Search

Inhibition of the B-cell receptor (BCR) signaling pathway is highly effective in B-cell neoplasia through Bruton tyrosine kinase inhibition by ibrutinib. Ibrutinib also disrupts cell adhesion between a tumor and its microenvironment. However, it is largely unknown how BCR signaling is linked to cell adhesion. We observed that intrinsic sensitivities of mantle cell lymphoma (MCL) cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion signatures were simultaneously downregulated by ibrutinib in the ibrutinib-sensitive, but not ibrutinib-resistant, cells. Among the differentially expressed genes, RAC2, part of the BCR signature and a known regulator of cell adhesion, was downregulated at both the RNA and protein levels by ibrutinib only in sensitive cells. RAC2 physically associated with B-cell linker protein (BLNK), a BCR adaptor molecule, uniquely in sensitive cells. RAC2 reduction using RNA interference and CRISPR impaired cell adhesion, whereas RAC2 overexpression reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib exhibited slower tumor growth, with reduced RAC2 expression in tissue. Finally, RAC2 was expressed in ∼65% of human primary MCL tumors, and RAC2 suppression by ibrutinib resulted in cell adhesion impairment. These findings, made with cell lines, a xenograft model, and human primary lymphoma tumors, uncover a novel link between BCR signaling and cell adhesion. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development.

Inhibition of the B-cell receptor (BCR) signaling pathway is highly effective in B-cell neoplasia through Bruton tyrosine kinase inhibition by ibrutinib. Ibrutinib also disrupts cell adhesion between a tumor and its microenvironment. However, it is largely unknown how BCR signaling is linked to cell adhesion. We observed that intrinsic sensitivities of mantle cell lymphoma (MCL) cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion signatures were simultaneously downregulated by ibrutinib in the ibrutinib-sensitive, but not ibrutinib-resistant, cells. Among the differentially expressed genes, RAC2, part of the BCR signature and a known regulator of cell adhesion, was downregulated at both the RNA and protein levels by ibrutinib only in sensitive cells. RAC2 physically associated with B-cell linker protein (BLNK), a BCR adaptor molecule, uniquely in sensitive cells. RAC2 reduction using RNA interference and CRISPR impaired cell adhesion, whereas RAC2 overexpression reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib exhibited slower tumor growth, with reduced RAC2 expression in tissue. Finally, RAC2 was expressed in ∼65% of human primary MCL tumors, and RAC2 suppression by ibrutinib resulted in cell adhesion impairment. These findings, made with cell lines, a xenograft model, and human primary lymphoma tumors, uncover a novel link between BCR signaling and cell adhesion. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development.

Chronic lymphocytic leukemia (CLL) is a malignancy of B cells with a variable clinical course. Prognostication is important to place patients into different risk categories for guiding decisions on clinical management, to treat or not to treat. Although several clinical, cytogenetic, and molecular parameters have been established, in the past decade, a tremendous understanding of molecular lesions has been obtained with the advent of high-throughput sequencing. Meanwhile, rapid advances in the understanding of the CLL oncogenic pathways have led to the development of small-molecule targeting signal transducers, Bruton tyrosine kinase and phosphatidylinositol 3-kinase, as well as anti-apoptotic protein BCL2 apoptosis regulator. After an initial response to these targeted therapies, some patients develop resistance and experience disease progression. Novel gene mutations have been identified that account for some of the drug resistance mechanisms. This article focuses on the prognostic and predictive molecular biomarkers in CLL relevant to the molecular pathology practice, beginning with a review of well-established prognostic markers that have already been incorporated into major clinical guidelines, which will be followed by a discussion of emerging biomarkers that are expected to impact clinical practice soon in the future. Special emphasis will be put on predictive biomarkers related to newer targeted therapies in hopes that this review will serve as a useful reference for molecular diagnostic professionals, clinicians, as well as laboratory investigators and trainees.

Introduction

Bruton tyrosine kinase inhibitors (BTKi) have generated remarkable clinical responses in patients with chronic lymphocytic leukemia (CLL) and other mature B-cell malignancies. However, deep, and durable response to BTK inhibition is uncommon. A significant number of patients with measurable residual disease relapse with resistance to therapies. Although mutational mechanisms responsible for resistance have been well described, it is not known what mechanism led to persistence of the minimal residual disease (MRD) in the first place.

The BTK inhibitor ibrutinib has demonstrated a remarkable therapeutic effect in mantle cell lymphoma (MCL). However, approximately one-third of patients do not respond to the drug initially. To identify the mechanisms underlying primary ibrutinib resistance in MCL, we analyzed the transcriptome changes in ibrutinib-sensitive and ibrutinib-resistant cell lines on ibrutinib treatment. We found that MYC gene signature was suppressed by ibrutinib in sensitive but not resistant cell lines. We demonstrated that MYC gene was structurally abnormal and MYC protein was overexpressed in MCL cells. Further, MYC knockdown with RNA interference inhibited cell growth in ibrutinib-sensitive as well as ibrutinib-resistant cells. We explored the possibility of inhibiting MYC through HSP90 inhibition. The chaperon protein is overexpressed in both cell lines and primary MCL cells from the patients. We demonstrated that MYC is a bona fide client of HSP90 in the context of MCL by both immunoprecipitation and chemical precipitation. Furthermore, inhibition of HSP90 using PU-H71 induced apoptosis and caused cell cycle arrest. PU-H71 also demonstrates strong and relatively specific inhibition of the MYC transcriptional program compared with other oncogenic pathways. In a MCL patient-derived xenograft model, the HSP90 inhibitor retards tumor growth and prolongs survival. Last, we showed that PU-H71 induced apoptosis and downregulated MYC protein in MCL cells derived from patients who were clinically resistant to ibrutinib. In conclusion, MYC activity underlies intrinsic resistance to ibrutinib in MCL. As a client protein of HSP90, MYC can be inhibited via PU-H71 to overcome primary ibrutinib resistance.

