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Archive for the ‘Circulating Progenitor Cells’ Category


  1. Lungs can supply blood stem cells and also produce platelets: Lungs, known primarily for breathing, play a previously unrecognized role in blood production, with more than half of the platelets in a mouse’s circulation produced there. Furthermore, a previously unknown pool of blood stem cells has been identified that is capable of restoring blood production when bone marrow stem cells are depleted.

 

  1. A new drug for multiple sclerosis: A new multiple sclerosis (MS) drug, which grew out of the work of UCSF (University of California, San Francisco) neurologist was approved by the FDA. Ocrelizumab, the first drug to reflect current scientific understanding of MS, was approved to treat both relapsing-remitting MS and primary progressive MS.

 

  1. Marijuana legalized – research needed on therapeutic possibilities and negative effects: Recreational marijuana will be legal in California starting in January, and that has brought a renewed urgency to seek out more information on the drug’s health effects, both positive and negative. UCSF scientists recognize marijuana’s contradictory status: the drug has proven therapeutic uses, but it can also lead to tremendous public health problems.

 

  1. Source of autism discovered: In a finding that could help unlock the fundamental mysteries about how events early in brain development lead to autism, researchers traced how distinct sets of genetic defects in a single neuronal protein can lead to either epilepsy in infancy or to autism spectrum disorders in predictable ways.

 

  1. Protein found in diet responsible for inflammation in brain: Ketogenic diets, characterized by extreme low-carbohydrate, high-fat regimens are known to benefit people with epilepsy and other neurological illnesses by lowering inflammation in the brain. UCSF researchers discovered the previously undiscovered mechanism by which a low-carbohydrate diet reduces inflammation in the brain. Importantly, the team identified a pivotal protein that links the diet to inflammatory genes, which, if blocked, could mirror the anti-inflammatory effects of ketogenic diets.

 

  1. Learning and memory failure due to brain injury is now restorable by drug: In a finding that holds promise for treating people with traumatic brain injury, an experimental drug, ISRIB (integrated stress response inhibitor), completely reversed severe learning and memory impairments caused by traumatic brain injury in mice. The groundbreaking finding revealed that the drug fully restored the ability to learn and remember in the brain-injured mice even when the animals were initially treated as long as a month after injury.

 

  1. Regulatory T cells induce stem cells for promoting hair growth: In a finding that could impact baldness, researchers found that regulatory T cells, a type of immune cell generally associated with controlling inflammation, directly trigger stem cells in the skin to promote healthy hair growth. An experiment with mice revealed that without these immune cells as partners, stem cells cannot regenerate hair follicles, leading to baldness.

 

  1. More intake of good fat is also bad: Liberal consumption of good fat (monounsaturated fat) – found in olive oil and avocados – may lead to fatty liver disease, a risk factor for metabolic disorders like type 2 diabetes and hypertension. Eating the fat in combination with high starch content was found to cause the most severe fatty liver disease in mice.

 

  1. Chemical toxicity in almost every daily use products: Unregulated chemicals are increasingly prevalent in products people use every day, and that rise matches a concurrent rise in health conditions like cancers and childhood diseases, Thus, researcher in UCSF is working to understand the environment’s role – including exposure to chemicals – in health conditions.

 

  1. Cytomegalovirus found as common factor for diabetes and heart disease in young women: Cytomegalovirus is associated with risk factors for type 2 diabetes and heart disease in women younger than 50. Women of normal weight who were infected with the typically asymptomatic cytomegalovirus, or CMV, were more likely to have metabolic syndrome. Surprisingly, the reverse was found in those with extreme obesity.

 

References:

 

https://www.ucsf.edu/news/2017/12/409241/most-popular-science-stories-2017

 

https://www.ucsf.edu/news/2017/03/406111/surprising-new-role-lungs-making-blood

 

https://www.ucsf.edu/news/2017/03/406296/new-multiple-sclerosis-drug-ocrelizumab-could-halt-disease

 

https://www.ucsf.edu/news/2017/06/407351/dazed-and-confused-marijuana-legalization-raises-need-more-research

 

https://www.ucsf.edu/news/2017/01/405631/autism-researchers-discover-genetic-rosetta-stone

 

https://www.ucsf.edu/news/2017/09/408366/how-ketogenic-diets-curb-inflammation-brain

 

https://www.ucsf.edu/news/2017/07/407656/drug-reverses-memory-failure-caused-traumatic-brain-injury

 

https://www.ucsf.edu/news/2017/05/407121/new-hair-growth-mechanism-discovered

 

https://www.ucsf.edu/news/2017/06/407536/go-easy-avocado-toast-good-fat-can-still-be-bad-you-research-shows

 

https://www.ucsf.edu/news/2017/06/407416/toxic-exposure-chemicals-are-our-water-food-air-and-furniture

 

https://www.ucsf.edu/news/2017/02/405871/common-virus-tied-diabetes-heart-disease-women-under-50

 

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Ido Sagi – PhD Student @HUJI, 2017 Kaye Innovation Award winner for leading research that yielded the first successful isolation and maintenance of haploid embryonic stem cells in humans.

Reporter: Aviva Lev-Ari, PhD, RN

 

Ido Sagi – PhD Student, Silberman Institute of Life Sciences, HUJI, Israel

  • Ido Sagi’s research focuses on studying genetic and epigenetic phenomena in human pluripotent stem cells, and his work has been published in leading scientific journals, including NatureNature Genetics and Cell Stem Cell.
  • Ido Sagi received BSc summa cum laude in Life Sciences from the Hebrew University, and currently pursues a PhD at the laboratory of Prof. Nissim Benvenisty at the university’s Department of Genetics in the Alexander Silberman Institute of Life Sciences.

The Kaye Innovation Awards at the Hebrew University of Jerusalem have been awarded annually since 1994. Isaac Kaye of England, a prominent industrialist in the pharmaceutical industry, established the awards to encourage faculty, staff and students of the Hebrew University to develop innovative methods and inventions with good commercial potential, which will benefit the university and society.

Publications – Ido Sagi

Comparable frequencies of coding mutations and loss of imprinting in human pluripotent cells derived by nuclear transfer and defined factors.
Cell Stem Cell 2014 Nov 6;15(5):634-42. Epub 2014 Nov 6.
The New York Stem Cell Foundation Research Institute, New York, NY 10032, USA; Naomi Berrie Diabetes Center & Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA. Electronic address:

November 2014

 



Stem cells: Aspiring to naivety.
Nature 2016 12 30;540(7632):211-212. Epub 2016 Nov 30.
The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
November 2016

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SOURCE

Other related articles on Genetic and Epigenetic phenomena in human pluripotent stem cells published by LPBI Group can be found in the following e-Books on Amazon.com

e-Books in Medicine

https://www.amazon.com/s/ref=dp_byline_sr_ebooks_9?ie=UTF8&text=Aviva+Lev-Ari&search-alias=digital-text&field-author=Aviva+Lev-Ari&sort=relevancerank

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    Etiologies of Cardiovascular Diseases: Epigenetics, Genetics and Genomics

    Nov 28, 2015 | Kindle eBook

    by Justin D. Pearlman MD ME PhD MA FACC and Stephen J. Williams PhD
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    Cancer Therapies: Metabolic, Genomics, Interventional, Immunotherapy and Nanotechnology in Therapy Delivery (Series C Book 2)

    May 13, 2017 | Kindle eBook

    by Larry H. Bernstein and Demet Sag
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    Perspectives on Nitric Oxide in Disease Mechanisms (Biomed e-Books Book 1)

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    Cancer Biology and Genomics for Disease Diagnosis (Series C: e-Books on Cancer & Oncology Book 1)

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    Genomics Orientations for Personalized Medicine (Frontiers in Genomics Research Book 1)

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    by Sudipta Saha PhD and Ritu Saxena PhD
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    Metabolic Genomics & Pharmaceutics (BioMedicine – Metabolomics, Immunology, Infectious Diseases Book 1)

    Jul 21, 2015 | Kindle eBook

    by Larry H. Bernstein MD FCAP and Prabodah Kandala PhD
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    Milestones in Physiology: Discoveries in Medicine, Genomics and Therapeutics (Series E: Patient-Centered Medicine Book 3)

    Dec 26, 2015 | Kindle eBook

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    Regenerative and Translational Medicine: The Therapeutic Promise for Cardiovascular Diseases

    Dec 26, 2015 | Kindle eBook

    by Justin D. Pearlman MD ME PhD MA FACC and Ritu Saxena PhD
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    Cardiovascular Original Research: Cases in Methodology Design for Content Co-Curation: The Art of Scientific & Medical Curation

    Nov 29, 2015 | Kindle eBook

    by Larry H. Bernstein MD FCAP and Aviva Lev-Ari PhD RN
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Blood forming precursors in bone marrow

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Blood stem cells study could pave the way for new cancer therapy

UNIVERSITY OF EDINBURGH

IMAGE

http://media.eurekalert.org/multimedia_prod/pub/web/110842_web.jpg

This image shows the formation of blood stem cells inside the embryonic vessel called dorsal aorta. In green is shown secreted molecule called NOGGIN, which plays an important role in this process. The University of Edinburgh

People with leukaemia could be helped by new research that sheds light on how the body produces its blood supply.

Scientists are a step closer to creating blood stem cells that could reduce the need for bone marrow transplants in patients with cancer or blood disorders.

Enabling scientists to grow the stem cells artificially from pluripotent stem cells could also lead to the development of personalised blood therapies, researchers say.

Blood stem cells are found in bone marrow and produce all blood cells in the body. These cells – known as haematopoietic stem cells (HSCs) – help to restore blood supply in patients who have been treated for leukaemia.

Researchers used a mouse model to pinpoint exactly how HSCs develop in the womb. They showed for the first time how three key molecules interact together to generate the cells, which are later found in adult bone marrow.

The discovery could help scientists to recreate this process in the lab, in the hope that HSCs could one day be developed for clinical use.

Scientists say this fundamental understanding of early development may also have an impact on other diseases that affect blood formation and supply.

###

The research has been published in Nature Communications.

Professor Alexander Medvinsky, of the University of Edinburgh’s MRC Centre for Regenerative Medicine said: “There is a pressing need to improve treatments for diseases like leukaemia and this type of research brings us a step closer to that milestone. The more we understand about how embryos develop these blood stem cells, the closer we come to being able to make them in the lab.”

http://www.ed.ac.uk/news/2016/stem-cells-100316

Céline Souilhol, Christèle Gonneau, Javier G. Lendinez, Antoniana Batsivari, Stanislav Rybtsov, Heather Wilson, Lucia Morgado-Palacin, David Hills, Samir Taoudi, Jennifer Antonchuk, Suling Zhao, Alexander Medvinsky. Inductive interactions mediated by interplay of asymmetric signalling underlie development of adult haematopoietic stem cells. Nature Communications, 2016; 7: 10784 DOI: 10.1038/ncomms10784

During embryonic development, adult haematopoietic stem cells (HSCs) emerge preferentially in the ventral domain of the aorta in the aorta–gonad–mesonephros (AGM) region. Several signalling pathways such as Notch, Wnt, Shh and RA are implicated in this process, yet how these interact to regulate the emergence of HSCs has not previously been described in mammals. Using a combination of ex vivo and in vivo approaches, we report here that stage-specific reciprocal dorso–ventral inductive interactions and lateral input from the urogenital ridges are required to drive HSC development in the aorta. Our study strongly suggests that these inductive interactions in the AGM region are mediated by the interplay between spatially polarized signalling pathways. Specifically, Shh produced in the dorsal region of the AGM, stem cell factor in the ventral and lateral regions, and BMP inhibitory signals in the ventral tissue are integral parts of the regulatory system involved in the development of HSCs.

Haematopoietic stem cells (HSCs) lie at the foundation of the adult haematopoietic system, and give rise to cells of all blood lineages throughout the lifespan of an organism. An important property of adult (definitive) haematopoietic stem cells (dHSCs) is that they are capable of long-term reconstitution of the haematopoietic system upon transplantation into irradiated recipients. In the mouse, such cells develop by embryonic stages E10–E11 in the aorta–gonad–mesonephros (AGM) region1, 2, 3, 4. An ex vivo approach showed that the AGM region has a robust autonomous capacity to generate dHSCs1. The AGM region comprises the dorsal aorta flanked on both sides by the urogenital ridges (UGRs), which contain embryonic rudiments of kidney and mesonephros. HSCs develop in a polarized manner, predominantly in the ventral floor of the dorsal aorta (AoV), more rarely in the dorsal domain of the dorsal aorta (AoD), and are absent in the UGRs2, 5, 6, 7. Localization of dHSCs to the AoV in mouse and human embryos was shown by long-term reconstitution experiments5, 6.

Abundant evidence indicates that during development, a specialized embryonic endothelial compartment known as haematogenic (or haemogenic) endothelium gives rise to haematopoietic stem and progenitors cells7, 8, 9, 10. The haematopoietic programme in various vertebrate models is executed predominantly in the AoV, and is recognized by the expression of essential haematopoietic transcription factors, for example, Runx1 and cKit, and the appearance of clusters of haematopoietic cells budding from the endothelium of the dorsal aorta6, 8, 9, 11, 12, 13, 14.

