Evolving frontline immunochemotherapy for mantle cell lymphoma and the impact on survival outcomes.

Because there have been a dvances in frontline treatment for mantle cell lymphoma (MCL) over the last 2 decades, we sought to characterize the changes in frontline treatment patterns and their association with outcomes. Patients with newly diagnosed MCL from September 2002 through June 2015 were enrolled in a prospective cohort study, and clinical characteristics, treatment, and clinical outcomes were compared between patients diagnosed from 2002 to 2009 (Era 1) compared with 2010 to 2015 (Era 2). Patient age, sex, and simplified MCL International Prognostic Index (sMIPI) score were similar between the 2 groups. In patients age 65 years or younger, there was less use of rituximab plus hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (R-Hyper-CVAD) (16.1% vs 8.8%) but more use of rituximab plus maximum-strength cyclophosphamide, doxorubicin, vincristine, and prednisone (R-maxi-CHOP) alternating with rituximab plus high-dose cytarabine (R-HiDAC), also known as the Nordic regimen, and R-CHOP alternating with rituximab plus dexamethasone, high-dose cytarabine, and cisplatin (R-DHAP) (1.1% vs 26.4%) and less use of R-CHOP or R-CHOP-like regimens (64.5% vs 35.2%) but more use of R-bendamustine (0% vs 12.1%) in Era 2 (P < .001). These changes were associated with improved event-free survival (EFS; 5-year EFS, 34.3% vs 50.0%; P = .010) and overall survival (OS; 5-year OS, 68.8% vs 81.6%; P = .017) in Era 2. In patients older than age 65 years, there was less use of R-CHOP or R-CHOP-like therapy (39.0% vs 14.3%) and nonstandard systemic therapy (36.6% vs 13.0%) but more use of R-bendamustine (0% vs 49.4%). These changes were associated with a trend for improved EFS (5-year EFS, 25.4% vs 37.5%; P = .051) in Era 2. The shift from R-CHOP or R-CHOP-like regimens to R-bendamustine was associated with improved EFS (5-year EFS, 25.0% vs 44.6%; P = .008) in Era 2. Results from this prospective cohort study provide critical real-world evidence for improved outcomes with evolving frontline patterns of care in patients with MCL.

Markers of Follicular Helper T Cells Are Occasionally Expressed in T-Cell or Histiocyte-Rich Large B-Cell Lymphoma, Classic Hodgkin Lymphoma, and Atypical Paracortical HyperplasiaA Diagnostic Pitfall For T-Cell Lymphomas of T Follicular Helper Origin.

OBJECTIVES: Follicular helper T cell (TFH) markers are expressed in angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma of the TFH phenotype (PTCL-TFH). However, differential expression and coexpression of these markers in benign and other malignant lymphoid proliferations have not been well studied. METHODS: We performed programmed death-1 (PD-1), C-X-C motif chemokine ligand 13 (CXCL13), inducible costimulator (ICOS), CD10, and B-cell lymphoma 6 protein (BCL-6) immunohistochemistry on AITL, PTCL not otherwise specified (PTCL-NOS), PTCL-TFH, T-cell or histiocyte-rich large B-cell lymphoma (THRLBCL), classic Hodgkin lymphoma (CHL), atypical paracortical hyperplasia (PCH), progressive transformation of germinal centers (PTGC), and reactive follicular hyperplasia (RFH). RESULTS: CXCL13 and ICOS were more sensitive but less specific for AITL than PD-1, CD10, and BCL-6. Moreover, 74% of AITL (none of PTCL-NOS or PTCL-TFH) coexpressed more than 2 TFH markers. In background T cells of THRLBCL, 70% of cases coexpressed more than 1 marker. The background T cells of CHL expressed all TFH markers except CD10 in all cases. In addition, 13% of PCH cases coexpressed more than 1 marker. In RFH and PTGC, all markers were expressed mainly in germinal centers with rare extrafollicular staining. CONCLUSIONS: AITL, PTCL-NOS, and PTCL-TFH show differential expression of TFH markers. AITL frequently coexpresses more than 2 TFH markers. TFH markers can be expressed in PCH and in background T cells of THRLBCL and CHL. Consequently, caution should be used before a diagnosis of AITL is established, particularly with limited samples.

