Archive for the ‘Acute lymphocytic leukemia’ Category

Cancer Companion Diagnostics

Curator: Larry H. Bernstein, MD, FCAP


Companion Diagnostics for Cancer: Will NGS Play a Role?

Patricia Fitzpatrick Dimond, Ph.D.

Companion diagnostics (CDx), in vitro diagnostic devices or imaging tools that provide information essential to the safe and effective use of a corresponding therapeutic product, have become indispensable tools for oncologists.  As a result, analysts expect the global CDx market to reach $8.73 billion by 2019, up from from $3.14 billion in 2014.

Use of CDx during a clinical trial to guide therapy can improve treatment responses and patient outcomes by identifying and predicting patient subpopulations most likely to respond to a given treatment.

These tests not only indicate the presence of a molecular target, but can also reveal the off-target effects of a therapeutic, predicting toxicities and adverse effects associated with a drug.

For pharma manufacturers, using CDx during drug development improves the success rate of drugs being tested in clinical trials. In a study estimating the risk of clinical trial failure during non-small cell lung cancer drug development in the period between 1998 and 2012 investigators analyzed trial data from 676 clinical trials with 199 unique drug compounds.

The data showed that Phase III trial failure proved the biggest obstacle to drug approval, with an overall success rate of only 28%. But in biomarker-guided trials, the success rate reached 62%. The investigators concluded from their data analysis that the use of a CDx assay during Phase III drug development substantially improves a drug’s chances of clinical success.

The Regulatory Perspective

According to Patricia Keegen, M.D., supervisory medical officer in the FDA’s Division of Oncology Products II, the agency requires a companion diagnostic test if a new drug works on a specific genetic or biological target that is present in some, but not all, patients with a certain cancer or disease. The test identifies individuals who would benefit from the treatment, and may identify patients who would not benefit but could also be harmed by use of a certain drug for treatment of their disease. The agency classifies companion diagnosis as Class III devices, a class of devices requiring the most stringent approval for medical devices by the FDA, a Premarket Approval Application (PMA).

On August 6, 2014, the FDA finalized its long-awaited “Guidance for Industry and FDA Staff: In Vitro Companion Diagnostic Devices,” originally issued in July 2011. The final guidance stipulates that FDA generally will not approve any therapeutic product that requires an IVD companion diagnostic device for its safe and effective use before the IVD companion diagnostic device is approved or cleared for that indication.

Close collaboration between drug developers and diagnostics companies has been a key driver in recent simultaneous pharmaceutical-CDx FDA approvals, and partnerships between in vitro diagnostics (IVD) companies have proliferated as a result.  Major test developers include Roche Diagnostics, Abbott Laboratories, Agilent Technologies, QIAGEN), Thermo Fisher Scientific, and Myriad Genetics.

But an NGS-based test has yet to make it to market as a CDx for cancer.  All approved tests include PCR–based tests, immunohistochemistry, and in situ hybridization technology.  And despite the very recent decision by the FDA to grant marketing authorization for Illumina’s MiSeqDx instrument platform for screening and diagnosis of cystic fibrosis, “There still seems to be a number of challenges that must be overcome before we see NGS for targeted cancer drugs,” commented Jan Trøst Jørgensen, a consultant to DAKO, commenting on presentations at the European Symposium of Biopathology in June 2013.

Illumina received premarket clearance from the FDA for its MiSeqDx system, two cystic fibrosis assays, and a library prep kit that enables laboratories to develop their own diagnostic test. The designation marked the first time a next-generation sequencing system received FDA premarket clearance. The FDA reviewed the Illumina MiSeqDx instrument platform through its de novo classification process, a regulatory pathway for some novel low-to-moderate risk medical devices that are not substantially equivalent to an already legally marketed device.

Dr. Jørgensen further noted that “We are slowly moving away from the ‘one biomarker: one drug’ scenario, which has characterized the first decades of targeted cancer drug development, toward a more integrated approach with multiple biomarkers and drugs. This ‘new paradigm’ will likely pave the way for the introduction of multiplexing strategies in the clinic using gene expression arrays and next-generation sequencing.”

The future of CDxs therefore may be heading in the same direction as cancer therapy, aimed at staying ahead of the tumor drug resistance curve, and acknowledging the reality of the shifting genomic landscape of individual tumors. In some cases, NGS will be applied to diseases for which a non-sequencing CDx has already been approved.

Illumina believes that NGS presents an ideal solution to transforming the tumor profiling paradigm from a series of single gene tests to a multi-analyte approach to delivering precision oncology. Mya Thomae, Illumina’s vice president, regulatory affairs, said in a statement that Illumina has formed partnerships with several drug companies to develop a universal next-generation sequencing-based oncology test system. The collaborations with AstraZeneca, Janssen, Sanofi, and Merck-Serono, announced in 2014 and 2015 respectively, seek to  “redefine companion diagnostics for oncology  focused on developing a system for use in targeted therapy clinical trials with a goal of developing and commercializing a multigene panel for therapeutic selection.”

On January 16, 2014 Illumina and Amgen announced that they would collaborate on the development of a next-generation sequencing-based companion diagnostic for colorectal cancer antibody Vectibix (panitumumab). Illumina will develop the companion test on its MiSeqDx instrument.

In 2012, the agency approved Qiagen’s Therascreen KRAS RGQ PCR Kit to identify best responders to Erbitux (cetuximab), another antibody drug in the same class as Vectibix. The label for Vectibix, an EGFR-inhibiting monoclonal antibody, restricts the use of the drug for those metastatic colorectal cancer patients who harbor KRAS mutations or whose KRAS status is unknown.

The U.S. FDA, Illumina said, hasn’t yet approved a companion diagnostic that gauges KRAS mutation status specifically in those considering treatment with Vectibix.  Illumina plans to gain regulatory approval in the U.S. and in Europe for an NGS-based companion test that can identify patients’ RAS mutation status. Illumina and Amgen will validate the test platform and Illumina will commercialize the test.

Treatment Options

Foundation Medicine says its approach to cancer genomic characterization will help physicians reveal the alterations driving the growth of a patient’s cancer and identify targeted treatment options that may not have been otherwise considered.

FoundationOne, the first clinical product from Foundation Medicine, interrogates the entire coding sequence of 315 cancer-related genes plus select introns from 28 genes often rearranged or altered in solid tumor cancers.  Based on current scientific and clinical literature, these genes are known to be somatically altered in solid cancers.

These genes, the company says, are sequenced at great depth to identify the relevant, actionable somatic alterations, including single base pair change, insertions, deletions, copy number alterations, and selected fusions. The resultant fully informative genomic profile complements traditional cancer treatment decision tools and often expands treatment options by matching each patient with targeted therapies and clinical trials relevant to the molecular changes in their tumors.

As Foundation Medicine’ s NGS analyses are increasingly applied, recent clinical reports describe instances in which comprehensive genomic profiling with the FoundationOne NGS-based assay result in diagnostic reclassification that can lead to targeted drug therapy with a resulting dramatic clinical response. In several reported instances, NGS found, among the spectrum of aberrations that occur in tumors, changes unlikely to have been discovered by other means, and clearly outside the range of a conventional CDx that matches one drug to a specific genetic change.

TRK Fusion Cancer

In July 2015, the University of Colorado Cancer Center and Loxo Oncology published a research brief in the online edition of Cancer Discovery describing the first patient with a tropomyosin receptor kinase (TRK) fusion cancer enrolled in a LOXO-101 Phase I trial. LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors.

While the authors say TRK fusions occur rarely, they occur in a diverse spectrum of tumor histologies. The research brief described a patient with advanced soft tissue sarcoma widely metastatic to the lungs. The patient’s physician submitted a tumor specimen to Foundation Medicine for comprehensive genomic profiling with FoundationOne Heme, where her cancer was demonstrated to harbor a TRK gene fusion.

Following multiple unsuccessful courses of treatment, the patient was enrolled in the Phase I trial of LOXO-101 in March 2015. After four months of treatment, CT scans demonstrated almost complete tumor disappearance of the largest tumors.

The FDA’s Elizabeth Mansfield, Ph.D., director, personalized medicine staff, Office of In Vitro Diagnostics and Radiological Health, said in a recent article,  “FDA Perspective on Companion Diagnostics: An Evolving Paradigm” that “even as it seems that many questions about co-development have been resolved, the rapid accumulation of new knowledge about tumor biology and the rapid evolution of diagnostic technology are challenging FDA to continually redefine its thinking on companion diagnostics.” It seems almost inevitable that a consolidation of diagnostic testing should take place, to enable a single test or a few tests to garner all the necessary information for therapeutic decision making.”

Whether this means CDx testing will begin to incorporate NGS sequencing remains to be seen.


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Management of Follicular Lymphoma

Curator: Larry H. Bernstein, MD, FCAP


Targeted Approaches to the Management of Follicular Lymphoma  

By Chaitra Ujjani, MD


Despite high rates of response to initial chemoimmunotherapy, patients with follicular lymphoma experience frequent relapses, and better treatment options are needed. Several novel biologic agents have been developed based on a greater understanding of the intrinsic factors driving the development of this heterogeneous disease. Such therapies target extracellular surface proteins and intracellular signaling pathways, as well as manipulate and engage the tumor microenvironment. Many of these agents have shown great promise in early-phase studies and are the focus of ongoing clinical investigations.


Introduction As the second most common form of non-Hodgkin lymphoma (NHL), follicular lymphoma affects thousands of new patients in the United States each year. Although follicular lymphoma is considered an indolent disease, its clinical course is highly variable. Asymptomatic patients with low tumor burden can be monitored closely with the “watch and wait” strategy, given that the early intervention of chemotherapy or immunotherapy has not demonstrated a survival benefit.[1,2] The most widely accepted indications for treatment are one or more of the following criteria from the Groupe d’Etude des Lymphomes Folliculaires (GELF): a single lesion > 7 cm, three nodal sites > 3 cm, splenomegaly, effusions, threat or evidence of organ compression, or constitutional symptoms.[3] Whereas patients with limited-stage disease have several treatment options—including single-agent rituximab, radiation, and chemoimmunotherapy, those with advanced-stage disease typically receive chemoimmunotherapy.[4,5] Both the German Study Group Indolent Lymphomas (StiL) NHL-2 study and the pharmaceutical company–sponsored Bendamustine Rituximab Investigational Non-Hodgkin’s Trial (BRIGHT) have established the front-line role of combination therapy with bendamustine and rituximab in the treatment of follicular lymphoma, based on comparable efficacy and better tolerability than standard regimens such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone).[6,7] Despite high response rates with initial therapy, follicular lymphoma is characterized by frequent relapses, and patients need improved treatment options.

Oncology (Williston Park). 29(10):760-768.

Table: Targeted Therapies in Development and FDA-Approved for the Treatment of Follicular Lymphomas


Since the discovery of rituximab, there has been significant innovation in drug development, based on a greater understanding of the pathogenesis of the disease. The multistep process leading to follicular lymphoma is theorized to begin with an initial genetic insult, the hallmark t(14;18) translocation, which results in overexpression of the anti-apoptotic B-cell lymphoma protein, BCL-2.[8] This translocation is not the sole factor in malignant transformation, as it is a naturally occurring anomaly, often identified in healthy individuals. Furthermore, preclinical studies have indicated a positive correlation between increasing numbers of genetic alterations and the progression from follicular lymphoma in situ to grade 3A follicular lymphoma.[9] The B-cell receptor is a critical cellular factor in the development of the disease. Its active tonic signaling leads to recruitment of the spleen tyrosine kinase (SYK) and activation of multiple downstream pathways, including phosphatidylinositol 3-kinase (PI3K), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein kinase (MAPK). Activation of these pathways ultimately results in the maturation, proliferation, and survival of malignant lymphocytes.[10] Other key components include immune cells such as T cells, dendritic cells, and reticular cells, which infiltrate among the centrocytes.[11] In addition to stimulating the B-cell receptor, these immune cells induce exhaustion of cytotoxic T cells and allow for T-cell evasion via disruption of synapses and secretion of interleukin-12.[12] Expression of the inhibitory receptor programmed cell death 1 (PD-1) is another contributing factor, as its presence is believed to affect the ability of T cells to mount appropriate antitumor responses.[13] Several novel therapies have been developed to target these various aspects of the disease in the hope of identifying more effective, yet tolerable, treatment options for patients with follicular lymphoma. (See the Table for a summary of therapies approved and in development for treatment of follicular lymphoma.)


Since the approval of rituximab, there has been significant investigation into the development of a superior anti-CD20 monoclonal antibody. Second- and third-generation versions vary in their structures and mechanisms of action. Two anti-CD20 antibodies, ofatumumab and obinutuzumab, have been approved by the US Food and Drug Administration (FDA) for indications in chronic lymphocytic leukemia (CLL). Ofatumumab, a type I human monoclonal antibody, was initially approved for patients whose disease is refractory to fludarabine and alemtuzumab.[14] Obinutuzumab was approved in combination with chlorambucil for patients with preexisting comorbidities that preclude conventional chemoimmunotherapy.[15] Although designed to induce stronger complement-dependent cytotoxicity than rituximab, ofatumumab demonstrated minimal activity in rituximab-refractory follicular lymphoma (overall response rate [ORR], 10% to 13%; median progression-free survival [PFS], 5.8 months).[16] In phase II trials of ofatumumab in combination with chemotherapy, including bendamustine and CHOP, results appeared comparable to those achieved with rituximab-based regimens, although no direct comparisons have been made.[17,18]

Obinutuzumab, a type II glycoengineered humanized antibody, is further in development for use in NHL. This agent, which works primarily by triggering antibody-dependent cellular cytotoxicity (ADCC) and apoptosis, has demonstrated superiority to rituximab in preclinical studies, including those employing whole blood B cell–depletion assays, human lymphoma xenograft mice models, and nonhuman primates.[19] When compared with rituximab in patients with indolent B-cell NHL, obinutuzumab produced a higher ORR by independent radiology review (45% vs 27%; P = .01); however, complete response (CR) and PFS rates were similar.[20] Like ofatumumab, treatment with obinutuzumab is associated with a higher rate of infusion-related reactions than rituximab (grade 1–4, 72% vs 49%; and grade 3/4, 11% vs 5%, respectively). In contrast to ofatumumab, it has efficacy in rituximab-refractory indolent NHL, producing an ORR of 50% and median PFS of 12 months among 10 patients.[21] The phase III GADOLIN study evaluated obinutuzumab in combination with bendamustine followed by maintenance obinutuzumab in the same disease setting (N = 413).[22] While the response rates by independent review were similar to those observed in the comparative arm of patients randomized to single-agent bendamustine (bendamustine-obinutuzumab ORR, 69% [CR, 11%] vs bendamustine alone ORR, 63% [CR, 12%]), the median PFS was significantly higher with the combination (not reached vs 14.9 months; P = .00011). Treatment with the combination of bendamustine and obinutuzumab was associated with a similar incidence of grade ≥ 3 adverse events compared with bendamustine monotherapy (68% vs 62%), which included neutropenia (33% vs 26.3%) and infusion-related reactions (9% vs 3.5%). While there was no difference in overall survival (OS) noted, the study did demonstrate the clinical benefit of obinutuzumab in rituximab-refractory disease. The role of the drug in this setting is becoming less clear, as more patients now receive bendamustine-rituximab as front-line therapy. In the phase Ib GAUDI study, bendamustine-obinutuzumab and obinutuzumab-CHOP produced similar response rates in patients with previously untreated follicular lymphoma, with ORRs of 93% (CR, 39%) and 95% (CR, 35%), respectively.[23] The incidences of grade 3/4 neutropenia and infection were similar to historical data on rituximab chemotherapy. These data prompted the front-line phase III GALLIUM study of chemotherapy (CHOP, CVP [cyclophosphamide, vincristine, and prednisone], or bendamustine) with obinutuzumab or rituximab followed by maintenance obinutuzumab or rituximab in advanced-stage indolent B-cell NHL ( identifier: NCT01332968). Newer monoclonal antibodies directed against CD20, such as ublituximab, and rituximab biosimilars are also in development.

Monoclonal antibodies to alternative targets

Monoclonal antibodies directed against other B-cell antigens have also been developed. Galiximab, a chimeric human-macaque anti-CD80 antibody, and epratuzumab, a humanized anti-CD22 antibody, were two of the first antibodies directed against these targets to be explored in follicular lymphoma. Both antibodies have shown activity as single agents and in combination with rituximab in follicular lymphoma. However, neither is being studied further due to the availability of newer, more promising therapies.[24-28] MEDI-551, an afucosylated humanized anti-CD19 antibody, induces cell death via ADCC and cytotoxic T-cell response. The lack of a fucose moiety on the Fc portion of the antibody is believed to enhance the activity of ADCC. Phase I studies of MEDI-551 in heavily pretreated follicular lymphoma have reported ORRs ranging from 31% to 82%.[29,30] The median PFS from the earlier study was nearly 10 months. MEDI-551 is currently being evaluated in aggressive lymphomas in combination with rituximab and salvage chemoimmunotherapy ( identifiers: NCT00983619 and NCT01453205). Also targeting CD19 is MOR208, a humanized monoclonal antibody that has been engineered to have a higher affinity to FcγRIIIa and FcγRIIa, resulting in stronger ADCC. In a phase II study of patients with relapsed and refractory B-cell NHL who had received a median of two prior therapies, the ORR was 26% among those with follicular lymphoma (n = 31).[31] The median duration of response (DOR) was 2.6 months; however, the longest DOR was 15.4 months. Upcoming trials with MOR208 include combination studies with lenalidomide in diffuse large B-cell lymphoma (DLBCL) and CLL ( identifiers: NCT02399085 and NCT02005289).


One of the first attempts to improve upon the efficacy of the naked monoclonal antibody was radioimmunotherapy, which produced ORRs of 65% to 74% in patients with relapsed and refractory indolent B-cell lymphomas.[32,33] 90Y-ibritumomab tiuxetan, the first radioimmunotherapy to receive FDA approval, was approved in February 2002 for relapsed or refractory low-grade, follicular, or transformed B-cell NHL. In 2009, it was granted expanded approval as consolidation therapy in previously untreated follicular lymphoma patients with a partial or complete response to first-line chemotherapy. 131I-tositumomab was approved in June 2003 (along with tositumomab) for CD20-positive follicular NHL, with and without transformation, in relapsed rituximab-refractory patients with relapse following chemotherapy. The use of 131I-tositumomab and 90Y-ibritumomab tiuxetan has declined significantly over the past several years; the manufacture and sale of 131I-tositumomab (marketed in the United States and Canada as Bexxar) was stopped in February 2014. Radioimmunotherapy can be difficult; there are strict hematologic criteria (< 25% lymphomatous marrow involvement, platelet count > 100 × 109, leukocyte count > 1.5 × 109), and its administration requires a certified nuclear medicine physician. In addition, the patient must not have had prior radiation to > 25% of the bone marrow nor undergone stem cell transplantation.[34]

Antibody-drug conjugates (ADCs)

Recent efforts in augmenting antibody-based therapy include the use of ADCs. Once bound to its target antigen, the ADC is engulfed via endocytosis, trafficked to the lysosome for degradation, and ultimately released, whereupon it causes damage to tubulin and DNA. The calicheamicin-bound anti-CD22, inotuzumab ozogamicin, was one of the first to be studied in patients with follicular lymphoma who were refractory to CD20-targeted therapy, yielding an ORR of 66%.[35] The ORR increased to 87% when inotuzumab ozogamicin was combined with rituximab in patients with relapsed and refractory follicular lymphoma, prompting a trial in which it was compared with combination treatment with rituximab plus chemotherapy.[36] The trial was closed early due to poor accrual. Inotuzumab ozogamicin is currently being studied in combination with the mammalian target of rapamycin (mTOR) inhibitor temsirolimus in relapsed and refractory CD22-expressing NHL ( identifier: NCT01535989). Pinatuzumab vedotin and polatuzumab vedotin, which target CD22 and CD79b, respectively, are ADCs linked to the anti-tubulin molecule monomethyl auristatin E. While both agents have demonstrated activity in indolent NHL (with reported ORRs of 50% and 47%, respectively), polatuzumab vedotin is being taken further in development.[37,38] When polatuzumab vedotin was administered at a higher dose (2.4 mg/kg) with rituximab in patients with relapsed and refractory follicular lymphoma (n = 25), the ORR was 76% (CR, 44%) and median PFS was 15 months.[39] The cohort of 20 patients treated at the lower rituximab dose (1.8 mg/kg) had a similar response rate (ORR, 70%; CR, 40%), and median PFS and DOR were not reached. Peripheral neuropathy, a common toxicity with ADCs, occurred less frequently with the lower dose of polatuzumab vedotin and was ameliorated in some patients by dose delay and reduction. Ongoing studies with polatuzumab vedotin include phase I/II combinations with bendamustine-rituximab or obinutuzumab-bendamustine in relapsed and refractory follicular lymphoma ( identifier: NCT02257567) and R-CHOP in B-cell NHL patients who have received less than one prior therapy ( identifier: NCT01992653). Coltuximab ravtansine (formerly SAR3419) is an anti-CD19 ADC that has also been associated with neurologic complications, primarily dose-limiting ocular toxicity.[40] IMGN529, which targets the overexpressed CD37 protein, is another B-cell–directed ADC in development.[41]

Despite high rates of response to initial chemoimmunotherapy, patients with follicular lymphoma experience frequent relapses, and better treatment options are needed. Several novel biologic agents have been developed based on a greater understanding of the intrinsic factors driving the development of this heterogeneous disease. Such therapies target extracellular surface proteins and intracellular signaling pathways, as well as manipulate and engage the tumor microenvironment. Many of these agents have shown great promise in early-phase studies and are the focus of ongoing clinical investigations.

Small-Molecule Inhibitors – PI3K inhibitors

In contrast to the various antibody-based therapies under investigation for treatment of follicular lymphoma, several small molecules have been designed to inhibit key intracellular pathways of the malignant B cell. The majority of these agents are directed against kinases downstream of the B-cell receptor, and many have been combined with bendamustine-rituximab, given this combination’s efficacy and tolerability. Idelalisib, a potent PI3K-δ inhibitor, was the first PI3K inhibitor to be approved by the FDA for follicular lymphoma. It received an indication for patients who have received at least two prior systemic therapies, based on results of a phase II study in rituximab-refractory indolent NHL[42] As reported at the 2015 American Society of Clinical Oncology Annual Meeting, of the 72 patients in the study who had follicular lymphoma, 54% were considered high-risk by the Follicular Lymphoma International Prognostic Index.[43] The patients had received a median of four prior therapies, and 86% had disease that was refractory to the last regimen they received. The ORR was 56% and the median PFS was 11 months. The median PFS for the 10 patients who achieved a CR was 27 months. Notable grade 3/4 adverse events included neutropenia (27% of all patients), diarrhea/colitis (16%), elevations in hepatic transaminases (13%), and pneumonia (7%).
Idelalisib was subsequently administered with rituximab, bendamustine, and bendamustine-rituximab in a phase I study of patients with relapsed (n = 79) and refractory (n = 59) indolent NHL, the majority of whom had follicular lymphoma.[44] The ORRs were similar between the arms (75% to 88%); however, treatment with bendamustine-rituximab-idelalisib was associated with the highest CR (43%) and longest median PFS (37.1 months). A phase III trial of bendamustine-rituximab with or without idelalisib in relapsed and refractory indolent B-cell NHL is ongoing ( identifier: NCT01732926). In phase I investigations, combined treatment with idelalisib and lenalidomide plus the second-generation SYK inhibitor entospletinib has demonstrated considerable toxicity, including hepatic transaminitis, sepsis, and refractory pneumonitis.[45,46] Second-generation PI3K inhibitors, including duvelisib (IPI-145), TGR-1202, and INCB040093, are in development. Duvelisib, a dual inhibitor of the delta and gamma isoforms of PI3K, has demonstrated an ORR of 69% (CR, 38%) in a heavily pretreated follicular lymphoma cohort (n = 13).[47] Based on these encouraging data, duvelisib is being administered with rituximab or obinutuzumab in patients with previously untreated follicular lymphoma ( identifier: NCT02391545) and with bendamustine and/or rituximab in those with relapsed B-cell malignancies ( identifier: NCT01871675).