It is broadly accepted that HSCs develop from the haematogenic endothelium within intra-aortic clusters. This transition involves several consecutive maturation steps of HSC precursors: pro-HSCsright arrowpre-HSC type Iright arrowpre-HSC type IIright arrowdHSC15, 16, 17. All these precursors express endothelial markers, such as vascular-endothelial cadherin (VC) and CD31, and sequentially upregulate haematopoietic surface markers: CD41 (pro-HSCs), CD43 (pre-HSC type I) and finally CD45 (pre-HSC type II). This maturation process occurs in the dorsal aorta between E9 and E11. Specifically, pro-HSCs emerge at E9, pre-HSCs Type I appear at E10 and pre-HSCs type II predominantly at E11. Unlike dHSCs, pre-HSCs cannot reconstitute the adult haematopoietic system by direct transplantation and require prior maturation in an embryonic or neonatal environment15, 16, 17, 18,19.

A number of signalling pathways (Notch, Wnt, retinoic acid, interleukin-3 and inflammatory) have been implicated in HSC development; however, a coherent picture is yet to be elucidated15, 17, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31. HSC precursors (pro-HSCs, pre-HSCs type I and pre-HSCs type II) express cKit17 from early developmental stages. A recent study has shown that the cKit ligand, known as stem cell factor (SCF), is a key regulator driving maturation of these HSC precursors into dHSCs in the AGM region17, which is in agreement with the marked decline of HSC activity in SCF mutant mice32, 33. In the adult, SCF is critically important for HSC maintenance in the bone marrow niche, mainly in the endothelial compartment32. Sonic Hedgehog (Shh) and bone morphogenetic protein 4 (BMP4) pathways are also important mediators; in zebrafish, these two morphogenes are involved in arterial specification and haematopoietic patterning, respectively34,35. In the mouse, subaortic BMP4 and Shh/Indian Hedgehog derived from gut were also proposed to be responsible for HSC development36, 37.

During development, interactions between spatially segregated compartments are essential for tissue patterning and specification, and are often mediated by gradients of secreted molecules38,39, 40. Molecules secreted by distant tissues, such as somites, can influence HSC development in the AGM region41, 42, 43, 44, 45. Developing HSCs are embedded in the complex AGM microenvironment, suggesting that HSC development may require signals derived from different compartments of the AGM region. We sought to test this hypothesis. However, the analysis of HSC development in vivo is significantly hampered by low accessibility of embryos developing in utero, fast maturation of dHSCs, lack of uniquely specific markers for HSC precursors and their low numbers in the AGM region. Therefore, we employed here a robust ex vivo culture system that models HSC development in the embryo in combination with functional HSC analysis using in vivolong-term reconstitution assay15, 16, 17. Specifically, to study interactions between AGM subregions, we took advantage of the in vitro reaggregation system that enables close juxtaposition of cell types15.

We show that interactions between three compartments of the AGM, the AoV, the AoD and the UGRs, are necessary for efficient generation of dHSCs. First, we show that dHSC activity in the isolated E10.5 AoV is limited but can be significantly enhanced by co-culture with the AoD, and that this is mediated at least partly by Shh, secreted dorsally in vivo. Second, while HSC activity in isolated E11.5 AoD is limited, co-culture with a competent AoV microenvironment activates dHSC generation in the AoD. We found that this effect is mediated by SCF, which is secreted abundantly by the AoV stroma in vivo as shown here. Third, we show that downregulation of BMP4 signalling by BMP antagonist Noggin, which is present at high levels in the AoV and especially in intra-aortic clusters as revealed here by in vivo observations, is required for HSC development. Fourth, UGRs, which express high levels of SCF, also enhance HSC development in the dorsal aorta.

Our results based on in vivo observations and ex vivo modelling strongly suggest that juxtaposed, anatomically distinct domains within the AGM region create a complex landscape of interactive signals that underpins HSC development.

Pre-HSCs localize preferentially to the AoV

As dHSCs mature from pre-HSCs, we investigated whether the emergence of dHSC predominantly in the AoV6 is a result of asymmetric (ventralized) distribution of pre-HSCs. Dorsal aortae were separated from UGRs and bisected into AoV and AoD (including notochord) as described previously6 (Supplementary Fig. 1a). The different domains were then directly transplanted into irradiated mice to detect dHSCs. We first confirmed our previous observation that at E11.5 dHSCs appear almost exclusively in the AoV, although some dHSCs were in the AoD and engrafted few recipients at high level (Supplementary Fig. 1b). Limiting dilution analysis showed that dHSCs are approximately four times more frequent in the AoV compared with AoD. UGRs did not contain HSCs in line with previous reports2, 6.

We then investigated the spatial distribution of pre-HSCs type I and pre-HSCs type II in E10.5–E11.5 embryos using the OP9 co-culture system supplemented with Il3+SCF+Flt3 (termed 3GF), which allows pre-HSCs (which do not engraft by direct transplantation) to mature into dHSC that become detectable by long-term repopulation assay as described previously16. Doses of transplanted cells (expressed in embryo equivalents, e.e.) were chosen based on the requirements of individual experiments (explained in Methods section). In these experiments (Fig. 1), the dose injected was high (1–2e.e.) to detect potentially low dHSC numbers in AoD and UGRs.

Figure 1: Localization of pre-HSCs in the AGM region.

Localization of pre-HSCs in the AGM region.

(a) E10.5 AoV, AoD and UGRs were co-aggregated with OP9 and cultured for 5 days, and the formation of dHSCs was then tested by transplantation into irradiated mice (2e.e. per recipient; AoV: six independent experiments; AoD: four independent experiments; UGRs: two independent experiments). Dashed line indicates the cutoff for high-level engraftment (>70% donor chimaerism). (b) E11.5 aortas and UGRs were transplanted after reaggregate culture (Ao: 0.2e.e. per recipient and UGRs: 1e.e. per recipient; two independent experiments). (c,d) Pre-HSCs type I (VC+CD45) (c) or type II (VC+CD45+) (d) sorted from E11.5 AoV and AoD were co-aggregated with OP9 cells and transplanted after culture (1e.e. per recipient; two independent experiments). (ad) Levels of engraftment are plotted, and number of repopulated versus total number of transplanted mice are shown in brackets. Number of embryo equivalents (ee) injected in each experiment are indicated on the graphs. (*P<0.05; ***P<0.005; Mann–Whitney U-test). In all these experiments, tissues were cultured with three growth factors (Flt3I, Il3 and SCF). AGM, aorta–gonad–mesonephros region; Ao, dorsal Aorta; AoV, ventral domain of the dorsal aorta; AoD, dorsal domain of the dorsal aorta; UGRs, urogenital ridges.

We have shown previously that E10.5 AGM region mainly contains type I pre-HSCs, whereas at E11.5, type I and type II pre-HSCs co-exist16. Dissected E10.5 AGM regions co-cultured with OP9 in 3GF for 5 days were transplanted into adult irradiated recipients. Out of 21 recipients that received cultured AoV, 20 showed high levels (>70%) of donor-derived long-term haematopoietic chimerism (Fig. 1a). In contrast, only 7 out of 16 recipients of cultured AoD were repopulated at high levels (>70%), while the remaining recipients showed lower or no repopulation (7 and 2, respectively). Cultured UGRs did not produce dHSCs (Fig. 1a). Thus, we conclude that the E10.5 AoD does contain pre-HSCs but at significantly lower numbers than the AoV.

We then investigated whether pre-HSCs localization changes in E11.5 embryos and found that pre-HSCs were still exclusively localized to the dorsal aorta; UGRs carefully separated from the lateral mesenchyme adjacent to the dorsal aorta did not give any repopulation after culture (Fig. 1b). To establish the location of pre-HSCs within the E11.5 dorsal aorta, cell populations enriched for pre-HSCs type I (VC+CD45) and pre-HSCs type II (VC+CD45+) were sorted from AoV and AoD, and co-cultured with OP9 stromal cells in the presence of 3GF as described previously16. We again were able to detect pre-HSC activity in AoD although at lower levels than in AoV. After maturation ex vivo, pre-HSCs type I from AoV and AoD repopulated 7 of 11 and 2 of 8 recipients, respectively (Fig. 1c). Similarly, cultured pre-HSCs type II from AoV and AoD repopulated 11 out of 12 and 4 out of 10 recipients, respectively (Fig. 1d). In all cases, multilineage engraftment was confirmed (Supplementary Fig. 2). These data show that pre-HSCs are significantly enriched in AoV.

Reciprocal inductive interactions between AoD and AoV

To explore hypothetical interactions between AoD and AoV, we made use of a dissociation–reaggregation system that recapitulates HSC development ex vivo15. This system allowed us to integrate AGM domains in a three-dimensional tissue-like organoid15 and study their interactions in HSC development. To track the origin of dHSCs, AoV and AoD from wild-type (WT) and green fluorescent protein (GFP) embryos with constitutive expression of GFP46 were co-aggregated (termed AoV//AoD co-aggregates) and cultured for 5 days in the presence of 3GF before transplantation (Fig. 2a). Mice transplanted with AoV//AoD co-aggregates can be reconstituted by dHSCs coming from AoD and AoV. The presence of GFP allowed the individual contributions of AoV and AoD to the total repopulation level within the same mouse to be assessed (Fig. 2b,c). This is presented in two separate columns in the graph. Namely, while columns 1 and 3 represent the same recipient mice, the former shows exclusively the contribution of the AoD and the latter shows exclusively the contribution of the AoV into each recipient. To assess the influence of AoD and AoV interaction on HSC development, the repopulation by co-aggregated AoD (column 1) or AoV (column 3) can then be compared with repopulation by independently cultured AoD (column 2) or AoV (column 4). All experiments included reciprocal use of WT and GFP tissues in AoV//AoD co-aggregates, and we observed no difference in repopulation properties between WT and GFP embryos. Homotypic AoV//AoV and AoD//AoD co-aggregates were always used as controls. Note that in these experiments, only 0.2e.e. were injected per recipient, to ensure that the repopulation levels were not saturated and to allow any inductive effects to be revealed.

Figure 2: Inductive interactions between AoV, AoD and UGRs as revealed by an ex vivomodel system.

Inductive interactions between AoV, AoD and UGRs as revealed by an ex vivo model system.

(a) Experimental design: the ventral domain (AoV) and the dorsal domain (AoD) of the aorta, and the urogenital ridges (UGRs) from wild-type (WT) and GFP+ embryos were subdissected, and chimeric reaggregates from tissues of these two genotypes were generated. Left column: to test interactions between AoV and AoD, chimeric AoV//AoD reaggregates were generated and transplanted into irradiated recipients after 4–5 days of culture (b,c). Right column: to test interactions between Ao and UGRs, chimeric Ao//UGR reaggregates were generated and transplanted into irradiated recipients after 4–5 days culture (d). GFP+ and/or GFP− donor-derived long-term repopulation allowed us to conclude whether dHSCs originated from AoV, AoD or UGRs. Accordingly, the tissue of origin of donor dHSCs is indicated below each graph. (b) E10.5 aortas from WT and GFP embryos were used to generate chimeric reaggregates as depicted schematically above plots. The reciprocal combination of WT and GFP tissues was used to generate AoV//AoD reaggregates. The tissue source of dHSCs is shown separately in the leftmost (AoD) and rightmost (AoV) columns as indicated below the plot (0.2e.e. per recipient; two independent experiments). (c) E11.5 aortas from WT and GFP embryos were used to generate chimeric reaggregates. The tissue source of dHSCs is shown separately in the leftmost (AoD) and rightmost (AoV) columns as indicated below the plot (0.2e.e. per recipient; two independent experiments). (d) E11.5 aortas (Ao) and UGRs from WT and GFP embryos were used to generate Ao//UGR chimeric reaggregates. As depicted schematically above the plot, the reciprocal combination of WT and GFP tissues was used to generate Ao//UGR reaggregates. The tissue source of dHSCs is shown separately in left (Ao) and right (UGRs) columns as indicated below the plot (0.01e.e. per recipient; six independent experiments). (e) Reaggregation of WT Ao with UGRs generate more dHSCs than Ao alone (0.05e.e. per recipient; two independent experiments). (be) In all these experiments, tissues were cultured with three growth factors.

Using this approach, we found that the E10.5 AoV generates more dHSCs when combined with AoD than on its own (Fig. 2b, compare two rightmost columns). One day later, E11.5 AoD had no positive influence on dHSC generation by AoV (Fig. 2c, compare two rightmost columns). Conversely, the E11.5 AoD produced more HSCs when reaggregated with the AoV than on its own (Fig. 2c, compare two leftmost columns). This inductive effect of AoV on AoD was not observed at E10.5 (Fig. 2b, compare two leftmost columns). These ex vivo modelling experiments revealed reciprocal stage-specific effects of AoV and AoD on HSC development, which could be explained by the differential release of factors by the two regions and/or by differences in the competency of the target cells to respond to signals.

UGRs enhance HSC development in the dorsal aorta  

SCF expression is involved in polarized HSC development

Figure 3: Involvement of polarized stem cell factor in HSC development.

Involvement of polarized stem cell factor in HSC development.