Utility of Flow Cytometry and Fluorescence In Situ Hybridization in Follow-up Monitoring of Plasma Cell Myeloma.

OBJECTIVES: We sought to investigate the clinical utility of flow cytometry (FC) and fluorescence in situ hybridization (FISH) in the workup of myeloma. METHODS: We retrospectively reviewed the reports of bone marrow biopsies received for myeloma evaluation between October 2015 and January 2019. RESULTS: A total of 1,708 biopsy specimens from 469 myeloma patients (mean age, 64.5 years [SD, 9.3]; female, 41.4%) were reviewed. Both FC and FISH had comparable detection rates at the time of initial diagnosis (97.6% vs 98.8%) and for follow-up cases (28.6% vs 28.2%). FC and FISH results were concordant in 98.8% of the initial diagnosis cases and 89.6% of the follow-up cases. The FISH-positive (FISH+)/FC-negative (FC-) discordance and FISH-/FC+ discordance occurred among 81 (5.0%) and 87 (5.4%) follow-up cases. In comparison with all concordant cases, FISH+/FC- discordant cases were more likely to have received treatment with daratumumab (P < .05). CONCLUSIONS: Plasma cell-enriched FISH and FC have comparable abnormal plasma cell detection rates, and approximately 10% of the follow-up cases have discordant FISH and FC results in which residual disease is detected by only one of these modalities. FISH testing should be considered for cases with negative FC, especially in patients who have received treatment with daratumumab or in cases in which there is concern about specimen adequacy.

Molecular profiling reveals a hypoxia signature in breast implant-associated anaplastic large cell lymphoma.

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a recently characterized T-cell malignancy that has raised significant patient safety concerns and led to worldwide impact on the implants used and clinical management of patients undergoing reconstructive or cosmetic breast surgery. Molecular signatures distinguishing BIA-ALCL from other ALCLs have not been fully elucidated and classification of BIA-ALCL as a WHO entity remains provisional. We performed RNA sequencing and gene set enrichment analysis comparing BIA-ALCLs to non-BIA-ALCLs and identified dramatic upregulation of hypoxia signaling genes including the hypoxia-associated biomarker CA9 (carbonic anyhydrase-9). Immunohistochemistry validated CA9 expression in all BIA-ALCLs, with only minimal expression in non-BIA-ALCLs. Growth induction in BIA-ALCL-derived cell lines cultured under hypoxic conditions was proportional to up-regulation of CA9 expression, and RNA sequencing demonstrated induction of the same gene signature observed in BIA-ALCL tissue samples compared to non-BIA-ALCLs. CA9 silencing blocked hypoxia-induced BIA-ALCL cell growth and cell cycle-associated gene expression, whereas CA9 overexpression in BIA-ALCL cells promoted growth in a xenograft mouse model. Furthermore, CA9 was secreted into BIA-ALCL cell line supernatants and was markedly elevated in human BIA-ALCL seroma samples. Finally, serum CA9 concentrations in mice bearing BIA-ALCL xenografts were significantly elevated compared to control serum. Together, these findings characterize BIA-ALCL as a hypoxia-associated neoplasm, likely attributable to the unique microenvironment in which it arises. These data support classification of BIA-ALCL as a distinct entity and uncover opportunities for investigating hypoxia-related proteins such as CA9 as novel biomarkers and therapeutic targets in this disease.