Bruton tyrosine kinase (BTK) inhibitors

Ibrutinib, a selective and irreversible inhibitor of BTK, may also have some impact on the tumor microenvironment via cytokine and chemokine inhibition.[48] Approved in CLL, mantle cell lymphoma, and Waldenström macroglobulinemia, it has demonstrated activity in a number of B-cell malignancies.[49-53] In a phase II study of relapsed and refractory follicular lymphoma, ibrutinib yielded an ORR of 30% (with one CR) and a median PFS of 9.9 months. The 40 enrolled patients had received a median of three prior therapies, and 36% were considered refractory to treatment. Common adverse events included mild diarrhea, rash, and fatigue; rare events included atrial fibrillation and bleeding.[54] Like idelalisib, ibrutinib has been combined with bendamustine-rituximab in the treatment of B-cell NHL.[55] This triplet produced an ORR of 90% (CR, 50%) in a cohort of 10 patients with previously treated follicular lymphoma. At the time these results were reported, the median PFS had not been reached. Notable grade 3/4 adverse events included neutropenia (33%), rash (25%), and thrombocytopenia (19%). Results from SELENE, a randomized phase III trial of ibrutinib with bendamustine-rituximab or R-CHOP in previously treated follicular lymphoma and marginal zone lymphoma, will provide more insight into the role of ibrutinib in the management of indolent NHL ( identifier: NCT01974440). Phase I trials of combinations with targeted agents include the Alliance for Clinical Trials in Oncology study of rituximab, lenalidomide, and ibrutinib in previously untreated follicular lymphoma[56] and the pharmaceutical-sponsored trial of combination therapy with ublituximab, TGR-1202, and ibrutinib in relapsed and refractory B-cell malignancies.[57] Second-generation BTK inhibitors, including ACP-196 and ONO-4059, are also in development.

B-cell lymphoma–2 (BCL-2) inhibitors

The chromosomal translocation t(14;18) allows for dysregulation of the BCL-2 oncogene and overexpression of the anti-apoptotic BCL-2 family of proteins, contributing to development of follicular lymphoma. Venetoclax, formerly known as ABT-199, is a second-generation selective BCL-2 inhibitor in the early stages of clinical investigation. When this agent was administered at a dose greater than 600 mg daily to six patients with relapsed and refractory follicular lymphoma, three patients achieved a response.[58] Common toxicities reported included mild nausea (34%) and diarrhea (25%), and grade 3/4 myelosuppression occurred in less than 15% of patients. Two patients (one with DLBCL and one with mantle cell lymphoma) in the entire NHL cohort (of 44 patients then enrolled in the study) developed laboratory evidence of grade 3 tumor lysis syndrome. When venetoclax was combined with bendamustine-rituximab in 21 patients with relapsed and refractory follicular lymphoma, the ORR was 71% (CR, 29%).[59] While a maximum tolerated dose was not reached, dose-limiting toxicities included thrombocytopenia, neutropenia, and Stevens-Johnson syndrome. Venetoclax is being evaluated in relapsed and refractory follicular lymphoma, in a three-arm phase II study of bendamustine-rituximab vs rituximab-venetoclax vs bendamustine-rituximab-venetoclax ( identifier: NCT02187861). It will be studied with ibrutinib in a phase I/II trial of relapsed follicular and marginal zone lymphoma (Ujjani C, principal investigator). Small-molecule inhibitors aimed at less well known targets are also under investigation, including selinexor (a selective inhibitor of nuclear export), MK-2206 (an AKT inhibitor), alisertib (an Aurora-A kinase inhibitor), and cerdulatinib (a dual SYK/Janus tyrosine kinase [JAK] inhibitor).


Loretta J. Nastoupil, MD
Department of Lymphoma/Myeloma, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center Houston, Texas

What Are the Challenges of Treating Follicular Lymphoma?

Follicular lymphoma, the most common indolent lymphoma, is characterized by high rates of initial response to chemoimmunotherapy, but it is not curable with standard therapy. The clinical course of the disease is highly variable and somewhat unpredictable. As a result, the optimal management of follicular lymphoma, including the most effective sequencing of therapy, is undefined. Identifying the subsets of patients at risk for early failure and those with indolent disease that remains quiescent would assist clinicians in tailoring therapy for individual patients. Given the heterogeneity of treatment options and possible clinical outcomes, improvement in risk stratification and personalization in follicular lymphoma is needed, particularly given the expanding treatment options outlined by Dr. Ujjani.

What Can We Expect in the Future?

Historically, prognostication for patients with follicular lymphoma has relied primarily on clinical characteristics. The Follicular Lymphoma International Prognostic Index (FLIPI) can distinguish patients with low or intermediate risk from those at high risk, but it is not routinely used to guide risk-adapted therapy. More recently, the development of m7-FLIPI, a multivariable risk model incorporating the mutation status of seven genes with established clinical relevance in follicular lymphoma, improved the ability to predict early treatment failure in patients receiving front-line chemoimmunotherapy. Identifying the follicular lymphoma patients at highest risk for early treatment failure with standard therapy allows for their prioritization to a clinical trial assessing some of the novel therapies outlined by Dr. Ujjani.

Given the number of therapeutic agents under investigation in follicular lymphoma, and the vast combinatorial possibilities, consideration of toxicity is as imperative as the need to conduct correlational studies to unravel the complexity of this disease.

Tumor Microenvironment
Immunomodulatory agents

As stated previously, the tumor microenvironment plays a critical role in the pathogenesis of follicular lymphoma. Approaches to promoting a functional immune system have allowed for effective treatment of the disease. The second-generation immunomodulatory agent lenalidomide has been the most extensively studied of these therapies. Its mechanisms of action include activation of natural killer cells and T cells, stimulation of apoptosis, and inhibition of tumor necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF).[60] In follicular lymphoma cell lines, lenalidomide has been shown to restore immunologic synapses between malignant lymphocytes and T cells and augment rituximab-mediated ADCC.[61] Lenalidomide has demonstrated modest activity as a single agent in relapsed or refractory follicular lymphoma (with ORRs ranging from 27% to 49%); however, when lenalidomide was added to rituximab the ORR improved to 76% and the median event-free survival time was 2 years.[62,63] The doublet (dubbed “R2,” for Revlimid [lenalidomide] plus rituximab) was evaluated by the Alliance study as a front-line regimen, producing an ORR of 93% (CR, 72%) and 2-year PFS of 89% (n = 65).[64] Minimal toxicity was noted with the regimen; common grade 3/4 adverse events included neutropenia (in 19% of patients), rash (8%), and infection (8%). In a similar study from the University of Texas MD Anderson Cancer Center, 35 of the 50 patients with follicular lymphoma achieved a CR (70%) and 5 had an unconfirmed CR.[65] The Swiss Group for Clinical Cancer Research and the Nordic Lymphoma Group reported an ORR of 78% (CR, 61%) with the R2 combination (n = 77).[66] The impressive activity noted in the Alliance and MD Anderson studies prompted the pharmaceutical-sponsored phase III RELEVANCE trial of R2 vs rituximab with chemotherapy (CVP, CHOP, or bendamustine) in previously untreated advanced-stage follicular lymphoma ( identifier: NCT01650701). R2 has been studied in combination with other regimens such as CHOP, producing an ORR of 94% (CR/unconfirmed CR, 74%) in patients with previously untreated follicular lymphoma.[67] These data are relatively comparable to previously reported results with R2 and call into question the need for CHOP. The Alliance has conducted subsequent studies of R2 with targeted agents including ibrutinib and idelalisib. Results with ibrutinib are pending; however, the trial of idelalisib was closed owing to considerable toxicity.[45,56] Lenalidomide has also been combined with obinutuzumab in the treatment of relapsed and refractory disease, yielding an ORR of 68% (CR, 35%) among the 20 patients enrolled in the phase I portion of a phase I/II study.[68]

Immune checkpoint modulators

In patients with follicular lymphoma, the overexpression of PD-1 in the intratumoral T cells results in an impairment in antitumor immune surveillance. Inhibition of PD-1 or its ligands, PD-L1 and PD-L2, has shown promise in the treatment of follicular lymphoma. Pidilizumab, a humanized PD-1 monoclonal antibody, was the first PD-1 inhibitor to be explored. Although minimally active as a single agent, when pidilizumab was administered in conjunction with rituximab in the setting of relapsed follicular lymphoma, the ORR was 66% and median PFS was 18.8 months (n = 29).[69,70] The majority of the responses were complete (52%), and the median PFS had not been reached for those who achieved a response. Nivolumab, a fully human monoclonal antibody approved for the treatment of melanoma and squamous non–small-cell lung cancer, has also demonstrated activity in relapsed and refractory follicular lymphoma. A phase I evaluation has reported an ORR of 40% (CR, 10%) in 10 patients.[71] Nivolumab is currently being studied in combination with ibrutinib in patients with relapsed B-cell malignancies ( identifier: NCT02329847). Pembrolizumab and MEDI-0680 are humanized PD-1 antibodies also under clinical investigation in CLL and other low-grade B-cell NHLs, as well as in relapsed and refractory aggressive B-cell lymphomas ( identifiers: NCT02332980 and NCT02271945, respectively). MEDI4736, a human anti–PD-L1 antibody, is also being studied with ibrutinib in patients with relapsed lymphoma ( identifier: NCT02401048). Similar to PD-1, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is a negative regulator of T-cell function. Inhibition of CTLA-4 with ipilimumab, also approved in melanoma, has demonstrated some activity in lymphoid malignancies, including in one patient with follicular lymphoma.[72]

Bispecific T-cell engager (BiTE)

The BiTE is a unique form of immunotherapy that stimulates T-cell function via binding simultaneously to CD3 on the surface of the T cell and a specific marker on the malignant B cell, resulting in caspase-mediated apoptosis. Blinatumomab, a CD19-specific BiTE approved for treatment of relapsed and refractory B-cell acute lymphoblastic leukemia (ALL), has demonstrated activity in other CD19-positive lymphoid diseases.[73] It produced an ORR of 100% in a phase I study of 13 patients with relapsed indolent NHL, the majority of whom had a follicular or mantle cell histology.[74] Four patients achieved a CR, and eight remained in remission at 13 months. A single cycle of blinatumomab requires a 4-week continuous IV infusion and is associated with significant, yet reversible, neurologic toxicity. The current focus of clinical investigations with this agent is ALL, and its role in follicular lymphoma is unclear.

Chimeric antigen receptor (CAR)-modified T cells

CAR-modified T cells are one of the newest, most intriguing, forms of immunotherapy. These autologous T cells have been genetically transduced using lentiviral vectors to express tumor cell–specific antigen receptors. Having demonstrated activity in CLL and ALL, CAR-modified T cells are now being explored in CD19-positive NHL. A phase II study at the University of Pennsylvania demonstrated an ORR of 100% among seven patients with relapsed and refractory follicular lymphoma who lacked curative treatment options.[75] Six patients achieved a CR by 6 months, and responses appeared to be durable. In all seven patients there was evidence of severe cytokine release syndrome (cytokine storm); in two of the patients this was a grade 3/4 toxicity. One patient developed grade 5 encephalopathy 2 months after completing therapy. Although quite promising, further investigation is necessary to fully understand this new method.

The treatment of follicular lymphoma has changed dramatically over the past several years. The availability of newer, novel forms of therapy has enabled the field to continue to evolve. In addition to having tumor-specific activity, these newer agents provide the possibility of a more favorable toxicity profile than conventional chemotherapy. Although chemoimmunotherapy has been the traditional front-line induction for patients with advanced-stage disease, this concept is being challenged by the remarkable efficacy of the R2 regimen (with ORR > 90%; CR, 61% to 72%).[64,66] If the phase III RELEVANCE trial demonstrates results with R2 that are even equivalent to those achieved with standard regimens such as R-CHOP or bendamustine-rituximab, a major paradigm shift will occur; R2 would then be the first chemotherapy-free option for the initial treatment of follicular lymphoma. Given that the attainment of a CR has been associated with a survival benefit in this setting, there is still room for improvement.[76] Although approved as a single agent, idelalisib is being studied in combination with rituximab in previously untreated and relapsed patients ( identifiers: NCT02258529 and NCT01732913). Ongoing clinical investigations, such as the Alliance phase I trial of R2 plus ibrutinib in previously untreated patients, are exploring the benefit of multitargeted agents in this population. Studies such as the phase II trial of bendamustine-rituximab vs rituximab-venetoclax vs bendamustine-rituximab-venetoclax are exploring the utility of other targeted agents in comparison to standard chemoimmunotherapy.

While the concept of multitargeted therapy is quite appealing, these regimens must be explored with caution. Early-phase investigations of idelalisib with R2 and entospletinib produced significant adverse events, requiring study closures.[45,46] In addition to understanding how to combine treatment with these agents safely and efficaciously, research efforts must incorporate sound correlative science. Through whole-exome sequencing, Woyach et al have already discovered mutations associated with resistance to ibrutinib in CLL.[77] The identification of other predictive biomarkers is imperative to tailor therapy effectively and to develop superior regimens for individual patients. Furthermore, this information may enable us to provide appropriate treatment options that are also financially prudent. Given the lengthy follow-up period required to achieve the traditional objectives of clinical trials, it is important to explore earlier, yet meaningful, surrogate endpoints. Residual positron emission tomography activity on post-induction imaging, the presence of minimal residual disease, and relapse within 2 years of chemoimmunotherapy have been associated with an inferior PFS and OS outcome; in contrast, the presence of a CR at 30 months has been correlated with a significantly reduced risk of progression in patients with follicular lymphoma.[78-81] By incorporating novel therapies into innovative clinical investigations, we may achieve significantly better outcomes and improve the outlook for patients with this incurable disease.

Financial Disclosure: Dr. Ujjani has served on advisory boards for Genentech, Inc., and Pharmacyclics, Inc.



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Human Genetics and Childhood Diseases

Larry H. Bernstein, Md, FCAP, Curator




Publication Roundup: HGMD

HGMD®, the Human Gene Mutation Database is used by scientists around the world to find information on reported genetic mutations. The papers below use the database to advance our understanding of disease, DNA dynamics, and more.

Local DNA dynamics shape mutational patterns of mononucleotide repeats in human genomes
First author: Albino Bacolla

Scientists in the US and UK published results in Nucleic Acids Research of a detailed analysis of single-base substitutions and indels in the human genome. Their findings show that certain base positions are more susceptible to mutagenesis than others. They used HGMD Professional to find mutations in specific genomic regions for analysis; the paper includes charts showing mutation patterns, germline SNPs, and more from HGMD data.

High prevalence of CDH23 mutations in patients with congenital high-frequency sporadic or recessively inherited hearing loss
First author: Kunio Mizutari

This Orphanet Journal of Rare Diseases paper from scientists in Japan sequenced 72 patients with unexplained hearing loss, finding several CDH23 mutations, some of which were novel. Mutations in the gene have been linked to Usher syndrome and other forms of hereditary hearing loss. The scientists used HGMD to find all known CDH23 mutations within nearly 70 coding regions.

Mutation analyses and prenatal diagnosis in families of X-linked severe combined immunodeficiency caused by IL2Rγ gene novel mutation
First author: Q.L. Bai

In Genetics and Molecular Research, scientists report the utility of mutation analysis of the interleukin-2 receptor gamma gene to assess carrier status and perform prenatal diagnosis for X-linked severe combined immunodeficiency. They studied two high-risk families, along with 100 controls, to evaluate the approach. Sequence variation was determined using HGMD Professional and an X-SCID database, and a new mutation was discovered in the project.

Impact of glucocerebrosidase mutations on motor and nonmotor complications in Parkinson’s disease
First author: Tomoko Oeda

Researchers from three hospitals in Japan published this Neurobiology of Aging report that may help stratify Parkinson’s disease patients by prognosis. They sequenced mutations in the GBA gene in 215 patients, finding that those who had mutations associated with Gaucher disease suffered dementia and psychosis much earlier than those who didn’t. The team found previously reported GBA mutations using HGMD Professional.

Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort
First author: Elisabet Selga

In this PLoS One publication, scientists from a number of institutions in Spain examined genetic variation among patients with Brugada syndrome, a rare genetic cardiac arrhythmia. They sequenced 14 genes in 55 patients, identifying 61 variants and finding the subset that appear pathogenic. Variants were filtered against a number of databases, including HGMD.



Local DNA dynamics shape mutational patterns of mononucleotide repeats in human genomes

Albino Bacolla1Xiao Zhu2Hanning Chen3Katy Howells4David N. Cooper4 and Karen M. Vasquez1

Nucl. Acids Res. (26 May 2015) 43(10): 5065-5080.

Single base substitutions (SBSs) and insertions/deletions are critical for generating population diversity and can lead both to inherited disease and cancer. Whereas on a genome-wide scale SBSs are influenced by cellular factors, on a fine scale SBSs are influenced by the local DNA sequence-context, although the role of flanking sequence is often unclear. Herein, we used bioinformatics, molecular dynamics and hybrid quantum mechanics/molecular mechanics to analyze sequence context-dependent mutagenesis at mononucleotide repeats (A-tracts and G-tracts) in human population variation and in cancer genomes. SBSs and insertions/deletions occur predominantly at the first and last base-pairs of A-tracts, whereas they are concentrated at the second and third base-pairs in G-tracts. These positions correspond to the most flexible sites along A-tracts, and to sites where a ‘hole’, generated by the loss of an electron through oxidation, is most likely to be localized in G-tracts. For A-tracts, most SBSs occur in the direction of the base-pair flanking the tracts. We conclude that intrinsic features of local DNA structure, i.e. base-pair flexibility and charge transfer, render specific nucleotides along mononucleotide runs susceptible to base modification, which then yields mutations. Thus, local DNA dynamics contributes to phenotypic variation and disease in the human population.


Changes in human genomic DNA in the form of base substitutions and insertions/deletions (indels) are essential to ensure population diversity, adaptation to the environment, defense from pathogens and self-recognition; they are also a critical source of human inherited disease and cancer. On a genome-wide scale, base substitutions result from the combined action of several factors, including replication fidelity, lagging versus leading strand DNA synthesis, repair, recombination, replication timing, transcription, nucleosome occupancy, etc., both in the germline and in cancer (14). On a much finer scale [(over a few base pairs (bp)], rates of base substitutions may be strongly influenced by interrelationships between base–protein and base–base interactions. For example, the mutator role of activation-induced deaminase (AID) in B-cells during class-switch recombination and somatic hypermutation (5) targets preferentially cytosines within WRC (W: A|T; R: A|G) sequences (6), whereas apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) overexpression displays a preference for base substitutions at cytosines in TCW contexts (7). Other examples, such as the induction of C→T transitions at CG:CG dinucleotides by cytosine-5-methylation and the role of UV light in promoting base substitutions at pyrimidine dimers have been well documented (reviewed in (4,8)). More recently, complex patterns of base substitution at guanosines in cancer genomes have been found to correlate with changes in guanosine ionization potentials as a result of electronic interactions with flanking bases (9), suggesting a role for electron transfer and oxidation reactions in sequence-dependent mutagenesis. However, despite these advances, the increasing number of sequence-dependent patterns of mutation noted in genome-wide sequencing studies has met with a lack of understanding of most of the underlying mechanisms (10). Thus, a picture is emerging in which mutations are often heavily dependent on sequence-context, but for which our comprehension is limited.

Mononucleotide repeats comprise blocks of identical base pairs (A|T or C|G; hereafter referred to as A-tracts and G-tracts) and display distinct features: they are abundant in vertebrate genomes; mutations within the tracts occur more frequently than the genome-wide average; mutations generally increase with increasing tract length; length instability is a hallmark of mismatch repair-deficiency in cancers; and sequence polymorphism within the general population has been linked to phenotypic diversity (1115). Thus, mononucleotide repeats appear ideal for addressing the question of sequence-dependent mutagenesis since base pairs within the tracts are flanked by identical neighbors. Both historic and recent investigations concur with the conclusion that a major source of mononucleotide repeat polymorphism is the occurrence of slippage (i.e. repeat misalignment) during semiconservative DNA replication, which gives rise to the addition or deletion of repeat units (11,12). An additional and equally important source of mutation has recently been suggested to arise from errors in DNA replication by translesion synthesis DNA polymerases, such as pol η and pol κ (13), also on slipped intermediates, leading to single base substitutions.

A key question that remains unanswered in these studies and which is relevant to the issue of sequence context-dependent mutagenesis is whether all base pairs within mononucleotide repeats display identical susceptibility to single base changes and whether indels (which are consequent to DNA breakage) occur randomly within the tracts.

Herein, we combine bioinformatics analyses on mononucleotide repeat variants from the 1000 Genomes Project and cancer genomes with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations to address the question of sequence-dependent mutagenesis within these tracts. We show that mutations along both A-tracts and G-tracts are highly non-uniform. Specifically, both base substitutions and indels occur preferentially at the first and last bp of A-tracts, whereas they are concentrated between the second and third G:C base pairs in G-tracts. These positions coincide with the most flexible base pairs for A-tracts and with the preferential localization of a ‘hole’ that results when one electron is lost due to an oxidation reaction anywhere along G-tracts. Thus, despite the uniformity of sequence composition, mutations occur in a sequence-dependent context at homopolymeric runs according to a hierarchy that is imposed by both local DNA structural features and long-range base–base interactions. We also show that the repair processes leading to base substitution must differ between A- and G-tracts, since in the former, but not in the latter, base substitutions occur predominantly in the direction of the base immediately flanking the tracts. Additional sequence-dependent patterns of mutation are likely to arise from studies of more heterogeneous sequence combinations, possibly involving other aspects intrinsic to the structure of DNA.



Mononucleotide repeat variation is defined by tract length and flanking base composition

We define mononucleotide repeats in the GRCh37/hg19 (hg19) human genome assembly as uninterrupted runs of A:T and G:C base pairs (hereafter referred to as A-tracts and G-tracts, respectively) from 4 to 13 base pairs in length (Figure 1A). We retrieved a total of 48,767,945 A-tracts and 13,633,781 G-tracts, both of which displayed a biphasic distribution with an inflection point between tract lengths of 8 and 9 (bp) and with the number of runs declining with length more dramatically for G-tracts than for A-tracts (Figure 1B), as noted previously (29). Both the number of short tracts and the extent of decline varied with flanking base composition, TA[n]T runs being two- to three-fold more abundant than CA[n]Cs (Supplementary Figure S1A) and AG[n]As declining the most rapidly (Supplementary Figure S1B). Thus, mononucleotide runs exist as a collection of separate pools of sequences in extant human genomes, each maintained at distinctive rates of sequence stability, as determined by factors such as bp composition (A:T versus G:C), tract length and flanking sequence composition.

Figure 1.

Mononucleotide repeat variation, evolutionary conservation and association with transcription. (A) The search algorithm was designed to retrieve runs of As or Ts (A-tracts) and Gs or Cs (G-tracts) length n (n = 4 to 13), along with their 5′ (n = 0) and 3′ (n = n + 1) nearest neighbors from hg19. Tract bases were numbered 5′ to 3′ with respect to the purine-rich sequence. The panel exemplifies the nomenclature for A- and G-tracts of length 4. (B) Logarithmic plot of the number of A-tracts (closed circles) and G-tracts (open circles) in hg19 as a function of length. (C) Normalized fractions of polymorphic tracts (F SNV) (number of SNVs divided by both hg19 number of tracts and n) from the 1KGP for A-tracts (closed circles) and G-tracts (open circles). (D) Radial plot of SNVs in the 1KGP at the 5′ and 3′ nearest neighbors of A-tracts. Periphery, tract length; horizontal axis, scale for the fraction of SNVs (F SNV). (E) Radial plot of SNVs in the 1KGP at the 5′ and 3′ nearest neighbors of G-tracts. (F) Percent difference in the numbers of A-tracts (closed circles) and G-tracts (open circles) between syntenic regions of hg19 and HN genomes. (G) The exponents of Benjamini-corrected P-values for A-tract-containing genes enriched in transcription-factor binding sites plotted as a function of A-tract length (triangles); each value represents the median of the top 11 USCS_TFBS terms. The percent A-tracts (closed circles) and G-tracts (open circles) intersecting genomic regions pulled-down by chromatin immunoprecipitation using antibodies against transcription factors are plotted as a function of tract length. (H) List of gene enrichment terms with a Benjamini-corrected P-value of <0.05 in common between genes containing A- and G-tracts of lengths 4–13, excluding the UCSC_TFBS terms.

 We examined the extent of sequence variation in the human population by mapping 38,878,546 single nucleotide variants (SNVs) from 1092 haplotype-resolved genomes (the 1000 Genomes Project, 1KGP) (30) to the hg19 A- and G-tracts. The normalized fractions of polymorphic tracts (F SNV) were greater for G-tracts than A-tracts and both displayed Gaussian-type distributions, with maxima of 0.067 for G-tracts of length 8 and 0.017 for A-tracts of length 9 (Figure 1C). CA[n]C and AG[n]A runs displayed the highest F SNV values for A- and G-tracts, respectively (Supplementary Figure S1C and D), with F SNV values for AG[n]As attaining ∼0.10 at length 8. We conclude that flanking base composition influences the rates of SNV within mononucleotide runs and, as a consequence, their representation in the reference human genome.

F SNV values at the flanking 5′ and 3′ bp were similar between A- and G-tracts, except for minor differences for the least represented (i.e. longest) tracts and did not exceed 0.02 (Supplementary Figure S1E). These fractions are expected to be greater than at more distant positions from the tracts, based on previous data (29). SNVs at G-tracts, but not at A-tracts, were more frequent than at flanking base pairs. F SNVs for base pairs flanking short (≤8 bp) tracts were at least twice as high as those flanking long tracts; F SNVs also displayed distinct sequence preference with most (∼0.1) variants occurring at Ts 3′ of G-tracts (Figure 1D and E). In summary, SNVs at mononucleotide runs do not increase monotonically with length but peak at 8–9 bp. This behavior mirrors the genomic distributions, both with respect to the total number of tracts (Figure 1B) and the subsets flanked by specific-sequence combinations (Supplementary Figure S1A–D). Variation at flanking base pairs also displayed a biphasic pattern centered at a length of 8–9 bp, with a greater chance of variation adjacent to G- than A-tracts and with characteristic sequence preferences.