(a) qRT–PCR on fresh AoV, AoD and UGRs at E10.5 and E11.5 showed high expression levels of stem cell factor (SCF) in AoV and UGRs, compared with AoD (data are mean±s.e.m; *P<0.05, **P<0.01, t-test; three independent experiments). No significant difference was observed between E10.5 and E11.5 expression level in any of the tissues. (b) Expression of SCF-GFP and CD31 determined by immunostaining on thick section (300μm) of SCF-GFP-positive E10.5 AGM region and on SCF-GFP-negative control. Bars, 50μm. (c) Expression of SCF in sorted populations from fresh E10.5–E11.5 AoV (V) and AoD (D) determined by qRT–PCR. Endo, endothelial population (VC+CD45CD43); type I, pre-HSCs type I (VC+CD45CD43+); type II, pre-HSCs type II (VC+CD45+); stroma, stromal population (VCCD45CD43). (*P<0.05, t-test; five independent experiments). (d) E10.5 AoD were cultured as reaggregates in the presence of Il3 and Flt3L with or without SCF and human SCF antagonist (SCF-Rh). (0.5e.e. per recipient; three independent experiments). (e,f) E11.5 AoD (two independent experiments) (e) and E10.5 AoV (two independent experiments) (f) were cultured as explants with or without SCF (no other cytokines); (0.2e.e. per recipient).

 

Shh signalling enhances dHSC generation

 

Figure 4: Sonic Hedgehog is a positive modulator of pre-HSC type I.

Sonic Hedgehog is a positive modulator of pre-HSC type I.

(a) Expression level of Sonic Hedgehog (Shh) in E10.5 and E11.5 AGM region determined by qRT–PCR. (data are mean±s.e.m; *P<0.05, t-test; E10.5: three independent experiments and E11.5: two independent experiments). (b) Patched1 and Gli1 expression in endothelial cells (endo: VC+CD45CD43), pre-HSCs type I (I: VC+CD45CD43+) and type II (II: VC+CD45+) sorted from E11.5 AoV and AoD (two independent experiments). (c) E10.5 AoV and AoD explants were cultured in presence of Shh recombinant protein before transplantation (AoV: 0.1e.e. per recipient; two independent experiments and AoD: 0.2e.e. per recipient; three independent experiments). (d) E10.5 AoV and doxycyline-inducible OP9-Shh were co-aggregated and cultured in presence or absence of doxycycline and/or Hedgehog (Hh) antagonist (200nM) before transplantation (0.2e.e. per recipient; two independent experiments). (e) 10.5 AoV and AoD co-aggregated with OP9 were cultured in presence of three growth factors with Hh antagonist before transplantation; (0.2e.e. per recipient; two independent experiments). (f) E11.5 AoV explants were cultured in presence of Shh recombinant protein before transplantation; (0.2e.e. per recipient; two independent experiments). (g): E11.5 AGM reaggregates were cultured in presence of Hh antagonist before transplantation; (0.1e.e. per recipient; two independent experiments). (c,d,f,g) In all these experiments, tissues were cultured without cytokines. Hh anta, Hh antagonist; Dox, doxycycline.

 

BMP signalling is downregulated in the dHSC lineage

Figure 5: Bone morphogenetic protein signalling is downregulated in dHSC lineage.

Bone morphogenetic protein signalling is downregulated in dHSC lineage.

(a) Expression of bone morphogenetic protein 4 (BMP4) at E10.5 determined by qRT–PCR; (data are mean±s.e.m.; *P<0.05, t-test; three independent experiments). (b) Expression of BMP4 in the E10.5 AGM region determined by immunostaining on frozen sections. Bars, 50μm. Zoomed image shows the subendothelial localization of BMP4 (arrowheads). Bars, 10μm. (c) Expression of phosphorylated-Smad (P-Smad) in the E10.5 AGM region determined by immunostaining on frozen sections. Bars, 50μm. (d) Id genes expression in endothelial cells, pre-HSCs type I and type II directly isolated from E10.5 and E11.5 AoV determined by qRT–PCR. Endo, endothelial population (VC+CD45CD43); type I, pre-HSCs type I (VC+CD45CD43+); type II, pre-HSCs type II (VC+CD45+); stroma, stromal population (VCCD45CD43). (Data are mean±s.e.m.; *P<0.05, **P<0.01; t-test; five independent experiments). (eg) Expression of P-Smad, CD31 and CD45 in the endothelium and haematopoietic clusters of E10.5 dorsal aorta. White arrowheads indicate cells with pre-HSC type II phenotype (CD31+CD45+); green arrows show (CD31+CD45−/low) cells budding out of the dorsal aorta and expressing P-Smad; asterisks indicate CD31+CD45 cells expressing P-Smad within the endothelium. Bars, 10μm. A positive control showing P-Smad staining in the dorsal part of the neural tube can be found in h.

 

Figure 6: Haematopoietic clusters are exposed to low concentration of BMP4 and high levels of Noggin.

Haematopoietic clusters are exposed to low concentration of BMP4 and high levels of Noggin.

(a) Expression of BMP antagonists at E10.5 determined by qRT–PCR (data are mean±s.e.m.; *P<0.05,***P<0.005; t-test; three independent experiments). (b) Expression of Noggin in the E10.5 AGM region determined by immunostaining on frozen sections. Note the expression of Noggin in the notochord (Nt) as expected. Bar, 50μm. (c) Expression of Noggin and BMP4 in intra-aortic clusters characterized by cKit and CD31 expression. Note that BMP4 is mainly expressed underneath the dorsal aorta (arrowheads), while Noggin is expressed in the cluster (arrows). Bars, 10μm. (d) Expression of Noggin in isolated populations from E10.5 and E11.5 AoV (V) and AoD (D) determined by qRT–PCR. (*P<0.05, t-test; five independent experiments). (e) Model showing downregulation of BMP activity in dHSC lineage. BMP4 is mainly expressed in the ventral mesenchyme, while Noggin is found in haematopoeitic clusters. Accordingly, BMP activity, assessed by the phosphorylation of Smad1,5 and 8 (P-Smad), is high in mesenchymal cells underneath the aortic endothelium and in some endothelial cells (CD31+CD45) of the aortic endothelium and decreases in the haematopoeitic clusters. While some pre-HSC type I cells (CD31+CD45−/low) exhibit BMP signalling at a low level, acquisition of CD45 (shown in red) is accompanied by a complete loss of BMP activity. EC, endothelial cells; MC, mesenchymal cells; I, pre-HSC type I; II, pre-HSC type II.

 

BMP signalling inhibits HSC development

http://www.nature.com/ncomms/2016/160308/ncomms10784/images_article/ncomms10784-f7.jpg

 

Interactions between SCF, Shh and BMP signalling pathways

Interplay between SCF, Shh and BMP pathways underpins inductive interactions in the AGM.

 

We have shown previously that during murine embryo development definitive HSCs emerge predominantly in the ventral domain of the dorsal aorta (AoV)6. This spatially polarized production of HSCs might be explained by different origins of dorsal and ventral endothelium and/or by asymmetric production of key factors involved in HSC development37, 52, 53 and we reasoned that directional inductive interactions between AGM compartments could be involved. Great insight into inductive interactions in various organs has previously been obtained through in vitro modelling39. Here we modelled interactions between AGM domains in a co-culture system, which supports HSC development15. Using this ex vivo system, we demonstrate that at early stages (E10.5) HSC maturation in the AoV region is enhanced by the presence of the AoD. One day later (E11.5), the AoV microenvironment is able to induce HSC development in the AoD, previously thought to be mostly devoid of HSC activity6. We also found that UGRs can enhance HSC production from the dorsal aorta, but cannot generate dHSCs themselves, even under influence of the dorsal aorta. Thus, our data strongly suggest that reciprocal stage-specific inductive AoD//AoV interactions and involvement of UGRs are required for execution of the robust development of HSCs in vivo.

Our data indicate that previously established dorso–ventrally polarized HSC development6 is defined by two main factors. First, our current data show that although the AoD contains pre-HSCs (both type I and type II), their numbers are lower than in AoV, in line with lower intra-aortic cluster formation previously described in mouse AoD6, 13. Second, as shown here, dHSCs can be induced in the AoD by the AoV, and therefore the dHSCs deficiency in AoD cannot be explained solely by asymmetric pre-HSC distribution, but may also be influenced by differences in the microenvironment.

To study this, we focused on SCF, Shh and BMP4, whose expressions are dorso–ventrally polarized in the AGM region36, 47, 49 (and current data). We found that SCF is an inductive signal that is expressed at high levels in the AoV and UGRs, and can stimulate HSC development in isolated AoD, a region which had previously been considered to be mostly devoid of HSC activity. This is in agreement with a key role of SCF in HSC maturation17. We found that the aortic endothelial compartment expresses high levels of SCF, suggesting its important role in HSC development comparable to the bone marrow microenvironment of adult HSCs32. Importantly, we found that the pre-HSC type I population expresses SCF suggesting a positive-autocrine loop, which could promote HSC development.

Shh signalling in zebrafish is required for aortic angioblast migration and subsequent arterial specification of the dorsal aorta34, 54. We found that in mouse Shh stimulates and a Hh antagonist inhibits the development of HSCs at E10.5 but not at E11.5, in keeping with a previous study37. The induction of dHSCs in AoV by AoD is also limited to the E10.5 stage. Since Shh is secreted by the notochord (which is included in AoD-dissected tissue), this stage specificity is likely defined by the predominant presence of pre-HSCs type I at E10.5, which express higher levels of Shh signalling components (Ptch1 and Gli1) compared with pre-HSCs type II. By E11.5, the pre-HSC population is mainly represented by type II cells15. Stage-specific loss of sensitivity to Hh signalling was also described in the developing neural tube55. Notably, the poor ability of AoD to develop HSCs despite abundant presence of Shh can also be explained by lower levels of Ptch1 and Gli1 detected in AoD- compared with AoV-derived pre-HSC type I. Our ex vivo modelling data indicate that AoD-derived Shh is an active inducer of HSC development in the AGM region. This conclusion does not exclude the possibility that Shh secreted by the gut could also reach the dorsal aorta37, although by E10.5 these sites are separated by an extended mesentery.

BMP4 signalling is a key factor involved during differentiation of ventral mesoderm and its further specification into haematopoietic cells. In zebrafish, BMP signalling is clearly required during the patterning of the dorsal aorta and for the emergence of dHSCs in the ventral wall34. Its role in mouse is less clear due to the early lethality of BMP mutants56. Several lines of evidence point to BMP4 as a good candidate regulating HSC development. Indeed, BMP4 is highly expressed in the ventral mesenchyme underneath the dorsal aorta34, 36, 49; some reports suggested its role in controlling dHSC emergence36, 57, 58. However, the in vitro systems used likely assayed the maintenance of dHSCs, rather than their maturation. It was also reported that BMP4 signalling can define their differentiation potential59. BMP4 is also involved in the regulation of essential haematopoietic transcription factors such as Scl/Gata2/Fli1 and Runx1 (refs 60, 61). Here we analysed BMP signalling activity in the dHSC lineage in the AGM region. We show that in vivo the pre-HSC type I to type II transition is accompanied by a downregulation of BMP targets (Id genes). This correlates with our data demonstrating that BMP activity is downregulated in intra-aortic clusters and the observations of others that Runx1 expression is attenuating in the developing HSC lineage60, 62, 63. How is this decrease of BMP activity achieved in vivo, despite the presence of BMP4 in AoV? It has previously been noted that in amphibian embryos several BMP inhibitors are also expressed in AoV34. Similarly, our analysis of the embryo showed high expression of a number of BMP antagonists as well as inhibitory Smad6 and Smad7 in mouse AoV that may counteract BMP4 action in HSC lineage. Furthermore, we found that in the AGM region BMP4 and Noggin are spatially segregated: Noggin being present in haematopoietic clusters and BMP4 being mainly expressed underneath the aortic endothelium. Therefore, maturing HSCs in clusters are exposed to low BMP4 concentration and high concentration of the BMP antagonist Noggin. Furthermore, our qRT–PCR analysis shows that the pre-HSC type I population expresses Noggin, which possibly creates a very effective shield that protects them from BMP4. Accordingly, our ex vivo analysis strongly suggests that downregulation of BMP signalling is functionally important for HSC development in the embryo. Indeed, forced BMP signalling activation by the addition of BMP4, strongly inhibits HSC development, and conversely the addition of Noggin stimulates HSC development in E10.5–E11.5 AGM cultures. These results are in line with recent observation that deletion of Smad4, a common transducer for BMP4/TGFβ signalling, markedly augments the formation of intra-aortic clusters64. Our data do not exclude the possibility that BMP4 is essential for specification of mouse dHSCs at earlier stages, as described in the zebrafish model, where BMP signalling is required for HSC development at stages closer to mouse E8.5 (ref. 34).

Our analysis indicates that all three signalling pathways studied can cooperate for HSC development (Fig. 8c). Notably, the interplay of Shh and BMP pathways is broadly involved in development. For example, counter gradients of polarized Shh and BMP signalling in the developing spinal cord specify neuronal subsets along the dorso–ventral axis65, and the dorsal aorta resembles the neural tube with inverse orientation of Shh- and BMP-secreting centres34. However, we detected an antagonistic relationship between Shh and BMP pathways. At the molecular level, Shh can induce Noggin and Smad6 expression, thus inhibiting BMP4 signalling. In turn, BMP4 suppresses and, accordingly, Noggin enhances Shh signalling. Cooperation between Shh and Noggin has been previously described as critically important for developmental specification of somitic, neural and hair follicle cells66, 67, 68. Our in vitro data suggest that the feed-forward loop Shhright arrowNoggin/Nogginright arrowShh is also involved in HSC development in vivo.