Clinical laboratory validation of the MCL35 assay for molecular risk stratification of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a clinically heterogeneous B cell malignancy for which a variety of prognostic factors have been proposed. Previously, a digital gene expression profiling "proliferation signature" capable of risk stratifying MCL was identified and subsequently developed into a multi-analyte prognostic assay, known as the "MCL35" assay. In this study, we sought to explore the performance characteristics of the MCL35 assay in a clinical laboratory and compare results with the Ki67 proliferation marker. The results describe the clinical validation of the MCL35 assay for molecular risk stratification of MCL including accuracy, sensitivity, specificity, use in acid-decalcified bone marrow core biopsies, fixatives, lower limit of RNA input, quality metrics, and other laboratory parameters. The resulting data indicate that this is a robust technique with outstanding reproducibility. Overall, the data support the concept of molecular signatures, as assessed with digital gene expression profiling, for improved standardization and reproducibility for proliferation assessment in MCL.

Recurrent MSC E116K mutations in ALK-negative anaplastic large cell lymphoma.

Anaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK)+ or ALK-, based on the presence or absence of ALK rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, MSC E116K, exclusively in ALK- ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of MSC E116K for ALK- ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting DUSP22 rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin-bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSCE116K acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of MYC and cell cycle inhibition. Chromatin immunoprecipitation-sequencing and transcriptome analysis identified the cell cycle regulatory gene E2F2 as a direct transcriptional target of musculin. MSCE116K reversed E2F2-induced cell cycle arrest and promoted expression of the CD30-IRF4-MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the MSC promoter. Finally, ALCL cells expressing MSC E116K were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent MSC mutation as a key driver of the CD30-IRF4-MYC axis and cell cycle progression in a unique subset of ALCLs.

Early event status informs subsequent outcome in newly diagnosed follicular lymphoma.

Recent advances in follicular lymphoma (FL) have resulted in prolongation of overall survival (OS). Here we assessed if early events as defined by event-free survival (EFS) at 12 and 24 months from diagnosis (EFS12/EFS24) can inform subsequent OS in FL. 920 newly diagnosed grade 1-3A FL patients enrolled on the University of Iowa/Mayo Clinic Lymphoma SPORE Molecular Epidemiology Resource (MER) from 2002-2012 were initially evaluated. EFS was defined as time from diagnosis to progression, relapse, re-treatment, or death due to any cause. OS was compared to age-and-sex-matched survival in the general US population using standardized mortality ratios (SMR) and 95% confidence intervals (CI). We used a cohort of 412 FL patients from two Lyon, France hospital registries for independent replication. Patients who failed to achieve EFS12 had poor subsequent OS (MER SMR = 3.72, 95%CI: 2.78-4.88; Lyon SMR = 8.74, 95%CI: 5.41-13.36). Conversely, patients achieving EFS12 had no added mortality beyond the background population (MER SMR = 0.73, 95%CI: 0.56-0.94, Lyon SMR = 1.02, 95%CI: 0.58-1.65). Patients with early events after immunochemotherapy had especially poor outcomes (EFS12 failure: MER SMR = 17.63, 95%CI:11.97-25.02, Lyon SMR = 19.10, 95%CI:9.86-33.36; EFS24 failure: MER SMR = 13.02, 95%CI:9.31-17.74, Lyon SMR = 7.22, 95%CI:4.13-11.74). In a combined dataset of all patients from both cohorts, baseline FLIPI was no longer informative in EFS12 achievers. Reassessment of patient status at 12 months from diagnosis in follicular lymphoma patients, or at 24 months in patients treated with immunochemotherapy, is a strong predictor of subsequent overall survival in FL. Early event status provides a simple, clinically relevant endpoint for studies assessing outcome in FL. Am. J. Hematol. 91:1096-1101, 2016. © 2016 Wiley Periodicals, Inc.

Personalized risk prediction for event-free survival at 24 months in patients with diffuse large B-cell lymphoma.