Long tracts are evolutionarily conserved and associated with high transcription

To assess whether more variable monosatellite runs (Figure 1C) might have undergone a greater reduction in number in extant humans relative to extinct hominids, we compared the number of A- and G-tracts between syntenic regions of five individuals comprising hg19 and three Neanderthal (HN) specimens (31). The difference between hg19 and HN was very small (<±2%) for the short tracts, but it displayed more negative values in hg19 with increasing tract length, which reached a maximum of −11.8 and −32.7% for A- and G-tracts, respectively, of length 9. Beyond this threshold, the numbers of tracts converged for A-tracts, whereas they were more abundant in hg19 for G-tracts >11 bp (Figure 1F). In summary, the largest difference in the number of mononucleotide runs between hg19 and HN sequences was centered at 9 bp for both A- and G-tracts, suggesting that the length distributions (Figure 1A and Supplementary Figure S1A and B) reflect distinct rates of evolutionary gains and losses due to differential sequence mutability (Figure 1C) as a function of length and flanking sequence composition (12).

The fact that long (>9 bp) mononucleotide runs display low variability in the human population (Figure 1C) and sequence conservation during evolutionary divergence (Figure 1F) raises the possibility that they might serve functional roles. Through gene enrichment analyses, we found that genes containing A- and G-tracts were enriched for genes associated with the term ‘UCSC_TFBS’, which pertains to transcripts harboring frequent transcription factor binding sites (32,33). For A-tract-containing genes, the median P-values for the top 11 UCSC_TFBS terms decreased from 2.95E-26 for tracts of length 4 to 5.22E-241 for tracts of length 13 (Figure 1G). The percent of A-tracts intersecting genomic fragments amplified from chromatin immunoprecipitation using transcription-factor binding antibodies (32,33) also increased from 8.7 to 9.9 from length 6 to 13, whereas it was constant (mean ± SD, 22.4 ± 1.1) for G-tracts (Figure1G). For gene classes excluding ‘UCSC_TFBS’, a search for categories enriched at P < 0.05 and common to all A- and G-tract-containing genes returned a set of 25 terms, 22 of which were associated with high levels of tissue-specific gene expression (Figure 1H). In summary, these analyses extend prior work (14) supporting a role for mononucleotide tracts in enhancing gene expression, a function that for A-tracts appears to increase with increasing tract length.

Repeat variability is highly skewed

Next we addressed whether bp along A- and G-tracts display equal probability and type of variation. In the 1KGP dataset, the number of SNVs at each position along both A- and G-tracts of length 4 was within a two-fold difference (144,000–240,000); for both types of sequence, transitions (i.e. A→G and G→A) were the predominant (51–78%) type of base substitution (Supplementary Figure S2A and B). However, with increasing length, the number of SNVs decreased up to 30-fold more drastically for G-tracts than for A-tracts, with increasing numbers of transversions (A→T and G→C|T) being predominant. Normalizing the data for the number of tracts genome-wide revealed that the extent of SNV varied by up to 10-fold, depending upon tract length and bp position. Specifically, the highest degree of variation was observed at the first and last A within the A-tracts (i.e. A1 and An), which underwent up to 61% A→T and 43% A→C transversions, respectively, at length 9 (Figure 2A). Likewise, for G-tracts, the most polymorphic sites were G3, followed by G2, for mid-size tracts of 8–10 bp, with 44% G→C transversions at G3 for tracts of length 8 (Figure2B). Thus, the extent of SNV at mononucleotide runs is grossly skewed in human genomes, both along the sequence itself and across tract length, which must account for the bell-shape behavior in F SNV for the tracts as a whole (Figure 1C).

Figure 2.

Population variation spectra. (A) Variation spectra of A-tracts. Percent (number of SNVs at each position divided by the number of tracts in hg19 × 100) of A→T (black), A→C (red) and A→G (green) SNVs in the 1KGP dataset (left). Percent SNVs at A1 as a function of tract length (right). (B) Variation spectra of G-tracts. As in panel A with G→T (black), G→C (red) and G→A (cyan) (left). Percent SNVs at G3 as a function of tract length (right). (C) Percent A→T, A→C and A→G transitions at each position along A-tracts (stars) preceded and followed by a T (TA[n]T, left), C (CA[n]C), center) and G (GA[n]G, right) as a function of tract length. (D) Percent G→T, G→C and G→A transitions at each position along G-tracts (stars) preceded and followed by a T (TG[n]T, left), C (CG[n]C), center) and A (AG[n]A, right) as a function of tract length. (E) Percent transitions at base pairs (stars) preceding or following A-tracts (left) and G-tracts (right) as a function of tract length (n). *, mutated position.

We assessed whether SNV hypervariability was associated with specific combinations of nearest neighbors. For A-tracts flanked 5′ by a T, C or G, the highest percentage of SNVs was observed at A1 when preceded by a T, which reached 7.9% for TA[n] tracts of length 9 (Supplementary Figure S2C). By contrast, for 3′ T, C or G, the greatest effect was elicited by a C, with the highest percentage (7.1%) of SNVs at An for A[n]C tracts of length 9 (Supplementary Figure S2D). Therefore, flanking base pairs play a critical role both in the spectra and frequencies of SNVs at A-tracts. More detailed plots along A-tracts either preceded (Supplementary Figure S2E), followed (Supplementary Figure S2F) or preceded and followed (Figure 2C) by a T, C or G revealed the dramatic and long-range (up to 9–10 bp for the longest tracts, higher than the value of 4 bp predicted by mathematical models of slippage (11)) influence of flanking base pairs on variation spectra, in which up to 95% of the changes were in the direction of the base flanking the tract. Because the number of A-tracts preceded or followed by a specific base varies by up to three-fold (Supplementary Figure S2G), we conclude that for A-tracts, the overall mutation fractions and spectra are the result of at least three variables; length, position along the tract, and base composition of the 5′ and 3′ nearest-neighbors.

For G-tracts flanked 5′ by a T, C or A, high percentages (10–12%) of SNVs were observed at G1 for tracts preceded by a C, an effect that decreased with increasing tract length (Supplementary Figure S3A). This result, together with an exceedingly low number of G→A transitions at G1 for tracts not preceded by a C (Supplementary Figure S3C) relative to all tracts (Supplementary Figure S2B), is consistent with the known high mutability of CG:CG dinucleotides as a result of cytosine-5 methylation (9). The hypermutability at G2 was observed preferentially for tracts preceded by an A, and to a lesser extent T, whereas that at G3 was insensitive to flanking sequence composition. Likewise, G-tracts flanked 3′ by a T, C or A did not display marked sequence-dependent effects (Supplementary Figure S3B). Detailed plots of the SNV spectra along G-tracts either preceded (Supplementary Figure S3D), followed (Supplementary Figure S3E), or preceded and followed (Figure 2D) by a T, C or A revealed a noticeable effect only for 5′ T in association with G→T substitutions at G1for tracts of length ≥8. Thus, despite a consistent over-representation of G-tracts flanked 5′ by a T (Supplementary Figures S3F and S1B), which must account for the high absolute number of SNVs at G1 for TG[n] relative to AG[n] and CG[n] (Supplementary Figure S3G), nearest-neighbor base composition seems to play a lesser role in SNV spectra at G-tracts than at A-tracts.

With respect to SNVs at the flanking 5′ and 3′ nearest positions, no B→A or H→G substitutions (Figure 1A) were found above a length threshold of 9 for A-tracts and 8 for G-tracts (Figure 2E, gray shading) out of 5969 SNVs, implying that tract expansion by recruiting flanking base pairs is disfavored at these lengths. In summary, base substitution along mononucleotide repeats is strongly skewed towards the edges of A-tracts and within the 5′ half of G-tracts, with frequencies that peak at midsize lengths (8–9 bp). For A-tracts ≥7 bp, base substitution occurred almost exclusively in the direction of the flanking nearest-neighbors. Finally, base substitution at flanking bases did not contribute to tract expansion for mononucleotide runs longer than 8–9 bp.

Insertions and deletions display length and positional preference

In addition to SNVs, mononucleotide runs are polymorphic in length as a result of indels. Herein, we consider separately two types of indels: one in which tract length changes by ±1 and flanking bp composition is not altered (slippage); the other comprising all other cases involving the addition or removal of 1–200 bp (indels). Slippage is a widely accepted mutational mechanism (1112,34), whereby DNA replication errors at reiterated DNA motifs cause changes in the number of motifs (most often +/−1). The normalized fractions of slippage in the 1KGP dataset peaked at lengths of 8 bp for A-tracts and 9 bp for G-tracts (Figure 3A), generating bell-shaped curves similar to those observed for SNVs (Figure1C) and with no differences in the highest fraction of ‘slipped’ tracts, which peaked at ∼0.02. By contrast, +1 slippage occurred more frequently than −1 slippage at A-tracts (Figure 3B). These results support recent studies on microsatellite repeats (12) and contrast with previous conclusions that slippage increases monotonically with tract length, and that the extent of slippage differs between A- and G-tracts (35,36).

Figure 3.

Population insertions and deletions. (A) Normalized fractions of A-tracts (closed circles) and G-tracts (open circles) displaying +/−1 bp slippage in the 1KGP dataset as a function of tract length. Data were obtained by dividing the number of events by both the number of hg19 tracts and tract length (n). (B) Ratio of the number of +1 to −1 slippage for A-tracts (closed circles) and G-tracts (open circles). (C) Indels at A-tracts. For positions along the tracts (‘Tract’), ‘F Indel’ is the ratio between the number of indels and the number of tracts in hg19 multiplied by tract length. For the positions immediately flanking the tracts genomic coordinates (‘Before tract’ and ‘After tract’), ‘F Indel’ is the ratio between the number of indels and the number of tracts in hg19. (D) Indels at G-tracts, calculated as described in panel C. (E) Heatmap representation of insertions along A-tracts. The percent insertions (i.e. the number of insertions at each position divided by the number of tracts in hg19) (y-axis) plotted as a function of location (x-axis) from position 0 (insertion between the bp 5′ to the tract and the first bp of the tract) to position n + 1 (insertion between the bp 3′ to the last bp of the tract and the following bp) (see Figure 1A) and as a function of tract length (z-axis). (F) Heatmap representation of insertions along G-tracts.

With respect to indels, the normalized fractions were low (<1 × 10−3) along short (4–6 bp) A- and G-tracts, but rose to a plateau for longer tracts as reported earlier (11); this plateau was 10-fold higher for G-tracts (∼0.03) than for A-tracts (∼0.003) (Figure 3C and D). Indels also occurred more frequently (up to six-fold for A-tracts of length 11) at nearest-neighboring base pairs (‘Before tract’ and ‘After tract’ in Figure 3C and D) than along the tracts. Thus, contrary to SNVs and slippage, indels increased to a plateau with mononucleotide tract length.

We analyzed in detail the locations of insertions along the tracts and the flanking positions with respect to the 5′ to 3′ orientation of the tracts (Figure 1A). The normalized fractions demonstrated that insertions peaked at the 3′, and to a lesser extent 5′, ends of the longest A-tracts (Figure 3E), but remained low. For G-tracts, insertions occurred most efficiently at two locations (G2–3 and G5) (Figure 3F), they increased with tract length (up to ∼0.04), and attained ∼10-fold higher values than for A-tracts. In conclusion, insertion sites at A- and G-tracts followed the patterns observed for SNVs (Figure 2A and B), suggesting that factors associated with local DNA dynamics sensitize specific bases along the tracts to genetic alteration, inducing both SBS and indels.

Base pair flexibility and charge localization map to sites of sequence changes

To elucidate elements of intrinsic DNA dynamics that may be responsible for the biases in SNV and insertion sites, we performed molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations on model A[6], A[9], G[6] and G[9] duplex DNA fragments. We focused on water bridge coordination (Figure 4A), bp step flexibility, and for the G[6] and G[9], charge localization, as these properties are known to impact the susceptibility of DNA to base damage, repair and mutation. The fractions of one water coordination increased along the A[9] and A[6] structures in a 5′ to 3′ direction, irrespective of flanking sequence composition, in concert with a decrease in minor groove width (Figure 4B and Supplementary Figure S4A) as predicted (37). Vstep, a measure of bp structural fluctuation, displayed a prominent peak of ∼40 Å3deg3 at the 5′-TA-3′ step for both structures (Figure 4C and Supplementary Figure S4B), which together with low water occupancy points to 5′-TA-3′ being a preferred location for base modification and mutation. In the G[9] and G[6] structures water coordination involved mostly two-water bridges due to wide (∼14 Å) minor grooves (Figure 4Dand Supplementary Figure S4C), whereas flexibility was modest (∼20–22 Å3deg3, Figure 4E and Supplementary Figure S4D). Thus, bp dynamics are likely to impact mutations at A-tracts to a greater extent than at G-tracts. Guanine has the lowest ionization potential (IP) of all four bases and IP further decreases at guanine runs, rendering them targets for electron loss, charge localization, oxidation and eventually mutation (4,38). Because after electron loss the ensuing charge (hole) can migrate along the DNA double-helix and relocalize at specific guanines, we addressed whether the preferred sites of mutation along G-tracts, i.e. G2–3 and G5, would also be preferred sites for charge localization. The QM/MM determinations indicated that whereas for the short G[6] fragment the difference in the density-derived atomic partial charges (DDAPC) (i.e. the hole) localized most often (∼50%) to the first position (Figure 4F), for the long G[9] fragment charge localization shifted downstream (mostly to the second, but also to positions 6–7, Figure 4G). Importantly, the charge was found exclusively around the guanine rings (Figure 4H). Thus, the two main sites of sequence change along G-tracts, i.e. G2–3 and G5, coincide with positions where charge localization and hence one-electron oxidation reactions is predicted to occur most frequently. In summary, bp flexibility at A-tracts and charge transfer at G-tracts likely represent intrinsic DNA features underlying the bias in SNV and insertions at mononucleotide runs in human genomes.

Figure 4.

MD and QM/MM simulations. (A) Molecular modeling of one (left) and two (right) minor groove water bridge coordination. (B) Fraction of one-water bridge occupancy (left axis) at A[9] DNA sequences flanked 5′ and 3′ by a T (black circles), C (red circles) or G (green circles). Minor groove widths (right axis), as determined from intrastrand phosphate-to-phosphate distances. (C) Vstep for A[9] DNA sequences, determined as the product of the square root of the eigenvalues (λi) described by the six bp step parameters shift, slide, rise, tilt, roll and twist; i.e. Vstep=6i=1λi−−√. (D) Fraction of one- (black circles) and two-water (red circles) bridge occupancy (left axis) at G[9] DNA sequences. Minor groove widths (right axis), as assessed from intrastrand phosphate-to-phosphate distances. (E) Vstep for G9 DNA sequences. (F) Average charge redistribution (open circles and right axis) for G[6] DNA structures upon vertical ionization, examined by calculating the difference on the density-derived atomic partial charges (DDAPC) for the neutral and negatively charged states. Histogram of the number of instances (left axis) in which the largest charge redistribution occurred at a specific position along the G[6] structures. (G) DDAPC for G[9] DNA structures (open circles and right axis) and histogram of the number of instances (left axis) in which the largest charge redistribution occurred at a specific position. (H) VMD rendering of a G[9] DNA structure displaying hole localization at G2. Capped base pairs were removed for clarity.

Position and orientation along nucleosome core particles modulate sequence variation

DNA wrapped around histones in nucleosomes is subject to local deformation (39), which may impact mutation. Thus, we analyzed the 1KGP SNVs at A- and G-tracts predicted to overlap with well-positioned nucleosome core particles (NCPs) (16). In hg19, the percentage of tracts that overlap with NCPs decreased moderately from ∼90% at length of 4 to 81% and 71% for A- and G-tracts of length 13, respectively (Figure 5A), suggesting that mononucleotide runs are not depleted in NCPs in human genomes as previously proposed (40). A-tracts of lengths 4–8 base pairs displayed distinctive peaks along the NCP surface in phase with the helical repeat of DNA (10.5 bp) and with minor grooves facing toward the inner protein core (lengths 4–5) (16) (Figure 5B and Supplementary Figure S5A). A-tracts of length of 9–13 bp exhibited only half (six) the peaks evident for the shorter tracts. For the G-tracts, only small peaks with no clear minor groove-inward-facing regions were detected (Supplementary Figure S5B).

Figure 5.

Positioning along nucleosome core particles. (A) Percent of A-tract (open circles) and G-tract (closed circles) base pairs in hg19 overlapping with well-positioned NCP genomic coordinates as a function of tract length. (B) Counts of base pairs in hg19 A-tracts of length 5 overlapping with NCPs genomic regions as a function of distance from the histone octamer dyad axis. Minor groove-inward-facing regions (gray) were derived from the X-ray crystal structure of NCP147 (41). (C) Percent SNVs in the 1KGP dataset (left axis) at every bp along A-tracts of length 5 for tracts centered at maxima (black) and minima (gray) along NCPs (Figure 5B). Percent increase (right axis) of SNVs at minima relative to maxima (green). P-values for paired t-tests: 0.013 (*), 0.002 (**) and 4.7 × 10−6 (***). (D) Whisker plots of%SNVs (left axis) at A1 for A-tracts of length 5 centered at maxima and minima (black) along NCPs (Figure 5B). Percent difference (right axis) in the number of A-tracts of length 5 in hg19 preceded by C, T or G (red) between those centered at minima and those centered at maxima (Figure5B). (E) C-containing/G-containing ratios (see text) for G-tracts of length 5 in hg19 as a function of distance from the NCP dyad axis (black) and location of core histones (maroon and green). Peaks correspond to negative iSAT (i.e. tilt parameters multiplied by the corresponding sin θ) values (gray) (39). Ratios of%SNV at G1 (upshifted by 0.5 for clarity) between C-containing (5′-CCCCCG-3′ sequences on the hg19 forward strand) and G-containing (5′-CGGGGG-3′ sequences on the hg19 forward strand) (Figure 1A) CG[5] tracts mapping NCP Chip-seq genomic intervals (red) fitted by a non-parametric local regression (loess; sampling proportion, 0.100; polynomial degree, 3). (F) VMD rendering (top) of TATTT residues 34–38 (yellow) and the complementary AAATA residues 672–753 (pink) from the 1EQZ pdb nucleosomal crystal structure, corresponding to peak area from −40 to −36 in Figure 5E. The switch in G-tract (lengths of 5 and 7) orientation along NCPs (bottom) serves to position the C-containing strand on the outside (yellow) and, correspondingly, the G-containing strand on the inside (pink).

 To assess if tract-positioning along NCPs influences SNVs, we selected A-tracts of lengths 5, 7 and 9 bp and G-tracts of lengths 5 and 7 bp whose central positions coincided with either the maxima or minima (41) (Figure 5B and Supplementary Figure S5A and B) and conducted pair-wiset-tests (330 total) between permutations of ‘categories’, including ‘tracts centered at maxima versus minima’, ‘position along the tracts’, ‘flanking sequence composition’, ‘specific NCP locations’ and ‘tract orientation’. For A-tracts, 79/207 (38%) significant pairs were found, 68 (86%) of which were related to differences between tracts centered at maxima versus minima, with a preponderance (63%) of tests displaying increased %SNVs at minima (Supplementary Figure S5C and E). For example, %SNVs at length 5 bp were greater at minima than at maxima at each position along the A-tracts (Figure 5C). A→C substitutions at A1 were more abundant at maxima than at minima (mean ± SD, 18.7 ± 0.7% at max and 17.6 ± 0.8% at min; P-value 0.001), whereas A→T substitutions at the same position displayed the opposite trend (mean ± SD, 18.4 ± 0.5% at max and 19.8 ± 1.1% at min; P-value 0.0005) (Figure 5D). A-tracts of length 7 also exhibited a similar pattern at A7 (Supplementary Figure S5H). The percentages of CA[5] and A[7]C tracts in hg19 centered at maxima were greater than at minima and the reverse was observed for the TA[5] and A[7T] tracts (Figure 5D and Supplementary Figure S5H). Thus, we conclude that positioning along the NCP surface of both the double-helical grooves and junctions with flanking base pairs influence SNVs along A-tracts. However, this influence is complex and for the most part, difficult to predict.

For G-tracts, most pairwise comparisons (18/34, 53%) indicated SNV variation according to sequence orientation (Supplementary Figure S5F and G). In hg19, the ratio of the numbers of G-tracts of lengths 5 and 7 for which the C-containing strand coincided with the forward sequence (downstream example sequence in Figure 1A) to the numbers of G-tracts for which the G-containing strand coincided with the forward sequence (upstream example sequence in Figure 1A) (C-containing/G-containing ratios) displayed a prominent 10.5-bp oscillation in phase with iSAT (Figure 5E), a measure of ‘inside’ and ‘outside’ bases, according to the bp step tilt parameter (39). Analysis of the helical path of a 146-bp DNA fragment wrapped around histones showed that the oscillation in the C-containing/G-containing ratios corresponds to a preference for guanine bases to face the protein core (Figure 5F). We analyzed the subset of G-tracts preceded by a 5′ C (i.e. CG[5]) to assess whether SNVs at G1, the position known to be mutable due to CpG methylation also oscillated with the C-containing/G-containing ratios. Oscillation in SNV-C-containing/SNV-G-containing values was evident, with peaks aligning to the hg19 troughs (Figure 5E) implying that the cytosines facing the protein surface harbor more variants than those facing away. We conclude that A- and G-tracts display preferential positioning (the former) and orientation (the latter) along NCPs, which in turn modulate the rate of sequence variation.

Mutations associated with human disease

Knowing that the first and last As of long A-tracts and G2–3 in G-tracts are the major sites of SNV in the human population, we addressed whether these features are also discernible in mutated mononucleotide tracts associated with human genetic disease. We collected 9,450,456 unique SBSs (both SBSs and SNVs refer to single base changes) from sequenced cancer genomes and normalized the percent mutations along A- and G-tracts to enable a direct comparison with the 1KGP dataset. For A-tracts (Figure 6A and Supplementary Figure S6A), SBSs displayed the same trend as the 1KGP data (Figure 2A) with respect to the bell-shape increase in mutations at A1 and An and the mutation spectra, although the susceptibility to mutation as a function of tract length attained greater values (6.36% for length 11 in cancer versus 4.15% for length 9 in the 1KGP datasets at A1). The first and last 3 bp also harbored more SBSs than in the 1KGP dataset for tracts >7 bp, a feature that we found to be due exclusively to a large cancer dataset (42) containing high-level microsatellite instability (MSI) samples (Supplementary Figure S6B and C), which are known to result from mismatch-repair deficiency (15). Thus, A-tracts display similar patterns of base substitution between the germline and somatic cancer tissues. For G-tracts, mutation spectra were characterized by G→T transversions at tract lengths >7, particularly at G1, the most frequently mutated position for tracts lengths up to 11 bp (Figure 6B and Supplementary Figure S6D). This trend persisted even when the high rates of methylation-mediated deamination mutations at the CG dinucleotide were removed (Supplementary Figure S6E). Thus, mutation patterns in cancer genomes contrast with those observed in the germline, both with respect to the most mutable position (G1 versus G2–3) and the types of base substitution (G→T in cancer genomes versus G→T and G→C in the germline).

Figure 6.

Mutation patterns in cancer genomes. (A) Mutation spectra for SBSs at A-tracts. Percent values were obtained by dividing the total number of SBSs at each position by the number of tracts in hg19 and then multiplying by 3.2516 to equalize the percentage of A-tracts of length 4 between the cancer genomes and the 1KGP datasets. (B) Mutation spectra for SBSs at G-tracts in cancer genomes. Percent values were obtained as in (A) using a multiplication factor of 3.7419. (C) Normalized fractions of A-tracts (closed circles) and G-tracts (open circles) displaying +/−1 bp slippage, obtained by dividing the number of events by both the number of tracts in hg19 and tract length. (D) Indels at A-tracts, calculated as described in Figure 3C. (E) Indels at G-tracts, calculated as described in Figure3C. (F) Heatmap representation of insertions along G-tracts, as described in Figure 3E.

 With respect to slippage, the fractions for A-tracts elicited an excess at lengths 9 and 10 bp relative to the 1KGP dataset, which was also due to the MSI-containing dataset. For G-tracts, the fractions peaked at length 8, as for the 1KGP dataset (Figures 3A and 6C), implying that the propensity to undergo slippage is indistinguishable between the germline and soma. Indels were also more abundant at flanking base pairs than along the tracts (Figure 6D and E), particularly for G-tracts of length >7, similar to the 1KGP dataset (Figure 3C and D). Detailed analyses of insertions revealed that both G1 and the preceding position were the most significant sites of mutation (F-values up to 0.08 at G1 for tracts of length 8) (Figure 6F). Thus, the 5′ end of long G-tracts is the most susceptible site for both SBSs and insertions in cancer genomes, in contrast to the germline where these occur within the runs, typically at G2–3.