Next-generation sequencing (NGS) diagnostic assays increasingly are becoming the standard of care in oncology practice. As the scale of an NGS laboratory grows, management of these assays requires organizing large amounts of information, including patient data, laboratory processes, genomic data, as well as variant interpretation and reporting. Although several Laboratory Information Systems and/or Laboratory Information Management Systems are commercially available, they may not meet all of the needs of a given laboratory, in addition to being frequently cost-prohibitive. Herein, we present the System for Informatics in the Molecular Pathology Laboratory (SIMPL), a free and open-source Laboratory Information System/Laboratory Information Management System for academic and nonprofit molecular pathology NGS laboratories, developed at the Genomic and Molecular Pathology Division at the University of Chicago Medicine. SIMPL was designed as a modular end-to-end information system to handle all stages of the NGS laboratory workload from test order to reporting. We describe the features of SIMPL, its clinical validation at University of Chicago Medicine, and its installation and testing within a different academic center laboratory (University of Colorado), and we propose a platform for future community co-development and interlaboratory data sharing.

Ibrutinib has generated remarkable responses in patients with chronic lymphocytic leukemia (CLL), including those with an unfavorable cytogenetic profile. However, patients develop resistance, with poor outcomes and no established treatment options. Mutations in BTKand PLCG2have emerged as main mechanisms of drug resistance, but not all patients carry these mutations. Further understanding of mechanisms of resistance is urgently needed and will support rational development of new therapeutic strategies. To that end, we characterized the genomic profiles of serial samples from 9 patients with ibrutinib-relapsed disease, including 6 who had Richter transformation. Mutations, indels, copy-number aberrations, and loss of heterozygosity were assessed using next-generation sequencing and single-nucleotide polymorphism array. We found that 18p deletion (del(18p)), together with del(17p)/TP53mutations, was present in 5 of 9 patients before ibrutinib therapy. In addition to BTKC481, we identified BTKT316A, a structurally novel mutation located in the SH2 domain of BTK. Minor BTKclones with low allele frequencies were captured in addition to major BTKclones. Although TP53loss predisposes patients for relapse, clone size of TP53loss may diminish during disease progression while mutant BTKclone expands. In patients who had Richter transformation, we found that the transformed cells were clonal descendants of circulating leukemia cells but continued to undergo evolution and drifts. Surprisingly, transformed lymphoma cells in tissue may acquire a different BTKmutation from that in the CLL leukemia cells. Collectively, these results provide insights into clonal evolution underlying ibrutinib relapse and prompt further investigation on genomic abnormalities that have clinical application potential.

Ibrutinib has generated remarkable responses in patients with chronic lymphocytic leukemia (CLL), including those with an unfavorable cytogenetic profile. However, patients develop resistance, with poor outcomes and no established treatment options. Mutations in BTK and PLCG2 have emerged as main mechanisms of drug resistance, but not all patients carry these mutations. Further understanding of mechanisms of resistance is urgently needed and will support rational development of new therapeutic strategies. To that end, we characterized the genomic profiles of serial samples from 9 patients with ibrutinib-relapsed disease, including 6 who had Richter transformation. Mutations, indels, copy-number aberrations, and loss of heterozygosity were assessed using next-generation sequencing and single-nucleotide polymorphism array. We found that 18p deletion (del(18p)), together with del(17p)/TP53 mutations, was present in 5 of 9 patients before ibrutinib therapy. In addition to BTKC481, we identified BTKT316A, a structurally novel mutation located in the SH2 domain of BTK. Minor BTK clones with low allele frequencies were captured in addition to major BTK clones. Although TP53 loss predisposes patients for relapse, clone size of TP53 loss may diminish during disease progression while mutant BTK clone expands. In patients who had Richter transformation, we found that the transformed cells were clonal descendants of circulating leukemia cells but continued to undergo evolution and drifts. Surprisingly, transformed lymphoma cells in tissue may acquire a different BTK mutation from that in the CLL leukemia cells. Collectively, these results provide insights into clonal evolution underlying ibrutinib relapse and prompt further investigation on genomic abnormalities that have clinical application potential.

Next-generation sequencing (NGS) genomic oncology profiling assays have emerged as key drivers of personalized cancer care and translational research. However, validation of these assays to meet strict clinical standards has been historically problematic because of both significant assay complexity and a scarcity of optimal validation samples. Herein, we present the clinical validation of 76 genes from a novel 1212-gene large-scale hybrid capture cancer sequencing assay (University of Chicago Medicine OncoPlus) using full-data comparisons against multiple clinical NGS amplicon-based assays to yield dramatic increases in per-sample data comparison efficiency compared with previously published validations. Using a sample set of 104 normal, solid tumor, and hematopoietic malignancy specimens, head-to-head NGS data analyses allowed for 6.8 million individual clinical base call comparisons, including 2729 previously confirmed variants, with 100% sensitivity and specificity. University of Chicago Medicine OncoPlus showed excellent performance for detection of single-nucleotide variants, insertions/deletions up to 52 bp, and FLT3internal tandem duplications of up to 102 bp or larger. Highly concordant copy number variant and ALK/RET/ROS1gene fusion detection were also observed. In addition to underlining the efficiency of NGS validation via full-data benchmarking against existing clinical NGS assays, this study also highlights the degree of performance similarity between hybrid capture and amplicon assays that is attainable with the application of strict quality control parameters and optimized computational analytics.