We recently defined event-free survival at 24 months (EFS24) as a clinically relevant outcome for patients with DLBCL. Patients who fail EFS24 have very poor overall survival, while those who achieve EFS24 have a subsequent overall survival equivalent to that of the age- and sex-matched general population. Here, we develop and validate a clinical risk calculator (IPI24) for EFS24. Model building was performed on a discovery dataset of 1,348 patients with DLBCL and treated with anthracycline-based immunochemotherapy. A multivariable model containing age, Ann Arbor stage, normalized serum LDH, ALC, ECOG performance status, bulky disease, and sex was identified. The model was then applied to an independent validation dataset of 1,177 DLBCL patients. The IPI24 score estimates the probability of failing to achieve the EFS24 endpoint for an individual patient. The IPI24 model showed superior discriminatory ability (c-statistic = 0.671) in the validation dataset compared to the IPI (c-statistic = 0.649) or the NCCN-IPI (c-statistic = 0.657). After recalibration of the model on the combined dataset, the median predicted probability of failing to achieve EFS24 was 36% (range, 12-88%), and the IPI24 showed an EFS24 gradient in all IPI groups. The IPI24 also identified a significant percentage of patients with high risk disease, with over 20% of patients having a 50% or higher risk of failing to achieve EFS24. The IPI24 provides an individual patient level probability of achieving the clinically relevant EFS24 endpoint. It can be used via electronic apps.

Primary Cutaneous Polymorphic EBV-Associated Posttransplant Lymphoproliferative Disorder After a Renal Transplant and Review of the Literature.

IMPORTANCE: Posttransplant lymphoproliferative disorder (PTLD) is an uncommon complication after solid organ transplants and hematopoietic stem cell transplants. Extranodal involvement in PTLD can involve several organ systems, including the central nervous system, bone marrow, lungs, gastrointestinal tract, and skin. Isolated involvement of the skin without systemic involvement in PTLD is rare. Primary cutaneous PTLD is generally categorized as either cutaneous T-cell lymphomas or cutaneous B-cell lymphomas, with variable Epstein-Barr virus (EBV) positivity. Herein, we describe an exceedingly uncommon case of a polymorphic variant of primary cutaneous PTLD. OBSERVATIONS: A woman in her 30s, who received an EBV+ deceased donor kidney transplant, presented with a 2-week history of 2 indurated patches over the lower abdomen. A skin biopsy revealed an atypical lymphoid proliferation with immunohistochemical stains demonstrating a mixed population of both B and T cells that stained strongly positive for EBV-encoded RNA. A bone marrow biopsy and positron emission tomography/computed tomography were negative for systemic involvement. The patient was treated with immunosuppression reduction and rituximab infusions. CONCLUSIONS AND RELEVANCE: This case highlights a rare polymorphic variant of primary cutaneous EBV-associated PTLD and increases awareness of this uncommon posttransplant complication. Cutaneous PTLD is reviewed, therefore dermatologists are aware of this uncommon disorder.

Relationships between chemotherapy, chemotherapy dose intensity and outcomes of follicular lymphoma in the immunochemotherapy era: a report from the University of Iowa/Mayo Clinic Lymphoma Specialized Program of Research Excellence Molecular Epidemiology Resource.

The optimal treatment of follicular lymphoma (FL) is not established. Rituximab's value potentially dilutes the impact of chemotherapy on FL. We reviewed 337 cases of FL treated initially with rituximab as monotherapy or with chemotherapy at the University of Iowa/Mayo Clinic from 2002 to 2009, investigating the association between chemotherapy delivery of cyclophosphamide or doxorubicin and survival. With median follow-up duration of 52.7 months, event-free survival (EFS) and overall survival (OS) were similar between the two groups, with a trend toward better EFS in the R-chemotherapy cohort (hazard ratio [HR]=1.24, p=0.28). In the R-chemotherapy group, increased total dose delivery and delivered dose intensity of doxorubicin were associated with improved EFS only in patients who did not receive R-maintenance (HR=0.81; p=0.02 and HR=0.94; p=0.04). Cyclophosphamide delivery was not associated with EFS. Thus, in the immunochemotherapy era, chemotherapy delivery strategy requires re-evaluation.

Event-free survival at 24 months is a robust end point for disease-related outcome in diffuse large B-cell lymphoma treated with immunochemotherapy.