We also extracted the mutated A- and G-tracts from the Human Gene Mutation Database (HGMD), a collection of >150,000 germline gene mutations associated with human inherited disease. A total of 1519 genes were mutated at A- or G-tracts out of a total of 3972 (38%); 3480 SBSs and 2866 slippage events were noted within these tracts, 85 and 46% of which were predicted to be disease-causing, respectively (Figure 7A and Supplementary Table S1). Ranking genes by the number of literature reports indicated that among the top 10 entries three were associated with cancer (BRCA1, BRCA2 and APC), two with hemophilia (F8 and F9), four with debilitating lesions of the skin (COL71A), muscle (DMD), lung (CFTR) and kidney (PKD1), with one causing hypercholesterolemia (LDLR) (Figure 7B). Thus, mutations within A- and G-tracts carry a high social burden by contributing to some of the most common human pathological conditions.

Figure 7.

Mutation patterns in HGMD and model for sequence context-dependent changes. (A) Number of germline SBSs and slippage events (Slip.) at A- and G-tracts in HGMD. Gene alterations were classified as disease-causing mutation (DM), likely disease-causing mutation (DM?), disease-associated and putatively functional polymorphism (DFP), disease-associated polymorphism with additional supporting functional evidence (DP) and invitro/laboratory orinvivo functional polymorphism (FP). Codon changes (SIFT predictor) were classified as damaging (d), null (n), tolerated (t) and low-confidence prediction (l). (B) The 10 most commonly reported genes in HGMD with mutations at A- and G-tracts. Various mutated tracts were generally reported for the same gene in different reports. (C) Mutation spectra for SBSs at A- (left) and G-tracts (right) in HGMD. Percent values were obtained by dividing the total number of SBSs at each position by the number of tracts in hg19 exons. A|G→T (black), A|G→C (red), A→G (green), G→A (cyan). (D) Normalized fractions of A-tracts (closed circles) and G-tracts (open circles) displaying +/−1 bp slippage, obtained by dividing the total number of events by the number of tracts in hg19 exons and by tract length. (E) Model for sequence context-dependent changes at A-tracts (left) and G-tracts (right). *, site of base modification.

 For both A- and G-tracts, SBSs occurred mostly at tract lengths of 4–7, with patterns more similar to those in the 1KGP than in the cancer datasets, both with respect to the location of the most mutable positions (first and last As and first/second Gs) and the types of base substitution (A→T and G→H) (Figure 7C and Supplementary Figure S6F). Likewise, slippage events peaked at tract lengths of 7–9 as observed in the 1KGP dataset (Figure 7D). In summary, the patterns of both SBSs and slippage in the HGMD dataset followed the trend observed in the 1KGP dataset, suggesting that germline variants at mononucleotide repeats leading to either population variation or human inherited disease may have arisen through similar mechanisms.

Why are specific A:T and G:C base pairs within A- and G-tracts more susceptible to sequence changes than their identical neighbors? For A-tracts, bp flexibility may play a role. Chemical damage to DNA, such as by hydroxyl radicals has been shown to be proportional to the geometrical solvent-accessible surface of the atomic groups, which increases with DNA flexibility (43). Along A-tracts flexibility is restricted, but it is high at both the 5′ and 3′ junctions. Thus, the fact that the highest rates of mutation coincide with the highest degree of flexibility at the 5′-TA-3′ bp step is consistent with the view that this position may be susceptible to DNA damage as a result of flexibility. Other sources of DNA dynamics are also likely to be relevant, such as sugar flexibility at the junctions, which increases with tract length (44). Chemical modification at these junctions may then lead to base substitution and indels, the latter as a result of strand breaks.

With respect to SNV mutation spectra, these were found mostly in the direction of flanking base composition above a length of 7–8 bp. We interpret this behavior in terms of DNA slippage along A-tracts when attempts are made during translesion synthesis (TLS) to bypass a damaged site (Figure 7Ei). Two scenarios may be considered to account for A→T transitions at A1. In the first, the last tract-template base would loop out into the polymerase active site permitting base-pairing and strand elongation (Figure 7Eii) using the tract-flanking base as a template (34,4546). In the second (Figure 7Eiii), slippage would occur behind the polymerase, prompting extension past the newly created A*:T mispair generated by primer/template misalignment. Either pathway would yield a common intermediate (Figure 7Eiv) that contains the base complementary to the junction across from the damaged site upon slippage resolution (34). Following DNA synthesis (S) and/or repair (R) (Figure 7Ev and vi), this mispair will generate a base change that is always identical to the tract-flanking base.

For G-tracts, the high rates of G→T transversions at G1 in cancer genomes are also consistent with preferred chemical attack at this site due to high flexibility (Figure 7F top). Direct chemical attack at a guanine is known to result in stable products, such as 8-oxo-G and Fapy-G, both of which are known to yield G→T transversions (4750). Thus, G1 may be the most susceptible site for such reactions for G-tracts of lengths ≥7 (Figure 7Fright), which in cancer genomes would become a mutation hotspot. In the germline, SNVs peaked inside G-tract base pairs, while mutational spectra were insensitive to flanking base composition; these events are inconsistent with a role for template misalignment and slippage as noted for A-tracts. Rather, the correspondence between hotspot mutations at G2–3 and G5 and the QM/MM simulations suggest a role for charge transfer. A large body of work during the past 20 years using computational, theoretical chemistry and biophysical techniques on short oligonucleotides, has shown that guanine is the most easily oxidizable base in DNA and that indeed a guanine radical cation can be generated through long-range hole transfer from an oxidant via one-electron oxidation mechanisms (5155). GGG triplets were found to act as the most effective traps in hole transfer by both experimental and theoretical work (5659), demonstrating that the resulting guanine radical cation (or its neutral deprotonated form) became rather delocalized, but it preferentially centered at the first and second G. These well-established patterns of chemical reactivity are consistent with our experimental observation of high mutation frequencies at G1 for short G-tracts and the results from QM/MM simulations on G6. For longer tracts, the downstream shift in mutation hotspots, i.e., G2–3 and G5, also correlate well with the charge localization predicted from QM/MM simulations, which explicitly included solvent effects and structural fluctuations. Thus, in conjunction with the constrained density functional theory (60), both the neutral and oxidized forms of a guanine nucleobase can be reliably constructed to infer the accurate determination of mutational patterns of mononucleotide repeats in human genomic DNA.

The compact organization of the sperm genome (61), and presumably low levels of oxidative stress in the germline, may enable guanine oxidization through one-electron oxidation reactions rather than by direct chemical attack, thereby favoring the formation of radical cations. A charge injected at G1 by electron loss would then migrate to neighboring guanines and localize at sites of low IP, such as G2 (Figure 7F left). Guanine radical cations are known to readily undergo further chemical modification leading to products such as 8-oxo-G, oxazolone, imidazolone, guanidinohydantoin, and spiroiminodyhydantoin (62) (M in Figure 7F), to yield G→T, G→C and G→A substitutions (4,63). Our model is in line with recent observations in which mutations at guanines within short G-runs (1–4 bp) correlate with sequence-dependent IPs at the target guanine in cancer genomes (9). Interestingly, these correlations were not observed in the germline (9). We interpret these composite observations as follows. The IP values for G-runs have been shown to decrease asymptotically with tract length, although the absolute values vary according to the methods and assumptions used (we obtained a value of 5.43 eV for both G[6] and G[9]) (64,65). We suggest that short G-runs with high IPs undergo one-electron oxidation reactions in the oxidative environment of cancer cells but would be refractory to such a mechanism in the germline (Figure 7Fright yellow and left white sectors). As length increases and IP values fall, G-runs would be attacked directly by oxidants abundant in tumor cells (Figure 7F orange sector), whereas oxidation will be limited to electron loss in the germline environment (Figure 7F left yellow sector).

These models (template misalignment for A-tracts and charge transfer for G-tracts) suggest a more complex scenario for mechanisms underlying mononucleotide repeat polymorphism in the human population than recently proposed (13), in which nucleotide misincorporation by error-prone polymerases is proposed as a primary source of mutations at both A- and G-tracts. As already stated, the directionality of SNVs toward tract-flanking bases in A-tracts and the hotspot mutations at G2–3, supports multiple and distinct mechanisms of base substitution at mononucleotide repeats.

Our analyses highlight additional information, including the lack of mutations in the direction of tract-base composition for base pairs flanking long tracts, the association with gene expression and the preference of guanines for the inner NCP surface, and extend prior observations (12) such as the bell-shape character of base substitution and slippage, whose mechanisms remain to be fully clarified. Finally, we document the contribution of mononucleotide mutagenesis to key aspects of human pathology beyond the well-established MSI instability in cancer (15), including hemophilia and tissue degeneration. Our collective work supports the conclusion that as the human genome undergoes evolutionary diversification and along the way suffers disease-associated mutations, oxidation reactions including charge transfer may play a prominent role.


Supplementary Data are available at NAR Online.



Mutation analyses and prenatal diagnosis in families of X-linked severe combined immunodeficiency caused by IL2Rγ gene novel mutation

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Genet. Mol. Res. 14 (2): 6164 – 6172   DOI: 10.4238/2015.June.9.2
Severe combined immunodeficiency diseases (SCIDs) are a group of primary immunodeficiency diseases characterized by a severe lack of T cells (or T cell dysfunction) caused by various gene abnormalities and accompanied by B cell dysfunction (WHO, 1992; Buckley et al., 1997). The incidence rates in infants were 1/75,000-1/10,0000 (WHO, 1992), but no morbidity statistics are available in China. The 2 genetic modes of SCID include X-linked recessive and autosomal recessive genetic inheritance. X-linked severe combined immunodeficiency (X-SCID) is the most common form, accounting for 50-60% of SCID cases (Noguchi et al., 1993). Immune system abnormalities in patients with X-SCID include T-B+NK-, in which T cells (CD3+) and natural killer (NK) cells (CD16+/CD56+) are absent or significantly reduced, and the number of B cells (CD19+) is normal or increased, causing reduced immunoglobulin production and class switching disorder (Buckley, 2004; Fischer et al., 2005). The IL- 2Rg gene mutation has been confirmed to be a major cause of X-SCID (Noguchi et al., 1993). In recent years, great progress has been made in understanding the pathogenesis of primary immunodeficiency disease and its application in clinical treatment, particularly regarding the development of critical care medicine and immune reconstruction technology. With timely control of infection and early bone marrow or stem cell transplantation, X-SCID patients can be treated, prolonging survival time. Therefore, early diagnosis of X-SCID is very important for patient treatment. Gene diagnosis has become a better early diagnosis or differential diagnosis method. In addition, familial X-SCID brings a great psychological burden to the relatives of patients. Ordinary chromosome analysis and immunological evaluation cannot be used for female carrier identification and fetal diagnosis, and gene diagnosis is the most effective method of carrier detection and prenatal diagnosis. In this study, we detected mutations in 2 families with X-SCID and identified 2 novel mutations, confirming the X-SCID pedigrees. Prenatal diagnosis was performed for the pregnant fetus in the mother of one of the probands based on gene diagnosis. Female individuals in this family were subjected to carrier detection.
IL2Rg gene mutation test Direct sequencing of 1-8 exons and the flanking region of the IL2Rg gene by PCR in family 1 showed that the 3rd exon of the proband contained the c.361-363delGAG heterozygous deletion mutation, which led to deletion of the 121st amino acid glutamate (p.E121del) in its coding product. There were no sequence variations in other coding regions or in the shear zone. The proband’s mother carried the same heterozygous mutation, while his father did not carry the mutation site (Figure 2a, b, c). This mutation was not observed in any cases of the control group, and this family was identified as an X-SCID family. The c.510-511insGAACT insertion heterozygous mutation was present in the 4th exon of the proband’s mother in family 2. This mutation was a 5-base repeat of GAACT, resulting in a change in amino acid 173 from tryptophan into a stop codon (p.W173X). While there were no sequence variations in other coding regions or in the shear zone, the patient’s father did not carry the mutation (see Figure 2d, e). We did not find this mutation in the healthy control group. We presumed that the 4th exon of the deceased child in family 2 contained the c.510-511insGAACT insertion mutation, leading to X-SCID symptoms, and thus we speculated that this family was an X-SCID pedigree. Prenatal diagnosis We verified the chorionic villus status of the fetus in family 1 using the PowerPlex 16 HS System kit. The results of prenatal diagnosis showed that the fetal tissue contained no maternal contamination and that this fetus was female. The results of prenatal diagnosis showed that there was no c.361-363delGAG (p.E121del) heterozygous mutation in the female fetus of family 1.
Figure 2. Sequencing graph of IL2Rg gene in 2 pedigrees with X-chain severe combined immunodeficiency. a.-c. Family 1. a. Normal control (rectangle indicates 3 edentulous bases of this patient). b. Proband carrying the c.361- 363delGAG (p.E121del) mutation (arrow indicates deletion of fragment connection sites). c. The proband’s mother contained a c.361-363delGAG (p.E121del) heterozygous mutation (arrow). d.-e. Family 2. d. The proband’s mother carried the c.510-511insGAACT (p.W173X) heterozygous mutation (arrow indicates that the reverse sequencing graph was positive). e. Normal control (rectangular box indicates 2 normal copies of GAACT (the mutation fragment was 3 copies). Carrier detection results For the c.361-363delGAG (p.E121del) site, the gene analysis results of the female individual in family 1 showed that I2 (proband’s grandmother) was a heterozygous carrier and that II3 (proband’s aunt) was a non-carrier and had no mutations.
IL-2 can combine with the IL-2 receptor (IL-2R) of the immune cell membrane. IL-2R is composed of 3 subunits, including the IL-2Ra chain (CD25), IL-2Rb chain (CD122), and IL- 2Rg chain (CD132). IL-2Rg functional units in common with IL-4, IL-7, IL-9, IL-15, IL-21, and other cytokine receptors, and these regions are referred to as the total chain (Li et al., 2000). The IL-2Rg chain can maintain the integrity of the IL-2R complex and is required for the internalization of the IL-2/IL-2R complex; it is also the link that contacts the cell membrane surface factor region and downstream cell signal transduction molecules. Therefore, the integrity of the IL-2Rg chain is vital for the immune function of an organism (Malka et al., 2008; Shi et al., 2009).
Mutations in the IL2Rg gene, which encodes IL-2Rg, were identified to be a major cause of X-SCID in 1993 (Noguchi et al., 1993). The IL2Rg gene is located on chromosome X q21.3-22, is 37.5 kb length, and contains 8 exons, which encode 369 IL-2Rg amino acids. The IL2Rg chain exhibits varying structural regions, such as the signal peptide [amino acids (AA) 1-22], extracellular domain (AA 23-262), transmembrane region (AA 263-283), and intracellular region (AA 284-369). The WSXWS motif is located in the extracellular region (AA 237-241), while Box 1 is located in the intracellular region (AA 286-294).
By the end of 2013, the Human Gene Mutation Database contained a total of 200 mutations in the IL2Rg gene (HGMD Professional 2013.4). The most common mutation types in the IL2Rg gene were the missense or nonsense mutations, which result from single base changes. A total of 100 missense or nonsense mutations have been identified, followed by insertion or deletion mutations in a total of 50 species. The 3rd most common type of mutations includes shear mutations in approximately 30 species. Eight exons contained mutations, and mutations in 3rd or 4th exons were the highest, accounting for a total mutation rate of 43% (86/200). According to the X-SCID gene database (IL2RGbase) (http://research.nhgri., the gene mutations in IL2Rg mainly occurred in the extracellular region of the IL2Rg chain (Fugmann et al., 1998). Zhang et al. (2013) reported that the IL2Rg gene mutations in 10 patients with X-SCID in China were located in the extracellular region. Two mutations reported in our study were also located in the extracellular region. The mutation of IL2Rg gene in family 1 was a codon mutation in the 3rd exon, resulting in a 3-base deletion. The c.361-363delGAG (p.E121del) mutation was located in the extracellular area of the IL- 2Rg subunit, and we inferred that the 121 glutamate deletion caused by the mutation would lead to changes in the structure of the peptide chain, affecting signal transmission and resulting in serious symptoms. The mutation of family 2 was a GAACT repeat of ILR2g gene; this repeat of 5 bases resulted in 173 codon changes from tryptophan into a stop codon. Generation of the peptide chain with the mutation lacked 196 amino acids compared to the normal chain, including the intracellular, transmembrane, and some extracellular regions, directly affecting the structure and function of receptors and causing disease. No studies have been reported regarding these 2 mutations. We combined with the mutation characteristics and clinical manifestations and diagnosed family 1 as X-SCID pedigrees. Although the patient in family 2 was deceased, it can be speculated that the 2 deceased patients in family 2 were X-SCID pedigrees caused by c.510-511insGAACT (W173X).
Prenatal diagnosis can accurately identify fetal situations and be used to avoid birth defects, which can also ease the anxiety of the pregnant mother. Gene diagnosis for pedigrees of patients based on DNA samples has advanced recently, particularly with the application of high-throughput sequencing technology (Alsina et al., 2013). We can now perform gene analysis for varied clinical infectious diseases for differential diagnosis. However, the effectiveness of prenatal diagnosis for pedigrees in which the proband is dead remains unclear. Because the gene mutations in the proband is unknown in these cases, the patient’s situation was only inferred by his mother’s genotypes. However, we considered that for the deceased, if we can define the mother was a pathogenic gene carrier, even if the proband is not X-SCID, the woman also has a risk of having X-SCID children and this pedigree may be X-linked recessive inheritance. Prenatal diagnosis may provide a choice for preventing the birth of patients in these families in the premise of informed consent.
Gene diagnosis of IL2Rg can also be used for carrier detection of suspected females in the family.
In the present study, we performed carrier detection of the patient’s grandmother and aunt in family 1 and determined that the patient’s pathogenic mutations were from his grandmother. His aunt did not inherit the pathogenic gene, and thus she was a non-carrier and her fertility will not be affected. In this study, we used direct sequencing of PCR products and identified IL2Rg gene mutations in 2 pedigrees with X-SCID. We found 2 unreported mutations in the IL2Rg gene, and prenatal diagnosis and carrier detection were conducted in 1 X-SCID family. Because the incidence rate of X-SCID is extremely low, it is difficult to promote the widespread use and application of genetic diagnosis. However, this study may provide some implications for the diagnosis of infants with immunodeficiency, and gene diagnosis techniques such as conventional or high-throughput sequencing should be used as soon as possible during pregnancy, which can be used to guide treatment. This method can also provide reliable prenatal diagnosis and carrier detection service for these families.
MEF2A gene mutations and susceptibility to coronary artery disease in the Chinese population
J. Li1 , H.-X. Chen2 , J.-G. Yang3 , W. Li3 , R. Du3 and L. Tian3       DOI
Coronary artery disease (CAD) has high morbidity and mortality rates worldwide. Thus, the pathogenesis of CAD has long been the focus of medical studies. Myocyte enhancer factor 2A (MEF2A) was first discovered as a CAD-related gene by Wang (2005) and Wang et al. (2003, 2005). Three mutation points in exon 7 of MEF2A were subsequently identified by Bhagavatula et al. (2004); however, Altshuler and Hirschhorn (2005) and Weng et al. (2005) predicted that the MEF2A gene lacked mutations. Zhou et al. (2006a,b) analyzed the mutations and polymorphisms in exons 7 and 11 of the MEF2A gene in the Han population in Beijing, and various rare mutations were found in exon 11 rather than in exon 7. The clinical significance of specific 21-bp deletions in MEF2A was also explored, and previous studies have shown mixed results. In this study, polymerase chain reaction-singlestrand conformation polymorphism (PCR-SSCP) and DNA sequencing were used to detect exon 11 of the MEF2A gene in samples collected from 210 CAD patients and 190 healthy controls and to investigate the function of the MEF2A gene in CAD pathogenesis and their correlation.
CAD, a common disease in China, is induced by multiple factors, such as genetics, the environment, and lifestyle. Thus, a multi-faceted approach is necessary in the study of CAD pathogenesis, particularly in molecular biology research, which is important for developing comprehensive treatment of CAD based on gene therapy. The MEF2A gene was first identified as a CAD-related gene through linkage analysis of a large family with CAD (9 of 13 patients developed MI) in 2003.
In this study, we found the following mutations: 1) codon 451G/T (147191) heterozygous or homozygous mutation; 2) loss of 1 (Q), 2 (QQ), 3 (QQP), 6 (425QQQQQQ430), and 7 (424QQQQQQQ430) amino acids (147108-147131); and 3) codon 435G/A (147143) heterozygous mutation. Among these mutations, the synonymous mutation at locus 147191 was confirmed by reference to the National Center for Biotechnology Information (NCBI) database to be a single nucleotide polymorphism, which was also demonstrated in our study by the extensive presence of this polymorphism in healthy controls. However, the heterozygous mutation at locus 147143 was only found in the genomes of CAD patients, and was therefore identified as a mutation.
Given that MEF2A is a CAD-related gene, the results of various studies are controversial among several countries. Weng et al. (2005) screened gene mutations in exon 11 of the MEF2A gene from 300 CAD patients and 1500 healthy controls. They hypothesized that the changes in 5-12 CAG repeats are genetic polymorphisms and that the 21-base deletion in exon 11 of the MEF2A gene did not induce autosomal dominant genetic CAD. Gonzalez et al. (2006) suggested that the CAG repeat polymorphism was independent of MI susceptibility in Spanish patients. Kajimoto et al. (2005) reported that the CAG repeat sequence was not correlated with MI susceptibility in Japanese patients. Horan et al. (2006) also found that the CAG repeat sequence was not associated with the susceptibility to early-onset familial CAD in an Irish population. Hsu et al. (2010) identified no correlation between the CAG repeat sequence and CAD susceptibility in the Taiwanese population. Dai et al. (2010) found that the structural change in exon 11 was not related to CAD in the Chinese Han population. Lieb et al. (2008) and Guella et al. (2009) hypothesized that MEF2A was independent of CAD. However, Yuan et al. (2006) and Han et al. (2007) suggested that the CAG repeat sequence was correlated with CAD because 9 CAG repeats was an independent predictor of CAD. Elhawari et al. (2010) and Maiolino et al. (2011) suggested that MEF2A is a susceptibility gene for CAD. Dai et al. (2013) showed that mutations in exon 12 are associated with the early onset of CAD in the Chinese population. Liu et al. (2012) failed to demonstrate a correlation between the CAG repeat sequence and CAD through case-control analysis, systematic review, and meta-analysis, but found that the 21- base deletion in exon 11 was strongly associated with CAD, and that genetic variations in MEF2A may be a relatively rare, but specific, pathogenic gene for CAD/MI. Kajimoto et al. (2005) reported 4-15 CAG repeats. However, only 4-11 CAG repeats were observed in our study, possibly because of genetic differences in patients in this study. Eleven CAG repeats were observed in most samples from the control group, and the proportion of 10, 9, and 8 repeats exceeded 1%. The heterozygous mutation at 147143, as well as the 4 and 5 CAG repeats, was only observed in CAD patients. Thus, we speculated that the CAG repeat sequence is correlated with CAD susceptibility, and the presence of 4 or 5 repeats may be a risk factor for CAD, which was inconsistent with the results obtained by Han et al. (2007). The inconsistency in these results may be explained by the differences in subjects and sample sizes among studies.
Impact of glucocerebrosidase mutations on motor and nonmotor complications in Parkinson’s disease

Homozygous and compound heterozygous mutations in GBA encoding glucocerebrosidase lead to Gaucher disease (GD). A link between heterozygous GBAmutations and Parkinson’s disease (PD) has been suggested ( Bembi et al., 2003,Goker-Alpan et al., 2004, Halperin et al., 2006, Machaczka et al., 1999, Neudorfer et al., 1996, Tayebi et al., 2001 and Tayebi et al., 2003). In 2009, a 16-center worldwide analysis of GBA revealed that heterozygous GBA mutation carriers have a strong risk of PD ( Sidransky et al., 2009).

In addition, heterozygote GBA mutations not only carry a risk for PD development but also the possibility of some risk burden on the progression of PD clinical course. In cross-sectional analyses of GBA mutations in PD patients, earlier disease onset, increased cognitive impairment, a greater family history of PD, and more frequent pain were reported in patients with mutations, compared with no mutations ( Chahine et al., 2013,Clark et al., 2007, Gan-Or et al., 2008, Kresojevic et al., 2015, Lwin et al., 2004, Malec-Litwinowicz et al., 2014, Mitsui et al., 2009, Neumann et al., 2009, Nichols et al., 2009,Seto-Salvia et al., 2012, Sidransky et al., 2009, Swan and Saunders-Pullman, 2013 and Wang et al., 2012). Recently, a few prospective studies have investigated clinical features of PD with GBA and showed a more rapid progression of motor impairment and cognitive decline in GBA mutation cases than in PD controls ( Beavan et al., 2015, Brockmann et al., 2015 and Winder-Rhodes et al., 2013). However, in terms of motor complications such as wearing-off and dyskinesia, no studies exist in the longitudinal course of PD with GBA mutations.