We propose a model where the polarized secreted factors form complex fields of gradients in vivo, which define an effector zone for optimal HSC development in the dorsal aorta and lead to the ventrally shifted appearance of dHSCs (Fig. 8c). Of interest, intra-aortic clusters are abundant in ventro–lateral positions69, which may reflect the position of this zone. The dissection close to such a zone could lead to accidental inclusion of powerful dHSCs in AoD samples observed here. Furthermore, it is possible that spatial segregation of co-operating and spatial overlap of antagonizing factors may also be important for adjustment of HSC development in vivo. Indeed, although the pool of pre-HSCs in the AGM region markedly expands during E9.5–11.5 (Rybtsov et al., submitted), complete maturation of the HSC pool is limited: while the majority of cells reach the pre-HSC type II stage, only one or two dHSCs are generated by the end of E11. Such controlled dynamics of HSC development may be needed to prevent a burst of active haematopoiesis in the AGM region. How exactly HSC maturation dynamics depend on overlapping concentrations of factors requires further analysis. Although ex vivo modelling is a powerful tool to dissect mechanisms of HSC development in vivo, there will likely be some variation in details. For example, spatial polarization in the developing HSC niche may define kinetics of HSC development in vivo.While we have demonstrated spatial polarization in vivo of the factors driving HSC development in our model system, it is currently unclear whether any factors become expressed in a polarized manner within the reaggregates and as such, whether polarization is also a pre-requisite for HSC maturation. Alternatively, if polarization is not required, the entire reaggregate may replicate the optimal zone for HSC development, resulting in massive generation of dHSCs. The distinction between these two scenarios will require further investigation.

In summary, our ex vivo modelling experiments suggest that HSC development in the embryo involves stage-dependent interactions between dorsal, ventral and lateral domains of the AGM region, mediated at least partly by the interplay of SCF, Shh, BMP4 and Noggin. Further detailed analysis will be required to better understand the complexity of the AGM signalling landscape in which HSC development takes place. Such knowledge may lead to development of novel protocols for the generation of definitive HSCs in vitro for clinical applications.

Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting

Jie He1Omar Abdel-Wahab2Michelle K. Nahas1Kai Wang1Raajit K. Rampal3Andrew M. Intlekofer4, et al.
http://www.bloodjournal.org/content/early/2016/03/10/blood-2015-08-664649March 10, 2016

Key Points

  • Novel clinically-available comprehensive genomic profiling of both DNA and RNA in hematologic malignancies.

  • Profiling of 3696 clinical hematologic tumors identified somatic alterations that impact diagnosis, prognosis, and therapeutic selection

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs) and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded (FFPE), blood and bone marrow samples with high accuracy in a clinically relevant timeframe, which is performed in our CLIA-certified CAP-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs and gene fusions, with similar accuracy to lower-throughput assays which focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically-relevant genomic alterations with therapeutic relevance.

Cohesin Ring Rules Blood Stem Cells, Binds Them to Renewal or Expansion

GEN News    http://www.genengnews.com/gen-news-highlights/cohesin-ring-rules-blood-stem-cells-binds-them-to-renewal-or-expansion/81252512/

A genome-wide RNAi screen was used to assess the effects of 15,000 genes on the balance between self-renewal and differentiation of human hematopoietic stem cells (HSCs). The screen identified candidate genes whose knockdown maintained the HSC phenotype during culture. Such findings could lead to better protocols to grow these cells outside the body, potentially making bone marrow transplants more available to patients suffering blood cancers, or even identifying novel genes to target during the treatment of leukemia (left and right panels). Four genes in particular implicated cohesin, a ring-like protein complex that binds to the DNA in all of our cells, in the control of self-renewal versus differentiation in HSCs. Deficiency of cohesin causes an increase in self-renewal and a decrease in differentiation of HSCs. [Cell Reports]

Best known for its ability to regulate the separation of sister chromatids during cell division, the cohesin protein complex, a ring-shaped structure, has shown that it has other powers, such as the facilitation of DNA repair and the modification of transcription. And now, according to scientists based at Lund University, there is evidence that the cohesin complex controls the growth of blood stem cells. More to the point, the cohesin complex determines whether blood stem cells self-renew or differentiate.

The new finding is significant because it can help scientists improve the expansion of blood stem cells outside the body, thus increasing the supply of blood stem cells to patients suffering leukemia or hereditary blood disorders. Besides making bone marrow transplant material more available, the new finding could point scientists to new points of attack for the treatment of blood cancer, which is a disruption between blood stem cell multiplication and maturation.

The Lund University scientists, led by Jonas Larsson, presented their results March 17 in the journal Cell Reports, in an article entitled “Genome-wide RNAi Screen Identifies Cohesin Genes as Modifiers of Renewal and Differentiation in Human HSCs.” The article describes how a genome-wide RNA interference (RNAi) screen was performed in primary human CD34+ cells. This screen enabled the scientists to identify candidate genes whose knockdown maintained the HSC phenotype during culture.

“A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, and SMC3) among the top 20 genes from the screen,” wrote the authors. “Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro.”

A similar expansion, the authors added, was observed in vivo following transplantation to immunodeficient mice.

“Transcriptome analysis of cohesin-deficient CD34+ cells showed an upregulation of HSC-specific genes,” the authors continued. This finding, the authors asserted, demonstrates that when cohesin is deficient, transcription shifts to a more stem cell–like pattern.

“The research is unique as the study of so many genes alongside one another is unprecedented,” said Dr. Larsson. “In addition, we have used human blood stem cells, which is difficult in itself as it is requires the gathering of a large amount of material.”

Of the 15,000 genes that were tested, the Lund team found around 20 candidates with a strong capacity to affect the balance of growth in the blood stem cells. What was striking was that four of these 20 genes were physically connected through cooperation in a protein complex.

“The discovery showed that this protein complex is crucial and has an overarching function in the growth of the blood stem cells,” emphasized Dr. Larsson.

The cohesin complex acts as a sort of brace that holds different parts of the DNA strand together in the cell. The researchers believe that this allows the cohesin complex to control access to the “on/off switches” in DNA and to change the impulses the blood stem cells receive from various genes, thereby affecting cell division. The blood stem cell either multiplies or matures to become a specialized cell with other tasks.

Independently of the Lund researchers’ discovery, other research in the field of blood cancer has recently identified mutations in exactly the same four genes in patients with various forms of blood cancer.

“This is incredibly exciting! Together with the results from our study, this indicates that the cohesin genes are directly and crucially significant in the development of blood cancer,” exclaimed the study’s lead author, Ph.D. candidate Roman Galeev. “Our findings entail a new understanding of how the expansion of blood stem cells is controlled. Eventually, this can lead to new ways of affecting the process, either to prevent the development of cancer or to expand the stem cells for transplant.”

UNPRECEDENTED PRECISION STUDY IDENTIFIES THE FOUR GENES RESPONSIBLE FOR BLOOD STEM CELL DEVELOPMENT.

  • A genome-wide RNAi screen was performed in primary human CD34+ cells
  • Several cohesin genes were identified as modifiers of renewal and differentiation
  • Cohesin-deficient HSCs show enhanced reconstitution capacity in vivo
  • Cohesin deficiency induces immediate HSC-specific transcriptional programs

Summary

To gain insights into the regulatory mechanisms of hematopoietic stem cells (HSCs), we employed a genome-wide RNAi screen in human cord-blood derived cells and identified candidate genes whose knockdown maintained the HSC phenotype during culture. A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, andSMC3) among the top 20 genes from the screen. Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro. A similar expansion was observed in vivo following transplantation to immunodeficient mice. Transcriptome analysis of cohesin-deficient CD34+ cells showed an upregulation of HSC-specific genes, demonstrating an immediate shift toward a more stem-cell-like gene expression signature upon cohesin deficiency. Our findings implicate cohesin as a major regulator of HSCs and illustrate the power of global RNAi screens to identify modifiers of cell fate.

Figure thumbnail fx1

Human hematopoiesis is maintained by a small number of hematopoietic stem cells (HSCs) that are capable of generating all blood cell lineages at an extremely rapid pace for the entire lifespan of a human being (Orkin and Zon, 2008). HSCs have been studied extensively during the last four decades and are probably the best functionally characterized adult stem cells. However, despite this, the regulatory mechanisms that govern different cellular fate options in HSCs have remained incompletely defined. In particular, it has been challenging to understand the molecular basis of the inherent ability of HSCs to self-renew and preserve their undifferentiated state, which has hampered efforts to expand HSCs ex vivo for therapeutic benefit (Dahlberg et al., 2011). Ex vivo expansion of HSCs would allow for critical improvements of bone marrow transplantation procedures in treatment of malignant and inherited hematological diseases (Chou et al., 2010). Defining the genetic and molecular basis of self-renewal of HSCs is thus important to enhance current cell-therapy strategies, but it is also essential in order to better understand mechanisms behind dysregulated hematopoiesis that may cause leukemia. Genes and pathways balancing cell-fate options between renewal and differentiation in stem cells are often key players in cancer development (Orkin and Zon, 2008).

Thumbnail image of Figure 1. Opens large image

Figure 1

Genome-wide RNAi Screen in Primitive Human Hematopoietic Cells Defines Genes and Pathways Associated with Cancer Progression and Cell Proliferation

(A) Overview of the experimental outline for the primary screen. 60 million cord blood-derived CD34+ cells were transduced with a pooled lentiviral library containing 75,000 shRNAs across six transduction replicates in total. A fraction of the cells were isolated after 72 hr, and proviral inserts were deep sequenced to determine the initial library distribution. Following 20 days of culture, CD34+ cells were magnetically isolated and proviral inserts were sequenced again to determine the changes in distribution for all shRNAs.

(B) Relative distribution of shRNAs following 20 days of in vitro culture, ranked from the most enriched to the most depleted. The y axis shows the average enrichment value across six replicate screens.

(C) Gene ontology analysis for all genes represented by multiple shRNAs in the most enriched (10%) fraction.

(D) KEGG pathway analysis showing strong enrichment for cancer-associated pathways among the top-scoring genes.

See also Figure S1 and Table S1.

We report here on the successful development of a genome-wide RNAi screening approach targeted to primary human hematopoietic stem and progenitor cells to define genes and pathways associated with self-renewal and differentiation. Based on findings from the screen, we implicate the cohesin complex as a crucial regulator of cell-fate decisions influencing self- renewal and differentiation in HSCs both in vitro and in vivo.

These efforts represent a genome-wide RNAi screen targeted to primary human HSPCs. The main limiting factor when performing functional screens in primary human cells is cell number. This obviously becomes even more challenging when rare cell subsets, such as stem and progenitor cells, are studied. Through unique access to cord blood with daily deliveries from several local hospitals, we were able to gather the necessary quantities to perform a screen in enriched primary HSPCs with reasonable coverage (300X).

 

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Targeting hematopoietic stem cells

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

New technology uncovered the stem cell niche in the bone marrow

HSCs, Stem cells, hematopoiesis

Hematopoietic stem cells (HSCs) are so rare that it’s difficult to comprehensively localize dividing and non-dividing HSCs. Thus, there has controversy about their specific location in the bone marrow. A recent Nature publication reported that the HSCs resides mainly in perisinusoidal niches through out the bone marrow and there are no spatially distinct niches for dividing and non-dividing blood-forming stem cells. This group of researchers at UT Southwestern Medical Center started the generation of a GFP knock-in for the gene Ctnnal1, a generic marker for HSCs in mice (α-catulinGFP mice) and confirmed that α-catulin-GFP+c-kit+ cells represent blood-forming HSCs by showing that α-catulin-GFP+c-kit+ cells gave long term multi-lineage reconstitution of irradiated mice. Using a tissue-clearing technique and deep confocal imaging, they were able to image thousands of α-catulin-GFP+c-kit+ cells and see their relation to other cells. This publication improved the understanding of the microenvironment of HSCs in the bone marrow, which would significantly improve the safety and effectiveness of bone marrow transplantation.

Melih Acar, etc. (October 2015) Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature

 

Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal

AcarKS. KocherlakotaMM. MurphyJG. PeyerH OguroCN. InraC JaiyeolaZ ZhaoK Luby-Phelps & Sean J. Morrison
Nature526,126–130(01 October 2015)
   
       doi:10.1038/nature15250

 

Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial1. HSCs are rare and few can be found in thin tissue sections2, 3 or upon live imaging4, making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as αcatulinGFP), we discover that αcatulinGFP is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of αcatulin−GFP+c-kit+ cells give long-term multilineage reconstitution of irradiated mice, indicating thatαcatulin−GFP+c-kit+ cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of αcatulin–GFP+c-kit+ cells and to digitally reconstruct large segments of bone marrow. The distribution of αcatulin–GFP+c-kit+ cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr+) and Cxcl12high niche cells, and approximately 85% of HSCs were within 10 μm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67+) and non-dividing (Ki-67), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr+Cxcl12high cells throughout the bone marrow.