Amplicon-based targeted next-generation sequencing assays are used widely to test for clinically relevant somatic mutations in cancer. However, accurate detection of large insertions and deletions (indels) via these assays remains challenging. Sequencing reads that cover these anomalies are, by definition, different from the reference sequence, and lead to variable performance of alignment algorithms. Reads with large indels may be aligned incorrectly, causing incorrect calls, or may remain unmapped and essentially ignored by downstream informatics pipeline sub-processes. Herein, we describe Amplicon Indel Hunter (AIH), a novel large (>5-bp) indel detection method that is reference genome independent and highly sensitive for the identification of somatic indels in amplicon-based, paired-end, next-generation sequencing data. We validated the algorithm for sensitivity and specificity using a set of clinical cancer samples with Clinical Laboratory Improvement Amendment–confirmed indels as well as in silicomutated data sets. The AIH detected 100% of tested large indels with relatively higher mutant allele frequencies compared with a variety of traditional aligners, which showed variably reduced sensitivity and specificity by comparison. The AIH especially outperformed alignment-based methods for detection of all tested FLT3internal tandem duplications up to 102 bp. Because AIH runs in parallel to traditional alignment-based informatics pathways, it can be readily incorporated into nearly any analysis pipeline for somatic mutation detection in paired-end amplicon-based data.

IMPORTANCE: Next-generation sequencing has identified new genetic markers that have altered prognosis for patients with chronic lymphocytic leukemia (CLL) at diagnosis. Understanding the significance of these prognostic indicators and recognizing their potential impact on treatment selection and patients’ outcomes is critical for clinicians and investigators. OBJECTIVE: To review novel prognostic factors at CLL diagnosis that have shown an impact on the prognosis and outcomes of this disease. EVIDENCE REVIEW: Literature from January 2004 through December 2014 was searched in PubMed, Cochrane Central Register of Controlled Trials, and Scopus to identify English-language, peer-reviewed articles on clinical and prognostic factors for CLL (TP53, ATM, NOTCH1, SF3B1, BIRC3, and MYD88). Reference lists were subsequently reviewed for additional articles. A total of 450 articles was identified, and 48 articles meeting inclusion criteria were reviewed. FINDINGS: Among prognostic markers reviewed, chromosomal aberrations have been validated and are currently used clinically to predict prognosis. Patients with 17p13.1 deletion have poor response to chemoimmunotherapy and are treated differently, with some undergoing allogeneic transplantation in first remission. CD38 and ZAP-70 status of malignant cells and unmutated immunoglobulin variable heavy chain gene have similarly been validated to predict adverse prognosis, but their implications on treatment selection have not been proven. The presence of TP53 and ATM mutations predicts worse prognosis, which has been corroborated in various studies. Patients with TP53 mutations have lower responses to chemoimmunotherapy. Furthermore, patients with TP53 and ATM mutations have inferior progression-free survival and overall survival, independent of other factors. Patients carrying the NOTCH1 and SF3B1 mutations have worse prognosis; patients with the NOTCH1 mutation have lower response rates to standard chemoimmunotherapy. The impact of BIRC3 on prognosis and survival requires further confirmation. CONCLUSIONS AND RELEVANCE: The heterogeneous clinical course of CLL is likely explained by underlying molecular prognostic factors. Moving forward, analyzing these factors at diagnosis is recommended for better prognostication.

Recurrent mutations in JAK2and MPLgenes are genetic hallmarks of BCR-ABL1–negative myeloproliferative neoplasms. Detection of JAK2and MPLmutations has been incorporated into routine diagnostic algorithms for these diseases. This Special Article summarizes results from a nationwide laboratory survey of JAK2and MPLmutation analysis. Based on the current practice pattern and the literature, this Special Article provides recommendations and guidelines for laboratory practice for detection of mutations in the JAK2and MPLgenes, including clinical manifestations for prompting the mutation analysis, current and recommended methodologies for testing the mutations, and standardization for reporting the test results. This Special Article also points to future directions for genomic testing in BCR-ABL1–negative myeloproliferative neoplasms.

PU-H71 is a purine-scaffold Hsp90 inhibitor that, in contrast to other Hsp90 inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is preferentially associated with oncogenic client proteins and enriched in tumor cells (teHsp90). This property allows PU-H71 to potently suppress teHsp90 without inducing toxicity in normal cells. We found that lymphoma cells infected by Epstein-Barr virus or Kaposi sarcoma-associated herpes virus (KSHV) are exquisitely sensitive to this compound. Using PU-H71 affinity capture and proteomics, an unbiased approach to reveal oncogenic networks, we identified the teHsp90 interactome in KSHV+ primary effusion lymphoma cells. Viral and cellular proteins were identified, including many involved in nuclear factor (NF)-κB signaling, apoptosis, and autophagy. KSHV vFLIP is a viral oncoprotein homologous to cFLIPs, with NF-κB–activating and antiapoptotic activities. We show that teHsp90 binds vFLIP but not cFLIPs. Treatment with PU-H71 induced degradation of vFLIP and IKKγ, NF-κB downregulation, apoptosis and autophagy in vitro, and more importantly, tumor responses in mice. Analysis of the interactome revealed apoptosis as a central pathway; therefore, we tested a BCL2 family inhibitor in primary effusion lymphoma cells. We found strong activity and synergy with PU-H71. Our findings demonstrate PU-H71 affinity capture identifies actionable networks that may help design rational combinations of effective therapies.