Here, we conducted a multicenter retrospective cohort analysis, and the data were investigated by survival time analysis to show the impact of GBA mutations on PD clinical course. We also investigated regional cerebral blood flow (rCBF) and cardiac sympathetic nerve degeneration of subjects with GBA mutations, compared with matched PD controls.

3.1. Subjects

Among the 224 eligible PD patients (the subjects were not related to each other), 9 subjects were excluded from the analysis (4 due to multiple system atrophy findings on subsequent brain MRI and 5 because of insufficient clinical information). Therefore, 215 PD patients [female, 52.1%; age, 66.7 ± 10.8 (mean ± standard deviation)] were analyzed. For non-PD healthy controls, 126 patients’ spouses (female, 58.7%; age, 67.3 ± 10.3) without a family history of PD or GD were enrolled.

3.2. GBA mutations and risk ratios for PD

In the PD subjects, we identified 10 nonsynonymous and 2 synonymous GBA variants. Within the nonsynonymous variants, 7 mutations were previously reported in GD [R120W, L444P-A456P-V460 (RecNciI), L444P, D409H, A384D, D380N, and444L(1447-1466 del 20, insTG)] as GD-associated mutations. Three nonsynonymous mutations have never been reported in GD patients [I(-20)V, I489V, and there was one novel mutation (Y11H)].

GD-associated GBA mutations were found in 19 of the 215 (8.8%) PD patients but none in the healthy controls. The risk of PD development relative to these GD-associated mutations was estimated as an OR of 25.1 [95% confidence interval (CI), 1.50–420,p = 0.0001] with 0-cell correction. The nonsynonymous mutations that were not reported in GD patients had no association with PD development (p = 0.506; OR, 1.3; 95% CI, 0.7–2.6) ( Table 1). Four subjects had double mutations. For subsequent analyses, 2 subjects with double mutations of I (-20)V and K466K were adopted to the group of mutations unreported in GD, and 2 subjects with double mutations of R120W and I(-20)V, and of R120W and L336L were adopted to the group of GD-associated mutations.

Table 1.Frequency of glucocerebrosidase gene allele in Parkinson’s disease patients and controls

Allele name PD (n = 215) Controls (n = 126) p Odds ratio (95% CI)
GD-associated mutations
 R120W 7a 0 0.050 9.1 (0.5–160.8)
 RecNciI (L444P-A456P-V460) 4 0
 L444P 4 0
 D409H 1 0
 A384D 1 0
 D380N 1 0
444L(1447-1466 del 20, insTG) 1 0
 Subtotal, n (%) 19 (8.8%) 0 (0%) <0.001 25.1 (1.5–419.8)b
Nonsynonymous mutations not reported in GD
 I(-20)V 27a 13 0.603 1.3 (0.6–2.5)
 I489V 3 0
 Y11Hc 0 1
 Subtotal, n (%) 30 (14.0%) 14 (11.1%) 0.506 1.3 (0.7–2.6)
Synonymous, n
 K466K 2a 1
 L336L 1a 0
Allele names refer to the processed protein (excluding the 39-residue signal peptide).

Key: CI, confidence interval; GD, Gaucher disease; PD, Parkinson’s disease.

a Four subjects had double mutations; 2 of I(-20)V and K466K, 1 of I(-20)V and R120W, and 1 of R120W and L336L.
b Odds ratio was calculated by adding 0.5 to each value.
c Novel mutation.
3.3. Clinical features of PD patients by GBA mutation groups

The clinical features of PD patients with GD-associated mutations, those with mutations unreported in GD, and those without mutations are shown in Table 2. In the GD-associated mutation group, females, those with a family history and those with dementia (DSM IV) were significantly more frequent than those in the no-mutation group (p = 0.047, 0.012, and 0.020, respectively). The age of PD onset was lower in patients with GD-associated mutations (55.2 ± 9.9 years ± standard deviation), compared with those without mutations (59.3 ± 11.5), although the statistical difference was not significant. There were no differences in clinical manifestations between subjects with mutations unreported in GD and those without mutations, except for dopamine agonist dosage (p = 0.026) ( Table 2).

Table 2.Epidemiological and clinical features of PD patients with Gaucher disease–associated GBA mutations, those with mutations previously unreported in GD and those without mutations

Variables Total n = 215 Mutation (-) GD-associated mutations

Mutations unreported in GD

167 19a pb 29c pd
Sex Female, n (%) 83 (49.7) 14 (73.7) 0.047 15 (51.7) ns
Age Mean (SD) 67.0 (10.8) 62.2 (10.7) 0.063e 67.5 (11.2) nsf
Disease duration (y) Mean (SD) 7.7 (5.5) 6.9 (4.6) nsf 7.2 (4.9) nsf
Onset age Mean (SD) 59.3 (11.5) 55.2 (9.9) ns 60.3 (11.8) ns
Family history Yes, n (%) 17 (11.0)g 6 (31.6) 0.012 0 (0.0) ns
Dementia (DSM-IV) Yes, n (%) 29 (17.4) 9 (47.4) 0.020 5 (17.2) ns
MMSE Mean (SD) 25.8 (5.4)h 23.3 (7.7) nsf 27.0 (3.4)i nsf
Onset symptom (tremor vs. others) Tremor, n (%) 78 (46.8) 9 (47.4) ns 15 (51.7) ns
Modified H-Y on (<3 vs. ≥3) ≥3, n (%) 82 (49.1) 14 (73.7) 0.042 16 (55.2) ns
UPDRS part 3 Mean (SD) 23.6 (12.2)j 28.5 (13.8) nsf 21.9 (8.7) nsf
Wearing off Yes, n (%) 70 (41.9) 9 (47.4) ns 13 (44.8) ns
Dyskinesia Yes, n (%) 49 (29.3) 8 (42.1) ns 8 (27.6) ns
Mood disorder Yes, n (%) 43 (25.7) 8 (42.1) ns 7 (24.1) ns
Orthostatic hypotension symptom Yes, n (%) 21 (12.6) 5 (26.3) ns 7 (24.1) ns
Psychosis history Yes, n (%) 59 (35.3) 10 (52.6) ns 7 (24.1) ns
ICD history Yes, n (%) 8 (4.8) 1 (5.3) ns 1 (3.4) ns
Stereotactic brain surgery for PD Yes, n (%) 4 (2.4) 0 (0.0) ns 0 (0.0) ns
Agonist LED mg/d Mean (SD) 92.8 (114.2) 72.1 (137.7) nse 163.7 (155.6) 0.026e
Levodopa LED mg/d Mean (SD) 400.7 (184.2) 456.7 (206.9) nsf 369.2 (230.3) nse
Total LED mg/d Mean (SD) 496.4 (233.7) 537.9 (258.9) nsf 525.7 (287.4) nsf
Categorical data were examined by Fisher’s exact test.

Key: DSM-IV, Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition; GBA, glucocerebrosidase gene; GD, Gaucher disease; H-Y, Hoehn and Yahr; ICD, impulse control disorder; LED, levodopa equivalent dose; ns, not significant; MMSE, Mini-Mental State Examination; PD, Parkinson’s disease; SD, standard deviation; UPDRS, Unified Parkinson’s Disease Rating Scale.

a Including a double-mutation subject (with a mutation unreported in GD).
b GD-associated mutations versus mutation (-).
c Two subjects with double mutation, including GD-associated mutations, were assigned to GD-associated mutation group.
d Other mutations versus mutation (-).
e Examined by Student t test after Levene’s test for equality of variances.
f Examined by Mann-Whitney U-test because of non-Gaussian distribution.
g    n = 155 due to 10 missing data.
h    n = 164 due to 3 missing data.
i     n = 28 due to 1 missing datum.
j     n = 165 due to 2 missing data.

3.4. Survival time analyses to develop dementia, psychosis, dyskinesia, and wearing-off

Time to develop clinical outcomes (dementia, psychosis, dyskinesia, and wearing-off) was compared in 19 subjects with GD-associated mutations, 29 with mutations unreported in GD, and 167 without mutation. The median observation time was 6.0 years. The subjects with GD-associated mutations showed a significantly earlier development of dementia and psychosis, compared with subjects without mutation (p < 0.001 and p = 0.017) ( Supplementary Table e-1, Fig. 1A and B). We rereviewed the clinical record of the subject who showed early dementia (defined by DSM IV) ( Fig. 1A) and made sure it did not satisfy the criteria of DLB ( McKeith et al., 2005).

Kaplan–Meier curves of dementia and psychosis in Parkinson's disease (PD) ...
Fig. 1.

Kaplan–Meier curves of dementia and psychosis in Parkinson’s disease (PD) patients with Gaucher disease (GD)-associated glucocerebrosidase gene (GBA) mutations and those without mutations. PD patients with GD-associated GBA mutations and those without GBA mutations were compared to investigate the time taken to develop dementia (A) and psychosis (B). Because of insufficient information in several patients, the numbers in each analysis were different. The patients with and without mutations were 17 and 165 (A), 18 and 165 (B) against a total of 19 and 167. DSM IV, Diagnostic and Statistical Manual of Mental Disorders, revised fourth edition. p-Values were calculated by log-rank tests.

The associations of GBA mutations and these symptoms were estimated as HRs, adjusting for sex and age at PD onset. HRs were 8.3 for dementia (95% CI, 3.3–20.9; p < 0.001) and 3.1 for psychosis (95% CI, 1.5–6.4; p = 0.002). The time until development of wearing-off and dyskinesia complications was not statistically significant, with HRs of 1.5 (95% CI, 0.8–3.1; p = 0.219) and 1.9 (95% CI, 0.9–4.1; p = 0.086) ( Table 3).

Table 3.Hazard ratios of GBA pathogenic mutations for clinical symptoms

Model Clinical feature Hazard ratio 95% CI p
1 Dementia (DSM-IV) 8.3 3.3–20.9 <0.001
2 Psychosis 3.1 1.5–6.4 0.002
3 Wearing-off 1.5 0.8–3.1 0.219
4 Dyskinesia 1.9 0.9–4.1 0.086
Each model was adjusted for sex and age at onset.

Key: CI, confidence interval; DSM-IV; The Diagnostic and Statistical Manual of Mental Disorders part 1IV; GBA, glucocerebrosidase.

Subjects with mutations unreported in GD did not show significant differences in time to develop all 4 outcomes, compared with no mutation subjects. Therefore, subjects with GD-unreported mutations were regarded as subjects without GBA mutations in further analyses.

3.5. rCBF on SPECT in patients with GD-associated GBA mutations

We conducted pixel-by-pixel comparisons of rCBF on SPECT between PD subjects with mutations (cases) and sex-, age-, and disease duration-matched PD subjects without any mutations in GBA (controls). Four controls were adopted for each case (except for a 34-year-old female case who was matched to a control), and in total 12 cases (female 50%, age at SPECT mean ± standard error (SE); 58.9 ± 3.3 years, disease duration at SPECT 7.3 ± 1.5 years) and 45 controls (female 64.4%, age at SPECT mean ± SE; 61.0 ± 1.3 years, disease duration at SPECT 7.1 ± 0.7 years) were analyzed. As a result, a significantly lower rCBF was seen in the cases compared to the controls in the bilateral parietal cortex, including the precuneus ( Fig. 2).

Regional cerebral blood flow in the group with GD-associated mutations compared ...
Fig. 2.

Regional cerebral blood flow in the group with GD-associated mutations compared with the matched Parkinson’s disease group without mutations. Regions with lower regional cerebral blood flow in the group with GD-associated mutations displayed on an anatomic reference map. Abbreviation: GD, Gaucher disease.

3.6. H/M ratios on MIBG scintigraphy in patients with GD-associated GBA mutations

Cardiac MIBG scintigraphy visualizes catecholaminergic terminals in vivo that are reduced as well as brain dopaminergic neurons in PD patients. We also investigated MIBG scintigraphy between 16 cases (female 68.8%, age at examination mean ± SE; 60.2 ± 2.6 years, disease duration at examination 6.2 ± 1.2 years) and sex-, age- and disease duration-matched 61 controls [(63.8 %, age 62.0 ± 1.1 years, disease duration 5.5 ± 0.6 years) (1:4 except for 1 young 34-year-old female case who was matched to a control)]. In the results, both early and late H/M ratios declined in both groups and did not show any significant differences (p = 0.309 and 0.244) ( Supplementary Table e-2).

4. Discussion

4.1. Contributions of GD-associated GBA mutations to the development of PD

In the analysis of 215 PD patients and 126 non-PD controls, we identified 10 nonsynonymous heterozygous GBA mutations, including 1 novel mutation. Among these mutations, 7 were GD-associated, and the patients carrying these mutations represented 8.8% of the PD cohort. No significant association was found between the GD-unreported mutations and PD development, which suggests that only the GD-associated mutations are a genetic risk for PD. According to a worldwide multicenter analysis of 1883 fully sequenced PD patients, 7% of the GD-associated mutations are found in non-Ashkenazi Jewish PD patients ( Sidransky et al., 2009). Although the mutation frequency in the present study was similar to previous results, the OR of GD-associated heterozygous mutations (25.1) was significantly greater than the OR (5.43) of other ethnic cohorts (Sidransky et al., 2009) and was consistent with an OR of 28.0 from a previous Japanese report ( Mitsui et al., 2009). These results, taken together, suggest the possibility thatGBA mutations are at a distinct risk for PD in the Japanese population. However, a larger Japanese cohort study is required to confirm this.

4.2. Cross-sectional clinical figures of PD with GBA mutations

Before the survival time analyses, we investigated clinical features at enrollment between mutation groups. The lower onset age, more frequent family history and dementia, and worse disease severity of PD in patients with GBA mutations, compared with those without mutations, were consistent with previous cross-sectional case-control reports ( Anheim et al., 2012, Brockmann et al., 2011, Chahine et al., 2013, Lesage et al., 2011, Li et al., 2013, Mitsui et al., 2009, Neumann et al., 2009, Seto-Salvia et al., 2012 and Sidransky et al., 2009). In contrast, female-predominance (73.7%, p = 0.047) in patients with mutations observed in the present study is inconsistent ( Neumann et al., 2009 and Seto-Salvia et al., 2012).

4.3. Impact of GBA mutations on the clinical course of PD

To investigate the impact of GBA mutations on the clinical course of PD, a prospective-designed study over a long period is preferred. Although there has been a few longitudinally designed study to date, follow-up clinical data for a median of 6 years of 121 PD cases from a community-based incident cohort was recently reanalyzed; results demonstrate that progression to dementia defined by DSM IV (HR 5.7) and Hoehn and Yahr stage 3 (HR 3.2) are significantly earlier in 4 GBA mutation-carrier patients compared with 117 patients with wild-type GBA ( Winder-Rhodes et al., 2013). A 2-year follow-up clinical report of 28 heterozygous GBA carriers who were recruited from relatives of GD-patients shows slight but significant deterioration of cognition and smelling, compared to healthy controls ( Beavan et al., 2015). Brockmann et al. (2015)assessed motor and nonmotor symptoms including cognitive and mood disturbances for 3 years in 20 PD patients with GBA mutations and showed a more rapid disease progression of motor impairment and cognitive decline in GBA mutation cases comparing to sporadic PD controls. The current long-term retrospective cohort study up to 12 years reinforced these results. It revealed that dementia and psychosis developed significantly earlier in subjects with GD-associated mutations compared with those without mutation, and the HRs of GBA mutations were estimated at 8.3 for dementia and 23.1 for psychosis, with adjustments for sex and PD onset age. In contrast, the results showed no significant difference in developing wearing-off and dyskinesia.

In this study, we also investigated whether GD-unreported mutations affected the clinical course of PD. In both cross-sectional and survival time analyses, the mutations unreported in GD carried no increased burden on clinical symptoms such as dementia, psychosis, wearing-off, and dyskinesia.

4.4. Reduced rCBF in PD with GBA mutations compared with matched PD controls

We found a significantly decreased rCBF, reflecting decreased synaptic activity, in the bilateral parietal cortex including the precuneus, in subjects with GD-associated mutations compared with matched subjects without mutations. The pattern of reduced rCBF was very similar to the pattern of H215O positron-emission tomography that Goker-Alpan (2012) reported, showing decreased resting rCBF in the lateral parietal association cortex and the precuneus bilaterally in GD subjects with parkinsonism (7 subjects with homozygous or compound heterozygous GBA mutations), compared with 11 PD without GBA mutations. Results suggest that PD with heterozygous GBAmutations and GD patients presenting parkinsonism had a common reduced pattern of rCBF. Interestingly, in their study, rCBF in the precuneus—but not in the lateral parietal cortex—correlated with IQ, suggesting that the involvement of the precuneus is critical for defining GBA-associated patterns.

4.5. Reduced cardiac MIBG H/M ratios as well as matched PD controls

We also showed that cardiac MIBG H/M ratios in subjects with GD-associated mutations were lower than the cutoff point for PD discrimination (Sawada et al., 2009), suggesting that postganglionic sympathetic nerve terminals to the epicardium were denervated, as well as in PD without mutations.

4.6. Mechanisms of impact on PD clinical course by GD-associated GBA mutations

Experimental studies suggesting a bidirectional pathogenic loop between α-synuclein and glucocerebrosidase have been accumulated (Fishbein et al., 2014, Gegg et al., 2012, Mazzulli et al., 2011, Noelker et al., 2015, Schondorf et al., 2014 and Uemura et al., 2015). Loss of glucocerebrosidase function compromises α-synuclein degradation in lysosome, whereas aggregated α-synuclein inhibits normal lysosomal function of glucocerebrosidase. The pathogenic loop may facilitate neurodegeneration in GD-associated PD brain, resulting in early development of dementia or psychosis as shown in the present study. Several recent researches propose the possibility that the similar mechanism as in PD with GBA mutations exists even in idiopathic PD brain ( Alcalay et al., 2015, Chiasserini et al., 2015, Gegg et al., 2012 and Murphy et al., 2014). On the other hand, the impacts of GD-associated GBA mutations for the development of motor complications such as wearing-off and dyskinesia were not statistically significant, suggesting other pathophysiological mechanisms in the striatal circuit brought out after long-term therapy especially by l-dopa.

4.7. Limitations

Our study has several limitations. In the design of the study, we assumed that the sample size was 215 (PD patients) for survival time analyses and investigated 224 PD patients. We assumed that the mutation prevalence would be 9.4%, and in fact, we found 19 patients with mutations (8.5%) of the 224 patients. Based on these figures, we estimated the risk ratios of heterozygous GBA mutations for the risk of PD development and PD clinical symptoms as ORs in the cross-sectional multivariate analyses, although the 95% CIs were broad. More of subject numbers will be needed to determine robust risk ratios.

Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort

PLOS   Published: July 14, 2015   DOI:

Brugada syndrome (BrS) was identified as a new clinical entity in 1992 [1]. Six years later, the first genetic basis for the disease was identified, with the discovery of genetic variations inSCN5A [2]. Nowadays, more than 300 pathogenic variations in this first gene are known to be associated with BrS [3]. SCN5A encodes for the α subunit of the cardiac voltage-dependent sodium channel (Nav1.5), which is responsible for inward sodium current (INa), and thus plays an essential role in phase 0 of the cardiac action potential (AP). Genetic variations in this gene can explain around 20–25% of BrS cases [3].

Since BrS was classified as a genetic disease, several other genes have been described to confer BrS-susceptibility [47]. Pathogenic variations have been mainly described in: 1) genes encoding proteins that modulate Nav1.5 function, and 2) other calcium and potassium channels and their regulatory subunits. All these proteins participate, either directly or indirectly, in the development of the cardiac AP. Although the incidence of pathogenic variations in these BrS-associated genes is low [6], it is considered that, among all of them, they could provide a genetic diagnosis for up to an extra 5–10% of BrS cases. Hence, altogether, a genetic diagnosis can be achieved approximately in 35% of clinically diagnosed BrS patients.
Other types of genetic abnormalities have been suggested to explain the remaining percentage of undiagnosed patients. Indeed, multiplex ligation-dependent probe amplification (MLPA) has allowed the detection of large-scale gene rearrangements involving one or several exons ofSCN5A in BrS cases. However, the low proportion of BrS patients carrying large genetic imbalances identified to date suggests that this type of rearrangements will provide a genetic diagnosis for a modest percentage of BrS cases [810].
BrS has been associated with an increased risk of sudden cardiac death (SCD), despite the reported variability in disease penetrance and expressivity [11]. The prevalence of BrS is estimated at about 1.34 cases per 100 000 individuals per year, with a higher incidence in Asia than in the United States and Europe [12]. However, the dynamic nature of the typical electrocardiogram (ECG) and the fact that it is often concealed, hinder the diagnosis of BrS. Therefore, an exhaustive genetic testing and subsequent family screening may prove to be crucial in identifying silent carriers. A large percentage of these pathogenic variation carriers are clinically asymptomatic, and may be at risk of SCD, which is, sometimes, the first manifestation of the disease [13].
In the present work, we aimed to determine the spectrum and prevalence of genetic variations in BrS-susceptibility genes in a Spanish cohort diagnosed with BrS, and to identify variation carriers among relatives, which would enable the adoption of preventive measures to avoid SCD in their families.

Study population 

Table 1. Demographics of the 55 Spanish BrS patients included in the study.

The table shows the demographic characteristics of all the patients included in the study. Numbers in parentheses represent the relative percentages for each condition. T1 ECG refers to Type 1 BrS diagnostic electrocardiogram (ECG), obtained either spontaneously, or after drug challenge. The information regarding both the electrophysiological studies (EPS) and the treatment was not available for all the patients. Two of the patients that didn’t receive any treatment died, and were not taken into account for the calculations of percentages (+2 dead). ICD, intracardiac cardioverter defibrillator.


Table 2. Characteristics of the Spanish BrS patients carrying rare genetic variations.

The table shows the clinical characteristics of the probands who carried rare genetic variations in SCN5A, SCN2B, or RANGRF. All of them are potentially pathogenic except that found in RANGRF, which is of unknown significance (see discussion). All the potentially pathogenic variations (PPVs) that had been previously reported, except p.P1725L and p.R1898C, had been identified in BrS patients. p.P1725L had been associated with Long QT Syndrome and p.R1898C was found in Exome Variant Server with a MAF of 0.0079%. No rare variations were identified in the control population. Patient’s age is expressed in years. Bold identifies the patients carrying variations that had not been described previously. M, male; F, female; S, syncope; ICD, intracardiac cardioverter defibrillator; UK, unknown; EPS, electrophysiological studies (+, positive response;-, negative response; N/P, not performed). The two patients who carried two PPVs each are identified by a and b, respectively.

Sequencing of genes associated with BrS

We performed a genetic screening of 14 genes (SCN5A, CACNA1C, CACNB2, GPD1L,SCN1B, SCN2B, SCN3B, SCN4B, KCNE3, RANGRF, HCN4, KCNJ8, KCND3, and KCNE1L), which allowed the identification of 61 genetic variations in our cohort. Of these, 20 were classified as potentially pathogenic variations (PPVs), one variation of unknown significance, and 40 common or synonymous variants considered benign.

The 20 PPVs were found in 18 of the 55 patients (32.7% of the patients, 83.3% males; Table 2). Sixteen patients (88.9%) carried one PPV, and two patients (11.1%) carried two different PPVs each. Nineteen out of the 20 PPVs identified were localized in SCN5A and one in SCN2B.

The vast majority of the PPVs identified were missense (70%). We also detected 2 nonsense variations (10%), 3 insertions or deletions causing frameshifts (15%), and one splicing variation (5%). The three frameshifts (p.R569Pfs*151, p.E625Rfs*95 and p.R1623Efs*7) were identified in SCN5A. These were not found in any of the databases consulted (see Methods), and were thus considered potentially pathogenic (see below). The other 16 rare variations identified inSCN5A had been previously described, and hence were also considered potentially pathogenic. Fourteen of them had been identified in BrS patients. Of these, 6 had also been identified in individuals diagnosed with other cardiac electric diseases (i.e. Sick Sinus Syndrome, Long QT Syndrome, Sudden Unexplained Nocturnal Death Syndrome or Idiopathic Ventricular Fibrillation [2,15,16,20,21,25]). The other 2, p.P1725L and p.R1898C, had only been associated with Long QT Syndrome or found in Exome Variant Server with a MAF of 0.0079%, respectively. Furthermore, we identified a variation in SCN2B (c.632A>G in exon 4 of the gene, resulting in p.D211G) which was considered pathogenic. This patient was included within our cohort, but the functional characterization of channels expressing SCN2B p.D211G was object of a previous study from our group [7]. We also identified a nonsense variation in RANGRFwhich has been formerly reported as rare genetic variation of unknown significance [29].

Additionally, we screened the relatives of those probands carrying a PPV. We analysed a total of 129 relatives, 69 of which (53.5%) were variation carriers. Genotype-phenotype correlations evidenced that 8 of the families displayed complete penetrance (S3 Table). Additionally, no relatives were available for one of the probands carrying a PPV, thus hampering genotype-phenotype correlation assessment. The other 12 families showed incomplete penetrance.


