp 779-967.
Rebulla P, Finazzi G, Marangoni F, et al.: The threshold for prophylactic platelet transfusions in adults with acute myeloid leukemia. Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto. N Engl J Med 337 (26): 1870-5, 1997. [PUBMED Abstract]
Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. The Trial to Reduce Alloimmunization to Platelets Study Group. N Engl J Med 337 (26): 1861-9, 1997. [PUBMED Abstract]
Geller RB: Use of cytokines in the treatment of acute myelocytic leukemia: a critical review. J Clin Oncol 14 (4): 1371-82, 1996. [PUBMED Abstract]
Rowe JM, Andersen JW, Mazza JJ, et al.: A randomized placebo-controlled phase III study of granulocyte-macrophage colony-stimulating factor in adult patients (> 55 to 70 years of age) with acute myelogenous leukemia: a study of the Eastern Cooperative Oncology Group (E1490). Blood 86 (2): 457-62, 1995. [PUBMED Abstract]
Stone RM, Berg DT, George SL, et al.: Granulocyte-macrophage colony-stimulating factor after initial chemotherapy for elderly patients with primary acute myelogenous leukemia. Cancer and Leukemia Group B. N Engl J Med 332 (25): 1671-7, 1995. [PUBMED Abstract]
Dombret H, Chastang C, Fenaux P, et al.: A controlled study of recombinant human granulocyte colony-stimulating factor in elderly patients after treatment for acute myelogenous leukemia. AML Cooperative Study Group. N Engl J Med 332 (25): 1678-83, 1995. [PUBMED Abstract]
Godwin JE, Kopecky KJ, Head DR, et al.: A double-blind placebo-controlled trial of granulocyte colony-stimulating factor in elderly patients with previously untreated acute myeloid leukemia: a Southwest oncology group study (9031). Blood 91 (10): 3607-15, 1998. [PUBMED Abstract]
Buchner T, Hiddemann W, Wormann B, et al.: GM-CSF multiple course priming and long-term administration in newly diagnosed AML: hematologic and therapeutic effects. [Abstract] Blood 84 (10 Suppl 1): A-95, 27a, 1994.
Löwenberg B, Boogaerts MA, Daenen SM, et al.: Value of different modalities of granulocyte-macrophage colony-stimulating factor applied during or after induction therapy of acute myeloid leukemia. J Clin Oncol 15 (12): 3496-506, 1997. [PUBMED Abstract]
Witz F, Sadoun A, Perrin MC, et al.: A placebo-controlled study of recombinant human granulocyte-macrophage colony-stimulating factor administered during and after induction treatment for de novo acute myelogenous leukemia in elderly patients. Groupe Ouest Est Leucémies Aiguës Myéloblastiques (GOELAM). Blood 91 (8): 2722-30, 1998. [PUBMED Abstract]

2.4.4.3 Treatment for CML

Chronic Myelogenous Leukemia Treatment (PDQ®)

http://www.cancer.gov/cancertopics/pdq/treatment/CML/Patient/page4

Treatment Option Overview

Key Points for This Section

There are different types of treatment for patients with chronic myelogenous leukemia.

Six types of standard treatment are used:

Targeted therapy
Chemotherapy
Biologic therapy
High-dose chemotherapy with stem cell transplant
Donor lymphocyte infusion (DLI)
Surgery

New types of treatment are being tested in clinical trials.

Patients may want to think about taking part in a clinical trial.

Patients can enter clinical trials before, during, or after starting their cancer treatment.

Follow-up tests may be needed.

There are different types of treatment for patients with chronic myelogenous leukemia.

Six types of standard treatment are used:

Targeted therapy

Imatinib mesylate, nilotinib, dasatinib, and ponatinib are tyrosine kinase inhibitors that are used to treat CML.

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

Chemotherapy

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

Biologic therapy

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

High-dose chemotherapy with stem cell transplant

See Drugs Approved for Chronic Myelogenous Leukemia for more information.

Donor lymphocyte infusion (DLI)

Surgery

Splenectomy

What`s new in chronic myeloid leukemia research and treatment?

http://www.cancer.org/cancer/leukemia-chronicmyeloidcml/detailedguide/leukemia-chronic-myeloid-myelogenous-new-research

Combining the targeted drugs with other treatments

Other studies are looking at combining other drugs, such as cyclosporine or hydroxychloroquine, with a TKI.

New drugs for CML

Because researchers now know the main cause of CML (the BCR-ABL gene and its protein), they have been able to develop many new drugs that might work against it.

Other drugs being studied in CML include the histone deacetylase inhibitor panobinostat and the proteasome inhibitor bortezomib (Velcade).

Several vaccines are now being studied for use against CML.

2.4.4.4. Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia Treatment (PDQ®)

General Information About Chronic Lymphocytic Leukemia

Key Points for This Section

Chronic lymphocytic leukemia is a type of cancer in which the bone marrow makes too many lymphocytes (a type of white blood cell).
Leukemia may affect red blood cells, white blood cells, and platelets.
Older age can affect the risk of developing chronic lymphocytic leukemia.
Signs and symptoms of chronic lymphocytic leukemia include swollen lymph nodes and tiredness.
Tests that examine the blood, bone marrow, and lymph nodes are used to detect (find) and diagnose chronic lymphocytic leukemia.
Certain factors affect treatment options and prognosis (chance of recovery).
Chronic lymphocytic leukemia is a type of cancer in which the bone marrow makes too many lymphocytes (a type of white blood cell).

http://www.cancer.gov/images/cdr/live/CDR755927-750.jpg

Leukemia may affect red blood cells, white blood cells, and platelets.