PU-H71 is a purine-scaffold Hsp90 inhibitor that, in contrast to other Hsp90 inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is preferentially associated with oncogenic client proteins and enriched in tumor cells (teHsp90). This property allows PU-H71 to potently suppress teHsp90 without inducing toxicity in normal cells. We found that lymphoma cells infected by Epstein-Barr virus or Kaposi sarcoma-associated herpes virus (KSHV) are exquisitely sensitive to this compound. Using PU-H71 affinity capture and proteomics, an unbiased approach to reveal oncogenic networks, we identified the teHsp90 interactome in KSHV+primary effusion lymphoma cells. Viral and cellular proteins were identified, including many involved in nuclear factor (NF)-κB signaling, apoptosis, and autophagy. KSHV vFLIP is a viral oncoprotein homologous to cFLIPs, with NF-κB–activating and antiapoptotic activities. We show that teHsp90 binds vFLIP but not cFLIPs. Treatment with PU-H71 induced degradation of vFLIP and IKKγ, NF-κB downregulation, apoptosis and autophagy in vitro, and more importantly, tumor responses in mice. Analysis of the interactome revealed apoptosis as a central pathway; therefore, we tested a BCL2 family inhibitor in primary effusion lymphoma cells. We found strong activity and synergy with PU-H71. Our findings demonstrate PU-H71 affinity capture identifies actionable networks that may help design rational combinations of effective therapies.

The BCR-ABL1translocation is a hallmark of chronic myeloid leukemia. Because patients treated with imatinib and other tyrosine kinase inhibitors achieve lower levels of detectable disease, quantitation of BCR-ABL1transcripts with quantitative RT-PCR has become an essential tool in chronic myeloid leukemia monitoring. The prognostic significance of molecular responses was recently established by large-scale clinical trials. Achieving defined levels of BCR-ABL1on the International Scale within specific time frames is an important measure for assessing patient response and probability for relapse and progression. However, extensive variation in quantitative RT-PCR procedures and reporting makes it difficult to interpret these results. More important, lack of standardization, particularly in the United States, prevents the comparison of individual patient results to the data from the clinical trials, which thereby prohibits the meaningful use of such results in the direction of patient care. In this article, we will present an overview of the clinical trial discoveries that drive the need for standardization, review the most updated monitoring guidelines by the National Comprehensive Cancer Network, and highlight recommendations for laboratory practice regarding internal controls and reference materials. Finally, we will provide an update on the recent efforts in the standardization of quantitative RT-PCR reporting using the International Scale.

The past 25 years have witnessed the field of molecular pathology evolving from an imprecisely defined discipline to a firmly established medical subspecialty that plays an essential role in patient care. During this time, the training, certification, and licensure requirements for directing and performing testing in a molecular pathology or molecular diagnostics laboratory have become better defined. The purpose of this document is to describe the various board certifications available to individuals seeking certification in molecular diagnostics at the level of laboratory director, supervisor, or technologist. Several national organizations offer certification in molecular pathology or molecular diagnostics for doctoral-level clinical scientists to function as the director of a molecular diagnostics laboratory. Furthermore, 12 states and Puerto Rico require licensing of medical technologists, including those working in molecular diagnostic laboratories. The information provided here updates a 2002 document by the Training and Education Committee of the Association for Molecular Pathology and has been expanded to include certification and licensing requirements for laboratory technologists.

We studied 24 spleens with extramedullary hematopoietic proliferation (EMHP), a key feature of advanced-stage Philadelphia chromosome-negative myeloproliferative neoplasms, obtained from 24 patients (14 primary myelofibrosis, 7 polycythemia vera and 3 unclassifiable). Hematoxylin and eosin, reticulin and trichrome stains, and immunohistochemical stains for myeloperoxidase, glycophorin-C, CD42b, CD34, CD117, CD8, nerve growth factor receptor and smooth muscle actin were evaluated. Clinical information was correlated with the morphological findings. Three distinct histological patterns of EMHP were recognized: diffuse (12), nodular (5), and mixed-nodular and diffuse (7). The preponderant lineage was granulocytic in diffuse, trilineage in nodular and erythroid in mixed EMHP. Erythropoiesis was largely intravascular, granulopoiesis was within the splenic cords and megakaryopoiesis was observed in both locations. The stromal changes paralleled the histological pattern with preservation of the splenic stromal and vascular architecture in the diffuse areas as opposed to areas of nodular EMHP. The morphological features of the splenic EMHP did not correlate with specific subtypes of myeloproliferative neoplasms. The mean duration of follow-up from initial diagnosis was 80 months. A total of 15 of the 24 patients died of disease: 8 of 12 (67%) with diffuse, 2 of 5 (40%) with nodular and 5 of 7 (71%) with mixed growth patterns. The mean duration from diagnosis to splenectomy was shorter in patients with diffuse (83 months) as compared with those with nodular EMHP (127 months). Our study demonstrates that splenic extramedullary hematopoietic proliferation in Philadelphia chromosome-negative myeloproliferative neoplasms shows distinct histological patterns that do not correlate with disease subtypes, but appear to suggest a trend between the histological patterns and clinical behavior. These results suggest a different biology of the disease in the nodular and diffuse extramedullary hematopoietic proliferation groups.