PURPOSE: Studies of diffuse large B-cell lymphoma (DLBCL) are typically evaluated by using a time-to-event approach with relapse, re-treatment, and death commonly used as the events. We evaluated the timing and type of events in newly diagnosed DLBCL and compared patient outcome with reference population data. PATIENTS AND METHODS: Patients with newly diagnosed DLBCL treated with immunochemotherapy were prospectively enrolled onto the University of Iowa/Mayo Clinic Specialized Program of Research Excellence Molecular Epidemiology Resource (MER) and the North Central Cancer Treatment Group NCCTG-N0489 clinical trial from 2002 to 2009. Patient outcomes were evaluated at diagnosis and in the subsets of patients achieving event-free status at 12 months (EFS12) and 24 months (EFS24) from diagnosis. Overall survival was compared with age- and sex-matched population data. Results were replicated in an external validation cohort from the Groupe d'Etude des Lymphomes de l'Adulte (GELA) Lymphome Non Hodgkinien 2003 (LNH2003) program and a registry based in Lyon, France. RESULTS: In all, 767 patients with newly diagnosed DLBCL who had a median age of 63 years were enrolled onto the MER and NCCTG studies. At a median follow-up of 60 months (range, 8 to 116 months), 299 patients had an event and 210 patients had died. Patients achieving EFS24 had an overall survival equivalent to that of the age- and sex-matched general population (standardized mortality ratio [SMR], 1.18; P = .25). This result was confirmed in 820 patients from the GELA study and registry in Lyon (SMR, 1.09; P = .71). Simulation studies showed that EFS24 has comparable power to continuous EFS when evaluating clinical trials in DLBCL. CONCLUSION: Patients with DLBCL who achieve EFS24 have a subsequent overall survival equivalent to that of the age- and sex-matched general population. EFS24 will be useful in patient counseling and should be considered as an end point for future studies of newly diagnosed DLBCL.

Rates and outcomes of follicular lymphoma transformation in the immunochemotherapy era: a report from the University of Iowa/MayoClinic Specialized Program of Research Excellence Molecular Epidemiology Resource.

PURPOSE: This study sought to characterize transformation incidence and outcome for patients with follicular lymphoma (FL) in a prospective observational series begun after diffusion of rituximab use. PATIENTS AND METHODS: Patients with newly diagnosed FL were prospectively enrolled onto the University of Iowa/Mayo Clinic Specialized Program of Research Excellence Molecular Epidemiology Resource from 2002 to 2009. Patients were actively followed for re-treatment, clinical or pathologic transformation, and death. Risk of transformation was analyzed via time to transformation by using death as a competing risk. RESULTS: In all, there were 631 patients with newly diagnosed grade 1 to 3a FL who had a median age at enrollment of 60 years. At a median follow-up of 60 months (range, 11 to 110 months), 79 patients had died, and 60 patients developed transformed lymphoma, of which 51 were biopsy proven. The overall transformation rate at 5 years was 10.7%, with an estimated rate of 2% per year. Increased lactate dehydrogenase was associated with increased risk of transformation. Transformation rate at 5 years was highest in patients who were initially observed and lowest in patients who initially received rituximab monotherapy (14.4% v 3.2%; P = .021). Median overall survival following transformation was 50 months and was superior in patients with transformation greater than 18 months after FL diagnosis compared with patients with earlier transformation (5-year overall survival, 66% v 22%; P < .001). CONCLUSION: Follicular transformation rates in the immunochemotherapy era are similar to risk of death without transformation and may be lower than reported in older series. Post-transformation prognosis is substantially better than described in older series. Initial management strategies may influence the risk of transformation.

Elevated serum free light chains are associated with event-free and overall survival in two independent cohorts of patients with diffuse large B-cell lymphoma.