Normally, the body makes blood stem cells (immature cells) that become mature blood cells over time. A blood stem cell may become a myeloid stem cell or a lymphoid stem cell.

A myeloid stem cell becomes one of three types of mature blood cells:

Red blood cells that carry oxygen and other substances to all tissues of the body.
White blood cells that fight infection and disease.
Platelets that form blood clots to stop bleeding.

A lymphoid stem cell becomes a lymphoblast cell and then one of three types of lymphocytes (white blood cells):

B lymphocytes that make antibodies to help fight infection.
T lymphocytes that help B lymphocytes make antibodies to fight infection.
Natural killer cells that attack cancer cells and viruses.

Blood cell development. CDR526538-750

http://www.cancer.gov/images/cdr/live/CDR526538-750.jpg

Blood cell development. A blood stem cell goes through several steps to become a red blood cell, platelet, or white blood cell.

This summary is about chronic lymphocytic leukemia. See the following PDQ summaries for more information about leukemia:

Adult Acute Lymphoblastic Leukemia Treatment.
Childhood Acute Lymphoblastic Leukemia Treatment.
Adult Acute Myeloid Leukemia Treatment.
Childhood Acute Myeloid Leukemia/Other Myeloid Malignancies Treatment.
Chronic Myelogenous Leukemia Treatment.
Hairy Cell Leukemia Treatment

Older age can affect the risk of developing chronic lymphocytic leukemia.

Being middle-aged or older, male, or white.
A family history of CLL or cancer of the lymph system.
Having relatives who are Russian Jews or Eastern European Jews.

Signs and symptoms of chronic lymphocytic leukemia include swollen lymph nodes and tiredness.

Painless swelling of the lymph nodes in the neck, underarm, stomach, or groin.
Feeling very tired.
Pain or fullness below the ribs.
Fever and infection.
Weight loss for no known reason.

Tests that examine the blood, bone marrow, and lymph nodes are used to detect (find) and diagnose chronic lymphocytic leukemia.

The following tests and procedures may be used:

Complete blood count (CBC) with differential : A procedure in which a sample of blood is drawn and checked for the following:
The number of red blood cells and platelets.
The number and type of white blood cells.
The amount of hemoglobin (the protein that carries oxygen) in the red blood cells.
The portion of the blood sample made up of red blood cells.

Results from the Phase 3 Resonate™ Trial

Significantly improved progression free survival (PFS) vs ofatumumab in patients with previously treated CLL

Patients taking IMBRUVICA® had a 78% statistically significant reduction in the risk of disease progression or death compared with patients who received ofatumumab¹
In patients with previously treated del 17p CLL, median PFS was not yet reached with IMBRUVICA® vs 5.8 months with ofatumumab (HR 0.25; 95% CI: 0.14, 0.45)¹

Significantly prolonged overall survival (OS) with IMBRUVICA® vs ofatumumab in patients with previously treated CLL

In patients with previously treated CLL, those taking IMBRUVICA® had a 57% statistically significant reduction in the risk of death compared with those who received ofatumumab (HR 0.43; 95% CI: 0.24, 0.79; P<0.05)¹

Typical treatment of chronic lymphocytic leukemia

http://www.cancer.org/cancer/leukemia-chroniclymphocyticcll/detailedguide/leukemia-chronic-lymphocytic-treating-treatment-by-risk-group

Initial treatment

In stronger and healthier patients, there are many options for treatment. Commonly used treatments include:

FCR: fludarabine (Fludara), cyclophosphamide (Cytoxan), and rituximab
Bendamustine (sometimes with rituximab)
FR: fludarabine and rituximab
CVP: cyclophosphamide, vincristine, and prednisone (sometimes with rituximab)
CHOP: cyclophosphamide, doxorubicin, vincristine (Oncovin), and prednisone
Chlorambucil combined with prednisone, rituximab, obinutuzumab, or ofatumumab
PCR: pentostatin (Nipent), cyclophosphamide, and rituximab
Alemtuzumab (Campath)
Fludarabine (alone)

Other drugs or combinations of drugs may also be also used.

Some people who have very high-risk disease (based on prognostic factors) may be referred for possible stem cell transplant (SCT) early in treatment.

Second-line treatment of CLL

If the leukemia responds, stem cell transplant may be an option for some patients.

Treating complications of CLL

CLL can cause problems with low blood counts and infections. Treatment of these problems were discussed in the section “Supportive care in chronic lymphocytic leukemia.”

2.4.4.5 Lymphoma treatment

Overview

http://www.emedicinehealth.com/lymphoma/page8_em.htm#lymphoma_treatment

The most widely used therapies are combinations of chemotherapy and radiation therapy.

Biological therapy, which targets key features of the lymphoma cells, is used in many cases nowadays.

When in remission, the lymphoma may come back. This is called recurrence.
The duration of remission depends on the type, stage, and grade of the lymphoma. A remission may last a few months, a few years, or may continue throughout one’s life.
Remission that lasts a long time is called durable remission, and this is the goal of therapy.
The duration of remission is a good indicator of the aggressiveness of the lymphoma and of the prognosis. A longer remission generally indicates a better prognosis.