We studied 24 spleens with extramedullary hematopoietic proliferation (EMHP), a key feature of advanced-stage Philadelphia chromosome-negative myeloproliferative neoplasms, obtained from 24 patients (14 primary myelofibrosis, 7 polycythemia vera and 3 unclassifiable). Hematoxylin and eosin, reticulin and trichrome stains, and immunohistochemical stains for myeloperoxidase, glycophorin-C, CD42b, CD34, CD117, CD8, nerve growth factor receptor and smooth muscle actin were evaluated. Clinical information was correlated with the morphological findings. Three distinct histological patterns of EMHP were recognized: diffuse (12), nodular (5), and mixed-nodular and diffuse (7). The preponderant lineage was granulocytic in diffuse, trilineage in nodular and erythroid in mixed EMHP. Erythropoiesis was largely intravascular, granulopoiesis was within the splenic cords and megakaryopoiesis was observed in both locations. The stromal changes paralleled the histological pattern with preservation of the splenic stromal and vascular architecture in the diffuse areas as opposed to areas of nodular EMHP. The morphological features of the splenic EMHP did not correlate with specific subtypes of myeloproliferative neoplasms. The mean duration of follow-up from initial diagnosis was 80 months. A total of 15 of the 24 patients died of disease: 8 of 12 (67%) with diffuse, 2 of 5 (40%) with nodular and 5 of 7 (71%) with mixed growth patterns. The mean duration from diagnosis to splenectomy was shorter in patients with diffuse (83 months) as compared with those with nodular EMHP (127 months). Our study demonstrates that splenic extramedullary hematopoietic proliferation in Philadelphia chromosome-negative myeloproliferative neoplasms shows distinct histological patterns that do not correlate with disease subtypes, but appear to suggest a trend between the histological patterns and clinical behavior. These results suggest a different biology of the disease in the nodular and diffuse extramedullary hematopoietic proliferation groups.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the role of SYK in its pathogenesis is not completely understood. Using tissue microarray, we demonstrated for the first time that SYK protein is activated in 27 of 61 (44%) primary human DLBCL tissues. Among DLBCL cell lines, 7 were sensitive and 3 were resistant to a highly specific SYK inhibitor, PRT060318. In sensitive DLBCL cells, SYK inhibition blocked the G1-S transition and caused cell-cycle arrest. This effect was reproduced by genetic reduction of SYK using siRNA. A detailed analysis of the BCR signaling pathways revealed that the consequence of SYK inhibition on PLCγ2 and AKT, as opposed to ERK1/2, was responsible for cell-cycle arrest. Genetic knock-down of these key molecules decelerated the proliferation of lymphoma cells. In addition, BCR signaling can be blocked by PRT060318 in primary lymphoma cells. Together, these findings provide insights into cellular pathways required for lymphoma cell growth and support the rationale for considering SYK inhibition as a potentially useful therapy for DLBCL. The results further suggest the possibility of using PLCγ2 and AKT as biomarkers to predict therapeutic response in prospective clinical trials of specific SYK inhibitors.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the role of SYK in its pathogenesis is not completely understood. Using tissue microarray, we demonstrated for the first time that SYK protein is activated in 27 of 61 (44%) primary human DLBCL tissues. Among DLBCL cell lines, 7 were sensitive and 3 were resistant to a highly specific SYK inhibitor, PRT060318. In sensitive DLBCL cells, SYK inhibition blocked the G1-S transition and caused cell-cycle arrest. This effect was reproduced by genetic reduction of SYK using siRNA. A detailed analysis of the BCR signaling pathways revealed that the consequence of SYK inhibition on PLCγ2 and AKT, as opposed to ERK1/2, was responsible for cell-cycle arrest. Genetic knock-down of these key molecules decelerated the proliferation of lymphoma cells. In addition, BCR signaling can be blocked by PRT060318 in primary lymphoma cells. Together, these findings provide insights into cellular pathways required for lymphoma cell growth and support the rationale for considering SYK inhibition as a potentially useful therapy for DLBCL. The results further suggest the possibility of using PLCγ2 and AKT as biomarkers to predict therapeutic response in prospective clinical trials of specific SYK inhibitors.

PRDM1/Blimp-1, a master regulator for B cell terminal differentiation, is a putative tumor suppressor in diffuse large B cell lymphomas (DLBCL). Inactivating mutations of PRDM1have been previously identified in a subset of nongerminal center B cell–like (GCB) DLBCL. We investigated the presence of alternative mechanisms of down-regulating PRDM1in a cohort of 25 primary DLBCL and six DLBCL cell lines. While some DLBCL, predominantly the GCB-type, showed low levels of both PRDM1α mRNA and protein, presumably as a result of direct transcription repression, discordant expressions between the two were identified in a subset of DLBCL without PRDM1mutations, the primarily non-GCB type, consistent with translational down-regulation. This subset of DLBCL exhibits relatively high PRDM1α mRNA levels but low levels of PRDM1. Data obtained from expression analysis, luciferase reporter assays, and transfection experiments support a role of targeting of PRDM1by microRNA let-7 family in mediating this down-regulation. Let-7, in particular let-7b, is overexpressed in DLBCL relative to normal GCB cells, suggesting that it is deregulated. Thus, abnormal epigenetic down-regulation of PRDM1by let-7 and other microRNAs may represent an alternative mechanism of reducing normal PRDM1 function in a subset of DLBCL with relatively high PRDM1α mRNA expression and unmutated PRDM1. These findings provide further evidence for an important role of impairment of terminal B cell differentiation in DLBCL pathogenesis.

An international basis for comparison of BCR-ABL mRNA levels is required for the common interpretation of data derived from individual laboratories. This will aid clinical decisions for individual patients with chronic myeloid leukemia (CML) and assist interpretation of results from clinical studies. We aligned BCR-ABL values generated by 38 laboratories to an international scale (IS) where a major molecular response (MMR) is 0.1% or less. Alignment was achieved by application of laboratory-specific conversion factors calculated by comparisons performed with patient samples against a reference method. A validation procedure was completed for 19 methods. We determined performance characteristics (bias and precision) for consistent interpretation of MMR after IS conversion. When methods achieved an average BCR-ABL difference of plus or minus 1.2-fold from the reference method and 95% limits of agreement within plus or minus 5-fold, the MMR concordance was 91%. These criteria were met by 58% of methods. When not met, the MMR concordance was 74% or less. However, irrespective of precision, when the bias was plus or minus 1.2-fold as achieved by 89% of methods, there was good agreement between the overall MMR rates. This indicates that the IS can deliver accurate comparison of molecular response rates between clinical trials when measured by different laboratories.