MLPA analysis

The 37 patients with negative results after the genetic screening of the 14 BrS-associated genes underwent MLPA analyses of SCN5A. This technique did not reveal any large exon deletion or duplication in this gene for any of the patients.


SCN5A p.R569Pfs*151 (c.1705dupC), a novel PPV

A 41-year-old asymptomatic male presented a type 3 BrS ECG which was suggestive of BrS. Flecainide challenge unmasked a type 1 BrS ECG (Fig 1A, left), which was also spontaneously observed sometimes during medical follow up. Sequencing of SCN5A revealed a duplication of a cytosine at position 1705 (c.1705dupC; Fig 1A, right), which originated a frameshift that lead to a truncated Nav1.5 channel (p.R569Pfs*151). The proband’s sister also carried this duplication, but had never presented signs of arrhythmogenesis. The proband’s twin daughters were also variation carriers, displayed normal ECGs and, to date, are asymptomatic (Fig 1A, middle). Thus, p.R569Pfs*151 represents a novel genetic alteration in the Nav1.5 channel that could potentially lead to BrS, but with incomplete penetrance.


Fig 1. Characteristics of the probands carrying non-reported potentially pathogenic variations (PPVs) in SCN5A and their families.

Left: Electrocardiograms of the probands: (A) patient carrying the p.R569Pfs*151 variation, showing the ST elevation characteristic of BrS in V1 at the time of the flecainide test; (B) patient carrying the p.E625Rfs*95 variation, showing the spontaneous ST elevation characteristic of BrS in V1 and V2; and (C) patient carrying the p.R1623Efs*7 variation, showing the spontaneous ST elevation characteristic of BrS in V1 and V2. Middle: Family pedigrees. Open symbols designate clinically normal subjects, filled symbols mark clinically affected individuals and question marks identify subjects without an available clinical diagnosis. Plus signs indicate the carriers of the PPVs and minus signs, non-carriers. The crosses mark deceased individuals and arrows identify the proband. Right: Detail of the electropherograms obtained after SCN5Asequence analysis of a control subject (left panels) and of the probands (right panels).

SCN5A p.E625Rfs*95 (c.1872dupA), a novel PPV

A 51-year-old asymptomatic male was diagnosed with BrS since he presented a spontaneous ST segment elevation in leads V1 and V2 characteristic of type 1 BrS ECG (Fig 1B, left). The sequencing of SCN5A evidenced an adenine duplication at position 1872 (c.1872dupA, Fig 1B, right). This genetic variation results in a truncated Nav1.5 channel (p.E625Rfs*95). The genetic analysis of the proband’s relatives proved that only her mother carried the variation (Fig 1B, middle). She was asymptomatic, but a BrS ECG was unmasked upon ajmaline challenge. The proband’s sister was found dead in her crib at 6 months of age, which suggests that her death might be compatible with BrS. Therefore, the p.E625Rfs*95 variation in the Nav1.5 channel represents a novel genetic alteration potentially causing BrS.

SCN5A p.R1623Efs*7 (c.4867delC), a novel PPV

The proband, a 31-year-old male, was admitted to hospital after suffering a syncope. His baseline 12-lead ECG showed a ST segment elevation in leads V1 and V2 that strongly suggested BrS type 1 (Fig 1C, left). A deletion of the cytosine at position 4867 (c.4867delC) was observed upon SCN5A sequencing (Fig 1C, right). This base deletion leads to a frameshift that originates a truncated Nav1.5 channel (p.R1623Efs*7). Genetic screening of his parents and sisters evidenced that none of them carried this novel variation (Fig 1C, middle). None of them had presented any signs of arrhythmogenicity, nor had a BrS ECG. Nevertheless, in uterogenetic analysis of one of his daughters proved that she had inherited the variation. She died when she was 1 year of age of non-arrhythmogenic causes. Hence, the p.R1623Efs*7 variation in the Nav1.5 channel is a novel genetic alteration originated de novo in the proband that could potentially lead to BrS.

Synonymous and common genetic variations portrayal

In our cohort, we identified 40 single nucleotide variations which were common genetic variants and/or synonymous variants (S2 Table). Twenty-nine had a minor allele frequency (MAF) over 1%, and were thus considered common genetic variants.

We also identified 11 variants with MAF less than 1%. Of them, 9 were synonymous variants, what made us assume that they were not disease-causing. Four of these synonymous variants were not found in any of the databases consulted, and thus their MAF was considered to be less than 1%. Each of these synonymous variations was identified in 1 patient of the cohort. A similar proportion of individuals carrying these novel variations was detected upon sequencing of 300 healthy Spanish individuals (600 alleles). The remaining 2 variants were missense, and although they had either a MAF of less than 1% or an unknown MAF according to the Exome Variant Server and dbSNP websites, they were common in our cohort (29.2 and 50%, respectively; S2 Table), and a similar MAF was detected in a Spanish cohort of healthy individuals (26.7% and 48.8%, respectively).

Influence of phenotype and age on PPV discovery

To assess if a connection existed between the probands’ phenotype and the PPV detection yield, we classified the patients in our cohort according to their ECG (spontaneous or induced type 1), the presence of BrS cases within their families, and the presence/absence of symptoms. Even though the overall PPV detection yield was 32.7%, it was even higher for symptomatic patients (Fig 2). Indeed, in this group of patients, having a family history of BrS was identified as a factor for increased PPV discovery yield. In the case of absence of BrS in the family, the variation discovery yield was almost double for those patients having a spontaneous type 1 BrS ECG than for patients with drug-induced type 1 ECG (45.5% vs 25%, respectively). In addition, we identified a PPV in 44.4% of the asymptomatic patients who presented family history of BrS and a spontaneous type 1 BrS ECG. When the patient presented drug-induced type 1 ECG or in the absence of family history of BrS, the PPV discovery yield was of around 15%.


Fig 2. Influence of the phenotype on PPV discovery yield.

Bar graph comparing the PPV detection yield in 8 different clinical categories (stated below the graph). Each bar shows the total number of patients for each clinical category divided in those with a PPV (black) and those without an identified PPV (white). The number of patients (in brackets) and percentages are given. Pos, positive; Neg, negative; Spont, spontaneous type 1 BrS ECG; Drug, drug-induced type 1 BrS ECG; n, number of patients.

We also investigated the role of age on the PPV occurrence. No significant age differences were observed between variation carriers and non-carriers (38.6±10.3 and 43.5±14.4, respectively, p = 0.16). However, the PPV discovery yield was higher for patients with ages between 30 and 50 years: out of the total of patients carrying a PPV, 83.3% of the patients were in this age range, while 11.1% were younger and 5.6% were older patients (Fig 3A, upper panel). The PPV discovery yield was significantly higher for symptomatic than for asymptomatic patients (42.3% vs 24.1%, respectively; Fig 3A, lower panels).


Fig 3. Influence of the age on PPVs discovery yield.

(A) Pie charts showing the distribution of patients in the overall population as well as in the categories of symptomatic and asymptomatic patients regarding PPV discovery. The percentage and the number of patients (in brackets) are given for each group. The small pie charts correspond to the age distribution of patients with an identified PPV. (B) Bar graphs of the PPV detection yields obtained for each of the age groups (< 30 years, 30–50 years and > 50 years). Numbers inside each bar correspond to the number of patients carrying a PPV for each category and the percentages represent the variation detection yield.

Noteworthy, in the 30–50 age range, 52.9% (9/17) of the symptomatic patients and 35.3% (6/17) of asymptomatic patients carried one PPV (Fig 3B, middle). Additionally, 40% (2/5) of the symptomatic young patients (< 30 years) were variation carriers, while no PPVs were identified in asymptomatic patients within this age range.

Overall, 55 unrelated Spanish patients clinically diagnosed with BrS were included in our study.Table 1 shows the demographics of this cohort, and Table 2 and S1 Table show the clinical and genetic characteristics of all the patients included in the study. The mean age at clinical diagnosis was of 41.9±13.3 years. Although the majority of patients were males (74.5%), their age at diagnosis was not different than that of females (41.8±12.1 years and 42.3±16.3 years, respectively; p = 0.92). A type 1 BrS ECG was present spontaneously in 37 patients (67.3%), and drug challenge revealed a type 1 BrS ECG for the remaining 18 patients (32.7%). Almost half of the patients had experienced symptoms, including 2 SCD and 4 aborted SCD. Patients who had not previously experienced any signs of arrhythmogenicity despite having a BrS ECG were considered asymptomatic. Comparison of symptomatic vs asymptomatic patients evidenced a similar percentage of males (73.1% and 75.9%, respectively). However, the mean age at diagnosis was different between the two groups of patients (37.7±14.3 and 45.7±11.4, respectively; p<0.05).


To the best of our knowledge, this is the first comprehensive genetic evaluation of 14 BrS-susceptibility genes and MLPA of SCN5A in a Spanish cohort. Well delimited BrS cohorts from Japan, China, Greece and even Spain have been genetically studied [24,3032]. Additionally, an international compendium of BrS genetic variations identified in more than 2100 unrelated patients from different countries was published in 2010 [3]. However, all these studies screenedSCN5A exclusively. In 2012, Crotti et al. reported the spectrum and prevalence of genetic variations in 12 BrS-susceptibility genes in a BrS cohort [5]. However, this study included patients of different ethnicity. Here, we report the analysis of 14 genes which has been conducted on a well-defined BrS cohort of the same ethnicity.

Our results confirm that SCN5A is still the most prevalent gene associated with BrS. Indeed,SCN5A-mediated BrS in our cohort (30.9%) is higher than the proportion described in other European reports [3,23], where a potentially causative variation is identified in only 20–25% of BrS patients. The reason for this discrepancy is unclear but could point towards a higher prevalence of SCN5A PPVs in the Spanish population or to selection bias. Additionally, we identified a genetic variation in SCN2B (c.632A>G, which results in p.D211G). We have formerly published the comprehensive electrophysiological characterization of this variation, and showed that indeed this variation could be responsible of the phenotype of the patient, thus linking SCN2B with BrS for the first time [7]. Also, we identified a variation in RANGRF. This variation (c.181G>T leading to p.E61X) had been previously reported in a Danish atrial fibrillation cohort [33]. Surprisingly, the authors reported an incidence of 0.4% for this variation in the healthy Danish population, which brought into question its pathogenicity. Our finding of this variation in an asymptomatic patient displaying a type 2 BrS ECG also points toward considering it as a rare genetic variation with a potential modifier effect on the phenotype but not clearly responsible for the disease [29].

No PPVs were identified in the other genes tested. Certainly, it is well accepted that the contribution of these genes to the disease is minor, and thus should only be considered under special circumstances [13,34]. In addition, recent studies have questioned the causality of variations identified in some of these minority genes [35].

We also used the MLPA technique for the detection of large exon duplications and/or deletions in SCN5A in patients without PPVs, and no large rearrangements were identified. This is in accordance with previous reports, which revealed that such imbalances are uncommon [810].

Kapplinger et al. [3] reported a predominance of PPVs in transmembrane regions of Nav1.5. Indeed, it has been proposed that most rare genetic variations in interdomain linkers may be considered as non-pathogenic [36]. In contrast, PPVs identified in this study are mainly located in extracellular loops and cytosolic linker regions of Nav1.5 (Fig 4). Additionally, 2 of our non-previously reported frameshifts are located in the DI-DII linker. These 2 genetic variations lead to truncated proteins, which would lack around 75% of the protein sequence, and thus are presupposed to be pathogenic.


Fig 4. Nav1.5 channel scheme showing the relative position of the SCN5A PPVs identified in our cohort.

Open symbols indicate already described variations and closed symbols locate novel variations reported in this study. DI to DIV designate the 4 domains of the protein, and numbers 1–6 identify the different segments within each domain. Crosses mark the voltage sensor.

In our cohort, we have identified 40 synonymous or common genetic variations, 4 of which have not been previously reported. These variations are gradually becoming more and more important in the explanation of certain phenotypes of genetic diseases. Only a few common variations identified here are already published as phenotypic modifiers [37,38]. The effect of these and other common variants identified in our cohort on BrS phenotype should be further studied.

Unexpectedly, almost 40% (7/18) of the PPV carriers did not present signs of arrhythmogenicity. We also performed genotype-phenotype correlations of the PPVs identified in the families (S3 Table). These studies uncovered relatives, most of whom were young individuals, who carried a familial variation but had never exhibited any clinical manifestations of the disease. This is in agreement with Crotti et al. and Priori et al. [5,23], who postulated that a positive genetic testing result is not always associated with the presence of symptoms. Indeed, the existence of asymptomatic patients carrying genetic variations described to cause a severe Nav1.5 channel dysfunction has been reported [39]. The identification of silent carriers is of paramount importance since it allows the adoption of preventive measures before any lethal episode takes place. Unknown environmental factors, medication and modifier genes have been suggested to influence and/or predispose to arrhythmogenesis [11]. Hence, this group of patients has to be cautiously followed in order to avoid fatal events.

Our studies on the connection between patients’ phenotype and the PPV detection yield highlighted the presence of symptoms as a factor for an increased variation discovery yield. Within the group of symptomatic individuals, a PPV was identified in a higher proportion of patients displaying a spontaneous type 1 BrS ECG than for patients showing a drug-induced ECG. Likewise, within the asymptomatic patients with family history of BrS, those who presented spontaneous type 1 BrS ECG carried a PPV more often than those with a drug-induced ECG (Fig 2). Referring to age, the vast majority (17/20, 85%) of the PPVs were identified in patients around their fourth decade of age (30–50 years). This is in accordance with the accepted mean age of disease manifestation. Moreover, in this age range, more than 50% of the patients who presented symptoms carried a variation that could be pathogenic (Fig 3). Importantly, 35.3% of asymptomatic patients of around 40 years of age also carried one of such variations. These data highlight the importance of performing a genetic test even in the absence of clinical manifestations of the disease, and particularly when in the 30–50 years range, which is in accordance with consensus recommendations [13,34].

In conclusion, we have analysed for the first time 14 BrS-susceptibility genes and performed MLPA of SCN5A in a Spanish BrS cohort. Our cohort showed male prevalence with a mean age of disease manifestation around 40 years. BrS in this cohort was almost exclusivelySCN5A-mediated. The mean PPV discovery yield in our Spanish BrS patients is higher than that described for other BrS cohorts (32.7% vs 20–25%, respectively), and is even higher for patients in the 30–50 years age range (up to 53% for symptomatic patients). All these evidences support the genetic testing, at least of SCN5A, in all clinically well diagnosed BrS patients.


Study Limitations

First of all, drug challenge tests were not performed for all the relatives who were asymptomatic variation carriers. This fact hampered their clinical diagnosis and represents an impediment to definitely assess the link between PPVs and BrS. These patients are nowadays under follow-up.

New PPVs have been identified in our cohort. The clinical information available for the families suggests that these new variations could be pathogenic. Still, in vitro studies of these variations are required in order to evaluate their functional effects and verify their pathogenic role. Additionally, genotyping in an independent cohort would help reduce the likelihood of type I (false positive) error in genetic variant discovery.

We have to acknowledge that the study set is relatively small. Consequently, the classification of patients according to the different clinical categories rendered rather small sub-groups, which may lead to over-interpretation of the results. Future studies will be directed to the genetic screening of additional Spanish BrS patients, which will probably reinforce the significance of the tendencies observed here.

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 Rituximab for a Variety of B-cell Malignancies

Curator: Larry H Bernstein, MD, FCAP



Impact of Rituximab (Rituxan) on the Treatment of B-Cell Non-Hodgkin’s Lymphoma

Non-Hodgkin’s lymphoma (NHL) is the most common hematological malignancy in adults, with B-cell lymphomas accounting for 85% of all NHLs. The most substantial advancement in the treatment of B-cell malignancies, since the advent of combination chemotherapy, has been the addition of the monoclonal anti-CD20 antibody rituximab (Rituxan). Since its initially reported single-agent activity in indolent lymphomas in 1997, the role of rituximab has expanded to cover both indolent and aggressive lymphomas.

This article focuses on the impact of rituximab on the treatment, survival, and long-term outcomes of patients with indolent and aggressive lymphomas over the past two decades.

Keywords: rituximab, non-Hodgkin’s lymphoma
Non-Hodgkin’s lymphoma (NHL) is the most common adult hematological cancer. In 2009, almost 66,000 cases were anticipated in the U.S. alone.1The incidence of NHL in the U.S. over the previous 15 years has increased by approximately 4% annually, despite the decline in age-adjusted incidence rates for all cancers combined.

NHL encompasses a heterogeneous group of lymphomas that have been classified in various ways. In 1995, the World Health Organization developed a classification that included a combination of morphology, immunotyping, genetic features, and clinical syndromes. The goal was to define disease entities of B cells, T cells, and natural killer (NK) cells that pathologists could recognize and that had clinical relevance. The lymphomas were further subdivided into categories based on their clinical behavior (indolent, aggressive, or highly aggressive).2 More recent updates of this classification have clarified some less common entities but have left the overall schema intact.3

B-cell lymphomas account for about 85% of all NHL diagnoses.4 Although many subtypes of NHL exist clinically, most are grouped as either indolent (characterized by a prolonged median survival but generally considered incurable) or aggressive (characterized by rapid growth but with the potential for cure). Because patients with indolent lymphoma eventually die with this disease if they do not die of intercurrent illness, new treatments are needed to prolong survival, with the ultimate goal to provide cure. For patients with aggressive lymphoma, unmet needs include higher initial cure rates, improved salvage chemotherapy options, and less toxic therapies for old and frail patients.

Conventional methods of treatment, including chemotherapy and radiation, are associated with toxicity and lack specific antitumor-targeted activity. Cell–surface proteins, such as CD19, CD20, and CD22, are highly expressed on B-cell lymphomas and represent key potential targets for treatment.

Antibody therapy directed against CD20 has had the most important clinical impact to date. CD20 is thought to be involved in the regulation of intracellular calcium, cell cycle, and apoptosis. CD20 is not shed, modulated, or internalized significantly upon antibody binding, thus making it an ideal target for passive immunotherapy.5

Over the past two decades, significant progress has been made in the development of new therapies for B-cell lymphoma. Perhaps the most important advance is the addition of rituximab (Rituxan, Genentech/Biogen Idec), which the FDA approved for use in the U.S. in 1997. Rituximab is a chimeric (mouse and human) monoclonal antibody directed against the B-cell antigen CD20. It depletes B cells by several mechanisms, including direct antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cell death, and signaling apoptosis.611

Phase 1 trials of two doses of rituximab (500 mg/m2 and 375 mg/m2 for four weeks) showed clinical responses with no dose-limiting toxicity.12 The weekly 375-mg/m2 dose, given for four weeks, was selected for further phase 2 evaluation and is currently the standard single-agent dose and schedule. Since this first reported activity, the role of rituximab has expanded to include both indolent and aggressive lymphomas.

This article addresses the effect of rituximab on survival and long-term outcomes in patients with NHL.

Indolent, Low-Grade Lymphomas

Unlike aggressive lymphomas, indolent B-cell lymphomas are not considered curable with conventional therapies. Many patients are observed for prolonged periods without requiring treatment.13 In one study, more than 50% of the patients remained untreated for a median period of almost six years after diagnosis.14 Treatment goals focus on maintaining good quality of life with minimal symptoms. The indications for treatment include the presence of B symptoms (fevers, night sweats, and weight loss), compromise of normal organ function, bulky disease, or the presence of cytopenias resulting from marrow involvement. Transformation to an aggressive histological pattern warrants treatment for the aggressive component.

Although many active therapies are available for indolent NHL, patients ultimately die of this disease, which is incurable. Additional therapeutic options with improved efficacy and reduced toxicity are still needed for patients with indolent NHL. In light of this unmet need, the FDA’s approval of rituximab for the treatment of relapsed or refractory CD20-positive (CD20+) NHL in 1997 was an important clinical advance. The approval was based on the pivotal trial reported by McLaughlin et al., in which single-agent rituximab brought about significant response rates in heavily pretreated patients with indolent lymphoma.15

Initial Therapy for Indolent (Follicular) Lymphoma

Rituximab as first-line therapy has been widely studied in patients with indolent lymphomas, both as a single agent and in combination with conventional chemotherapy (Table 1). Witzig et al. evaluated the use of single-agent rituximab, 375 mg/m2 weekly for four doses, as an initial therapy for patients with stage III or IV grade 1 follicular lymphoma (FL). In this small phase 2 trial of only 37 patients, the reported objective response rate (ORR) was 72% and the complete remission rate (CRR) was 36%.16 Similarly, a phase 2 study by Hainsworth et al., which evaluated initial therapy in patients with indolent lymphomas, showed response rates in the range of 50%.17

Table 1

Randomized Trials Using Rituximab in First-Line and Relapsed Settings in Indolent Lymphomas

Using rituximab as a first-line therapy in patients with low-tumor-burden, indolent NHL, Colombat et al. reported an ORR of 73%.18 Long-term follow-up results of the completed randomized phase 3 Rituximab ExtendedSchedule Or Re-treatment Trial (RESORT, ECOG 4402 [Eastern Cooperative Oncology Group]) are still pending. In this randomized study, patients received four weekly rituximab treatments. Retreatment is then given as a single dose of rituximab every three months or upon disease progression with four weekly doses. The aim of the study is to define the benefit of maintenance therapy or re-treatment with rituximab (in terms of time to requiring a therapy other than rituximab) when progressive disease is documented.

The benefit of adding rituximab to combination chemotherapy during the initial treatment of FL has been documented in multiple clinical trials over the past decade. The phase 3 trial by Marcus et al. compared cyclophosphamide, vincristine, and prednisone (CVP), with and without rituximab, in 318 previously untreated patients with stage III and IV CD20+ FL.19 The addition of rituximab to CVP (R-CVP) significantly improved time to disease progression (34 months with R-CVP vs. 15 months with CVP, respectively; P < 0.0001) and duration of response (38 months vs. 14 months, respectively; P < 0.0001). Disease-free survival was 21 months with CVP but has not yet been determined in the group receiving R-CVP.

The East German Study Group evaluated the combination of rituximab with mitoxantrone, chlorambucil, and prednisone (MCP), followed by maintenance interferon in treatment-naive patients with stage III/IV CD20+ FL.20 The ORR was 92% with rituximab and 75% with chemotherapy alone (P = 0.0009).

Rituximab was also tested in combination with cyclophosphamide, hydroxydaunorubicin (doxorubicin), Oncovin (vincristine), and prednisone (R-CHOP) as first-line therapy in 428 patients with FL in a randomized phase 3 study with three years of follow-up.21 This combination showed a significant prolongation of time to treatment failure (P < 0.001) and prolonged duration of remission (P = 0.001) with the addition of rituximab. A higher ORR was observed in the group receiving R-CHOP (96%), compared with CHOP alone (90%) (P = 0.011). Even with a short follow-up, overall survival rates improved in the group receiving chemotherapy and rituximab (P = 0.016).

Similar results were seen in the GELA–GOELAMS FL 2000 trial (Groupe d’Etude des Lymphomes de l’Adulte/Groupe Ouest Est des Leucémies etAutres Maladies du Sang). This study was designed to examine the combination of rituximab with cyclophosphamide, hydroxydaunorubicin (doxorubicin), etoposide (VP-16), and prednisolone (CHVP) plus interferon-2 .22 A significant improvement in event-free survival at five years was noted for the rituximab patients (37% vs. 53%, respectively; P = 0.0004).

A meta-analysis of seven randomized controlled trials assessed the value of adding rituximab to conventional chemotherapy for 1,943 patients with FL, mantle-cell lymphoma, and other indolent lymphomas.23 This analysis demonstrated improved overall survival with the combination, as follows:

  • hazard ratio (HR) for mortality, 0.65
  • 95% confidence interval (CI), 0.51–0.79
  • disease control (HR for the disease event, 0.62; 95% CI, 0.55–0.71)
  • response rates (relative risk for response 1.21; 91% CI, 1.16–1.27)

Specifically in FL, overall survival was better with rituximab plus chemotherapy (HR for mortality, 0.60; 95% CI, 0.37–0.98).23 The study authors concluded that the combination of rituximab and chemotherapy for patients with indolent lymphomas was superior to chemotherapy alone with respect to overall survival, disease-free survival, and response rates.23

Relapsed/Refractory Indolent Non-Hodgkin’s Lymphoma

The pivotal trial upon which the initial approval of rituximab was based showed the drug’s efficacy as a single agent in relapsed/refractory indolent NHL.15 Re-treatment with rituximab alone in 57 patients with low-grade FL who had previously responded to single-agent rituximab yielded a response rate of 40% and a similar duration of response, indicating sensitivity to re-treatment with the same agent.24

Davis et al. studied the use of single-agent rituximab in patients with bulky lesions (larger than 10 cm) and relapsed NHL.25 Patients receiving rituximab 375 mg/m2 weekly for four doses had an ORR of 43%. Among patients with a partial response, lesion size decreased by 76%.