Remission can also be partial. This means that the tumor shrinks after treatment to less than half its size before treatment.

The following terms are used to describe the lymphoma’s response to treatment:

Improvement: The lymphoma shrinks but is still greater than half its original size.
Stable disease: The lymphoma stays the same.
Progression: The lymphoma worsens during treatment.
Refractory disease: The lymphoma is resistant to treatment.

The following terms to refer to therapy:

Induction therapy is designed to induce a remission.
If this treatment does not induce a complete remission, new or different therapy will be initiated. This is usually referred to as salvage therapy.
Once in remission, one may be given yet another treatment to prevent recurrence. This is called maintenance therapy.

Chemotherapy

ABVD: Doxorubicin (Adriamycin), bleomycin (Blenoxane), vinblastine (Velban, Velsar), and dacarbazine (DTIC-Dome). ABVD chemotherapy is usually given every two weeks for two to eight months.

The type of chemotherapy, number of cycles of chemotherapy, and the additional use of radiation therapy are based on the stage of the Hodgkin lymphoma and the type and number of prognostic factors.

Adult Non-Hodgkin Lymphoma Treatment (PDQ®)

http://www.cancer.gov/cancertopics/pdq/treatment/adult-non-hodgkins/Patient/page1

Key Points for This Section

Adult non-Hodgkin Lymphoma is a disease in which malignant (cancer) cells form in the lymph system.

Because lymph tissue is found throughout the body, adult non-Hodgkin lymphoma can begin in almost any part of the body. Cancer can spread to the liver and many other organs and tissues.

There are many different types of lymphoma.

Age, gender, and a weakened immune system can affect the risk of adult non-Hodgkin lymphoma.

If cancer is found, the following tests may be done to study the cancer cells:

Immunohistochemistry : A test that uses antibodies to check for certain antigens in a sample of tissue. The antibody is usually linked to a radioactive substance or a dye that causes the tissue to light up under a microscope. This type of test may be used to tell the difference between different types of cancer.
Cytogenetic analysis : A laboratory test in which cells in a sample of tissue are viewed under a microscope to look for certain changes in the chromosomes.
Immunophenotyping : A process used to identify cells, based on the types of antigens ormarkers on the surface of the cell. This process is used to diagnose specific types of leukemia and lymphoma by comparing the cancer cells to normal cells of the immune system.

Certain factors affect prognosis (chance of recovery) and treatment options.

The prognosis (chance of recovery) and treatment options depend on the following:

The stage of the cancer.
The type of non-Hodgkin lymphoma.
The amount of lactate dehydrogenase (LDH) in the blood.
The amount of beta-2-microglobulin in the blood (for Waldenström macroglobulinemia).
The patient’s age and general health.
Whether the lymphoma has just been diagnosed or has recurred (come back).

Stages of adult non-Hodgkin lymphoma may include E and S.

Adult non-Hodgkin lymphoma may be described as follows:

E: “E” stands for extranodal and means the cancer is found in an area or organ other than the lymph nodes or has spread to tissues beyond, but near, the major lymphatic areas.

S: “S” stands for spleen and means the cancer is found in the spleen.

Stage I adult non-Hodgkin lymphoma is divided into stage I and stage IE.

Stage I: Cancer is found in one lymphatic area (lymph node group, tonsils and nearby tissue, thymus, or spleen).
Stage IE: Cancer is found in one organ or area outside the lymph nodes.

Stage II adult non-Hodgkin lymphoma is divided into stage II and stage IIE.

Stage II: Cancer is found in two or more lymph node groups either above or below the diaphragm (the thin muscle below the lungs that helps breathing and separates the chest from the abdomen).
Stage IIE: Cancer is found in one or more lymph node groups either above or below the diaphragm. Cancer is also found outside the lymph nodes in one organ or area on the same side of the diaphragm as the affected lymph nodes.

Stage III adult non-Hodgkin lymphoma is divided into stage III, stage IIIE, stage IIIS, and stage IIIE+S.

Stage III: Cancer is found in lymph node groups above and below the diaphragm (the thin muscle below the lungs that helps breathing and separates the chest from the abdomen).
Stage IIIE: Cancer is found in lymph node groups above and below the diaphragm and outside the lymph nodes in a nearby organ or area.
Stage IIIS: Cancer is found in lymph node groups above and below the diaphragm, and in the spleen.
Stage IIIE+S: Cancer is found in lymph node groups above and below the diaphragm, outside the lymph nodes in a nearby organ or area, and in the spleen.

In stage IV adult non-Hodgkin lymphoma, the cancer:

is found throughout one or more organs that are not part of a lymphatic area (lymph node group, tonsils and nearby tissue, thymus, or spleen), and may be in lymph nodes near those organs; or
is found in one organ that is not part of a lymphatic area and has spread to organs or lymph nodes far away from that organ; or
is found in the liver, bone marrow, cerebrospinal fluid (CSF), or lungs (other than cancer that has spread to the lungs from nearby areas).

Adult non-Hodgkin lymphomas are also described based on how fast they grow and where the affected lymph nodes are in the body. Indolent & aggressive.