An international basis for comparison of BCR-ABLmRNA levels is required for the common interpretation of data derived from individual laboratories. This will aid clinical decisions for individual patients with chronic myeloid leukemia (CML) and assist interpretation of results from clinical studies. We aligned BCR-ABLvalues generated by 38 laboratories to an international scale (IS) where a major molecular response (MMR) is 0.1% or less. Alignment was achieved by application of laboratory-specific conversion factors calculated by comparisons performed with patient samples against a reference method. A validation procedure was completed for 19 methods. We determined performance characteristics (bias and precision) for consistent interpretation of MMR after IS conversion. When methods achieved an average BCR-ABLdifference of plus or minus 1.2-fold from the reference method and 95% limits of agreement within plus or minus 5-fold, the MMR concordance was 91%. These criteria were met by 58% of methods. When not met, the MMR concordance was 74% or less. However, irrespective of precision, when the bias was plus or minus 1.2-fold as achieved by 89% of methods, there was good agreement between the overall MMR rates. This indicates that the IS can deliver accurate comparison of molecular response rates between clinical trials when measured by different laboratories.

An acquired mutation in Janus kinase 2 (JAK2), V617F, has recently been identified in human myeloproliferative disorders. Detection of the mutation is helpful in differential diagnosis, prognosis, and predication of therapeutic response. Because the mutation can be present in a small proportion of granulocytic populations in myeloproliferative disorder patients, a highly sensitive detection method is required. In this study, we systematically optimized the reaction conditions of a published amplification refractory mutation system-polymerase chain reaction research protocol to make it a robust clinical diagnostic test. The modifications led to a clear demonstration of the V617F mutation in a patient who would have been easily missed by the original amplification refractory mutation system-polymerase chain reaction assay. The test detects the V617F mutation not only with a high analytic sensitivity of 0.05 to 0.1% but also with a high diagnostic specificity of 99%. In addition, the assay has the ability to distinguish cases with only mutant alleles from cases with mixed normal and mutant alleles. The assay is fast and easy to perform, and no special equipment other than thermocyclers is required. All these features make the assay readily and broadly applicable in clinical molecular diagnostic laboratories.

Peroxisome proliferator-activated receptor γ (PPARγ) is a metabolic regulator involved in maintaining glucose and fatty acid homeostasis. Besides its metabolic functions, the receptor has also been implicated in tumorigenesis. Ligands of PPARγ induce apoptosis in several types of tumor cells, leading to the proposal that these ligands may be used as antineoplastic agents. However, apoptosis induction requires high doses of ligands, suggesting the effect may not be receptor-dependent. In this report, we show that PPARγ is expressed in human primary T-cell lymphoma tissues and activation of PPARγ with low doses of ligands protects lymphoma cells from serum starvation-induced apoptosis. The prosurvival effect of PPARγ was linked to its actions on cellular metabolic activities. In serum-deprived cells, PPARγ attenuated the decline in ATP, reduced mitochondrial hyperpolarization, and limited the amount of reactive oxygen species (ROS) in favor of cell survival. Moreover, PPARγ regulated ROS through coordinated transcriptional control of a set of proteins and enzymes involved in ROS metabolism. Our study identified cell survival promotion as a novel activity of PPARγ. These findings highlight the need for further investigation into the role of PPARγ in cancer before widespread use of its agonists as anticancer therapeutics.

Quantitative monitoring of breakpoint cluster region(BCR)-Abelson kinase (ABL) transcripts has become indispensable in the clinical care of patients with chronic myelogenous leukemia. Because quantity and quality of RNA in clinical samples are highly variable, a suitable internal normalization control is required for accurate BCR-ABLquantification. However, few studies have examined suitability of the control genes using criteria relevant to residual disease testing. In this study, we evaluated a number of control genes with the application of several novel criteria, including control gene performance on serial patient sample testing and in a residual disease model. We also examined expression of the control genes in BCR-ABL-positive K562 cells in response to Gleevec treatment. We found that β-glucuronidase is the best control gene among those studied. Importantly, ABL, a widely used control gene, generates misleading BCR-ABLchanges that potentially affect the clinical management of chronic myelogenous leukemia patients.

Monitoring breakpoint cluster region-Abelson kinase (BCR-ABL) levels in patients treated for chronic myelogenous leukemia (CML) has become an integral part of patient management. Real-time reverse transcriptase- polymerase chain reaction is the method of choice for this purpose because of its high analytical sensitivity and reproducibility. Given the variation of RNA quality and quantity in clinical specimens, accurate quantitative assessment of BCR-ABL depends on normalization of the BCR-ABLsignal to an appropriate internal reference. However, the controls used by different laboratories vary, and there is no clear consensus on an ideal reference due to limited investigations. In this study, we compared nine commonly used control genes for three criteria: mRNA abundance, levels in CML and non-CML cells, and their degradation kinetics in comparison with BCR-ABL. We found that β-glucuronidase (GUSB) is the most suitable among the nine genes tested. Although ABL is most widely used, our data suggest that the amount of ABL is different in CML and non-CML cells. Moreover, ABLlevels are regulated by cellular stress. These findings have a direct impact on current clinical laboratory practice and patient care because the use of a proper control gene affects the reported levels of BCR-ABL transcripts used for patient management decisions.

The translocation t(14;18) resulting in fusion of the BCL2and the immunoglobulin heavy chain genes (BCL2-IGH)is present in 80% to 90% of follicular lymphomas and 20% to 30% of diffuse large B-cell lymphomas. Polymerase chain reaction (PCR) analysis for the translocation products suffers from low analytic specificity. As a result, either nested PCR or probe hybridization is required to aid in the identification of the specific translocation products. These added procedures are undesirable in clinical laboratories because nested procedures increase the possibility of contamination and probe hybridization increases assay turnaround time. To simplify the BCL2-IGHassay procedure, we attempted to eliminate the nonspecific PCR products by optimizing the annealing temperatures of the PCR assays using a gradient thermocycler. We showed that gradually increasing the annealing temperature from 55°C to 67°C significantly enhanced the intensity of the specific PCR products while eliminating the nonspecific ones. We compared the simplified procedure with a PCR-probe hybridization method on 68 patient specimens. The simplified procedure had increased analytic and diagnostic specificities with comparable sensitivities. With significantly improved analytic specificity, one round of PCR is sufficient to detect the BCL2-IGHgene rearrangements without further confirmation.

