Eric D. Hsi

Pralatrexate in Patients With Relapsed or Refractory Peripheral T-Cell Lymphoma: Results From the Pivotal PROPEL Study

PURPOSE Peripheral T-cell lymphoma (PTCL) is a poor prognosis subtype of non-Hodgkins lymphoma with no accepted standard of care. This study evaluated the efficacy and tolerability of pralatrexate, a novel antifolate with promising activity. PATIENTS AND METHODS Patients with independently confirmed PTCL who progressed following ≥ 1 line of prior therapy received pralatrexate intravenously at 30 mg/m(2)/wk for 6 weeks in 7-week cycles. Primary assessment of response was made by independent central review using the International Workshop Criteria. The primary end point was overall response rate. Secondary end points included duration of response, progression-free survival (PFS), and overall survival (OS). RESULTS Of 115 patients enrolled, 111 were treated with pralatrexate. The median number of prior systemic therapies was three (range, 1 to 12). The response rate in 109 evaluable patients was 29% (32 of 109), including 12 complete responses (11%) and 20 partial responses (18%), with a median DoR of 10.1 months. Median PFS and OS were 3.5 and 14.5 months, respectively. The most common grade 3/4 adverse events were thrombocytopenia (32%), mucositis (22%), neutropenia (22%), and anemia (18%). CONCLUSION To our knowledge, PROPEL (Pralatrexate in Patients with Relapsed or Refractory Peripheral T-Cell Lymphoma) is the largest prospective study conducted in patients with relapsed or refractory PTCL. Pralatrexate induced durable responses in relapsed or refractory PTCL irrespective of age, histologic subtypes, amount of prior therapy, prior methotrexate, and prior autologous stem-cell transplant. These data formed the basis for the US Food and Drug Administration approval of pralatrexate, the first drug approved for this disease.

MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

Diffuse large B-cell lymphoma (DLBCL) is stratified into prognostically favorable germinal center B-cell (GCB)-like and unfavorable activated B-cell (ABC)-like subtypes based on gene expression signatures. In this study, we analyzed 893 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). We show that MYC/BCL2 protein coexpression occurred significantly more commonly in the ABC subtype. Patients with the ABC or GCB subtype of DLBCL had similar prognoses with MYC/BCL2 coexpression and without MYC/BCL2 coexpression. Consistent with the notion that the prognostic difference between the 2 subtypes is attributable to MYC/BCL2 coexpression, there is no difference in gene expression signatures between the 2 subtypes in the absence of MYC/BCL2 coexpression. DLBCL with MYC/BCL2 coexpression demonstrated a signature of marked downregulation of genes encoding extracellular matrix proteins, those involving matrix deposition/remodeling and cell adhesion, and upregulation of proliferation-associated genes. We conclude that MYC/BCL2 coexpression in DLBCL is associated with an aggressive clinical course, is more common in the ABC subtype, and contributes to the overall inferior prognosis of patients with ABC-DLBCL. In conclusion, the data suggest that MYC/BCL2 coexpression, rather than cell-of-origin classification, is a better predictor of prognosis in patients with DLBCL treated with R-CHOP.

Genetic heterogeneity of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-κB (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.

The genetic landscape of mutations in Burkitt lymphoma

Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.

Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation: UTX , EZH2 , and DNMT3A

Chronic myelomonocytic leukemia (CMML), a myelodysplastic/myeloproliferative neoplasm, is characterized by monocytic proliferation, dysplasia, and progression to acute myeloid leukemia. CMML has been associated with somatic mutations in diverse recently identified genes. We analyzed 72 well-characterized patients with CMML (N = 52) and CMML-derived acute myeloid leukemia (N = 20) for recurrent chromosomal abnormalities with the use of routine cytogenetics and single nucleotide polymorphism arrays along with comprehensive mutational screening. Cytogenetic aberrations were present in 46% of cases, whereas single nucleotide polymorphism array increased the diagnostic yield to 60%. At least 1 mutation was found in 86% of all cases; novel UTX, DNMT3A, and EZH2 mutations were found in 8%, 10%, and 5.5% of patients, respectively. TET2 mutations were present in 49%, ASXL1 in 43%, CBL in 14%, IDH1/2 in 4%, KRAS in 7%, NRAS in 4%, and JAK2 V617F in 1% of patients. Various mutant genotype combinations were observed, indicating molecular heterogeneity in CMML. Our results suggest that molecular defects affecting distinct pathways can lead to similar clinical phenotypes.