The treatment plan depends mainly on the following:

The type of non-Hodgkin’s lymphoma
Its stage (where the lymphoma is found)
How quickly the cancer is growing
The patient’s age
Whether the patient has other health problems
If there are symptoms present such as fever and night sweats (see above)

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Hematological Malignancy Diagnostics

Posted in Academic Publishing, Acute lymphocytic leukemia, Acute myelocytic leukemia, Anemia, BioIT: BioInformatics, Biomarkers & Medical Diagnostics, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Cancer Screening, Cell Biology, Clinical & Translational, Clinical Diagnostics, Clinical Genomics, Curation, Curation methodology, Discovery process, Disease Biology, Experimental validation, Explanatory, Genetics & Pharmaceutical, Genome Biology, Genomic Expression, Genomic Testing: Methodology for Diagnosis, Hematology, Hematopoiesis, Lymphoma, Myelodysplasia, Myelofibrosis, Neutropenia, Neutrophilia, Platelet count disorder, tagged diagnostics, Flow cytometry, genomic analysis, hematological disorders on August 11, 2015| Leave a Comment »

Hematological Malignancy Diagnostics

Author and Curator: Larry H. Bernstein, MD, FCAP

2.4.3 Diagnostics

2.4.3.1 Computer-aided diagnostics

Back-to-Front Design

Robert Didner
Bell Laboratories

Decision-making in the clinical setting
Didner, R Mar 1999 Amer Clin Lab

Mr. Didner is an Independent Consultant in Systems Analysis, Information Architecture (Informatics) Operations Research, and Human Factors Engineering (Cognitive Psychology), Decision Information Designs, 29 Skyline Dr., Morristown, NJ07960, U.S.A.; tel.: 973-455-0489; fax/e-mail: bdidner@hotmail.com

A common problem in the medical profession is the level of effort dedicated to administration and paperwork necessitated by various agencies, which contributes to the high cost of medical care. Costs would be reduced and accuracy improved if the clinical data could be captured directly at the point they are generated in a form suitable for transmission to insurers or machine transformable into other formats. Such a capability could also be used to improve the form and the structure of information presented to physicians and support a more comprehensive database linking clinical protocols to outcomes, with the prospect of improving clinical outcomes. Although the problem centers on the physician’s process of determining the diagnosis and treatment of patients and the timely and accurate recording of that process in the medical system, it substantially involves the pathologist and laboratorian, who interact significantly throughout the in-formation-gathering process. Each of the currently predominant ways of collecting information from diagnostic protocols has drawbacks. Using blank paper to collect free-form notes from the physician is not amenable to computerization; such free-form data are also poorly formulated, formatted, and organized for the clinical decision-making they support. The alternative of preprinted forms listing the possible tests, results, and other in-formation gathered during the diagnostic process facilitates the desired computerization, but the fixed sequence of tests and questions they present impede the physician from using an optimal decision-making sequence. This follows because:

People tend to make decisions and consider information in a step-by-step manner in which intermediate decisions are intermixed with data acquisition steps.
The sequence in which components of decisions are made may alter the decision outcome.
People tend to consider information in the sequence it is requested or displayed.
Since there is a separate optimum sequence of tests and questions for each cluster of history and presenting symptoms, there is no one sequence of tests and questions that can be optimal for all presenting clusters.
As additional data and test results are acquired, the optimal sequence of further testing and data acquisition changes, depending on the already acquired information.

Therefore, promoting an arbitrary sequence of information requests with preprinted forms may detract from outcomes by contributing to a non-optimal decision-making sequence. Unlike the decisions resulting from theoretical or normative processes, decisions made by humans are path dependent; that is, the out-come of a decision process may be different if the same components are considered in a different sequence.

Proposed solution

This paper proposes a general approach to gathering data at their source in computer-based form so as to improve the expected outcomes. Such a means must be interactive and dynamic, so that at any point in the clinical process the patient’s presenting symptoms, history, and the data already collected are used to determine the next data or tests requested. That de-termination must derive from a decision-making strategy designed to produce outcomes with the greatest value and supported by appropriate data collection and display techniques. The strategy must be based on the knowledge of the possible outcomes at any given stage of testing and information gathering, coupled with a metric, or hierarchy of values for assessing the relative desirability of the possible outcomes.

A value hierarchy

The numbered list below illustrates a value hierarchy. In any particular instance, the higher-numbered values should only be considered once the lower- numbered values have been satisfied. Thus, a diagnostic sequence that is very time or cost efficient should only be considered if it does not increase the likelihood (relative to some other diagnostic sequence) that a life-threatening disorder may be missed, or that one of the diagnostic procedures may cause discomfort.
Minimize the likelihood that a treatable, life-threatening disorder is not treated.
Minimize the likelihood that a treatable, discomfort-causing disorder is not treated.
Minimize the likelihood that a risky procedure(treatment or diagnostic procedure) is inappropriately administered.
Minimize the likelihood that a discomfort-causing procedure is inappropriately administered.
Minimize the likelihood that a costly procedure is inappropriately administered.
Minimize the time of diagnosing and treating thepatient.8.Minimize the cost of diagnosing and treating the patient.

The above hierarchy is relative, not absolute; for many patients, a little bit of testing discomfort may be worth a lot of time. There are also some factors and graduations intentionally left out for expository simplicity (e.g., acute versus chronic disorders).This value hierarchy is based on a hypothetical patient. Clearly, the hierarchy of a health insurance carrier might be different, as might that of another patient (e.g., a geriatric patient). If the approach outlined herein were to be followed, a value hierarchy agreed to by a majority of stakeholders should be adopted.