Thiazolidinediones (TZDs) are widely used for treatment of type 2 diabetes mellitus. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is the molecular target of TZDs and is believed to mediate the apoptotic effects of this class of drugs in a variety of cell types, including B and T lymphocytes. The finding that TZDs induce lymphocyte death has raised concerns regarding whether TZDs might further impair immune functions in diabetics. To address this issue, we investigated the roles of PPAR gamma and TZDs in lymphocyte survival. PPAR gamma was up-regulated upon T cell activation. As previously reported, PPAR gamma agonists induced T cell death in a dose-dependent manner. However, the concentrations of TZD needed to cause T cell death were above those needed to induce PPAR gamma-dependent transcription. Surprisingly, at concentrations that induce optimal transcriptional activation, TZD activation of PPAR gamma protected cells from apoptosis following growth factor withdrawal. The survival-enhancing effects depended on both the presence and activation of PPAR gamma. Measurements of mitochondrial potential revealed that PPAR gamma activation enhanced the ability of cells to maintain their mitochondrial potential. These data indicate that activation of PPAR gamma with TZDs can promote cell survival and suggest that PPAR gamma activation may potentially augment the immune responses of diabetic patients.

Background

Wang: Incyte Research Institute: Ended employment in the past 24 months.

Approximately 95% of individuals with spinal muscular atrophy (SMA) lack both copies of the SMNt gene at 5q13. The presence of a nearly identical centromeric homolog of the SMNt gene, SMNc, necessitates a quantitative polymerase chain reaction approach to direct carrier testing. Adapting a radioactivity-based method described previously, multiplex polymerase chain reaction was performed using fluorescently labeled primers followed by analysis on an ABI 373a DNA sequencer. The SMNt copy number was calculated from ratios of peak areas using both internal and genomic standards. Samples from 60 presumed carriers (50 parents of affected individuals and 10 relatives implicated by linkage analysis) and 40 normal control individuals were tested. Normalized results (to the mean of five or more control samples harboring two copies of the SMNt gene) were consistently within the ranges of 0.4 to 0.6 for carriers (one copy) and 0.8 to 1.2 for normal controls (two copies), without overlap. Combining linkage analyses with direct carrier test results demonstrated de novo deletions associated with crossovers, unaffected individuals carrying two SMNt gene copies on one chromosome and zero SMNt gene copies on the other chromosome, and unaffected individuals with three copies of the SMNt gene. This report demonstrates that fluorescence-based carrier testing for SMA is accurate, reproducible, and useful for genetic risk assessment, and that carrier testing may need to be combined with linkage analysis in certain circumstances. Am. J. Med. Genet. 85:463–469, 1999. © 1999 Wiley-Liss, Inc.

Using genomic DNA from patients with follicular lymphoma, we performed polymerase chain reaction (PCR) amplifications to detect t(14;18) translocations. Unexpectedly large products of ∼1 kilobase (kb) were detected by gel electrophoresis in 2 of 50 positive cases. In these 2 cases, sequence analyses showed novel breakpoints in the 3' untranslated region of bcl-2, ∼800 bp downstream of the major breakpoint region (mbr). The breakpoints in IgH occurred in JH4 in one patient and JH5 in the other. Sequences just upstream of the new bcl-2 breakpoints suggest a mechanism of translocation that may include minisatellite core-mediated recombination. In one of our two patients with novel bcl-2 breakpoints, the ∼1 kb product obtained using conventional mbr primers was detectable only when a nested PCR was performed. These findings have important implications for diagnosis and minimal residual disease detection in t(14;18)-positive lymphomas.

Three common alleles of apolipoprotein E (apoE) have been identified and are expressed codominantly to generate six genotypes. Different apoE genotypes are implicated in several cardiovascular and neurologic disorders. Testing for apoE genotypes has increasing diagnostic importance, particularly in the risk assessment of coronary artery disease. A reproducible and cost-effective assay was developed.

The objective of this work was to study the prevalence of the Factor V Leiden mutation in an obstetrical clinic largely comprised of African‐American women. A cross‐sectional study was performed evaluating a total of 231 consecutive women of African‐American origin. Of these patients, 21 were considered at high risk for thrombosis, but none was found to carry the mutation. One patient (0·4 per cent) of the total was heterozygous for the Factor V Leiden mutation. African‐American women do not appear to be at an increased risk of being heterozygous or homozygous for the Leiden mutation. This low prevalence may be confounded by ascertainment bias in a population of pregnant women. © 1998 John Wiley & Sons, Ltd.

The objective of this work was to study the prevalence of the Factor V Leiden mutation in an obstetrical clinic largely comprised of African-American women. A cross-sectional study was performed evaluating a total of 231 consecutive women of African-American origin. Of these patients, 21 were considered at high risk for thrombosis, but none was found to carry the mutation. One patient (0·4 per cent) of the total was heterozygous for the Factor V Leiden mutation. African-American women do not appear to be at an increased risk of being heterozygous or homozygous for the Leiden mutation. This low prevalence may be confounded by ascertainment bias in a population of pregnant women. © 1998 John Wiley & Sons, Ltd.