 

Figure 1: Deep imaging of αcatulin−GFP+ HSCs in digitally reconstructed bone marrow.close

 

Deep imaging of [agr]-catulin-GFP+ HSCs in digitally reconstructed bone marrow.

a, Only 0.021 ± 0.006% of αcatulinGFP/+ bone marrow cells were GFP+ (n = 14 mice in 11 independent experiments). b, Nearly allαcatulin−GFP+c-kit+ bone marrow cells were CD150+CD48 (n = 9 mice in 3 independent experiments;

 

Extended Data Figure 3: αcatulin−GFP expression among haematopoietic cells is highly restricted to HSCs.

 

[agr]-catulin-GFP expression among haematopoietic cells is highly restricted to HSCs.

 

a, The frequency of αcatulin−GFP+ bone marrow cells in negative control αcatulin+/+ (WT) mice and α-catulinGFP/+ mice (n = 14 mice per genotype in 11 independent experiments). In all cases in this figure, percentages refer to the frequency of each population as a percentage of WBM cells. b, αcatulin−GFP+c-kit+ cells from Fig. 1b are shown (blue dots) along with all other bone marrow cells in the same sample (red dots). c, CD150+CD48LSK HSCs express αcatulin−GFP but CD150CD48LSK MPPs do not (n = 17 mice in 12 independent experiments). A minority of the αcatulin−GFP+c-kit+ cells had high forward scatter, lacked reconstituting potential, and were gated out when isolating HSCs by flow cytometry and when identifying HSCs during imaging (see Extended Data Fig. 5for further explanation). d, Linc-kitlowSca-1lowCD127+CD135+ common lymphoid progenitors (CLPs), Linc-kit+Sca-1CD34+CD16/32 common myeloid progenitors (CMPs), Linc-kit+Sca-1CD34+CD16/32+ granulocyte-macrophage progenitors (GMPs), and Linc-kit+Sca-1CD34CD16/32 megakaryocyte-erythroid progenitors (MEPs) did not express αcatulin−GFP. αcatulinGFP/+ and control cell populations had similar levels of background GFP signals that accounted for fewer than 1% of the cells in each population (n = 9 mice per genotype in 2 independent experiments).

 

Extended Data Figure 7: HSC density is higher in the diaphysis as compared to the metaphysis.

HSC density is higher in the diaphysis as compared to the metaphysis.

a, Schematic of a femur showing the separation of epiphysis/metaphysis from diaphysis. We divided metaphysis from diaphysis at the point where the central sinus branched (see red line in panels a, f,and i). This is also the point at wh…

 

 

Extended Data Figure 9: Bone marrow blood vessel types can be distinguished based on vessel diameter, continuity of basal lamina, morphology, and position; and no difference in the distribution of HSCs in the bone marrow of male and female mice was detected.close

Bone marrow blood vessel types can be distinguished based on vessel diameter, continuity of basal lamina, morphology, and position; and no difference in the distribution of HSCs in the bone marrow of male and female mice was detected.

a, b, Schematic (a) and properties (b) of blood vessels in the bone marrow. Blood enters the marrow through arterioles that branch as they become smaller in diameter and approach the endosteum, where they connect to smaller diameter tra…

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Reporter: Danut Dragoi, PhD

Scientists at MIT and Massachusetts General Hospital have discovered how cancer cells latch onto blood vessels and invade tissues to form new tumors — a finding that could help them develop drugs that inhibit this process and prevent cancers from metastasizing.

Cancer cells circulating in the bloodstream can stick to blood vessel walls and construct tiny “bridges” through which they inject genetic material that transforms the endothelial cells lining the blood vessels, making them much more hospitable to additional cancer cells, according to the new study.

The researchers also found that they could greatly reduce metastasis in mice by inhibiting the formation of these nanobridges. Endothelial cells line every blood vessel and are the first cells in contact with any blood-borne element. They serve as the gateway into and out of tumors and have been the focus of intense research in vascular and cancer biology.

Building bridges

Metastasis is a multistep process that allows cancer to spread from its original site and form new tumors elsewhere in the body. Certain cancers tend to metastasize to specific locations; for example, lung tumors tend to spread to the brain, and breast tumors to the liver and bone.

To metastasize, tumor cells must first become mobile so they can detach from the initial tumor. Then they break into nearby blood vessels so they can flow through the body, where they become circulating tumor cells (CTCs). These CTCs must then find a spot where they can latch onto the blood vessel walls and penetrate into adjacent tissue to form a new tumor.

Blood vessels are lined with endothelial cells, which are typically resistant to intruders.

The researchers first spotted tiny bridges between cancer cells and endothelial cells while using electron microscopy to study the interactions between those cell types. They speculated that the cancer cells might be sending some kind of signal to the endothelial cells.

Once we saw that these structures allowed for a ubiquitous transfer of a lot of different materials, microRNAs were an obvious interesting molecule because they’re able to very broadly control the genome of a cell in ways that we don’t really understand,” Connor says. “That became our focus.”

MicroRNA, discovered in the early 1990s, helps a cell to fine-tune its gene expression. These strands of RNA, about 22 base pairs long, can interfere with messenger RNA, preventing it from being translated into proteins.

In this case, the researchers found, the injected microRNA makes the endothelial cells “sticky.” That is, the cells begin to express proteins on their surfaces that attract other cells to adhere to them. This allows additional CTCs to bind to the same site and penetrate through the vessels into the adjacent tissue, forming a new tumor.

Non-metastatic cancer cells did not produce these invasive nanobridges when grown on endothelial cells.

Shutting down metastasis

The nanobridges are made from the proteins actin and tubulin (NB-the protein actin is abundant in all eukaryotic cells. It was first discovered in skeletal muscle, where actin filaments slide along filaments of another protein called myosin to make the cells contract. In non-muscle cells, actin filaments are less organized and myosin is much less prominent, and a tubulin is a protein that is the main constituent of the micro-tubules of living cells, which also form the cytoskeleton that gives cells their structure). The researchers found that they could inhibit the formation of these nanobridges, which are about 300 microns long, by giving low doses of drugs that interfere with actin.

When the researchers gave these drugs to mice with tumors that normally metastasize, the tumors did not spread.

Sengupta’s lab is now trying to figure out the mechanism of nanobridge formation in more detail, with an eye toward developing drugs that act more specifically to inhibit the process.

 The SEM picture below,

Cancer_cell_Vein

is a rounded cancer cell (top left) that sends out nanotubes connecting with endothelial cells. Genetic material can be injected via these nanotubes, transforming the endothelial cells and making them more hospitable to additional cancer cells. Image credit: Sengupta Lab. The second picture below,

Cancer_Cell_Vein_Penetration

is showing a cancer cell (bottom center) that creates a gap and enters the endothelial tube. Another cancer cell (middle right) sends out nanotubes to connect with endothelial cells. Both image are credited to Sengupta Lab

An interesting comment on why plants do not develop cancer is given here,  The article states that in plants, as in animals, most cells that constitute the organism limit their reproductive potential in order to provide collective support for the immortal germ line. And, as in animals, the mechanisms that restrict the proliferation of somatic cells in plants can fail, leading to tumors. There are intriguing similarities in tumorigenesis between plants and animals, including the involvement of the retinoblastoma pathway as well as overlap with mechanisms that are used for stem cell maintenance. However, plant tumors are less frequent and are not as lethal as those in animals. The authors of the article argue that fundamental differences between plant and animal development make it much more difficult for individual plant cells to escape communal controls.

The structure of the endothelium, the thin layer of cells that line our arteries and veins, is visible here. The endothelium is like a gatekeeper, controlling the movement of materials into and out of the bloodstream. Endothelial cells are held tightly together by specialized proteins that function like strong ropes (red) and others that act like cement (blue). In the picture here, the cell is preparing to divide. Two copies of each chromosome (blue) are lined
up next to each other in the center of the cell. Next, protein strands (red) will pull apart these paired
chromosomes and drag them to opposite sides of the cell. The cell will then split to form two daughter cells, each with a single, complete set of chromosomes. It is interesting that protein strands (red) in this picture are implicated on pulling apart the two copies of chromosomes in the same way the proteins actin and tubules do in the cancer cell interaction with the veins and arteries. It looks like the proteins shaped as strings, the nanobridges, due their shape to the tension development between cancer cells and the veins.

Source
1.  http://news.mit.edu/2015/cancer-cells-escape-blood-vessels-1216
2.  http://www.nature.com/nrc/journal/v10/n11/full/nrc2942.html

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Blocking Differentiation to Produce Stem Cells

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Blocking Differentiation is Enough to Turn Mature Cells into Stem Cells

 

 

ID3 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein [ Homo sapiens (human) ]

Gene ID: 3399, updated on 15-Nov-2015

http://www.ncbi.nlm.nih.gov/gene?Db=gene

 

Official Symbol ID3 provided by HGNC 

Official Full Name inhibitor of DNA binding 3, dominant negative helix-loop-helix protein provided by HGNC

Primary source HGNC:HGNC:5362 See related Ensembl:ENSG00000117318; HPRD:02608; MIM:600277; Vega:OTTHUMG00000003229

Gene type protein coding

RefSeq status REVIEWED

OrganismHomo sapiens

LineageEukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo

Also known as HEIR-1; bHLHb25

Summary The protein encoded by this gene is a helix-loop-helix (HLH) protein that can form heterodimers with other HLH proteins. However, the encoded protein lacks a basic DNA-binding domain and therefore inhibits the DNA binding of any HLH protein with which it interacts. [provided by RefSeq, Aug 2011]

Orthologs mouse all

 

Location:
1p36.13-p36.12
Exon count:
3
Annotation release Status Assembly Chr Location
107 current GRCh38.p2 (GCF_000001405.28) 1 NC_000001.11 (23557930..23559794, complement)
105 previous assembly GRCh37.p13 (GCF_000001405.25) 1 NC_000001.10 (23884421..23886285, complement)

Chromosome 1 – NC_000001.11Genomic Context describing neighboring genes

Related articles in PubMed

 

Induced Developmental Arrest of Early Hematopoietic Progenitors Leads to the Generation of Leukocyte Stem Cells

Tomokatsu Ikawa, Kyoko Masuda, Mirelle J.A.J. Huijskens, Rumi Satoh, Kiyokazu Kakugawa, Yasutoshi Agata, Tomohiro Miyai, Wilfred T.V. Germeraad, Yoshimoto Katsura, Hiroshi Kawamoto
Stem Cell Reports Nov 10, 2015; Volume 5, Issue 5, 716–727.   DOI: http://dx.doi.org/10.1016/j.stemcr.2015.09.012
Highlights
  • Overexpression of ID3 endows hematopoietic progenitors with self-renewal activity
  • A simple block of cell differentiation is sufficient to induce stem cells
  • Induced leukocyte stem (iLS) cells exhibit robust multi-lineage reconstitution
  • Equivalent progenitors were produced from human cord blood hematopoietic stem cells

Self-renewal potential and multipotency are hallmarks of a stem cell. It is generally accepted that acquisition of such stemness requires rejuvenation of somatic cells through reprogramming of their genetic and epigenetic status. We show here that a simple block of cell differentiation is sufficient to induce and maintain stem cells. By overexpression of the transcriptional inhibitor ID3 in murine hematopoietic progenitor cells and cultivation under B cell induction conditions, the cells undergo developmental arrest and enter a self-renewal cycle. These cells can be maintained in vitro almost indefinitely, and the long-term cultured cells exhibit robust multi-lineage reconstitution when transferred into irradiated mice. These cells can be cloned and re-expanded with 50% plating efficiency, indicating that virtually all cells are self-renewing. Equivalent progenitors were produced from human cord blood stem cells, and these will ultimately be useful as a source of cells for immune cell therapy.

Figure thumbnail fx1

http://www.cell.com/cms/attachment/2040173852/2053709392/fx1.jpg

 

Somatic tissues with high turnover rates, such as skin, intestinal epithelium, and hematopoietic cells, are maintained by the activity of self-renewing stem cells, which are present in only limited numbers in each organ (Barker et al., 2012,Copley et al., 2012, Fuchs and Chen, 2013). For example, the frequency of hematopoietic stem cells (HSCs) in the mouse is about 1 in 105 of total bone marrow (BM) cells (Spangrude et al., 1988). Once HSCs begin the differentiation process, their progeny cells have hardly any self-renewal capacity, indicating that self-renewal is a special feature endowed only to stem cells.

Cells such as embryonic stem (ES) cells that retain self-renewal potential and multipotency only in vitro can also be included in the category of stem cells. Such stemness of ES cells is thought to be maintained by formation of a core transcriptional network and an epigenetic status unique to ES cells (Lund et al., 2012, Meissner, 2010, Ng and Surani, 2011). A stem cell equivalent to ES cells, called induced pluripotent stem (iPS) cells, can be produced from somatic cells by overexpression of only a few specific transcription factors (OCT3/4, SOX2, KLF4, and C-MYC), which are thought to be the essential components in forming the core network of transcriptional factors that define the status of ES cells (Takahashi et al., 2007, Takahashi and Yamanaka, 2006, Yamanaka, 2012). It is thus generally conceived that acquisition of such a network for a somatic cell depends on the reprogramming of the epigenetic status of that cell.