PURPOSE: The serum free light chain (FLC) assay quantitates free kappa (κ) and free lambda (λ) immunoglobulin light chains. This assay has prognostic value in plasma cell proliferative disorders. There are limited data on serum FLC in B-cell malignancies. PATIENTS AND METHODS: The association of pretreatment FLC with event-free survival (EFS) and overall survival (OS) in diffuse large B-cell lymphoma (DLBCL) was evaluated in 76 patients from the North Central Cancer Treatment Group trial N0489 (NCT00301821) and 219 patients from the University of Iowa/Mayo Clinic Specialized Program of Research Excellence Molecular Epidemiology Resource (MER). Published reference ranges were used to define an elevated FLC or an abnormal κ:λ FLC ratio. RESULTS: Elevated FLC or abnormal κ:λ FLC ratio was present in 32% and 14% of patients, respectively. Patients with elevated FLC had an inferior OS and EFS in both cohorts compared with patients with normal FLC (N0489: EFS hazard ratio [HR], 3.06; OS HR, 3.16; both P < .02; MER: EFS HR, 2.42; OS HR, 3.40; both P < .001; combined EFS HR, 2.57; OS HR, 3.74; both P < .001). All associations remained significant for EFS and OS after adjusting for the International Prognostic Index (IPI). Abnormal κ:λ FLC ratio was modestly associated with outcome in the combined group (EFS HR, 1.61; OS HR, 1.67; both P = .07), but not in patients without corresponding elevated κ or λ. Elevated FLC was the strongest predictor of outcome in multivariable models with the IPI components. CONCLUSION: Increased serum FLC is an independent, adverse prognostic factor for EFS and OS in DLBCL and warrants further evaluation as a biomarker in DLBCL.

An enhanced antigen-retrieval protocol for immunohistochemical staining of formalin-fixed, paraffin-embedded tissues.

Formalin is the most commonly used fixative for light microscopy because of its preservation of -morphological details. A major adverse effect of formalin fixation is formation of cross-linkages between epitopes (amino acid residues) and unrelated proteins by formaldehyde groups. The great majority of monoclonal and polyclonal antibodies used for immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) tissues necessitate unmasking antigens for antigen retrieval. There are currently two major antigen-retrieval procedures based on treatment of deparaffinized tissue sections with heat or, less commonly, with enzymatic digestion. The use of various antigen-retrieval solutions and heating sources does not allow standardization of IHC staining and minimalization of interlaboratory discrepancies. We developed a novel modified antigen-retrieval protocol for reversing the effect of -formalin fixation. The key feature of this protocol is treatment of deparaffinized tissue sections at reduced constant heat (97(o)C in a water bath) for 40 min in 25 mM Tris-HCl (pH 8.5), 1 mM EDTA, and 0.05% SDS (Tris-EDTA-SDS) buffer. Sections are then immunostained with primary and secondary antibodies conjugated with polymer-labeled Horse Radish Peroxidase. Compared to conventional antigen-retrieval procedures, this protocol more efficiently reverses the effect of formalin fixation of a wide variety of cellular antigens and in most instances decreases the use of primary antibody by 2-40 times, resulting in cost savings. Moreover, this protocol eliminates the need for using different antigen-retrieval methods in the laboratory, which reduces both time and labor for medical technologists.

Independent diagnostic accuracy of flow cytometry obtained from fine-needle aspirates: a 10-year experience with 451 cases.

Although the topic is somewhat contentious, fine-needle aspiration (FNA) is frequently used in conjunction with flow cytometry (FC) to evaluate lymphoid proliferations. Despite the fact that the FNA and FC are often analyzed independently, no previous large-scale study has independently analyzed FC of FNA specimens. FC reports of 511 FNAs were retrospectively reviewed and FC diagnoses categorized as monoclonal, atypical, normal/reactive, or insufficient cellularity (3.9%). Abnormal immunophenotype was considered a positive test result. "Gold standard" diagnoses were established by histologic examination, treatment based on FNA, or clinical features. In 92.2% (451/489), there was adequate follow-up. The diagnostic accuracy of FC was 88.4%, sensitivity was 85.8%, and specificity was 92.9%. In addition, FC accuracy for classes of non-Hodgkin lymphoma was assessed. We conclude that FC is an independently accurate ancillary test in the evaluation of FNA. However, the presence of false-negative and false-positive cases supports the common practice of correlating FC with cytomorphologic findings even if performed independently.