Ibrutinib (Ibr) is a specific inhibitor of Bruton tyrosine kinase, a component of the B-cell receptor (BCR) signaling. Venetoclax (Veneto) inhibits the anti-apoptotic protein BCL2. Both drugs are highly effective as monotherapy against chronic lymphocytic leukemia (CLL) (Byrd NEJM, Roberts, NEJM, 2015 and Roberts, Blood, 2019) and clinical trials using the combination therapy are ongoing. An interesting clinical observation is that the tumor cell sensitivity to these two drugs differ between different anatomic compartments. While lymph node-resident CLL cells are more sensitive to ibr, circulating CLL cells in the peripheral blood are more sensitive to the action of veneto. When these two drugs are used in combination to treat relapsed/refractory (Hillman ASH 2018 and Kater EHA 2019) or previously untreated CLL patients (Jain NEJM 2019), rate of complete response significantly increases compared to single drug alone. Moreover, negativity of bone marrow minimal residual disease continues to improve over time.

Wang: Incyte Reaserch Institute: Employment. Ma:Abbvie: Research Funding; Xeme: Research Funding; Novartis: Research Funding; Bioverativ: Consultancy; Janssen: Consultancy, Speakers Bureau; Beigene: Research Funding; Kite: Consultancy; Gilead: Research Funding; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Acerta: Research Funding; Astra Zeneca: Consultancy, Research Funding, Speakers Bureau; Genentech: Consultancy; Incyte: Research Funding; Juno: Research Funding. Larson:Celgene: Consultancy; Agios: Consultancy; Novartis: Honoraria, Other: Contracts for clinical trials.

Wang: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Research Funding; AstraZeneca: Consultancy, Research Funding; MoreHealth: Consultancy; Pharmacyclics: Honoraria, Research Funding; Novartis: Research Funding; Dava Oncology: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Honoraria, Research Funding.

Background:

Background: B-cell receptor (BCR) signaling pathway is recognized as a crucial pathway for the pathogenesis of neoplastic B-cells. Inhibition of the BCR signaling and the downstream pathway is highly effective in B-cell malignancy through Bruton tyrosine kinase inhibition by ibrutinib. In addition to cell proliferation inhibition, ibrutinib disrupts cell adhesion between tumor and its microenvironment through unknown molecular mechanisms, resulting in peripheral lymphocytosis with accompanying lymphadenopathy reduction in patients who receive ibrutinib.

Stock: Jazz Pharmaceuticals: Consultancy. Riedell:Bayer: Consultancy, Speakers Bureau; Novartis: Consultancy; Kite Pharmaceuticals: Consultancy, Speakers Bureau. Smith:BMS: Consultancy; Portola: Honoraria.

Background:Mantle cell lymphoma (MCL) is an incurable non-Hodgkin B-cell lymphoma. B-cell receptor (BCR) signaling is chronically active in several mature B-cell malignancies including mantle cell lymphoma. Inhibition of the BCR and downstream pathway is highly effective in B-cell neoplasia and can be achieved at the cellular level through BTK inhibition by ibrutinib, for example. Ibrutinib has generated remarkable patients' response in large clinical trials and has been approved by FDA for the treatment of relapsed/refractory MCL. Ibrutinib targets Bruton tyrosine kinase primarily by inhibition of B-cell receptor signaling and cell proliferation, and secondarily by induction of tumor apoptosis. In addition, the drug inhibits the interaction of tumor cells with their microenvironment by disruption of cell adhesion. The effect on cell adhesion appears to be a mechanism shared by many BCR-targeted therapies including inhibitors of LYN (Leuk Res 38, 34, 2014), SYK (Blood, 115, 2578, 2010; Blood 125, 2336, 2015), and PI3K (idelalisib) (NEJM, 370, 997, 2014; Blood, 125, 2306). This class action raised a question regarding how the BCR pathway is connected to the cell adhesion phenotype. We set out to investigate the underlying mechanisms that are largely unknown at the present time.

Background:18F-fluorodeoxyglucose (FDG) positron emission tomography with computed tomography (PET-CT) has been widely used in lymphoma patients for diagnosis, staging, assessment of response and surveillance. And the standardized uptake value (SUV), a commonly used indicator in PET-CT scan, can quantitatively reflect the glucose uptake and glycolysis of the tumor. Diffuse large B-cell lymphoma (DLBCL), one of the most common subtypes of aggressive lymphomas, is supposed to exhibit high standardized uptake value (SUV) in 18F-FDG PET-CT due to high rate of glucose (18F-FDG) uptake and high level of glycolysis. Nevertheless, PET-CT “invisible” DLBCL cases have also been described, which featuring a relatively low 18F-FDG uptake denoted by low SUV. Herein, we analyzed this subgroup of under-estimated disease.

Background: B-cell receptor signaling (BCR) plays a central role in mantle cell lymphoma (MCL) and inhibition of BCR signaling through the BTK inhibitor, ibrutinib, has generated remarkable responses in patients. However, approximately one-third of the patients do not respond well to the drug, and disease relapse on ibrutinib is nearly universal. Alternative therapeutic strategies aimed to prevent and overcome ibrutinib resistance are needed. Selinexor is a first-in-class selective inhibitor of nuclear export. It binds and inhibits exportin XPO-1, which mediates the nuclear export of proteins and mRNAs. Inactivation of XPO-1 increases the retention of tumor suppressors in the nuclei to enhance suppression of tumor cell growth. Inactivation of exportin also decreases the the expression of oncogenes of which translation is eIF4E-dependent. Since many of the oncogenes and tumor suppressor genes in MCL also shuttle between nuclei and cytoplasm, we speculate that selinexor would be active in MCL. As selinexor acts on a cellular process rather than a particular molecular target, we thus postulate selinexor may have the potential to act broadly in both ibrutinib-sensitive and -resistant mantle lymphoma cells. In this study, we compared and contrasted the intrinsic cellular response of MCL cells to ibrutinib versus selinexor. We aim to identify molecular mechanisms underlying differential cellular response to these two drugs in order to obtain a deeper understanding of what underlies tumor cells' susceptibility to drug intervention.