The addition of rituximab to standard chemotherapy was found to be beneficial in the treatment of FL patients with relapsed/refractory NHL (see Table 1). An international trial by van Oers et al. evaluated the combination of six cycles of CHOP with rituximab 375 mg/m2 given intravenously on day 1 of each cycle, compared with chemotherapy alone in 465 patients with advanced disease.26 The ORR was higher with the addition of rituximab (85% with R-CHOP vs. 72% with CHOP alone; P < 0.001), and the median progression-free survival rate was also significantly improved in the rituximab group (33.1 vs. 20 months; P < 0.001). The addition of rituximab to the combination of fludarabine, cyclophosphamide, and mitoxantrone (FCM) in a similar group of patients also showed superior responses.27

Rituximab with bendamustine (Treanda, Cephalon) was studied in a phase 2 trial in patients with relapsed disease. This combination was found to be very effective, with an ORR of 92%.28

Maintenance Therapy for Follicular Lymphoma

Some authors consider rituximab to be an ideal medication to use as maintenance therapy for an incurable disease such as FL because of its minimal toxicity and long half-life, which obviates the need for frequent administration.29 The use of rituximab as maintenance therapy after induction treatment has been the subject of several studies (Table 2) and is being evaluated by two large phase 3 trials: Primary Rituximab and Maintenance (PRIMA) and RESORT.30,31

Table 2

Trials Showing Benefit for Maintenance Rituximab after Induction Therapy for Indolent Lymphomas

In the PRIMA trial, patients with previously untreated FL requiring therapy received a rituximab–chemotherapy regimen designated by the participating center as R-CHOP, R-CVP, or R-FCM. Responding patients were then randomly assigned to receive observation or scheduled re-treatment with rituximab as a single dose every eight weeks for two years. The RESORT trial was designed for asymptomatic patients with low-tumor-burden, indolent NHL (as discussed in detail on page 149).

The Swiss Group for Clinical Cancer Research (SAKK) evaluated maintenance rituximab following induction with rituximab monotherapy in a phase 3 trial in patients with newly diagnosed NHL and in previously treated patients with FL.32 In this study, the maintenance schedule consisted of four infusions at two-month intervals. Event-free survival was significantly longer among patients who received this maintenance schedule.

A phase 2 trial by the Minnie Pearl Cancer Research Network compared maintenance rituximab (four weekly doses repeated every six months for two years) with re-treatment using rituximab upon disease progression.33The study showed significant prolongation of progression-free survival in the maintenance therapy group (31.3 vs. 7.4 months, respectively; P = 0.007), although no difference in overall survival or duration benefit from rituximab was observed between the two cohorts.

ECOG 1496 was a study that compared the use of maintenance rituximab with observation after induction with a non-rituximab chemotherapy regimen (CVP) in 282 patients with newly diagnosed FL.34 Improvement in progression-free survival at three years (68% with rituximab vs. 33% with CVP; P < 0.001) and overall survival at three years (91% vs. 86%, respectively; P = 0.08) were noted in the maintenance arm. In the relapsed setting, the prolonged use of rituximab was found to be beneficial with improved progression-free survival when it was used after CHOP or R-CHOP and after treatment with R-FCM.26,35

Although significant evidence exists for the improved progression-free survival with the use of maintenance therapy for FL, the benefit in terms of overall survival is still controversial. Moreover, because different dosing schedules were used in these studies, no data are available for the optimal dosing schedule of maintenance therapy and the recommended duration of this treatment.

Rituximab plus Chemotherapy: Effect on Survival In Follicular Lymphoma

There is no doubt that the clinical development of rituximab has been a significant breakthrough in the field of indolent lymphomas. However, its effect on overall survival in this group of patients is still open to debate.

An analysis of survival in patients 15 years of age and older with NHL diagnosed between 1990 and 2004, using data from the Surveillance, Epidemiology and End Results (SEER) program, revealed a markedly improved outcome for patients with NHL in recent years. This finding may be related, in part, to the addition of rituximab.36

A large retrospective analysis by Swenson et al. was conducted to examine survival rates of 14,564 patients with FL diagnosed between 1978 and 1999 in the U.S.37 Improvement in survival was noted over the past 25 years, and a reduction in the relative risk of death by 1.8% per year was observed from 1983 to1999.

In a second analysis, the Southwest Oncology Group (SWOG) looked at the survival of patients with FL on three large randomized clinical trials between 1974 and 2000.38 Overall survival rates improved over this period of time. The greatest improvement was observed with the most recent treatment approach consisting of CHOP with an anti-CD20 monoclonal antibody.

The study by Marcus et al., published in 2008, showed improved survival rates among untreated patients receiving CVP plus rituximab when compared with CVP alone (four-year survival, 83% vs. 77%, respectively; P= 0.029).19 Other studies, including a Cochrane meta-analysis, have shown similar trends toward improved survival.23,26,34

These observations can be attributed to multiple factors, including improved supportive care measures, enhancements in education of physicians and patients, and better treatments of relapsed and transformed cases.39 Despite these uncertainties regarding its effect on overall survival, it is clear that rituximab has substantially advanced the treatment of indolent lymphomas in the last decade.

Diffuse Large B-Cell Lymphoma

As the most common high-grade form of NHL, diffuse large B-cell lymphoma (DLBCL) accounts for more than 30% of new diagnoses. The median age of presentation is 60 years. Unlike indolent lymphomas, DLBCL is an aggressive lymphoma; if it is untreated, survival can be measured in months. More than 70% of patients with DLBCL present at an advanced stage, and systemic chemotherapy is the foundation of treatment. Since its development in the 1970s, CHOP has been the mainstay of treatment for this group of patients.

A milestone phase 3 trial found that complex regimens that included the addition of other chemotherapy agents to CHOP did not demonstrate any significant difference in overall survival, disease-free survival, or remission rates over CHOP.4043 Moreover, CHOP was associated with significantly less toxicity and cost.

Based on these results, CHOP remained the gold standard of therapy for DLBCL. Nonetheless, long-term remission occurred in only about 45% of patients, so that more than half of patients relapsed with the best therapy possible in the early 1990s. A relatively small percentage of relapsed DLBCL patients (25%–50%) might have been “salvaged” with high-dose chemotherapy and stem-cell support, yet many patients were not even eligible for such therapy.

Thus, in the early 1990s, the addition of more chemotherapy drugs into complex regimens had not improved results with CHOP, and there was a sense that future improvements in therapy would not come from additional “standard” drugs. While rituximab was approved for treatment of low-grade lymphoma in 1997, several trials combining rituximab with CHOP (R-CHOP) for aggressive lymphomas began prior to that time. Because rituximab-related toxicities were not overlapping with those of CHOP, both CHOP and rituximab could be administered at full doses. Results from large international, randomized trials have demonstrated the significant benefits of the addition of rituximab to standard chemotherapy for DLBCL. These trials are summarized next.

Previously Untreated Diffuse Large B-Cell Lymphoma

Based on the efficacy of rituximab in low-grade lymphomas, Vose et al. conducted a phase 2 study of rituximab with CHOP chemotherapy in 33 previously untreated patients with advanced-stage, aggressive B-cell lymphoma.44 Rituximab at a dose of 375 mg/m2 was administered on day 1 of each of six cycles of CHOP. The ORR was 94%; 61% of patients had complete responses (CRs), and 33% had partial responses (PRs). This was the first report that demonstrated an improved efficacy of the combination without worsening toxicity.

GELA investigators randomized previously untreated elderly patients (60–80 years of age) to eight cycles of CHOP alone (197 patients) or eight cycles of R-CHOP given on day 1 of each cycle (202 patients).45 The rate of CRs was significantly higher in the rituximab group (76% vs. 63% receiving CHOP alone, P = 0.005). Sixty percent of patients exhibited features of poor risk, with age-adjusted International Prognostic Index (aaIPI) scores of 2 to 3. With a median follow-up of two years, event-free survival rates (57% vs. 38%; P < 0.001) and overall survival rates (70% vs. 57%; P = 0.007) were significantly higher with rituximab (Table 3). Furthermore, toxicity was not greater with the addition of rituximab.

Table 3

Trials Using Rituximab for Diffuse Large B-Cell Lymphomas in the First-Line Setting

A long-term analysis at seven years has confirmed the benefit of the addition of rituximab.46 Event-free survival (42% with R-CHOP vs. 25%; P < 0.0001), progression-free survival (52% vs. 29%, respectively; P < 0.0001) and disease-free survival (66% vs. 42% respectively, P = 0.0001) were all statistically better for patients treated with combination therapy.

A retrospective analysis of the GELA trial suggested that R-CHOP increased overall survival preferentially in bcl-2–positive patients compared with CHOP alone.47 These data suggested that rituximab may overcome chemotherapy resistance associated with bcl-2 in patients with DLBCL. However, other retrospective analyses have led to conflicting results on whether the benefit of R-CHOP is primarily or only observed in bcl-2expressing DLBCL.

Habermann et al. randomly assigned patients older than 60 years of age to receive CHOP or R-CHOP, with a second random assignment to maintenance rituximab therapy or observation in responders (see Table 3).48This study demonstrated the benefit of the addition of rituximab to CHOP using a modified schedule of rituximab administration. Three-year failure-free survival rates were 53% and 46% (P = 0.04). Failure-free survival was higher for patients who received maintenance therapy with rituximab after CHOP but not for patients who received R-CHOP initially.

The trials described above established R-CHOP as standard first-line therapy for elderly patients with DLBCL. With respect to younger patients, the MabThera (rituximab) International Trial (MInT) confirmed the benefit of adding rituximab to standard chemotherapy in 824 patients (18 to 60 years of age) with only zero (0) to one risk factor, as assessed by the IPI (seeTable 3).49 Patients with stage II to IV or stage I disease with bulky lymphadenopathy were randomly assigned to six cycles of CHOP-like chemotherapy with or without the addition of rituximab. Radiation therapy was subsequently administered to initial sites of bulky disease. Three-year event-free survival rates (79% vs. 59%; P < 0.0001) and overall survival rates (93% vs. 84%; P = 0.00001) were both significantly higher for patients treated with the addition of rituximab. There were no additional major adverse effects.

Sehn et al. compared outcomes during a three-year period; 18 months pre- and post-inclusion of rituximab in standard treatment protocols guided care for patients with newly diagnosed advanced-stage DLBCL in British Columbia.50 All age and risk factor groups were included. Adding rituximab resulted in dramatic improvement in both progression-free survival and overall survival (Figure 1). These studies have indicated significant benefit for the addition of rituximab to chemotherapy for the treatment of DLBCL in a wide range of patient ages and risk categories. Although adding other cytotoxic chemotherapy agents to CHOP failed to improve outcomes, R-CHOP is now the gold standard for treating DLBCL in all subgroups.43

Figure 1

Overall survival according to treatment regimen in diffuse large B-cell lymphoma. Results of a randomized trial of non-rituximab containing regimens as historical controls for British Columbia outcome data with R-CHOP. MACOP-B = methotrexate, Adriamycin,

Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Coiffier et al. conducted a randomized phase 2 trial to evaluate the efficacy and tolerability of rituximab in patients with relapsed/refractory DLBCL, mantle-cell lymphoma, or other intermediate-grade or high-grade B-cell lymphomas and previously untreated patients older than 60 years of age.51 Fifty-four patients received eight weekly infusions of rituximab 375 mg/m2 in arm A or one infusion of 375 mg/m2, followed by seven weekly infusions of 500 mg/m2in arm B. A total of five complete responses and 12 partial responses were observed among the 54 enrolled patients, with no difference between the two doses. The ORR was 31%. An analysis of prognostic factors showed that response rates were lower in patients with refractory disease, in patients with lymphoma not classified as DLBCL, and patients with a tumor larger than 5 cm in diameter. Single-agent rituximab is active in aggressive NHL but not as active as in indolent NHL. This finding led to the use of combinations of rituximab plus chemotherapy in such patients.

The combination of rituximab, ifosfamide, carboplatin, and etoposide (R-ICE) was evaluated in relapsed DLBCL for cytoreduction prior to autologous hematopoietic stem cell transplantation (HSCT).52 Thirty-six eligible patients received rituximab plus ICE (RICE), and 34 patients received all three planned cycles. The CR rate was 53%, significantly better than the 27% CR rate (P = 0.01) achieved among 147 similar consecutive historical control patients with DLBCL treated with ICE; the PR rate was 25%. Progression-free survival in patients who underwent transplantation after RICE was marginally better than for 95 consecutive historical controls who underwent transplantation after ICE alone, but the results did not reach statistical significance (54% with RICE vs. 43% with ICE alone at two years, respectively; P = 0.25). Preliminary results of the CORAL study (Collaborative Trial in Relapsed Aggressive Lymphoma) demonstrated a decreased response to rituximab in the salvage setting of patients previously treated with rituximab-containing regimens.53

In a phase 2 study, rituximab was evaluated in addition to etoposide, prednisone, Oncovin (vincristine), doxorubicin, and cyclophosphamide (EPOCH) in patients with relapsed or refractory aggressive NHL.54 The ORR of 68% included 28% of patients in complete remission. At three years, event-free survival and overall survival rates were 28% and 38%, respectively.

Few studies have explored the use of rituximab as an adjunct to autologous HSCT after high-dose chemotherapy in patients with relapsed DLBCL.5557 These studies have reported positive results with rituximab in this setting. Larger, randomized trials are needed to establish a definitive role for rituximab in these patients. While overall the data on rituximab efficacy in aggressive NHL is not as strong in relapsed patients as for initial R-CHOP, rituximab is active and additional confirmatory studies are needed in various relapsed settings.

Maintenance Therapy for Diffuse Large B-Cell Lymphoma

Unlike the situation with indolent lymphomas, there is no apparent benefit to maintenance rituximab in DLBCL. In an ECOG trial, responding patients were randomly assigned to receive maintenance rituximab or to observation alone.48 Two-year failure-free survival was 76% for maintenance therapy and 61% for observation alone, but these figures were confounded depending on whether rituximab was used initially. No significant differences in survival were seen when rituximab was included either as maintenance or as induction therapy. Failure-free survival was prolonged with maintenance therapy after CHOP but not after R-CHOP. This study confirmed the role of R-CHOP as standard first-line therapy in older DLBCL patients, with maintenance therapy to be used only for patients not previously treated with rituximab.


Rituximab is usually well tolerated, and toxicities are generally mild.12,24,58Common side effects include pruritus, nausea, vomiting, dizziness, headaches, fevers, and rigors. A major concern is the potential for an infusion-related reaction, such as rigors, chills, anaphylactic reactions potentially leading to myocardial infarction and cardiogenic shock. These reactions occur most commonly during the first administration of rituximab. Although infusion reactions are rarely fatal, predisposing cardiac conditions can increase the risk of death. Pre medication with acetaminophen and antihistamines is recommended prior to infusion. Reactions usually abate if the infusion is discontinued and can then be restarted at a slower rate. The benefit of premedication with glucocorticoids is not entirely clear, but they are useful if a reaction occurs. Mucocutaneous reactions, including Stevens–Johnson syndrome, have also been reported within one to 13 weeks following rituximab exposure.

Tumor lysis syndrome has also occurred in patients with bulky lymphoma. Hepatitis B reactivation with fulminant hepatitis, hepatic failure, and death have been reported in patients with previous hepatitis B infection who have been treated with rituximab. Consultation with a hepatologist and administration of antiviral therapy should be considered if hepatitis B antigen is detectable. The risk of reactivation of hepatitis C is not well defined. The use of live vaccines, including those against herpes zoster, is not recommended during rituximab therapy secondary to the risk of causing an active infection. Rituximab-treated patients are also at risk for other viral infections, including cytomegalovirus, herpes simplex, parvovirus B19, and West Nile virus.

Late-onset neutropenia has been described as a possible complication of adding rituximab to chemotherapy.59 In a retrospective review, patients who received chemotherapy plus rituximab for CD20+, B-cell NHL had a higher rate of late-onset neutropenia compared with historical controls receiving chemotherapy alone.

A study published in 2009 reported 57 cases of progressive multifocal leukoencephalopathy (PML) following the administration of rituximab, usually with additional therapy, in HIV-negative patients.60 PML, a viral infection that affects the white matter of the brain, is usually fatal. This cohort of patients was treated with a median of six doses of rituximab. The median time from last rituximab dose to PML diagnosis was 5.5 months, and median survival after the diagnosis of PML was two months. In accordance with these data, the FDA issued a boxed (black-box) warning.

Reversible posterior leukoencephalopathy (RPLE), a subacute neurological syndrome manifested as headaches, cortical blindness, and seizures with a characteristic appearance on magnetic resonance imaging (MRI), has also been described in rare cases.61,62 It is not clear whether these events are directly related to rituximab, because most of these patients have received multiple therapies, but RPLE has also been reported after other antibody and small-molecule therapeutics. Cardiac arrhythmias, renal toxicity, and bowel obstruction with perforation have also been reported.57

Rituximab induces B-cell depletion, which may compromise the immune system; however, recovery of the normal B-cell population usually occurs six to nine months after discontinuation of therapy.15 Despite this depletion, rituximab has not been definitively shown to cause a significant decrease in circulating immunoglobulin levels, although this may occur with more prolonged maintenance strategies. Stable immunoglobulin levels are likely to reflect that plasma cells are long-lived and do not express CD20.

In a prospective study, van der Kolk et al. investigated the effect of rituximab on the humoral immune response to two primary antigens and two recall antigens.63 After rituximab treatment, the humoral immune response to the recall antigens was significantly decreased when compared with the response before treatment.


Attempts to improve upon rituximab have focused on antibody engineering, including humanized instead of chimeric antibodies, stronger binding affinity for CD20, or enhancing effector functions such as antibody-dependent, cell-mediated cytotoxicity (ADCC) or complement activation. Ofatumumab (Arzerra, GlaxoSmithKline) is a humanized monoclonal anti-CD20 antibody that targets a small loop epitope of CD20. Compared with rituximab, in the laboratory it delivers stronger complement-dependent cytotoxicity, even in lymphoma cells with low expression of CD20. Approved by the FDA in October 2009 for the treatment of fludarabine and alemtuzumab–refractory chronic lymphocytic leukemia (CLL), the drug also showed activity in relapsed/refractory FL.64,65

Additional humanized antibodies under development include some with enhanced ADCC, stronger binding to low-affinity polymorphisms of FcgRIII, or targeting other epitopes on the CD20 molecule. Whether these agents are more effective, less immunogenic, or faster to infuse with fewer infusion reactions resulting may be difficult to determine.

Other proteins on the surface of B cells are also potential antibody targets. CD22 has a pattern of expression similar to that of CD20 on normal and malignant B lymphocytes, and it is targeted by epratuzumab (UCB/Immunomedics).66,67 Because CD22 is internalized upon antibody binding, it might be better suited for delivering toxins inside CD22+ cells. Examples of this approach include inotuzumab ozogamicin (CMC-544, Wyeth), an anti-CD22 immunoconjugate with the antitumor antibiotic calicheamicin, and CAT-3888 (Cambridge Antibody Technology), formerly called BL22, which uses a Pseudomonas exotoxin fragment.68,69


Rituximab (Rituxan) has changed the treatment paradigms and outcomes for all CD20+ NHL and represents arguably the most noteworthy advance in lymphoma treatment over the past decade. In patients with NHL, the addition of rituximab to standard treatment significantly enhanced response to therapy and overall outcomes. Rituximab is currently approved for treatment of relapsed and refractory indolent lymphomas as single-agent therapy and as initial therapy in combination with standard chemotherapy regimens. In patients with DLBCL, it is approved for use as initial therapy with CHOP or other anthracycline-based chemotherapy. The drug was also recently approved for use with chemotherapy in previously treated and untreated patients with CLL.

Benefits have been sustained among all age groups, and the drug has been safe and well tolerated in elderly patients as well. Overall survival of patients with NHL has improved over the last two decades. While some of this improvement may stem from earlier or more precise diagnosis and better supportive care, the results of many trials reviewed in this article indicate significant improvement in outcomes with the addition of rituximab to the therapeutic armamentarium.

Despite these advances, questions remain, mainly in the field of indolent lymphomas. More research is under way to establish the optimal schedule, timing, and duration for maintenance rituximab. Reports of clinical trials demonstrating longer follow-up of indolent lymphoma are eagerly awaited in an attempt to clarify the effect of rituximab on overall survival.

Rituximab represents a paradigm shift in treatment of B-cell NHL; it marks the beginning of a new age of targeted therapies in oncology, being the first approved therapeutic monoclonal antibody for cancer. In the years to come, we anticipate more clinical trials combining rituximab with targeted treatments that might further improve outcomes while minimizing toxicity.

1. Jemal A, Siegel R, Ward E. Cancer statistics, 2008. CA Cancer J Clin.2008;58(2):71–96. [PubMed]
2. Rudiger T, Muller-Hermelink HK. WHO classification of malignant lymphomas. Radiologe. 2002;42(12):936–942. [PubMed]
3. Harris NL, Jaffe ES, Diebold J, et al. The World Health Organization Classification of Neoplastic Diseases of the Hematopoietic and Lymphoid Tissues. Report of the Clinical Advisory Committee meeting. Airlie House, Virginia, November 1997; Ann Oncol; 1999. pp. 1419–1432. [PubMed]
4. Armitage JO, Weisenburger DD. New approach to classifying non-Hodgkin’s lymphomas: Clinical features of the major histologic subtypes. Non-Hodgkin’s Lymphoma Classification Project. J Clin Oncol.1998;16(8):2780–2795. [PubMed]
5. Tedder TF, Engel P. CD20: A regulator of cell–cycle progression of B lymphocytes. Immunol Today. 1994;15(9):450–454. [PubMed]
6. Silverman GJ, Weisman S. Rituximab therapy and autoimmune disorders: Prospects for anti-B cell therapy. Arthritis Rheum. 2003;48(6):1484–1492.[PubMed]
7. Shan D, Ledbetter JA, Press OW. Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood. 1998;91(5):1644–1652. [PubMed]
8. Reff ME, Carner K, Chambers KS, et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood.1994;83(2):435–445. [PubMed]
9. Golay J, Zaffaroni L, Vaccari T, et al. Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood. 2000;95(12):3900–3908.[PubMed]
10. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat Med.2000;6(4):443–446. [PubMed]
11. Alas S, Bonavida B. Rituximab inactivates signal transducer and activation of transcription 3 (STAT3) activity in B–non-Hodgkin’s lymphoma through inhibition of the interleukin 10 autocrine/paracrine loop and results in down-regulation of Bcl-2 and sensitization to cytotoxic drugs.Cancer Res. 2001;61(13):5137–5144. [PubMed]
12. Maloney DG, Liles TM, Czerwinski DK, et al. Phase I clinical trial using escalating single-dose infusion of chimeric anti-CD20 monoclonal antibody (Idec-C2B8) in patients with recurrent B-cell lymphoma. Blood.1994;84(8):2457–2466. [PubMed]
13. Horning SJ, Rosenberg SA. The natural history of initially untreated low-grade non-Hodgkin’s lymphomas. N Engl J Med. 1984;311(23):1471–1475. [PubMed]
14. Advani R, Rosenberg SA, Horning SJ. Stage I and II follicular non-Hodgkin’s lymphoma: Long-term follow-up of no initial therapy. J Clin Oncol. 2004;22(8):1454–1459. [PubMed]
15. McLaughlin P, Grillo-Lopez AJ, Link BK, et al. Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: Half of patients respond to a four-dose treatment program. J Clin Oncol.1998;16(8):2825–2833. [PubMed]

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Platelet Transfusions

Larry H. Bernstein, MD, FCAP, Curator


Platelet Transfusion: A Clinical Practice Guideline From the AABB

Richard M. Kaufman, MD; Benjamin Djulbegovic, MD, PhD; Terry Gernsheimer, MD; Steven Kleinman, MD,
Alan T. Tinmouth, MD; Kelley E. Capocelli, MD; Mark D. Cipolle, MD, PhD; Claudia S. Cohn, MD, PhD; et al.

Ann Intern Med. 2015;162(3):205-213.

Annals of Internal Medicine 3 February 2015, Vol 162, No. 3>

Approximately 2.2 million platelet doses are transfused annually in the United States (1). A high proportion of these platelet units are transfused prophylactically to reduce the risk for spontaneous bleeding in patients who are thrombocytopenic after chemotherapy or hematopoietic progenitor cell transplantation (HPCT) (13). Unlike other blood components, platelets must be stored at room temperature, limiting the shelf life of platelet units to only 5 days because of the risk for bacterial growth during storage. Therefore, maintaining hospital platelet inventories is logistically difficult and highly resource-intensive (45). Platelet transfusion is associated with several risks to the recipient (Table 1), including allergic reactions and febrile nonhemolytic reactions. Sepsis from a bacterially contaminated platelet unit represents the most frequent infectious complication from any blood product today (8). In any situation where platelet transfusion is being considered, these risks must be balanced against the potential clinical benefits.

Background: The AABB (formerly, the American Association of Blood Banks) developed this guideline on appropriate use of platelet transfusion in adult patients.