STAT3 mutations unify the pathogenesis of chronic lymphoproliferative disorders of NK cells and T-cell large granular lymphocyte leukemia

Chronic lymphoproliferative disorders of natural killer cells (CLPD-NKs) and T-cell large granular lymphocytic leukemias (T-LGLs) are clonal lymphoproliferations arising from either natural killer cells or cytotoxic T lymphocytes (CTLs). We have investigated for distribution and functional significance of mutations in 50 CLPD-NKs and 120 T-LGL patients by direct sequencing, allele-specific PCR, and microarray analysis. STAT3 gene mutations are present in both T and NK diseases: approximately one-third of patients with each type of disorder convey these mutations. Mutations were found in exons 21 and 20, encoding the Src homology 2 domain. Patients with mutations are characterized by symptomatic disease (75%), history of multiple treatments, and a specific pattern of STAT3 activation and gene deregulation, including increased expression of genes activated by STAT3. Many of these features are also found in patients with wild-type STAT3, indicating that other mechanisms of STAT3 activation can be operative in these chronic lymphoproliferative disorders. Treatment with STAT3 inhibitors, both in wild-type and mutant cases, resulted in accelerated apoptosis. STAT3 mutations are frequent in large granular lymphocytes suggesting a similar molecular dysregulation in malignant chronic expansions of NK and CTL origin. STAT3 mutations may distinguish truly malignant lymphoproliferations involving T and NK cells from reactive expansions.

Comprehensive gene expression profiling and immunohistochemical studies support application of immunophenotypic algorithm for molecular subtype classification in diffuse large B-cell lymphoma: a report from the International DLBCL Rituximab-CHOP Consortium Program Study

Gene expression profiling (GEP) has stratified diffuse large B-cell lymphoma (DLBCL) into molecular subgroups that correspond to different stages of lymphocyte development–namely germinal center B-cell like and activated B-cell like. This classification has prognostic significance, but GEP is expensive and not readily applicable into daily practice, which has lead to immunohistochemical algorithms proposed as a surrogate for GEP analysis. We assembled tissue microarrays from 475 de novo DLBCL patients who were treated with rituximab-CHOP chemotherapy. All cases were successfully profiled by GEP on formalin-fixed, paraffin-embedded tissue samples. Sections were stained with antibodies reactive with CD10, GCET1, FOXP1, MUM1 and BCL6 and cases were classified following a rationale of sequential steps of differentiation of B cells. Cutoffs for each marker were obtained using receiver-operating characteristic curves, obviating the need for any arbitrary method. An algorithm based on the expression of CD10, FOXP1 and BCL6 was developed that had a simpler structure than other recently proposed algorithms and 92.6% concordance with GEP. In multivariate analysis, both the International Prognostic Index and our proposed algorithm were significant independent predictors of progression-free and overall survival. In conclusion, this algorithm effectively predicts prognosis of DLBCL patients matching GEP subgroups in the era of rituximab therapy.

Mutations of E3 Ubiquitin Ligase Cbl Family Members Constitute a Novel Common Pathogenic Lesion in Myeloid Malignancies