Efficiency

Once the higher values are satisfied, the time and cost of diagnosis and treatment should be minimized. One way to do so would be to optimize the sequence in which tests are performed, so as to minimize the number, cost, and time of tests that need to be per-formed to reach a definitive decision regarding treatment. Such an optimum sequence could be constructed using Claude Shannon’s information theory.

According to this theory, the best next question to ask under any given situation (assuming the question has two possible outcomes) is that question that divides the possible outcomes into two equally likely sets. In the real world, all tests or questions are not equally valuable, costly, or time consuming; therefore, value(risk factors), cost, and time should be used as weighting factors to optimize the test sequence, but this is a complicating detail at this point.

A value scale

For dynamic computation of outcome values, the hierarchy could be converted into a weighted value scale so differing outcomes at more than one level of the hierarchy could be readily compared. An example of such a weighted value scale is Quality Adjusted Life Years (QALY).

Although QALY does not incorporate all of the factors in this example, it is a good conceptual starting place.

The display, request, decision-making relationship

For each clinical determination, the pertinent information should be gathered, organized, formatted, and formulated in a way that facilitates the accuracy, reliability, and efficiency with which that determination is made. A physician treating a patient with high cholesterol and blood pressure (BP), for example, may need to know whether or not the patient’s cholesterol and BP respond to weight changes to determine an appropriate treatment (e.g., weight control versus medication). This requires searching records for BP, certain blood chemicals (e.g., HDLs, LDLs, triglycerides, etc.), and weight from several

sources, then attempting to track them against each other over time. Manually reorganizing this clinical information each time it is used is extremely inefficient. More important, the current organization and formatting defies principles of human factors for optimally displaying information to enhance human information-processing characteristics, particularly for decision support.

While a discussion of human factors and cognitive psychology principles is beyond the scope of this paper, following are a few of the system design principles of concern:

Minimize the load on short-term memory.
Provide information pertinent to a given decision or component of a decision in a compact, contiguous space.
Take advantage of basic human perceptual and pat-tern recognition facilities.
Design the form of an information display to com-plement the decision-making task it supports.

F i g u re 1 shows fictitious, quasi-random data from a hypothetical patient with moderately elevated cholesterol. This one-page display pulls together all the pertinent data from six years of blood tests and related clinical measurements. At a glance, the physician’s innate pattern recognition, color, and shape perception facilities recognize the patient’s steadily increasing weight, cholesterol, BP, and triglycerides as well as the declining high-density lipoproteins. It would have taken considerably more time and effort to grasp this information from the raw data collection and blood test reports as they are currently presented in independent, tabular time slices.

Design the formulation of an information display to complement the decision-making task.

The physician may wish to know only the relationship between weight and cardiac risk factors rather than whether these measures are increasing or decreasing, or are within acceptable or marginal ranges. If so, Table 1 shows the correlations between weight and the other factors in a much more direct and simple way using the same data as in Figure 1. One can readily see the same conclusions about relations that were drawn from Figure 1.This type of abstract, symbolic display of derived information also makes it easier to spot relationships when the individual variables are bouncing up and down, unlike the more or less steady rise of most values in Figure 1. This increase in precision of relationship information is gained at the expense of other types of information (e.g., trends). To display information in an optimum form then, the system designer must know what the information demands of the task are at the point in the task when the display is to be used.

Present the sequence of information display clusters to complement an optimum decision-making strategy.

Just as a fixed sequence of gathering clinical, diagnostic information may lead to a far from optimum outcome, there exists an optimum sequence of testing, considering information, and gathering data that will lead to an optimum outcome (as defined by the value hierarchy) with a minimum of time and expense. The task of the information system designer, then, is to provide or request the right information, in the best form, at each stage of the procedure. For ex-ample, Figure 1 is suitable for the diagnostic phase since it shows the current state of the risk factors and their trends. Table 1, on the other hand, might be more appropriate in determining treatment, where there may be a choice of first trying a strict dietary treatment, or going straight to a combination of diet plus medication. The fact that Figure 1 and Table 1 have somewhat redundant information is not a problem, since they are intended to optimally provide information for different decision-making tasks. The critical need, at this point, is for a model of how to determine what information should be requested, what tests to order, what information to request and display, and in what form at each step of the decision-making process. Commitment to a collaborative relationship between physicians and laboratorians and other information providers would be an essential requirement for such an undertaking. The ideal diagnostic data-collection instrument is a flexible, computer-based device, such as a notebook computer or Personal Digital Assistant (PDA) sized device.

Barriers to interactive, computer-driven data collection at the source

As with any major change, it may be difficult to induce many physicians to change their behavior by interacting directly with a computer instead of with paper and pen. Unlike office workers, who have had to make this transition over the past three decades, most physicians’ livelihoods will not depend on converting to computer interaction. Therefore, the transition must be made attractive and the changes less onerous. Some suggestions follow:

Make the data collection a natural part of the clinical process.
Ensure that the user interface is extremely friendly, easy to learn, and easy to use.
Use a small, portable device.
Use the same device for collection and display of existing information (e.g., test results and his-tory).
Minimize the need for free-form written data entry (use check boxes, forms, etc.).
Allow the entry of notes in pen-based free-form (with the option of automated conversion of numeric data to machine-manipulable form).
Give the physicians a more direct benefit for collecting data, not just a means of helping a clerk at an HMO second-guess the physician’s judgment.
Improve administrative efficiency in the office.
Make the data collection complement the clinical decision-making process.
Improve information displays, leading to better outcomes.
Make better use of the physician’s time and mental effort.