Methods: These guidelines are based on a systematic review of randomized, clinical trials and observational studies (1900 to September 2014) that reported clinical outcomes on patients receiving prophylactic or therapeutic platelet transfusions. An expert panel reviewed the data and developed recommendations using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework.

Recommendation 1: The AABB recommends that platelets should be transfused prophylactically to reduce the risk for spontaneous bleeding in hospitalized adult patients with therapy-induced hypoproliferative thrombocytopenia. The AABB recommends transfusing hospitalized adult patients with a platelet count of 10 × 109 cells/L or less to reduce the risk for spontaneous bleeding. The AABB recommends transfusing up to a single apheresis unit or equivalent. Greater doses are not more effective, and lower doses equal to one half of a standard apheresis unit are equally effective. (Grade: strong recommendation; moderate-quality evidence)

Recommendation 2: The AABB suggests prophylactic platelet transfusion for patients having elective central venous catheter placement with a platelet count less than 20 × 109 cells/L. (Grade: weak recommendation; low-quality evidence)

Recommendation 3: The AABB suggests prophylactic platelet transfusion for patients having elective diagnostic lumbar puncture with a platelet count less than 50 × 109 cells/L. (Grade: weak recommendation; very-low-quality evidence)

Recommendation 4: The AABB suggests prophylactic platelet transfusion for patients having major elective nonneuraxial surgery with a platelet count less than 50 × 109 cells/L. (Grade: weak recommendation; very-low-quality evidence)

Recommendation 5: The AABB recommends against routine prophylactic platelet transfusion for patients who are nonthrombocytopenic and have cardiac surgery with cardiopulmonary bypass. The AABB suggests platelet transfusion for patients having bypass who exhibit perioperative bleeding with thrombocytopenia and/or evidence of platelet dysfunction. (Grade: weak recommendation; very-low-quality evidence)

Recommendation 6: The AABB cannot recommend for or against platelet transfusion for patients receiving antiplatelet therapy who have intracranial hemorrhage (traumatic or spontaneous). (Grade: uncertain recommendation; very-low-quality evidence)

Table 1. Approximate Per-Unit Risks for Platelet Transfusion in the United States



Clinical and laboratory aspects of platelet transfusion therapy
Literature review current through: Sep 2015. | This topic last updated: Jun 12, 2015.

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.

(See “Approach to the adult patient with a bleeding diathesis”.)

(See “Refractoriness to platelet transfusion therapy”.)

(See “Clinical manifestations, evaluation, and management of neonatal thrombocytopenia”, section on ‘Platelet transfusion’.)

PLATELET COLLECTION — There are two ways that platelets can be collected: by isolation from a unit of donated blood, or by apheresis from a donor in the blood bank.

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 1010platelets 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 1011platelets) [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. (See ‘Ordering platelets’ below.)

Issues related to the effects of platelet pheresis on the donor are covered elsewhere. (See “Blood donor screening: Procedures and processes to enhance safety for the blood recipient and the blood donor”, section on ‘Apheresis platelet donors’.)

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’ below.)

Several strategies are used to prevent the complications associated with WBC and plasma contamination of platelets. (See ‘Ordering platelets’ below.)

Platelets concentrates also contain a small number of red blood cells (RBCs) that express Rh antigens on their surface (platelets do not express Rh antigens). The small numbers of RBCs in apheresis platelets negates the issue of Rh alloimmunization in most patients. However, blood banks avoid giving platelets from Rh+ donors to Rh female patients because of the potential risk of Rh alloimmunization and subsequent hemolytic disease of the newborn. (See “Overview of Rhesus D alloimmunization in pregnancy”.)

PLATELET STORAGE AND PATHOGEN REDUCTION — Platelets are stored at room temperature, because cold induces clustering of von Willebrand factor receptors on the platelet surface and morphological changes of the platelets, leading to enhanced clearance by hepatic macrophages and reduced platelet survival in the recipient [3-6].

All cells are more metabolically active at room temperature, so platelets are stored in bags that allow oxygen and carbon dioxide gas exchange. Citrate is included to prevent clotting and maintain proper pH, and dextrose is added as an energy source [2].

A disadvantage of room temperature storage is the increased growth of bacteria compared with blood products stored in the refrigerator or freezer. (See‘Complications of platelet transfusion’ below.)

Strategies for reducing exposure to contaminating pathogens include:

Donor screening for bloodborne pathogens (see “Blood donor screening: Laboratory testing”, section on ‘Infectious disease screening’ and “Blood donor screening: Procedures and processes to enhance safety for the blood recipient and the blood donor”, section on ‘Protection of the recipient’)

Proper skin sterilization techniques during collection, and discarding the first 15 to 30 mL of blood collected, which is most likely to be contaminated by skin bacteria

Performing tests to screen for bacterial contamination, such as automated culture-based assays, and rapid point-of-issue tests (see “Transfusion-transmitted bacterial infection”, section on ‘Detection of contamination’)

Using blood products that have been subjected to pathogen inactivation or reduction treatment (not available in the United States) (see “Pathogen inactivation of blood products”, section on ‘Pathogen inactivation of platelets’and “Preparation of blood components”, section on ‘Pathogen reduction’)

The shelf life of platelets stored at room temperature is five days because of the bacterial infection risk that increases in relationship to the storage duration. This short shelf life contributes to the greater sensitivity of platelet inventory to shortages.

INDICATIONS FOR PLATELET TRANSFUSION — Platelets can be transfused therapeutically (ie, to treat active bleeding or in preparation for an invasive procedure that would cause bleeding), or prophylactically (ie, to prevent spontaneous bleeding).

Actively bleeding patient — Actively bleeding patients with thrombocytopenia should be transfused with platelets immediately to keep platelet counts above50,000/microL in most bleeding situations, and above 100,000/microL if there is disseminated intravascular coagulation or central nervous system bleeding. (See“Clinical features, diagnosis, and treatment of disseminated intravascular coagulation in adults”, section on ‘Treatment’ and “Spontaneous intracerebral hemorrhage: Treatment and prognosis”, section on ‘Initial treatment’.).

Other factors contributing to bleeding should also be addressed. These include:

Surgical or anatomic defect


Infection or inflammation


Acquired or inherited platelet function defect

The dose and frequency of platelet transfusions will depend on the platelet count and the severity of bleeding. (See ‘Dose’ below.)

Preparation for an invasive procedure — Platelets are transfused in preparation for an invasive procedure if the thrombocytopenia is severe and the risks of bleeding are deemed high. Most of the data used to determine bleeding risk come from retrospective studies of patients who are afebrile and have thrombocytopenia but not coagulopathy [7]. Typical platelet count thresholds that are used for some common procedures are as follows:

Neurosurgery or ocular surgery – 100,000/microL

Most other major surgery – 50,000/microL

Endoscopic procedures – 50,000/microL for therapeutic procedures;20,000/microL for low risk diagnostic procedures (see “Endoscopic procedures in patients with disorders of hemostasis”, section on ‘Procedure-related bleeding risk’)

Central line placement – 20,000/microL [8]

Lumbar puncture – 10,000 to 20,000/microL in patients with hematologic malignancies and greater than 40,000 to 50,000 in patients without hematologic malignancies, but lower in patients with immune thrombocytopenia (ITP) [9-11]

Epidural anesthesia – 80,000/microL [11]

Bone marrow aspiration/biopsy20,000/microL

Prevention of spontaneous bleeding — Prophylactic transfusion is used to prevent spontaneous bleeding in patients at high risk of bleeding. The threshold for prophylactic transfusion varies depending on the patient and on the clinical scenario. (See ‘Specific clinical scenarios’ below.)

Predicting spontaneous bleeding — There are no ideal tests for predicting who will bleed spontaneously [12]. Studies of patients with thrombocytopenia suggest that patients can bleed even with platelet counts greater than 50,000/microL [13]. However, bleeding is much more likely at platelet counts less than 5000/microL. Among individuals with platelet counts between 5000/microL and 50,000/microL,clinical findings can be helpful in decision-making regarding platelet transfusion.

The platelet count at which a patient bled previously can be a good predictor of future bleeding.

Petechial bleeding and ecchymoses are generally not thought to be predictive of serious bleeding, whereas mucosal bleeding and epistaxis (so-called “wet” bleeding) are thought to be predictive.

Coexisting inflammation, infection, and fever also increase bleeding risk.

The underlying condition responsible for a patient’s thrombocytopenia also may help in estimating the bleeding risk. As an example, some patients with ITP often tolerate very low platelet counts without bleeding, while patients with some acute leukemias that are associated with coagulopathy (eg, acute promyelocytic leukemia) can have bleeding at higher platelet counts (eg, 30,000 to 50,000/microL). (See ‘Specific clinical scenarios’ below.)

Compared with adults, children with bone marrow suppression may be more likely to experience bleeding at the same degree of thrombocytopenia. In a secondary subgroup analysis of the PLADO trial, in which patients were randomly assigned to different platelet doses, children had more days of bleeding, more severe bleeding, and required more platelet transfusions than adults with similar platelet counts [14]. However, these findings do not suggest a different threshold for platelet transfusion in children, as the increased risk of bleeding was distributed across a wide range of platelet counts.

Tests for platelet-dependent hemostasis (ie, bleeding time, thromboelastography, and other point of care tests) are generally not used to predict bleeding in thrombocytopenic patients. (See “Platelet function testing”, section on ‘The in vivo bleeding time’ and “Platelet function testing”, section on ‘Instruments that simulate platelet function in vitro’.)

Therapeutic versus prophylactic transfusion — By convention, most authors use the term “therapeutic transfusion” to refer both to transfusion of platelets to treat active bleeding and transfusion of platelets in preparation for an invasive procedure that could cause bleeding. The term “prophylactic transfusion” is used to refer to platelet transfusion given to prevent spontaneous bleeding.

We use prophylactic platelet transfusion to prevent spontaneous bleeding in most afebrile patients with platelet counts below 10,000/microL due to bone marrow suppression. We use higher thresholds (ie, 30,000/microL) in patients who are febrile or septic. Patients with acute promyelocytic leukemia (APL) have a coexisting coagulopathy, and we use a platelet transfusion threshold of 30,000 to 50,000/microLfor them. (See ‘Leukemia and chemotherapy’ below.)

Patients with platelet consumption disorders (eg, immune thrombocytopenia [ITP], disseminated intravascular coagulation) and platelet function disorders are typically transfused only for bleeding or, in some cases, invasive procedures. Platelets should not be withheld in bleeding patients with these conditions due to fear of “fueling the fire” of thrombus formation. (See ‘Immune thrombocytopenia (ITP)’ below and ‘TTP or HIT’ below and ‘Platelet function defects’ below.)

Given the need to balance the risk of spontaneous bleeding with the potential complications of unnecessary platelet transfusion, the decision of whether to transfuse platelets based upon a clinical event (ie, for active bleeding or invasive procedures) or at a particular threshold (ie, to prevent spontaneous bleeding) is challenging. Standard practice has evolved to transfusion of platelets at a threshold platelet count of 10,000 to 20,000/microL for most patients with severe hypoproliferative thrombocytopenia due to hematologic malignancies, cytotoxic chemotherapy, and hematopoietic cell transplant (HCT) [15]. However, the risks and benefits of reserving platelet transfusion for active bleeding episodes in these patients continue to be evaluated [7,16-19].

In a randomized trial, 400 patients with acute myeloid leukemia (AML; patients with APL were excluded) and patients undergoing autologous HCT for hematologic malignancies were assigned to receive platelet transfusions when morning platelet counts were ≤10,000/microL or only for active bleeding [20]. Patients transfused only for active bleeding received fewer platelet transfusions during the 14-day period after induction or consolidation chemotherapy (1.63 versus 2.44 per patient, a 33.5 percent reduction). However, among patients with AML who were transfused only for active bleeding, there were more episodes of major bleeding (six cerebral, four retinal, and one vaginal) and there were two fatal intracranial hemorrhages compared with four retinal hemorrhages among patients transfused for a platelet count ≤10,000/microL. Patients undergoing HCT also experienced more bleeding episodes when transfused only for active bleeding, but most of these were minor.

In another randomized trial, 600 patients with hematologic malignancies receiving chemotherapy, autologous, or allogeneic HCT were assigned to receive platelet transfusion for a platelet count ≤10,000/microL or only for active bleeding (the Trial of Prophylactic Platelets [TOPPS]) [21-23]. Compared with those who received prophylactic transfusions, patients transfused only for active bleeding received fewer platelet transfusions during the 30-day period after randomization, but had a higher incidence of major bleeding (50 versus 43 percent) and a shorter time to first bleed (1.2 versus 1.7 days) [24]. There were no differences in the duration of hospitalization, and no deaths due to bleeding. In a predefined subgroup analysis, patients undergoing autologous HCT had similar rates of major bleeding whether they were transfused for a platelet count≤10,000/microL or only for active bleeding (45 and 47 percent).

The findings from these trials support continued use of prophylactic transfusion for patients with hematologic malignancies and HCT until further data become available. Although the findings suggest that reserving platelet transfusion for active bleeding may be safe for some adults undergoing autologous HCT, such a strategy requires intensive monitoring and the ability to perform immediate imaging for suspected CNS or ocular bleeding. We do not recommend reserving platelet transfusion for active bleeding in patients with HCT outside of highly specialized centers with the ability to support this level of vigilance.

SPECIFIC CLINICAL SCENARIOS — There are several common clinical scenarios that raise the questions of whether to transfuse patients prophylactically to prevent bleeding, and, if prophylactic transfusion is used, of what platelet count is the best threshold for transfusion.

Leukemia and chemotherapy — Patients with leukemia, hematopoietic cell transplant (HCT), or those being treated with cytotoxic chemotherapy have a suppressed bone marrow that cannot produce adequate platelets. We use prophylactic transfusion in these settings. The thresholds suggested below apply to patients with thrombocytopenia who are afebrile and without active infection. If fever or sepsis is present, higher thresholds may be needed.

Acute myeloid leukemia (AML) – Patients with AML can have suppressed bone marrow from AML, chemotherapy, or HCT. We use standard dose prophylactic transfusion of these patients at a threshold platelet count of10,000/microL, and transfusion for any bleeding greater than petechial bleeding. (See ‘Dose’ below.)

This approach is in line with the 2001 American Society for Clinical Oncology (ASCO) guidelines (table 1) and a practice guideline from the AABB [25]. It is supported by randomized trials comparing prophylactic (ie, threshold-based) and therapeutic platelet transfusion, in which patients who did not receive prophylactic transfusion had more severe bleeding [20,24,26]. (See ‘Therapeutic versus prophylactic transfusion’ above and “Overview of the complications of acute myeloid leukemia”, section on ‘Bleeding’.)

Acute promyelocytic leukemia (APL) – Patients with APL differ from other patients with AML because they often have an associated coagulopathy that puts them at high risk for disseminated intravascular coagulation and bleeding. We prophylactically transfuse these patients at a platelet count of 30,000 to50,000/microL, and treat any sign of bleeding, especially central nervous system bleeding, with immediate platelet transfusion. (See “Clinical manifestations, pathologic features, and diagnosis of acute promyelocytic leukemia in adults”, section on ‘Disseminated intravascular coagulation’ and“Initial treatment of acute promyelocytic leukemia in adults”, section on ‘Control of coagulopathy’.)

Acute lymphoblastic leukemia (ALL) – Patients with ALL have thrombocytopenia from bone marrow suppression. In addition, these patients are often treated with L-asparaginase, which causes severe hypofibrinogenemia. However, the risk of life-threatening bleeding is low. As an example, in over 2500 children with ALL, only two intracranial hemorrhages occurred, and they were associated with hyperleukocytosis in one case and intracerebral fungal infection in the other [9]. We transfuse adults with ALL at a threshold platelet count of 10,000/microL. The use of platelet transfusion in children with ALL is discussed separately. (See “Overview of the treatment of acute lymphoblastic leukemia in children and adolescents”, section on ‘Bleeding’.)

Chemotherapy for solid tumors – Cancer chemotherapy often makes patients thrombocytopenic from bone marrow suppression. Randomized trials of platelet transfusion threshold in this population have not been performed. Observational studies support a prophylactic platelet transfusion threshold of 10,000/microL[26]. A threshold of 20,000/microL may be appropriate for patients with necrotic tumors. These recommendations are generally consistent with the ASCO 2001 Guidelines (table 1) [26].

Hematopoietic cell transplant (HCT) – Chemotherapy and radiation therapy administered as part of the conditioning regimen for HCT can be highly bone marrow suppressive, depending on the doses used. We use standard dose prophylactic transfusion of these patients at a threshold platelet count of10,000/microL, and therapeutic transfusion for any bleeding greater than petechial bleeding. (See “Hematopoietic support after hematopoietic cell transplantation”, section on ‘Platelet transfusion’.)

Aplastic anemia – Patients with aplastic anemia do not have a malignancy, but they may have severe thrombocytopenia, and they may be candidates for HCT. Issues related to platelet transfusion in these patients are discussed separately. (See “Treatment of aplastic anemia in adults”.)

Prophylactic platelet transfusion for a platelet count ≤10,000/microL in hospitalized patients with thrombocytopenia from therapy-induced bone marrow suppression is consistent with a practice guideline from the AABB [25].

Immune thrombocytopenia (ITP) — Individuals with immune thrombocytopenia produce anti-platelet antibodies that destroy circulating platelets and megakaryocytes in the bone marrow. Circulating platelets in patients with ITP tend to be highly functional, and platelet counts tend to be well above 30,000/microL. Bleeding is rare even in patients with severe thrombocytopenia (ie, platelet count <30,000/microL). (See “Immune thrombocytopenia (ITP) in adults: Clinical manifestations and diagnosis”, section on ‘Pathogenesis’.)

Our general approach to platelet transfusion in patients with ITP is to transfuse for bleeding rather than at a specific platelet count. (See “Immune thrombocytopenia (ITP) in adults: Initial treatment and prognosis”, section on ‘Indications for treatment’.)

TTP or HIT — Thrombotic thrombocytopenic purpura (TTP) and heparin-induced thrombocytopenia (HIT) are disorders in which platelet consumption causes thrombocytopenia and an increased risk of bleeding; but the underlying platelet activation in these conditions also increases the risk of thrombosis.

Platelet transfusions can be helpful or even life-saving in patients with these conditions who are bleeding and/or have anticipated bleeding due to a required invasive procedure (eg, placement of a central venous catheter), and platelet transfusion should not be withheld from a bleeding patient due to concerns that platelet transfusion will exacerbate thrombotic risk. However, platelet transfusions may cause a slightly increased risk of thrombosis in patients with these conditions; thus, we do not use prophylactic platelet transfusions routinely in patients with TTP or HIT in the absence of bleeding or a required invasive procedure.

Support for this approach comes from a large retrospective review of hospitalized patients with TTP and HIT, in which platelet transfusion was associated with a very slight increased risk of arterial thrombosis but not venous thromboembolism [27]. In contrast, the review found that patients with immune thrombocytopenia (ITP) had no increased risk of arterial or venous thrombosis with platelet transfusion. Of note, this was a retrospective study in which sicker patients were more likely to have received platelets, and the temporal relationships between platelet transfusions and thromboses were not assessed.

TTP – Of 10,624 patients with TTP in the large review mentioned above, approximately 10 percent received a platelet transfusion [27]. Arterial thrombosis occurred in 1.8 percent of patients who received platelets, versus 0.4 percent of patients who did not (absolute increase, 1.4 percent; adjusted odds ratio [OR], 5.8; 95% CI, 1.3-26.6). The rate of venous thrombosis was not different in those who received platelets and those who did not (adjusted OR 1.1; 95% CI 0.5-2.2).

In contrast, a systematic review of patients with TTP who received platelet transfusions, which included retrospective data for 358 patients and prospective data for 54 patients, did not find clear evidence that platelet transfusions were associated with adverse outcomes [28].

HIT – Of 6332 patients with HIT in the large review mentioned above, approximately 7 percent received a platelet transfusion [27]. Arterial thrombosis occurred in 6.9 percent of patients who received platelets, versus 3.1 percent of patients who did not (absolute increase, 3.8 percent; adjusted OR, 3.4; 95% CI, 1.2-9.5). The rate of venous thrombosis was not different in those who received platelets and those who did not (adjusted OR 0.8; 95% CI 0.4-1.7).

In a series of four patients with HIT who received platelet transfusions, two of three with active bleeding had cessation of bleeding following platelet transfusion, and no thromboses occurred; a literature review was not able to identify any complications clearly attributable to platelet transfusion [29].

Management of TTP and HIT is discussed in detail separately. (See “Acquired TTP: Initial treatment” and “Management of heparin-induced thrombocytopenia”.)

Liver disease and DIC — Patients with liver disease and DIC have a complex mixture of procoagulant and anticoagulant defects along with thrombocytopenia, and therefore they are at risk for thrombosis and bleeding. There is no evidence to support the administration of platelets in these patients if they are not bleeding. However, platelet transfusion is justified in patients who have serious bleeding, are at high risk for bleeding (eg, after surgery), or require invasive procedures. (See “Clinical features, diagnosis, and treatment of disseminated intravascular coagulation in adults”, section on ‘Prevention/treatment of bleeding’ and “Hemostatic abnormalities in patients with liver disease”, section on ‘Bleeding’.)

Platelet function defects — Platelet function defects can be inherited or acquired, and may be associated with thrombocytopenia or a normal platelet count. Platelet transfusion in these settings is typically reserved for bleeding.

Inherited diseases Platelet function is impaired in Wiskott-Aldrich syndrome, Glanzmann thrombasthenia, and Bernard-Soulier syndrome. Bleeding in patients with these conditions is treated with platelet transfusion, along with other hemostatic agents discussed below. (See “Congenital and acquired disorders of platelet function”, section on ‘Inherited disorders of platelet function’ and‘Alternatives to platelet transfusion’ below.)

Acquired conditions – Uremia, diabetes mellitus, myeloproliferative disorders, and other medical conditions can impair platelet function. Bleeding risk can be reduced by treating the underlying condition. Platelet transfusion is typically reserved for major bleeding in these conditions. (See “Congenital and acquired disorders of platelet function”, section on ‘Acquired platelet functional disorders’.)

Patients who are febrile or septic can have impaired platelet function. We transfuse these patients for bleeding. We also use a higher threshold for when fever or sepsis coexist with thrombocytopenia (eg, in patients with leukemia). (See ‘Leukemia and chemotherapy’ above.)

Antiplatelet agentsAspirin, nonsteroidal antiinflammatory drugs (NSAIDs),dipyridamole, ADP receptor (P2Y12) inhibitors (eg, clopidogrel, ticlopidine), andGPIIb/IIIa antagonists (eg, abciximab, eptifibatide) are used to prevent thrombosis by interfering with normal platelet function. The antiplatelet effects of these agents are weakest with aspirin and more potent with the P2Y12inhibitors. (See “Platelet biology”, section on ‘Drugs with antiplatelet actions’.)

Typically, the approach to treating mild bleeding in a patient taking an antiplatelet agent is to discontinue the drug, assuming a favorable risk-benefit ratio. Although data are limited, platelet transfusion appears to be the best option in patients taking antiplatelet agents who experience severe bleeding [30].

Patients taking these agents may also require urgent surgical procedures (eg, coronary artery bypass grafting, neurosurgical interventions, and others). The role of platelet transfusion in this setting is not well defined. Some clinicians give prophylactic platelet transfusions to patients taking antiplatelet drugs who require major surgery, while other clinicians use platelet transfusion only to treat excessive surgical bleeding [30,31]. These cases can be complex, and we favor an individualized approach based on the complete clinical picture.

Other medications – Other medications may impair platelet function. As an example, the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib inhibits platelet aggregation by interfering with activation signals. The role of platelet transfusion in patients with ibrutinab-associated bleeding despite a sufficient platelet count is unknown, and decisions are individualized according to the platelet count and the severity and site of bleeding.

Massive blood loss — Patients with massive blood loss from surgery or trauma are transfused with red blood cells (RBC), resulting in partial replacement of the blood volume with a product lacking platelets and clotting factors. In this setting, we transfuse RBC, fresh frozen plasma (FFP), and random donor platelet units in a 1:1:1 ratio. As an example, a patient transfused with six units of RBC would also receive six units of pooled platelets or one apheresis unit (both of which provide approximately 5 x 1011 platelets) and six units of FFP. (See “Initial evaluation and management of shock in adult trauma”, section on ‘Transfusion of blood products’.).

Cardiopulmonary bypass — Patients who undergo prolonged cardiopulmonary bypass can have thrombocytopenia and impaired platelet function. The use of platelet transfusion in the cardiopulmonary bypass setting is discussed separately. (See“Congenital and acquired disorders of platelet function”, section on ‘Cardiopulmonary bypass’ and “Early noncardiac complications of coronary artery bypass graft surgery”, section on ‘Bleeding’.)

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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

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

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

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

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. 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.

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.

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.

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®)

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]


  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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Treatments other than Chemotherapy for Leukemias and Lymphomas

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

2.5.1 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

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.

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

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

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)

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;jsessionid=A6001D9D865EA1EBC667EF98382EF20C.f03t01

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

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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