PURPOSE Acquired somatic uniparental disomy (UPD) is commonly observed in myelodysplastic syndromes (MDS), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), or secondary acute myelogenous leukemia (sAML) and may point toward genes harboring mutations. Recurrent UPD11q led to identification of homozygous mutations in c-Cbl, an E3 ubiquitin ligase involved in attenuation of proliferative signals transduced by activated receptor tyrosine kinases. We examined the role and frequency of Cbl gene family mutations in MPN and related conditions. METHODS We applied high-density SNP-A karyotyping to identify loss of heterozygosity of 11q in 442 patients with MDS, MDS/MPN, MPN, sAML evolved from these conditions, and primary AML. We sequenced c-Cbl, Cbl-b, and Cbl-c in patients with or without corresponding UPD or deletions and correlated mutational status with clinical features and outcomes. RESULTS We identified c-Cbl mutations in 5% and 9% of patients with chronic myelomonocytic leukemia (CMML) and sAML, and also in CML blast crisis and juvenile myelomonocytic leukemia (JMML). Most mutations were homozygous and affected c-Cbl; mutations in Cbl-b were also found in patients with similar clinical features. Patients with Cbl family mutations showed poor prognosis, with a median survival of 5 months. Pathomorphologic features included monocytosis, monocytoid blasts, aberrant expression of phosphoSTAT5, and c-kit overexpression. Serial studies showed acquisition of c-Cbl mutations during malignant evolution. CONCLUSION Mutations in the Cbl family RING finger domain or linker sequence constitute important pathogenic lesions associated with not only preleukemic CMML, JMML, and other MPN, but also progression to AML, suggesting that impairment of degradation of activated tyrosine kinases constitutes an important cancer mechanism.

LMO2 Protein Expression Predicts Survival in Patients With Diffuse Large B-Cell Lymphoma Treated With Anthracycline-Based Chemotherapy With and Without Rituximab

PURPOSE The heterogeneity of diffuse large B-cell lymphoma (DLBCL) has prompted the search for new markers that can accurately separate prognostic risk groups. We previously showed in a multivariate model that LMO2 mRNA was a strong predictor of superior outcome in DLBCL patients. Here, we tested the prognostic impact of LMO2 protein expression in DLBCL patients treated with anthracycline-based chemotherapy with or without rituximab. PATIENTS AND METHODS DLBCL patients treated with anthracycline-based chemotherapy alone (263 patients) or with the addition of rituximab (80 patients) were studied using immunohistochemistry for LMO2 on tissue microarrays of original biopsies. Staining results were correlated with outcome. RESULTS In anthracycline-treated patients, LMO2 protein expression was significantly correlated with improved overall survival (OS) and progression-free survival (PFS) in univariate analyses (OS, P = .018; PFS, P = .010) and was a significant predictor independent of the clinical International Prognostic Index (IPI) in multivariate analysis. Similarly, in patients treated with the combination of anthracycline-containing regimens and rituximab, LMO2 protein expression was also significantly correlated with improved OS and PFS (OS, P = .005; PFS, P = .009) and was a significant predictor independent of the IPI in multivariate analysis. CONCLUSION We conclude that LMO2 protein expression is a prognostic marker in DLBCL patients treated with anthracycline-based regimens alone or in combination with rituximab. After further validation, immunohistologic analysis of LMO2 protein expression may become a practical assay for newly diagnosed DLBCL patients to optimize their clinical management.

Mutational profile and prognostic significance of TP53 in diffuse large B-cell lymphoma patients treated with R-CHOP: Report from an International DLBCL Rituximab-CHOP Consortium Program Study

TP53 mutation is an independent marker of poor prognosis in patients with diffuse large B-cell lymphoma (DLBCL) treated with cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (CHOP) therapy. However, its prognostic value in the rituximab immunochemotherapy era remains undefined. In the present study of a large cohort of DLBCL patients treated with rituximab plus CHOP (R-CHOP), we show that those with TP53 mutations had worse overall and progression-free survival compared with those without. Unlike earlier studies of patients treated with CHOP, TP53 mutation has predictive value for R-CHOP-treated patients with either the germinal center B-cell or activated B-cell DLBCL subtypes. Furthermore, we identified the loop-sheet-helix and L3 motifs in the DNA-binding domain to be the most critical structures for maintaining p53 function. In contrast, TP53 deletion and loss of heterozygosity did not confer worse survival. If gene mutation data are not available, immunohistochemical analysis showing > 50% cells expressing p53 protein is a useful surrogate and was able to stratify patients with significantly different prognoses. We conclude that assessment of TP53 mutation status is important for stratifying R-CHOP-treated patients into distinct prognostic subsets and has significant value in the design of future therapeutic strategies.