Conclusion

The medical profession is facing a crisis of information. Gathering information is costing a typical practice more and more while fees are being restricted by third parties, and the process of gathering this in-formation may be detrimental to current outcomes. Gathered properly, in machine-manipulable form, these data could be reformatted so as to greatly improve their value immediately in the clinical setting by leading to decisions with better outcomes and, in the long run, by contributing to a clinical data warehouse that could greatly improve medical knowledge. The challenge is to create a mechanism for data collection that facilitates, hastens, and improves the outcomes of clinical activity while minimizing the inconvenience and resistance to change on the part of clinical practitioners. This paper is intended to provide a high-level overview of how this may be accomplished, and start a dialogue along these lines.

References

Tversky A. Elimination by aspects: a theory of choice. Psych Rev 1972; 79:281–99.
Didner RS. Back-to-front design: a guns and butter approach. Ergonomics 1982; 25(6):2564–5.
Shannon CE. A mathematical theory of communication. Bell System Technical J 1948; 27:379–423 (July), 623–56 (Oct).
Feeny DH, Torrance GW. Incorporating utility-based quality-of-life assessment measures in clinical trials: two examples. Med Care 1989; 27:S190–204.
Smith S, Mosier J. Guidelines for designing user interface soft-ware. ESD-TR-86-278, Aug 1986.
Miller GA. The magical number seven plus or minus two. Psych Rev 1956; 65(2):81–97.
Sternberg S. High-speed scanning in human memory. Science 1966; 153: 652–4.

Table 1

Correlation of weight with other cardiac risk factors

Cholesterol	0.759384
HDL	0.53908
LDL	0.177297
BP-syst.	0.424728
BP-dia.	0.516167
Triglycerides	0.637817

Figure 1 Hypothetical patient data.

(not shown)

Realtime Clinical Expert Support

http://pharmaceuticalintelligence.com/2015/05/10/realtime-clinical-expert-support/

Regression: A richly textured method for comparison and classification of predictor variables

http://pharmaceuticalintelligence.com/2012/08/14/regression-a-richly-textured-method-for-comparison-and-classification-of-predictor-variables/

Converting Hematology Based Data into an Inferential Interpretation

Larry H. Bernstein, Gil David, James Rucinski and Ronald R. Coifman
In Hematology – Science and Practice
Lawrie CH, Ch 22. Pp541-552.
InTech Feb 2012, ISBN 978-953-51-0174-1
https://www.researchgate.net/profile/Larry_Bernstein/publication/221927033_Converting_Hematology_Based_Data_into_an_Inferential_Interpretation/links/0fcfd507f28c14c8a2000000.pdf

A model for Thalassemia Screening using Hematology Measurements

https://www.researchgate.net/profile/Larry_Bernstein/publication/258848064_A_model_for_Thalassemia_Screening_using_Hematology_Measurements/links/0c9605293c3048060b000000.pdf

2.4.3.2 A model for automated screening of thalassemia in hematology (math study).

Kneifati-Hayek J, Fleischman W, Bernstein LH, Riccioli A, Bellevue R.
Lab Hematol. 2007; 13(4):119-23. http://dx.doi.org:/10.1532/LH96.07003.

The results of 398 patient screens were collected. Data from the set were divided into training and validation subsets. The Mentzer ratio was determined through a receiver operating characteristic (ROC) curve on the first subset, and screened for thalassemia using the second subset. HgbA2 levels were used to confirm beta-thalassemia.

RESULTS: We determined the correct decision point of the Mentzer index to be a ratio of 20. Physicians can screen patients using this index before further evaluation for beta-thalassemia (P < .05).

CONCLUSION: The proposed method can be implemented by hospitals and laboratories to flag positive matches for further definitive evaluation, and will enable beta-thalassemia screening of a much larger population at little to no additional cost.

Measurement of granulocyte maturation may improve the early diagnosis of the septic state.

2.4.3.3 Bernstein LH, Rucinski J. Clin Chem Lab Med. 2011 Sep 21;49(12):2089-95.
http://dx.doi.org:/10.1515/CCLM.2011.688.

2.4.3.4 The automated malnutrition assessment.

David G, Bernstein LH, Coifman RR. Nutrition. 2013 Jan; 29(1):113-21.
http://dx.doi.org:/10.1016/j.nut.2012.04.017

2.4.3.5 Molecular Diagnostics

Genomic Analysis of Hematological Malignancies

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy that occurs in children. Although more than 90% of children with ALL now survive to adulthood, those with the rarest and high-risk forms of the disease continue to have poor prognoses. Through the Pediatric Cancer Genome Project (PCGP), investigators in the Hematological Malignancies Program are identifying the genetic aberrations that cause these aggressive forms of leukemias. Here we present two studies on the genetic bases of early T-cell precursor ALL and acute megakaryoblastic leukemia.

Early T-Cell Precursor ALL Is Characterized by Activating Mutations
The CBFA2T3-GLIS2Fusion Gene Defines an Aggressive Subtype of Acute Megakaryoblastic Leukemia in Children

Early T-cell precursor ALL (ETP-ALL), which comprises 15% of all pediatric T-cell leukemias, is an aggressive disease that is typically resistant to contemporary therapies. Children with ETP-ALL have a high rate of relapse and an extremely poor prognosis (i.e., 5-year survival is approximately 20%). The genetic basis of ETP-ALL has remained elusive. Although ETP-ALL is associated with a high burden of DNA copy number aberrations, none are consistently found or suggest a unifying genetic alteration that drives this disease.