On the other hand, it could be envisioned that the self-renewing status of cells represents a state in which their further differentiation is inhibited. It is known, for example, that to maintain ES/iPS cells, factors such as leukemia inhibitory factor and basic fibroblast growth factor, for mouse and human cultures, respectively (Williams et al., 1988, Xu et al., 2005), are required, and these factors are thought to block further differentiation of the cells. In this context, it has previously been shown that systemic disruption of transcription factors essential for the B cell lineage, such as PAX5, E2A, and EBF1, leads to the emergence of self-renewing multipotent hematopoietic progenitors, which can be maintained under specific culture conditions (Ikawa et al., 2004a, Nutt et al., 1999, Pongubala et al., 2008). It has recently been shown that the suppression of lymphoid lineage priming promotes the expansion of both mouse and human hematopoietic progenitors (Mercer et al., 2011, van Galen et al., 2014). Therefore, it would seem theoretically possible to make a stem cell by inducing inactivation of these factors at particular developmental stages. Conditional depletion of PAX5 in B cell lineage committed progenitors, as well as mature B cells, resulted in the generation of T cells from the B lineage cells (Cobaleda et al., 2007, Nutt et al., 1999, Rolink et al., 1999). These studies, however, were mainly focused on the occurrence of cell-fate conversion by de-differentiation of target cells. Therefore, the minimal requirement for the acquisition of self-renewal potential remains undetermined.

Our ultimate goal is to obtain sufficient number of stem cells by expansion to overcome the limitation of cell numbers for immune therapies. We hypothesize that stem cells can be produced by simply blocking differentiation. As mentioned earlier, self-renewing multipotent progenitors (MPPs) can be produced by culturing E2A-deficient hematopoietic progenitors in B cell-inducing conditions (Ikawa et al., 2004a). Because it remains unclear at which developmental stage the acquisition of self-renewing potential has occurred in the case of such a systemic deletion, we thought to develop a method in which E2A function could be inactivated and reactivated in an inducible manner. We decided to use the ID3 protein for this purpose, because it is known that ID proteins serve as dominant-negative inhibitors of E proteins (Norton et al., 1998, Sayegh et al., 2003). Here we show that the overexpression of ID3 into HSCs or hematopoietic progenitor cells (HPCs) in both mouse and human induces the stemness of the progenitors and that the cells acquire the self-renewal activity. The ID3-expressing cells can be maintained in vitro as MPPs with T, B, and myeloid lineage potentials.

 

Results

Jump to Section
Introduction
Results
  Generation of ID3-Transduced Hematopoietic Progenitors
  IdHP Cells Are Multipotent, Maintaining T, B, and Myeloid Lineage Potentials
  IdHP Cells Are Multipotent at a Clonal Level
  Generation of IdHP Cells from Mouse BM
  Generation of Inducible IdHP Cells Using ID3-ER Retrovirus
  Generation of IdHP Cells from Human Cord Blood Hematopoietic Progenitors
Discussion
Experimental Procedures
  Mice
  Antibodies
  Growth Factors
  Isolation of Hematopoietic Progenitors
  Retroviral Constructs, Viral Supernatants, and Transduction
  In Vitro Differentiation Culture System
  Cloning of mIdHP Cells
  Colony-Forming Unit in Culture Assay
  Cell Cycle Assay
  Adoptive Transfer of mIdHP and hIdHP Cells
  PCR Analysis of Igh Gene Rearrangement
  RNA Extraction and qRT-PCR
  Microarray Analysis
Author Contributions
Supplemental Information
References

Generation of ID3-Transduced Hematopoietic Progenitors

IdHP Cells Are Multipotent, Maintaining T, B, and Myeloid Lineage Potentials

IdHP Cells Are Multipotent at a Clonal Level

Generation of IdHP Cells from Mouse BM

Generation of Inducible IdHP Cells Using ID3-ER Retrovirus

Generation of IdHP Cells from Human Cord Blood Hematopoietic Progenitors

Thumbnail image of Figure 1. Opens large image

http://www.cell.com/cms/attachment/2040173852/2053709390/gr1.jpg

 

Identification of cellular and molecular events regulating self-renewal or differentiation of the cells is a fundamental issue in the stem cell biology or developmental biology field. In the present study, we revealed that the simple inhibition of differentiation in HSCs or HPCs by overexpressing ID proteins and culturing them in suitable conditions induced the self-renewal of hematopoietic progenitors and allowed the extensive expansion of the multipotent cells. The reduction of ID proteins in MPPs resulted in the differentiation of the cells into lymphoid and myeloid lineage cells. Thus, it is possible to manipulate the cell fate by regulating E-protein or ID-protein activities. This inducible system will be a useful tool to figure out the genetic and epigenetic program controlling the self-renewal activity of multipotent stem cells.

Previous studies have shown that hematopoietic progenitors deficient for E2A, EBF1, and PAX5 maintain multilineage differentiation potential without losing their self-renewing capacity (Ikawa et al., 2004a, Nutt et al., 1999, Pongubala et al., 2008), indicating that the inhibition of the differentiation pathway at certain developmental stages leads to the expansion of multipotent stem cells. However, the MPPs were not able to differentiate into B cells because they lacked the activities of transcription factors essential for the initiation of the B lineage program. In addition, a restriction point regulating the lineage-specific patterns of gene expression during B cell specification remained to be determined because of the lack of an inducible system that regulates B cell differentiation. Here we have established the multipotent iLS cells using ID3-ER retrovirus, which can be maintained and differentiated into B cells in an inducible manner by simply adding or withdrawing 4-OHT. The data indicated the essential role of E2A for initiation of the B cell program that restricts other lineage potentials, because the depletion of 4-OHT from the culture immediately leads to the activation of E proteins, such as E2A, HEB, and E2-2, that promote B cell differentiation. This strategy is useful in understanding the cues regulating the self-renewal or differentiation of uncommitted progenitors to the B cell pathway. Analysis of genome-wide gene expression patterns and histone modifications will determine the exact mechanisms that underlie the B cell commitment process.

The iLS cells can also be generated from human CB hematopoietic progenitors. Human iLS cells exhibited differentiation potential and self-renewal activity similar to those of murine iLS cells, suggesting a similar developmental program during human B cell fate specification. Our data are consistent with a study demonstrating the critical role of the activity of ID and E proteins for controlling the status of human HSCs and progenitors (van Galen et al., 2014). This study reported that the overexpression of ID2 in human CB HSCs enhanced the myeloid and stem cell program at the expense of lymphoid commitment. Specifically, ID2 overexpression resulted in a 10-fold expansion of HSCs, suggesting that the inhibition of E-protein activities promotes the self-renewal of HSCs by antagonizing the differentiation. This raises a question about the functional differences between ID2 and ID3. Id3 seems to suppress the B cell program and promote the myeloid program more efficiently than does ID2, because the ID2-expressing HPCs appear to retain more B cell potential than ID3-expressing iLS cells (Mercer et al., 2011, van Galen et al., 2014). The self-renewal activity and differentiation potential of ID2-HPCs derived from murine HSCs in the BM seemed to be limited both in vivo and in vitro analysis (Mercer et al., 2011). In our study, the iLS cells retained more myeloid potential and self-renewal capacity during the culture. Strikingly, the multipotent iLS cells enormously proliferated (>103-fold in 1 month) as long as the cells were cultured in undifferentiated conditions. This could be due to the functional differences among Id family genes. Alternatively, combination with additional environmental signals, such as cytokines or chemokines, may affect the functional differences of ID proteins, although any ID proteins can repress the activation of the E2A targets, such as Ebf1 and Foxo1, that are essential for B cell differentiation. ID1 and ID3, but not ID2, are demonstrated to be negative regulators of the generation of hematopoietic progenitors from human pluripotent stem cells (Hong et al., 2011). Further analysis is required to determine the physiological role of ID proteins in regulating hematopoietic cell fate. It also remains to be determined whether the ID3-ER system can be applied to human progenitors. It would be informative to compare the regulatory networks that control B cell differentiation in mouse and human.

Immune cell therapy has become a major field of interest in the last two decades. However, the required high cell numbers restrain the application and success of immune reconstitution or anti-cancer treatment. For example, DCs are already being used in cell therapy against tumors. One of the major limitations of DC vaccine therapy is the difficulty in obtaining sufficient cell numbers, because DCs do not proliferate in the currently used systems. The method of making iLS cells could be applied to such cell therapies. Taken together, the simplicity of this method and the high expansion rate and retention of multilineage potential of the cells make this cell source appealing for regenerative medicine or immune cell therapy.

In summary, we showed that an artificially induced block of differentiation in uncommitted progenitors is sufficient to produce multipotent stem cells that retain self-renewal activity. Once the differentiation block is released, the cells start differentiating into mature cells both in vivo and in vitro. Thus, this method could be applicable for establishing somatic stem cells from other organs in a similar manner, which would be quite useful for regenerative medicine. The relative ease of making stem cells leads us to conceive that a block in differentiation is essential not only in other types of artificially engineered stem cells, such as ES cells and iPS cells, but also in any type of physiological somatic stem cell. In this context, it is tempting to speculate that it could have been easy for a multicellular organism to establish somatic stem cells by this mechanism during evolution.

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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 K1Luznik LGojo 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 GJ1
Hematology 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.[13]

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%, = .082) for patients aged 55 to 64 years.[15][Level of evidence: 1iiDii]

References

  1. 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.
  2. 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.
  3. 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]
  4. 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]
  5. 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.
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. 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.
  14. 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]
  15. 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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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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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, (/ɨˌrɪθrɵˈpɔɪ.ɨtɨn/UK /ɛˌrɪθr.pˈtɪn/) 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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Hematological Cancer Classification

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

 

 

Introduction to leukemias and lymphomas

 

2.4.1 Ontogenesis of the blood elements: hematopoiesis

http://www.britannica.com/EBchecked/topic/69747/blood-cell-formation

Blood cells are divided into three groups: the red blood cells (erythrocytes), the white blood cells (leukocytes), and the blood platelets (thrombocytes). The white blood cells are subdivided into three broad groups: granulocytes, lymphocytes, and monocytes.

Blood cells do not originate in the bloodstream itself but in specific blood-forming organs, notably the marrow of certain bones. In the human adult, the bone marrow produces all of the red blood cells, 60–70 percent of the white cells (i.e., the granulocytes), and all of the platelets. The lymphatic tissues, particularly the thymus, the spleen, and the lymph nodes, produce the lymphocytes (comprising 20–30 percent of the white cells). The reticuloendothelial tissues of the spleen, liver, lymph nodes, and other organs produce the monocytes (4–8 percent of the white cells). The platelets, which are small cellular fragments rather than complete cells, are formed from bits of the cytoplasm of the giant cells (megakaryocytes) of the bone marrow.

In the human embryo, the first site of blood formation is the yolk sac. Later in embryonic life, the liver becomes the most important red blood cell-forming organ, but it is soon succeeded by the bone marrow, which in adult life is the only source of both red blood cells and the granulocytes. Both the red and white blood cells arise through a series of complex, gradual, and successive transformations from primitive stem cells, which have the ability to form any of the precursors of a blood cell. Precursor cells are stem cells that have developed to the stage where they are committed to forming a particular kind of new blood cell.

In a normal adult the red cells of about half a liter (almost one pint) of blood are produced by the bone marrow every week. Almost 1 percent of the body’s red cells are generated each day, and the balance between red cell production and the removal of aging red cells from the circulation is precisely maintained.

Cells-in-the-Bone-Marrow-1024x747

http://interactive-biology.com/wp-content/uploads/2012/07/Cells-in-the-Bone-Marrow-1024×747.png

Erythropoiesis

http://www.interactive-biology.com/3969/erythropoiesis-formation-of-red-blood-cells/

Erythropoiesis – Formation of Red Blood Cells

Because of the inability of erythrocytes (red blood cells) to divide to replenish their own numbers, the old ruptured cells must be replaced by totally new cells. They meet their demise because they don’t have the usual specialized intracellular machinery, which controls cell growth and repair, leading to a short life span of 120 days.

This short life span necessitates the process erythropoiesis, which is the formation of red blood cells. All blood cells are formed in the bone marrow. This is the erythrocyte factory, which is soft, highly cellar tissue that fills the internal cavities of bones.

Erythrocyte differentiation takes place in 8 stages. It is the pathway through which an erythrocyte matures from a hemocytoblast into a full-blown erythrocyte. The first seven all take place within the bone marrow. After stage 7 the cell is then released into the bloodstream as a reticulocyte, where it then matures 1-2 days later into an erythrocyte. The stages are as follows:

  1. Hemocytoblast, which is a pluripotent hematopoietic stem cell
  2. Common myeloid progenitor, a multipotent stem cell
  3. Unipotent stem cell
  4. Pronormoblast
  5. Basophilic normoblast also called an erythroblast.
  6. Polychromatophilic normoblast
  7. Orthochromatic normoblast
  8. Reticulocyte

These characteristics can be seen during the course of erythrocyte maturation:

  • The size of the cell decreases
  • The cytoplasm volume increases
  • Initially there is a nucleus and as the cell matures the size of the nucleus decreases until it vanishes with the condensation of the chromatin material.

Low oxygen tension stimulates the kidneys to secrete the hormone erythropoietin into the blood, and this hormone stimulates the bone marrow to produce erythrocytes.