Through the efforts of the PCGP, Jinghui Zhang, PhD (Computational Biology), James R. Downing, MD (Pathology), Charles G. Mullighan, MBBS(Hons), MSc, MD (Pathology), and colleagues analyzed the whole-genome sequences of leukemic cells and matched normal DNA from 12 pediatric patients with ETP-ALL. The identified genetic mutations were confirmed in a validation cohort of 52 ETP-ALL specimens and 42 non-ETP T-lineage ALLs (T-ALL).

In the journal Nature, the investigators reported that each ETP-ALL sample carried an average of 1140 sequence mutations and 12 structural variations. Of the structural variations, 51% were breakpoints in genes with well-established roles in hematopoiesis or leukemogenesis (e.g., MLH2,SUZ12, and RUNX1). Eighty-four percent of the structural variations either caused loss of function of the gene in question or resulted in the formation of a fusion gene such as ETV6-INO80D. The ETV6 gene, which encodes a protein that is essential for hematopoiesis, is frequently mutated in leukemia. Among the DNA samples sequenced in this study, ETV6 was altered in 33% of ETP-ALL but only 10% of T-ALL cases.

Next-generation sequencing in hematologic malignancies: what will be the dividends?

Jason D. Merker, Anton Valouev, and Jason Gotlib
Ther Adv Hematol. 2012 Dec; 3(6): 333–339.
http://dx.doi.org:/10.1177/2040620712458948

The application of high-throughput, massively parallel sequencing technologies to hematologic malignancies over the past several years has provided novel insights into disease initiation, progression, and response to therapy. Here, we describe how these new DNA sequencing technologies have been applied to hematolymphoid malignancies. With further improvements in the sequencing and analysis methods as well as integration of the resulting data with clinical information, we expect these technologies will facilitate more precise and tailored treatment for patients with hematologic neoplasms.

Leveraging cancer genome information in hematologic malignancies.

Rampal R¹, Levine RL.
J Clin Oncol. 2013 May 20; 31(15):1885-92.
http://dx.doi.org:/10.1200/JCO.2013.48.7447

The use of candidate gene and genome-wide discovery studies in the last several years has led to an expansion of our knowledge of the spectrum of recurrent, somatic disease alleles, which contribute to the pathogenesis of hematologic malignancies. Notably, these studies have also begun to fundamentally change our ability to develop informative prognostic schema that inform outcome and therapeutic response, yielding substantive insights into mechanisms of hematopoietic transformation in different tissue compartments. Although these studies have already had important biologic and translational impact, significant challenges remain in systematically applying these findings to clinical decision making and in implementing new technologies for genetic analysis into clinical practice to inform real-time decision making. Here, we review recent major genetic advances in myeloid and lymphoid malignancies, the impact of these findings on prognostic models, our understanding of disease initiation and evolution, and the implication of genomic discoveries on clinical decision making. Finally, we discuss general concepts in genetic modeling and the current state-of-the-art technology used in genetic investigation.

p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies

E Wattel, C Preudhomme, B Hecquet, M Vanrumbeke, et AL.
Blood, (Nov 1), 1994; 84(9): pp 3148-3157
http://www.bloodjournal.org/content/bloodjournal/84/9/3148.full.pdf

We analyzed the prognostic value of p53 mutations for response to chemotherapy and survival in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and chronic lymphocytic leukemia (CLL). Mutations were detected by single-stranded conformation polymorphism (SSCP) analysis of exons 4 to 10 of the P53 gene, and confirmed by direct sequencing. A p53 mutation was found in 16 of 107 (15%) AML, 20 of 182 (11%) MDS, and 9 of 81 (11%) CLL tested. In AML, three of nine (33%) mutated cases and 66 of 81 (81%) nonmutated cases treated with intensive chemotherapy achieved complete remission (CR) (P = .005) and none of five mutated cases and three of six nonmutated cases treated by low-dose Ara C achieved CR or partial remission (PR) (P = .06). Median actuarial survival was 2.5 months in mutated cases, and 15 months in nonmutated cases (P < lo-‘). In the MDS patients who received chemotherapy (intensive chemotherapy or low-dose Ara C), 1 of 13 (8%) mutated cases and 23 of 38 (60%) nonmutated cases achieved CR or PR (P = .004), and median actuarial survival was 2.5 and 13.5 months, respectively (P C lo-’). In all MDS cases (treated and untreated), the survival difference between mutated cases and nonmutated cases was also highly significant. In CLL, 1 of 8 (12.5%) mutated cases treated by chemotherapy (chlorambucil andlor CHOP andlor fludarabine) responded, as compared with 29 of 36 (80%) nonmutated cases (P = .02). In all CLL cases, survival from p53 analysis was significantly shorter in mutated cases (median 7 months) than in nonmutated cases (median not reached) (P < IO-’). In 35 of the 45 mutated cases of AML, MDS, and CLL, cytogenetic analysis or SSCP and sequence findings showed loss of the nonmutated P53 allele. Our findings show that p53 mutations are a strong prognostic indicator of response to chemotherapy and survival in AML, MDS, and CLL. The usual association of p53 mutations to loss of the nonmutated P53 allele, in those disorders, ie, to absence of normal p53 in tumor cells, suggests that p53 mutations could induce drug resistance, at least in part, by interfering with normal apoptotic pathways in tumor cells.