Rarely, a malignancy or cancer of erythropoiesis occurs. It is referred to as erythroleukemia. This most likely arises from a common myeloid precursor, and it may occur associated with a myelodysplastic syndrome.

Summary of erythrocyte maturation

White blood cell series: myelopoiesis

http://www.nlm.nih.gov/medlineplus/ency/presentations/100151_3.htm

http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/15220.jpg

There are various types of white blood cells (WBCs) that normally appear in the blood: neutrophils (polymorphonuclear leukocytes; PMNs), band cells (slightly immature neutrophils), T-type lymphocytes (T cells), B-type lymphocytes (B cells), monocytes, eosinophils, and basophils. T and B-type lymphocytes are indistinguishable from each other in a normal slide preparation. Any infection or acute stress will result in an increased production of WBCs. This usually entails increased numbers of cells and an increase in the percentage of immature cells (mainly band cells) in the blood. This change is referred to as a “shift to the left” People who have had a splenectomy have a persistent mild elevation of WBCs. Drugs that may increase WBC counts include epinephrine, allopurinol, aspirin, chloroform, heparin, quinine, corticosteroids, and triamterene. Drugs that may decrease WBC counts include antibiotics, anticonvulsants, antihistamine, antithyroid drugs, arsenicals, barbiturates, chemotherapeutic agents, diuretics and sulfonamides.   (Updated by: David C. Dugdale, III, MD)

https://www.med-ed.virginia.edu/courses/path/innes/nh/wcbmaturation.cfm

Note that the mature forms of the myeloid series (neutrophils, eosinophils, basophils), all have lobed (segmented) nuclei. The degree of lobation increases as the cells mature.

The earliest recognizable myeloid cell is the myeloblast (10-20m dia) with a large round to oval nucleus. There is fine diffuse immature chromatin (without clumping) and a prominant nucleolus.

The cytoplasm is basophilic without granules. Although one may see a small golgi area adjacent to the nucleus, granules are not usually visible by light microscopy. One should not see blast cells in the peripheral blood.

myeloblast x100b

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20myeloblast%20x100b.jpeg

The promyelocyte (10-20m) is slightly larger than a blast. Its nucleus, although similar to a myeloblast shows slight chromatin condensation and less prominent nucleoli. The cytoplasm contains striking azurophilic granules or primary granules. These granules contain myeloperoxidase, acid phosphatase, and esterase enzymes. Normally no promyelocytes are seen in the peripheral blood.

At the point in development when secondary granules can be recognized, the cell becomes a myelocyte.

promyelocyte x100

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20promyelocyte%20×100%20a.jpeg

Myelocytes (10-18m) are not normally found in the peripheral blood. Nucleoli may not be seen in the late myelocyte. Primary azurophilic granules are still present, but secondary granules predominate. Secondary granules (neut, eos, or baso) first appear adjacent to the nucleus. In neutrophils this is the “dawn” of neutrophilia.

Metamyelocytes (10-18m) have kidney shaped indented nuclei and dense chromatin along the nuclear membrane. The cytoplasm is faintly pink, and they have secondary granules (neutro, eos, or baso). Zero to one percent of the peripheral blood white cells may be metamyelocytes (juveniles).

metamyelocyte x100

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20metamyelocyte%20×100.jpeg

Bands, slightly smaller than juveniles, are marked by a U-shaped or deeply indented nucleus.

band neutrophilx100a

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20band%20x100a.jpeg

Segmented (segs) or polymorphonuclear (PMN) leukocytes (average 14 m dia) are distinguished by definite lobation with thin thread-like filaments of chromatin joining the 2-5 lobes. 45-75% of the peripheral blood white cells are segmented neutrophils.

https://www.med-ed.virginia.edu/courses/path/innes/images/nhjpeg/nh%20neutrophil%20×100%20d.jpeg

Thrombocytogenesis

The incredible journey: From megakaryocyte development to platelet formation

Kellie R. Machlus1,2 and Joseph E. Italiano Jr
JCB 2013; 201(6): 785-796
http://dx.doi.org:/10.1083/jcb.201304054

Large progenitor cells in the bone marrow called megakaryocytes (MKs) are the source of platelets. MKs release platelets through a series of fascinating cell biological events. During maturation, they become polyploid and accumulate massive amounts of protein and membrane. Then, in a cytoskeletal-driven process, they extend long branching processes, designated proplatelets, into sinusoidal blood vessels where they undergo fission to release platelets.

megakaryocyte production of platelets

http://dm5migu4zj3pb.cloudfront.net/manuscripts/26000/26891/medium/JCI0526891.f4.jpg

platelets and the immune continuum nri2956-f3

http://www.nature.com/nri/journal/v11/n4/images/nri2956-f3.jpg

2.4.2 Classification of hematological malignancies
Practical Diagnosis of Hematologic Disoreders. 4th edition. Vol 2.
Kjeldsberg CR, Ed.  ASCP Press.  2006. Chicago, IL.

2.4.2.1 Primary Classification

Acute leukemias

Myelodysplastic syndromes

Acute myeloid leukemia

Acute lymphoblastic leukemia

Myeloproliferative Disorders

Chronic myeloproliferative disorders

Chronic myelogenous leukemia and related disorders

Myelofibrosis, including chronic idiopathic

Polycythemia, including polycythemia rubra vera

Thrombocytosis, including essential thrombocythemia

Chronic lymphoid leukemia and other lymphoid leukemias

Lymphomas

Non-Hodgkin Lymphoma

Hodgkin lymphoma

Lymphoproliferative disorders associated with immunodeficiency

Plasma Cell dyscrasias

Mast cell disease and Histiocytic neoplasms

2.4.2.2 Secondary Classification

2.4.2.3 Nuance – PathologyOutlines
Nat Pernick, Ed.

Leukemia – Acute

Primary referencesacute leukemia-generalAML generalAML classificationtransient abnormal myelopoiesis

Recurrent genetic abnormalities: AML with t(6;9)AML with t(8;21)AML with 11q23 abnormalitiesAML with inv(16) or t(16;16)AML with Down syndromeAML with FLT3 mutationsAML with myelodysplastic related changesAML therapy relatedAPL microgranular variantAPL with t(15;17)APL with t(V;17)APL therapy related

AML not otherwise categorized: minimally differentiated (M0)without maturation (M1)with maturation (M2)M3myelomonocyticmonoblastic and monocyticerythroidmegakaryoblasticCD13/CD33 negativebasophilicmyeloid sarcomaacute panmyelosis with myelofibrosiswith Philadelphia chromosomewith pseudo Chediak-Higashi anomalyhypocellular

ALL: generalWHO classificationwith eosinophilia

PreB ALL: generalt(9;22)t(v;11q23)t(1;19)t(5;14)t(12;21)hyperdiploidyhypodiploidymature B ALL/Burkitt

Other ALL: T ALLambiguous lineagemixed phenotype

AML and related malignancies

Acute myeloid leukemias with recurrent genetic abnormalities:

  • AML with t(8;21)(q22;q22); RUNX1-RUNX1T1
  • AML with inv(16)(p13.1;q22) or t(16;16)(p13.1;q22); CBF&beta-MYH11
  • Acute promyelocytic leukemia with t(15;17)(q22;q12); PML/RAR&alpha and variants
  • AML with t(9;11)(p22;q23); MLLT3-MLL
  • AML with t(6;9)(p23;q34); DEK-NUP214
  • AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1
  • AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1
  • AML with mutated NPM1*
  • AML with mutated CEBPA*

* provisional

Acute myeloid leukemia with myelodysplasia related changes

Therapy related acute myeloid leukemia

  • Alkylating agent related
  • Topoisomerase II inhibitor related (some maybe lymphoid)

Acute myeloid leukemia not otherwise categorized:

  • AML minimally differentiated (M0)
  • AML without maturation (M1)
  • AML with maturation (M2)
  • Acute myelomonocytic leukemia (M4)
  • Acute monoblastic and monocytic leukemia (M5a, M5b)
  • Acute erythroid leukemia (M6)
  • Acute megakaryoblastic leukemia (M7)
  • Acute basophilic leukemia
  • Acute panmyelosis with myelofibrosis

Myeloid Sarcoma

Myeloid proliferations related to Down syndrome:

  • Transient abnormal myelopoeisis
  • Myeloid leukemia associated with Down syndrome

Blastic plasmacytoid dentritic cell neoplasm:

Acute leukemia of ambiguous lineage:

  • Acute undifferentiated leukemia
  • Mixed phenotype acute leukemia with t(9;22)(q34;q11.2); BCR-ABL1
  • Mixed phenotype acute leukemia with t(v;11q23); MLL rearranged
  • Mixed phenotype acute leukemia, B/myeloid, NOS
  • Mixed phenotype acute leukemia, T/myeloid, NOS
  • Mixed phenotype acute leukemia, NOS, rare types
  • Other acute leukemia of ambiguous lineage
  • References: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue (IARC, 2008), Discovery Medicine 2010, eMedicine

Acute lymphocytic leukemia

General
=================================================================

  • WHO classification system includes former FAB classifications ALL-L1 and L2
    ● FAB L3 is now considered Burkitt lymphoma

WHO classification of acute lymphoblastic leukemia
=================================================================

Precursor B lymphoblastic leukemia / lymphoblastic lymphoma:
● ALL with t(9;22)(q34;q11.2); BCR-ABL (Philadelphia chromosome)
● ALL with t(v;11q23) (MLL rearranged)
● ALL with t(1;19)(q23;p13.3); TCF3-PBX1 (E2A-PBX1)
● ALL with t(12;21)(p13;q22); ETV6-RUNX1 (TEL-AML1)
● Hyperdiploid > 50
● Hypodiploid
● t(5;14)(q31;q32); IL3-IGH

Precursor T lymphoblastic leukemia / lymphoma

Additional references
=================================================================

Mixed phenotype acute leukemia (MPAL)

General
=================================================================

  • De novo acute leukemia containing separate populations of blasts of more than one lineage (bilineal or bilineage), or a single population of blasts co-expressing antigens of more than one lineage (biphenotypic)Excludes:
    ● Acute myeloid leukemia (AML) with recurrent translocations t(8;21), t(15;17) or inv(16)
    ● Leukemias with FGFR1 mutations
    ● Chronic myelogenous leukemia (CML) in blast crisis
    ● Myelodysplastic syndrome (MDS)-related AML and therapy-related AML, even if they have MPAL immunophenotypeCriteria for biphenotypic leukemia:
    ● Score of 2 or more for each of two separate lineages:The European Group for the Immunological Classification of Leukemias (EGIL) scoring system2008 WHO classification of acute leukemias of ambiguous lineage 

Prognosis
=================================================================

  • Poor, overall survival of 18 months
    ● Young age, normal karyotype and ALL induction therapy are associated with favorable survival
    ● Ph+ is a predictor for poor prognosis
    ● Bone marrow transplantation should be considered in first remission

Major Categories

MPAL with t(9;22)(q34;q11.2); BCR-ABL1
=================================================================

  • 20% of all MPAL
    ● Blasts with t(9;22)(q34;q11.2) translocation or BCR-ABL1 rearrangement (Ph+) without history of CML
    ● Majority in adults
    ● High WBC counts● Most of the cases B/myeloid phenotype
    ● Rare T/myeloid, B and T lineage, or trilineage leukemiasMorphology:
    ● Many cases show a dimorphic blast population, one resembling myeloblasts and the other lymphoblastsCytogenetic abnormalities:
    ● Conventional karyotyping for t(9;22), FISH or PCR for BCR-ABL1 translocation
    ● Additional complex karyotypes
    ● Ph+ is a poor prognostic factor for MPAL, with a reported median survival of 8 months
    ● Worse than patients of all other types of MPAL

MPAL with t(v;11q23); MLL rearranged
=================================================================

  • Meeting the diagnostic criteria for MPAL with blasts bearing a translocation involving the 11q23 breakpoint (MLL gene)
    ● MPAL with MLL rearranged rare
    ● More often seen in children and relatively common in infancy
    ● High WBC counts
    ● Poor prognosis
    ● Dimorphic blast population, with one resembling monoblasts and the other resembling lymphoblasts
    ● Lymphoblast population often shows a CD19+, CD10- B precursor immunophenotype, frequently CD15+
    ● Expression of other B markers usually weak
    ● Translocations involving MLL gene include t(4;11)(q21;q23), t(11;19)(q23;p13), and t(9;11)(p22;q23)
    ● Cases with chromosome 11q23 deletion should not be classified in this category

B cell acute lymphoblastic leukemia (ALL) / lymphoblastic lymphoma (LBL)

General

=================================================================

  • Current 2008 WHO classification: B lymphoblastic leukemia / lymphoma, NOS or B lymphoblastic leukemia / lymphoma with recurrent genetic abnormalities
  • See also lymphomas: B cell chapter
  • Also called B cell acute lymphoblastic leukemia / lymphoblastic lymphoma, pre B ALL / LBL
  • Usually children
  • B acute lymphoblastic leukemia presents with pancytopenia due to extensive marrow involvement, stormy onset of symptoms, bone pain due to marrow expansion, hepatosplenomegaly due to neoplastic infiltration, CNS symptoms due to meningeal spread and testicular involvement
  • B acute lymphoblastic lymphoma often presents with cutaneous nodules, bone or nodal involvement, < 25% lymphoblasts in bone marrow and peripheral blood; aleukemic cases are usually asymptomatic
  • Depending on specific leukemia, arises in either hematopoietic stem cell or B-cell progenitor
  • Tumors are derived from pre-germinal center naive B cells with unmutated VH region genes
  • Have multiple immunophenotyping aberrancies relative to normal B cell precursors (hematogones); at relapse, 73% show loss of 1+ aberrance and 60% show new aberrancies (Am J Clin Pathol 2007;127:39)

Prognostic features

=================================================================

  • Favorable prognosis: age 1-10 years, female, white; preB phenotype, hyperdiploidy>50, t(12,21), WBC count at presentation <50×108/L, non-traumatic tap with no blasts in CNS, rapid response to chemotherapy < 5% blasts on morphology on day 15, remission status after induction <5% blasts on morphology and <0.01% blast on flow or PCR, CD10+
  • Intermediate prognosis: hyperdiploidy 47-50, diploid, 6q- and rearrangements of 8q24
  • Unfavorable prognosis: under age 1 (usually have 11q23 translocations) or over age 10; t(9;22) (but not if age 59+ years, Am J Clin Pathol 2002;117:716); male, > 50×108/L WBC at presentation, hypodiploidy, near tetraploidy, 17p- and MLL rearrangements t(v;11q23); CD10-; non-traumatic tap with > 5% blasts or traumatic tap (7%); also increased microvessel staining using CD105 in children (Leuk Res 2007;31:1741), MDR1 expression in children (Oncol Rep 2004;12:1201) and adults (Blood 2002;100:974), 25%+ blasts on morphology on day 15, remission status after induction ≥ 5% blasts on morphology and ≥ 0.1% blasts on flow or PCR

Case reports

=================================================================

  • 12 month old girl and 13 month old boy with mature phenotype but no translocations (Arch Pathol Lab Med 2003;127:1340)
  • 56 year old man with ALL arising from follicular lymphoma (Arch Pathol Lab Med 2002;126:997)
  • 76 year old man with basal cell carcinoma (Diagn Pathol 2007;2:32)
  • With hemophagocytic lymphohistiocytosis (Pediatr Blood Cancer 2008;50:381)

Treatment

================================================================

  • Chemotherapy cures more children than adults; adolescents benefit from intensive regimens (Hematology Am Soc Hematol Educ Program 2005:123)

Micro description

=================================================================

  • Bone marrow smears: small to intermediate blast-like cells with scant, variably basophilic cytoplasm, round / oval or convoluted nuclei, fine chromatin and indistinct nucleoli; frequent mitotic figures; may have “starry sky” appearance similar to Burkitt lymphoma; may have large lymphoblasts with 1-4 prominent nucleoli resembling myeloblasts; usually no sclerosis
  • Bone marrow biopsy: usually markedly hypercellular with reduction of trilinear maturation; cells have minimal cytoplasm, medium sized nuclei that are often convoluted, moderately dense chromatin and indistinct nucleoli, brisk mitotic activity
  • Other tissues: may have “starry sky” appearance similar to Burkitt lymphoma; collagen dissection, periadipocyte growth pattern and single cell linear filing

Chronic Leukemia

Chronic Myeloid Neoplasms

Myelodysplastic syndromes (MDS): general, WHO classification, childhood, refractory anemia, refractory anemia with ringed sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess blasts, 5q-syndrome, therapy related, unclassified, arsenic toxicity

Myeloproliferative neoplasms (MPN): general, WHO classification, chronic eosinophilic leukemia, chronic myelogenous leukemia, chronic neutrophilic leukemia, essential thrombocythemia, hypereosinophilic syndrome, mast cell disease, polycythemia vera, primary myelofibrosis, unclassifiable

MDS/MPN: general, WHO classification, atypical CML, chronic myelomonocytic leukemia (CMML), chronic myelomonocytic leukemia with eosinophilia, juvenile myelomonocytic leukemia, unclassifiable

Myeloid neoplasms associated with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1: PDGFRA, PDGFRB, FGFR1

Miscellaneous: transient myeloproliferative disorder of Downís syndrome

Lymphoma and plasma cell neoplasms

Lymph nodes: normal development-generalB cellsT cellsNK cellsnormal histologygrossing lymph nodesfeatures to report

Molecular testing: theoryFISHnorthern blotPCRsouthern blot

Non-Hodgkin lymphoma: generalcytogeneticsstagingstaging-pediatricmorphologic clueshemophagocytic syndromechemotherapeutic atypia

B cell disorders: generalpost-rituximabbone marrow biopsyclassification-historicalWHO classification

B cell lymphoma subtypes: age-related EBV-associatedALK positive large cellBurkittunclassifiable-intermediate between Burkitt and diffuse large B cell lymphomaCLL
diffuse large B cell: 
diffuse-NOSCD5+T cell / histiocyte richprimary cutaneous-generalprimary cutaneous-legprimary sites-other
follicular: 
generalchildhoodcutaneousGI
hairy cell leukemiaHCL variantintravascular large B celllymphomatoid granulomatosislymphoplasmacyticmantle cell-classicmantle cell-blastoidmarginal zone-generalmarginal zone-MALTMALT-primary sitesmarginal zone-nodalmediastinal (thymic)plasmablasticpre B lymphoblastic leukemia/lymphomaprimary effusionprolymphocytic leukemiapyothorax associatedSLLsplenic marginal zonesplenic lymphoma with villous lymphocytes

Plasma cell neoplasms: generalmyelomaplasmacytomaheavy chain diseaseprimary amyloidosisMGUSOsteosclerotic myeloma (POEMS)cryoglobulinemia

T/NK cell disorders: generalWHO classificationadult T cellaggressive NK cell leukemiaanaplastic large cell ALK+ALK-angioimmunoblastic T cellblastic plasmacytoidchronic lymphoproliferative disorders of NK cellscutaneous CD4+ small/medium sized T cell lymphomacutaneous CD30 positive T cell lymphoproliferative disorderscutaneous gamma delta T cell lymphomaenteropathyepidermotropic CD8+ T cell lymphomahepatosplenicindolent T cell proliferationsmycosis fungoidesNK/T cell lymphoma-nasal typenodal CD8+ cytotoxic T cellnonB nonT lymphoblasticperipheral T cell lymphoma, NOSprimary effusion lymphomaSezary syndromestagingsubcutaneous panniculitis-likeT cell large granular lymphocytic leukemiaT cell lymphoblastic leukemia/lymphomaT cell prolymphocytic leukemia

Hodgkin lymphoma: general/stagingclassiclymphocyte depletedlymphocyte rich classicalmixed cellularitynodular lymphocyte predominantnodular sclerosis

Post-transplantation: generalWHO classificationplasmacytic hyperplasia/IM-like lesionspolymorphic B cell lymphoproliferative disordersmonomorphic B cell lymphoproliferative disordersothergraft versus host disease

Other: AIDS associated-generalAIDS associated-examplesEBV+ T cell lymphoproliferative disorders of childhoodprimary immune disorders related

Myeloproliferative neoplasms (MPN)

WHO 2008 – Myeloproliferative neoplasms (MPN) 

General
=================================================================

  • Chronic myelogenous leukemia
    ● Polycythemia vera
    ● Essential thrombocythemia
    ● Primary myelofibrosis
    ● Chronic neutrophilic leukemia
    ● Chronic eosinophilic leukemia, not otherwise categorized
    ● Mast cell disease
    ● MPNs, unclassifiable

WHO 2001 – Chronic myeloproliferative diseases 

Definition
=================================================================

  • Chronic myelogenous leukemia (Philadelphia chromosome, t(9;22)(q34;q11), BCR-ABL positive)
    ● Chronic neutrophilic leukemia
    ● Chronic eosinophilic leukemia (and the hypereosinophilic syndrome)
    ● Polycythemia vera
    ● Chronic idiopathic myelofibrosis (with extramedullary hematopoiesis)
    ● Essential thrombocythemia
    ● Chronic myeloproliferative disease, unclassifiable

Additional references
=================================================================

The World Health Organization (WHO) classification of the myeloid neoplasms  James W. Vardiman, Nancy Lee Harris, and Richard D. Brunning
Blood 2002; 100(7)  http://dx.doi.org/10.1182/blood-2002-04-1199

Lymphoma – Non B cell neoplasms

T/NK cell disorders/WHO classification (2008)

Principles of classification
=================================================================

  • Based on all available information (morphology, immunophenotype, genetics, clinical)
    ● No one antigenic marker is specific for any neoplasm (except ALK1)
    ● Immune profiling less helpful in subclassification of T cell lymphomas then B cell lymphomas
    ● Certain antigens commonly associated with specific disease entities but not entirely disease specific
    ● CD30: common in anaplastic large cell lymphoma but also classic Hodgkin lymphoma and other B and T cell lymphomas
    ● CD56: characteristic for nasal NK/T cell lymphoma, but also other T cell neoplasms and plasma cell disorders
    ● Variation of immunophenotype within a given disease (hepatosplenic T cell lymphoma: usually γδ but some are αβ)
    ● Recurrent genetic alterations have been identified for many B cell lymphomas but not for most T cell lymphomas
    ● No attempt to stratify lymphoid malignancies by grade
    ● Recognize the existence of grey zone lymphomas
    ● This multiparameter approach has been validated in international studies as highly reproducible

WHO 2008 classification of tumors of hematopoietic and lymphoid tissues (T/NK)
=================================================================

Precursor T-lymphoid neoplasms
● T lymphoblastic leukemia/lymphoma, 9837/3

Mature T cell and NK cell neoplasms
● T cell prolymphocytic leukemia, 9834/3
● T cell large granular lymphocytic leukemia, 9831/3
● Chronic lymphoproliferative disorder of NK cells, 9831/3
● Aggressive NK cell leukemia, 9948/3
● Systemic EBV-positive T cell lymphoproliferative disease of childhood, 9724/3
● Hydroa vacciniforme-like lymphoma, 9725/3
● Adult T cell leukemia/lymphoma, 9827/3
● Extranodal NK/T cell lymphoma, nasal type, 9719/3
● Enteropathy-associated T cell lymphoma, 9717/3
● Hepatosplenic T cell lymphoma, 9716/3
● Subcutaneous panniculitis-like T cell lymphoma, 9708/3
● Mycosis fungoides, 9700/3
● Sézary syndrome, 9701/3
● Primary cutaneous CD30-positive T cell lymphoproliferative disorders
● Lymphomatoid papulosis, 9718/1
● Primary cutaneous anaplastic large cell lymphoma, 9718/3
● Primary cutaneous gamma-delta T cell lymphoma, 9726/3
● Primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T cell lymphoma, 9709/3
● Primary cutaneous CD4-positive small/medium T cell lymphoma, 9709/3
● Peripheral T cell lymphoma, NOS, 9702/3
● Angioimmunoblastic T cell lymphoma, 9705/3
● Anaplastic large cell lymphoma, ALK-positive, 9714/3
● Anaplastic large cell lymphoma, ALK-negative, 9702/3

Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia Staging
Author: Sandy D Kotiah, MD; Chief Editor: Jules E Harris, MD
Medscape Sep 6, 2013
http://emedicine.medscape.com/article/2006578-overview

General considerations in the staging of chronic lymphocytic leukemia (CLL) and the revised Rai (United States) and Binet (Europe) staging systems for CLL are provided below.[1, 2, 3]

See Chronic Leukemias: 4 Cancers to Differentiate, a Critical Images slideshow, to help detect chronic leukemias and determine the specific type present.

General considerations

  • CLL and small lymphocytic lymphoma (SLL) are different manifestations of the same disease; SLL is diagnosed when the disease is mainly nodal, and CLL is diagnosed when the disease is seen in the blood and bone marrow
  • CLL is diagnosed by > 5000 monoclonal lymphocytes/mm3 for longer than 3mo; the bone marrow usually has more than 30% monoclonal lymphocytes and is either normocellular or hypercellular
  • Monoclonal B lymphocytosis is a precursor form of CLL that is defined by a monoclonal B cell lymphocytosis < 5000 monoclonal lymphocytes/mm3; all lymph nodes smaller than 1.5 cm; no anemia; and no thrombocytopenia

Revised Rai staging system (United States)

Low risk (formerly stage 0)[1] :

  • Lymphocytosis, lymphocytes in blood > 15000/mcL, and > 40% lymphocytes in the bone marrow

Intermediate risk (formerly stages I and II):

  • Lymphocytosis as in low risk with enlarged node(s) in any site, or splenomegaly or hepatomegaly or both

High risk (formerly stages III and IV):

  • Lymphocytosis as in low risk and intermediate risk with disease-related anemia (hemoglobin level < 11.0 g/dL or hematocrit < 33%) or platelets < 100,000/mcL

Binet staging system (Europe)

Stage A:

  • Hemoglobin ≥ 10 g/dL, platelets ≥ 100,000/mm3, and < 3 enlarged areas

Stage B:

  • Hemoglobin ≥ 10 g/dL, platelets ≥ 100,000/mm3, and ≥ 3 enlarged areas

Stage C:

  • Hemoglobin < 10 g/dL, platelets < 100,000/mm3, and any number of enlarged areas

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