Genomic approaches to hematologic malignancies

Benjamin L. Ebert and Todd R. Golub
Blood. 2004; 104:923-932
https://www.broadinstitute.org/mpr/publications/projects/genomics/Review%20Genomics%20of%20Heme%20Malig,%20Blood%202004.pdf

In the past several years, experiments using DNA microarrays have contributed to an increasingly refined molecular taxonomy of hematologic malignancies. In addition to the characterization of molecular profiles for known diagnostic classifications, studies have defined patterns of gene expression corresponding to specific molecular abnormalities, oncologic phenotypes, and clinical outcomes. Furthermore, novel subclasses with distinct molecular profiles and clinical behaviors have been identified. In some cases, specific cellular pathways have been highlighted that can be therapeutically targeted. The findings of microarray studies are beginning to enter clinical practice as novel diagnostic tests, and clinical trials are ongoing in which therapeutic agents are being used to target pathways that were identified by gene expression profiling. While the technology of DNA microarrays is becoming well established, genome-wide surveys of gene expression generate large data sets that can easily lead to spurious conclusions. Many challenges remain in the statistical interpretation of gene expression data and the biologic validation of findings. As data accumulate and analyses become more sophisticated, genomic technologies offer the potential to generate increasingly sophisticated insights into the complex molecular circuitry of hematologic malignancies. This review summarizes the current state of discovery and addresses key areas for future research.

2.4.3.6 Flow cytometry

Introduction to Flow Cytometry: Blood Cell Identification

Dana L. Van Laeys
https://www.labce.com/flow_cytometry.aspx

No other laboratory method provides as rapid and detailed analysis of cellular populations as flow cytometry, making it a valuable tool for diagnosis and management of several hematologic and immunologic diseases. Understanding this relevant methodology is important for any medical laboratory scientist.

Whether you have no previous experience with flow cytometry or just need a refresher, this course will help you to understand the basic principles, with the help of video tutorials and interactive case studies.

Basic principles include:

Immunophenotypic features of various types of hematologic cells
Labeling cellular elements with fluorochromes
Blood cell identification, specifically B and T lymphocyte identification and analysis
Cell sorting to isolate select cell population for further analysis
Analyzing and interpreting result reports and printouts

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Treatment of Acute Leukemias

Treatments and drugs

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Steroids, Inflammation, and CAR-T Therapy [6.3.8] (UPDATED)

1. Evolution of Chimeric Antigen Receptors

A brief history of construction of 2nd and 3rd generation CAR-T cells given by cancer.gov:

Constructing a CAR T Cell (from cancer.gov)

2. Consideration for Design of Trials and Mitigating Toxicities

3. Case Reports of Adverse Events and Their Amelioration During CAR-T Therapy

CAR-T Therapy have Had reports of Serious Adverse Events

From FierceBiotech UPDATED: Two deaths force MSK to hit the brakes on engineered T cell cancer study

Keynote presentation by Carl H. June, recipient of The Richard V. Smalley MD 2013 Award

CRISPR takes first steps in humans

Structured Abstract

INTRODUCTION

RATIONALE

RESULTS

CONCLUSION

UPDATED 10/04/2021 (Advances in CAR-T therapy for solid tumors)

NK cells expressing a chimeric activating receptor eliminate MDSCs and rescue impaired CAR-T cell activity against solid tumors

Abstract

INTRODUCTION

MATERIALS AND METHODS

Cytokines, cell lines, and antibodies.

CAR-encoding retroviral vectors.

Expansion and retroviral transduction of human NK and T cells.

Induction and enrichment of human MDSCs.

In vitro T-cell suppression assay.

In vitro CAR-T chemotaxis assay.

In vivo tumor microenvironment model.

Immunohistochemistry of neuroblastoma xenografts.

Analysis of intra-tumoral MDSCs from patients with neuroblastoma.

Statistics.

RESULTS

NKG2D.ζ NK cells expand and have cytotoxicity against target cells.

Transgenic NKG2D.ζ is unaffected by TGFβ or soluble NKG2D ligands.

Human MDSCs express NKG2D ligands and are killed by NKG2D.ζ–NK cells.

NKG2D.ζ–NK cells eliminate intra-tumoral MDSCs and reduce tumor burden.

NKG2D.ζ–NK cells secrete chemokines that recruit GD2.CAR-T cells.

NKG2D.ζ NK cells improve GD2.CAR-T cell trafficking to tumor sites.

Elimination of MDSCs increases antitumor activity of GD2.CAR-T cells.

DISCUSSION

Other posts on this site on Immunotherapy and Cancer include

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Adult non-Hodgkin lymphoma is a disease in which malignant (cancer) cells form in the lymph system.

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Treatments other than Chemotherapy for Leukemias and Lymphomas

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Treatments for Lymphomas and Leukemias

2.4.4 Treatments for leukemia by type

Treatments and drugs

Adult non-Hodgkin Lymphoma is a disease in which malignant (cancer) cells form in the lymph system.

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Hematological Malignancy Diagnostics

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A brief history of construction of 2^nd and 3^rd generation CAR-T cells given by cancer.gov: