CD46 and CD59 inhibitors enhance complement-dependent cytotoxicity of anti-CD38 monoclonal antibodies daratumumab and isatuximab in multiple myeloma and other B-cell malignancy cells.

Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage. Remarkable progress has been made in the treatment of MM with anti-CD38 monoclonal antibodies such as daratumumab and isatuximab, which can kill MM cells by inducing complement-dependent cytotoxicity (CDC). We showed that the CDC efficacy of daratumumab and isatuximab is limited by membrane complement inhibitors, including CD46 and CD59, which are upregulated in MM cells. We recently developed a small recombinant protein, Ad35K++, which is capable of transiently removing CD46 from the cell surface. We also produced a peptide inhibitor of CD59 (rILYd4). In this study, we tested Ad35K++ and rILYd4 in combination with daratumumab and isatuximab in MM cells as well as in cells from two other B-cell malignancies. We showed that Ad35K++ and rILYd4 increased CDC triggered by daratumumab and isatuximab. The combination of both inhibitors had an additive effect in vitro in primary MM cells as well as in vivo in a mouse xenograft model of MM. Daratumumab and isatuximab treatment of MM lines (without Ad35K++ or rILYd4) resulted in the upregulation of CD46/CD59 and/or survival of CD46high/CD59high MM cells that escaped the second round of daratumumab and isatuximab treatment. The escape in the second treatment cycle was prevented by the pretreatment of cells with Ad35K++. Overall, our data demonstrate that Ad35K++ and rILYd4 are efficient co-therapeutics of daratumumab and isatuximab, specifically in multi-cycle treatment regimens, and could be used to improve treatment of multiple myeloma.

Pembrolizumab With R-CHOP in Previously Untreated DLBCL: Sustained, High Efficacy, and Safety With Long-Term Follow-Up.

BACKGROUND: While generally ineffective in relapsed diffuse large B cell lymphoma (DLBCL), immune checkpoint inhibitors (ICIs) may hold greater promise in untreated, immunocompetent patients. We previously reported safety and early efficacy of pembrolizumab plus rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (PR-CHOP) in a phase I trial of untreated DLBCL, noting responses in 90% of patients (complete response 77%) and a 2-year progression-free survival (PFS) of 83%. We herein report long-term safety and efficacy at 5-year follow up. PATIENTS AND METHODS: Adult patients with untreated DLBCL or grade 3b follicular lymphoma, intended to receive 6 cycles of R-CHOP were eligible. Patients (N = 30) were treated with pembrolizumab 200 mg IV and R-CHOP in 21-day cycles for 6 cycles. RESULTS: At median follow up of 4.8 years, 5-year PFS was 71% (CI, 54%-94%) and 5-year overall survival was 83% (CI, 71%-98%). Immune-related adverse events (IRAEs) occurred in 7 (23%) patients (10% grade 3/4). Three IRAEs (rash, thyroiditis, rheumatoid arthritis) occurred beyond 3 months of treatment completion. PD-L1 tumor expression was documented in 19 of 23 (83%) tested patients. None of the 19 patients who had any PD-L1 expression have relapsed, whereas 2 out of the 4 patients with no PD-L1 expression have relapsed. CONCLUSION: PR-CHOP has led to durable responses in most patients, with the best outcomes in PD-L1-expressing disease. Furthermore, the safety profile was manageable, with no consistent pattern of late events. These data support ongoing strategies incorporating ICIs in frontline DLBCL therapy and confirmation of predictive biomarkers including tumor PD-L1 expression.

A Review of the Flow Cytometric Findings in Classic Hodgkin Lymphoma, Nodular Lymphocyte Predominant Hodgkin Lymphoma and T Cell/Histiocyte-Rich Large B Cell Lymphoma.

Classic Hodgkin lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and T cell/histiocyte-rich large B cell lymphoma form a unique set of lymphomas with similar morphologic growth patterns (occasional neoplastic cells within a prominent cellular cell background) that are pathobiologically related. Distinguishing these entities has been historically difficult by flow cytometry; however, our laboratory has developed antibody-fluorochrome combinations capable of immunophenotyping these lymphomas. Additionally, characterization of the background reactive lymphocytes can aid in narrowing the differential diagnosis. This review summarizes the immunophenotypic features and insights of the neoplastic and reactive populations found in this unique group of lymphomas.

Peripheral lymphocytes and lactate dehydrogenase correlate with response and survival in head and neck cancers treated with immune checkpoint inhibitors.

BACKGROUND: Little is known regarding associations between peripheral blood biomarkers (PBBMs) and survival, response, and toxicity in recurrent/metastatic head and neck squamous cell carcinomas (R/M HNSCC) treated with immune checkpoint inhibitors (ICIs). METHODS: In this single-institution retrospective cohort study, a dataset of patients with R/M HNSCC treated with ICIs between 08/2012-03/2021 was established, including demographic and clinicopathologic characteristics. Pretreatment PBBMs were collected and evaluated for associations with grade ≥3 adverse events (G ≥ 3AE) by CTCAEv5, objective response (ORR) by RECIST 1.1, overall survival (OS), and progression-free survival (PFS). Multivariable models for each outcome were created using elastic net variable selection. RESULTS: Our study included 186 patients, with 51 (27%) demonstrating complete or partial response to immunotherapy. Multivariable models adjusted for ECOG performance status (PS), p16, and smoking demonstrated that pretreatment higher LDH and absolute neutrophils, as well as lower percent lymphocytes correlated with worse OS and PFS. Higher LDH and lower % lymphocytes also correlated with worse ORR. CONCLUSIONS: In the largest study to date examining PBBMs in ICI-treated R/M HNSCCs, our variable selection method revealed PBBMs prognostic for survival and response to immunotherapy. These biomarkers warrant further investigation in a prospective study along with validation with CPS biomarker.

Neutrophil to lymphocyte ratio and peripheral blood biomarkers correlate with survival outcomes but not response among head and neck and salivary cancer treated with pembrolizumab and vorinostat.

BACKGROUND: Associations between peripheral blood biomarkers and oncologic outcomes were explored in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HN) and salivary gland cancer (SGC) treated with pembrolizumab and vorinostat on a phase II trial (NCT02538510). EXPERIMENTAL DESIGN: Twenty-five HN and 25 SGCs were treated with pembrolizumab and vorinostat. Baseline peripheral blood was available in 21 HN and 20 SGCs and evaluated for associations with grade ≥3 adverse events (G ≥ 3AE) by CTCAEv4, objective response rate (ORR), overall survival (OS), and progression-free survival (PFS). RESULTS: Higher pretreatment neutrophil-to-lymphocyte ratio (NLR) and neutrophils, as well as lower pretreatment lymphocytes and T helper cells correlated with worse OS and PFS. Higher NLR further predicted increased rates of G ≥ 3AEs. No correlations with ORR were observed. CONCLUSIONS: In a prospectively evaluated cohort of HN and SGCs treated with pembrolizumab and vorinostat, we observed novel associations between peripheral blood biomarkers and oncologic outcomes and toxicities.

Predictors of risk of relapse in classic Hodgkin lymphoma.

Using multiparametric flow cytometric analysis, in a cohort of 62 patients with classic Hodgkin lymphoma having a median follow-up period of 69.5 months, we found-patients who experienced primary resistance or disease relapse (DR) had lower percentage of rosetted Hodgkin Reed-Sternberg cells (HRS-cells) as compared with patients who achieved sustained complete remission (SCR) (p=0.022); patients >35 years of age had higher percentage of HRS-cells (p=0.017) and lower percentage of B cells (p=0.017) and the nodular sclerosis subtype had higher percentage of B-cells (p=0.046) and activated B-cells (p=0.03). The proportion of SCR and DR subsets did not differ by histological subtypes, disease stage or age groups.

Efficacy, safety, and molecular response predictors of oral ixazomib and short-course rituximab in untreated iNHL.

Patients with indolent B-cell non-Hodgkin lymphoma (iNHL) generally require treatment but experience normal survival, emphasizing the need for simpler, safer therapies. Proteasome inhibitors target aberrant signaling pathways within iNHL and have manageable toxicities. We evaluated the oral proteasome inhibitor ixazomib as initial monotherapy, and combined with rituximab, for first-line treatment of iNHL. Treatment-naïve patients with iNHL needing therapy received oral ixazomib 4 mg weekly until progressive disease or unacceptable adverse events. A 4-week course of rituximab was added during month 7. The primary end point was overall response rate (ORR) during the ixazomib monotherapy window. Correlations included gene expression profiling and response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. Thirty-three patients with follicular lymphoma (FL) (n = 20), marginal zone lymphoma (n = 7), and other iNHL were treated with a median follow-up of 30.3 months. During the 6-month ixazomib window, the ORR was 24%, including 35% in FL. The best ORR over the entire study period was 52% overall and 65% in FL; complete response was achieved in 33% and 45%, respectively. The median duration of response was 25.8 months (range, 0-49.7), and the 24-month progression-free and overall survival rates were 51% (95% confidence interval [CI], 32-67) and 91% (95% CI, 74-97), respectively. Ixazomib was well tolerated. Baseline downregulation of proteasome genes, PSMB9 (P = .03) and PSMB8 (P = .007), were associated with response. All evaluated patients generated anti-S antibodies to SARS-CoV-2 vaccination, with a median of 254.9 binding arbitrary unit per mL. Ixazomib demonstrated efficacy alone and with short-course rituximab in untreated iNHL while exhibiting favorable toxicity, convenience, and retention of the B-cell immune response. This trial is registered at www.clinicaltrials.gov as NCT02339922.

Diffuse large B-cell lymphoma (DLBCL) is infiltrated with activated CD8+ T-cells despite immune checkpoint signaling.

Background: B-cell non-Hodgkin lymphomas (NHL) are hematologic malignancies that arise in the lymph node. Despite this, the malignant cells are not cleared by the immune cells present. The failure of anti-tumor immunity may be due to immune checkpoints such as the PD-1/PDL-1 axis, which can cause T-cell exhaustion. Unfortunately, unlike Hodgkin lymphoma, checkpoint blockade in NHL has shown limited efficacy. Methods: We performed an extensive functional analysis of malignant and non-malignant lymph nodes using high dimensional flow cytometry. We compared follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and lymph nodes harboring reactive hyperplasia (RH). Results: We identified an expansion of CD8+PD1+ T-cells in the lymphomas relative to RH. Moreover, we demonstrate that these cells represent a mixture of activated and exhausted T-cells in FL. In contrast, these cells are nearly universally activated and functional in DLBCL. This is despite expression of counter-regulatory molecules such as PD-1, TIM-3, and CTLA-4, and the presence of regulatory T-cells. Conclusions: These data may explain the failure of single-agent immune checkpoint inhibitors in the treatment of DLBCL. Accordingly, functional differences of CD8+ T-cells between FL and DLBCL may inform future therapeutic targeting strategies.

Clinicopathologic Findings in Patients With Initial Diagnosis of Extranodal Marginal Zone B-Cell Lymphoma of Mucosa-Associated Lymphoid Tissue (MALT) in Colorectal Mucosa.

OBJECTIVES: To evaluate clinicopathologic features, management, and behavior of colorectal extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT). METHODS: Clinical data, laboratory studies, and radiographic records were reviewed (2005-2018), and fluorescence in situ hybridization studies were performed. RESULTS: Eleven patients were identified, six of whom were discovered as an incidental finding on endoscopy. Morphologic and immunophenotypic features were similar to MALT lymphomas at other sites except that lymphoepithelial lesions were uncommon. Three of nine patients were positive for BIRC3/MALT1 fusions, two of whom had identical B-cell clones identified in subsequent gastric biopsy specimens. Eight of 10 patients had no clinically evaluable disease after observation (±antibiotics; n = 4) or radiation/chemotherapy (n = 4). CONCLUSIONS: Patients with incidental and localized colonic MALT lymphoma demonstrated an excellent prognosis with conservative management, although longer follow-up and data based on consistent staging and surveillance methods (including gastric evaluation) are necessary for informed management.

A comparison and review of the flow cytometric findings in classic Hodgkin lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, T cell/histiocyte rich large B cell lymphoma, and primary mediastinal large B cell lymphoma.

The "Hodgkin-like" lymphomas including classic Hodgkin lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, T cell/histiocyte rich large B cell lymphoma, and primary mediastinal large B cell lymphoma have been shown to be pathobiologically related. With the exception of primary mediastinal large B cell lymphoma, these lymphomas have similar morphologic growth patterns with occasional neoplastic cells within a prominent reactive cell background. Historically, distinguishing these entities was difficult by flow cytometry; however, over the past 15 years, our laboratory has developed antibody-fluorochrome combinations capable of accurately distinguishing these entities by their immunoprofile. Additionally, an algorithmic approach based on characterization of the background reactive B-cell and T-cell populations can aid in narrowing the differential diagnosis. This review summarizes both the morphologic and immunophenotypic features and the current flow cytometric insights of the neoplastic and reactive populations found in this unique subset of lymphomas.

Evaluation of Preclinical Activity of Isatuximab in Patients with Acute Lymphoblastic Leukemia.

This study reports the pharmacologic effects of isatuximab, a CD38 mAb, on T- and B-cell acute lymphoblastic leukemia (ALL). We analyzed CD38 expression in 50-T-ALL and 50 B-ALL clinical samples, and 16 T-ALL and 11 B-ALL cell lines. We primarily focused on in vitro assessments of isatuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In vivo assessment of isatuximab activity was performed in several ALL xenograft models, including disseminated and subcutaneous tumor models in female C.B-17 severe combined immunodeficiency mice. Our study reveals that most patients (90%-100%) carried CD38+ blasts independent of disease burden. The median CD38 receptor density on abnormal lymphoblasts is 41,026 copies/cell on T-ALL and 28,137 copies/cell on B-ALL, respectively. In patients with T-ALL, there is a significant increase of CD38 expression in abnormal blasts compared with normal T cells. High-level CD38 receptor density (RD) is critical to trigger effective isatuximab-mediated ADCC against target ALL cells. In addition, a correlation between CD38 RD and isatuximab-mediated ADCP is demonstrated. In the disseminated CD38+, T-ALL, and B-ALL xenograft models, isatuximab is able to induce robust antitumor activity, even at low doses. This study shows that isatuximab has significant in vitro and in vivo activity against ALL cells with robust ADCC and ADCP effects that are associated with CD38 expression levels in both T-ALL and B-ALL.

Dose-dense brentuximab vedotin plus ifosfamide, carboplatin, and etoposide for second-line treatment of relapsed or refractory classical Hodgkin lymphoma: a single centre, phase 1/2 study.

BACKGROUND: Relapsed or refractory classical Hodgkin lymphoma could be treated with multiagent salvage chemotherapy followed by autologous haematopoietic stem-cell transplantation. The aim of this study is to establish the safety and activity of dose-dense brentuximab vedotin combined with ifosfamide, carboplatin, and etoposide (BV-ICE) chemotherapy in second-line treatment of classical Hodgkin lymphoma. METHODS: We conducted a single-arm, open-label, phase 1/2 study of dose-dense BV-ICE at the Seattle Cancer Care Alliance, University of Washington (Seattle, WA, USA). Eligibility criteria were age 18 years or older; diagnosis of first relapse, primary refractory classical Hodgkin lymphoma after one previous line of therapy; measurable disease of at least 1 cm in the longest axis, CT of chest, abdomen, and pelvis with PET within the past 28 days; Eastern Cooperative Oncology Group performance status of 0-1; and adequate organ function. A 3 + 3 dose escalation study was done for the phase 1 part of the trial to establish the maximum tolerated dose to be used for the phase 2 study. Brentuximab vedotin was delivered on days 1 and 8 at either 1·2 mg/kg (dose level 1) or 1·5 mg/kg (dose level 2) intravenously (capped at 150 mg) with standard dosing of ICE on days 1-3 (ifosfamide 5 g/m2 plus mesna 5 g/m2 intravenously over 24 h on day 2, carboplatin area under the curve 5 on day 2 in one intravenous injection, and etoposide 100 mg/m2 on days 1-3 in one intravenous injection per day) for two 21-day cycles. The primary endpoint was to establish the recommended phase 2 dose (phase 1 part) and complete response rate after two cycles, with a prespecified target of 78% (phase 2 part). Safety analysis was done in all enrolled participants and the primary activity analysis was done in all patients with evaluable response data. This study is registered with ClinicalTrials.gov (NCT02227199); enrolment and study treatment are complete. FINDINGS: Between Oct 16, 2014, and Feb 10, 2020, we enrolled 45 patients with a median age of 31 years (IQR 28-45). The recommended phase 2 dose of brentuximab vedotin was established to be 1·5 mg/kg. After a median follow-up of 3·1 years (IQR 1·7-4·1), 32 (74%; 95% CI 58·8-86·5) of 43 evaluable patients had complete responses after two cycles of treatment. Grade 3-4 haematological toxic effects were common, including neutropenia (33 [73%]), anaemia (six [13%]), and thrombocytopenia (36 [80%]). The most common grade 3-4 non-haematological toxic effects were febrile neutropenia (four [9%]), sepsis (six [13%]), increased alanine aminotransferase (five [11%]), hyperglycaemia (three [7%]), pulmonary embolism (two [4%]), and increased aspartate aminotransferase (two [4%]). There was one (2%) on-treatment death due to multisystem organ failure that was considered treatment related. Serious adverse events occurred in 13 (29%) patients. INTERPRETATION: Our data suggest that dose-dense BV-ICE is a rapidly administered and active salvage regimen for patients with relapsed or refractory classical Hodgkin lymphoma despite a complete response in this trial lower than the prespecified phase 2 target. Although cross-trial comparisons should be made with caution, activity results seem to be similar to previously presented brentuximab vedotin chemotherapy salvage combinations delivered over much longer durations and can be considered in young (<60 years), transplantation-eligible patients for second-line therapy. FUNDING: Seagen, Lymphoma Research Foundation, National Institutes of Health/National Cancer Institute, and generous philanthropic donations to the University of Washington from numerous individuals and families in support of lymphoma research.

De Novo Identification and Visualization of Important Cell Populations for Classic Hodgkin Lymphoma Using Flow Cytometry and Machine Learning.

OBJECTIVES: Automated classification of flow cytometry data has the potential to reduce errors and accelerate flow cytometry interpretation. We desired a machine learning approach that is accurate, is intuitively easy to understand, and highlights the cells that are most important in the algorithm's prediction for a given case. METHODS: We developed an ensemble of convolutional neural networks for classification and visualization of impactful cell populations in detecting classic Hodgkin lymphoma using two-dimensional (2D) histograms. Data from 977 and 245 clinical flow cytometry cases were used for training and testing, respectively. Seventy-eight nongated 2D histograms were created per flow cytometry file. Shapley additive explanation (SHAP) values were calculated to determine the most impactful 2D histograms and regions within histograms. SHAP values from all 78 histograms were then projected back to the original cell data for gating and visualization using standard flow cytometry software. RESULTS: The algorithm achieved 67.7% recall (sensitivity), 82.4% precision, and 0.92 area under the receiver operating characteristic. Visualization of the important cell populations for individual predictions demonstrated correlations with known biology. CONCLUSIONS: The method presented enables model explainability while highlighting important cell populations in individual flow cytometry specimens, with potential applications in both diagnosis and discovery of previously overlooked key cell populations.

Creating Virtual Hematoxylin and Eosin Images using Samples Imaged on a Commercial CODEX Platform.

Multiparametric fluorescence imaging through CODEX allows the simultaneous imaging of many biomarkers in a single tissue section. While the digital fluorescence data thus obtained can provide highly specific characterizations of individual cells and microenvironments, the images obtained are different from those usually interpreted by pathologists (i.e., hematoxylin and eosin [H&E] slides and 3,3'-diaminobenzidine-stained immunohistochemistry slides). Having the fluorescence data plus coregistered H&E or similar data could facilitate the adoption of multiparametric imaging into regular workflows, as well as facilitate the transfer of algorithms and machine learning previously developed around H&E slides. Since commercial CODEX instruments do not produce H&E-like images by themselves, we developed a staining protocol and associated image processing to make "virtual H&E" images that can be incorporated into the CODEX workflow. While there are many ways to achieve virtual H&E images, including the use of a fluorescent nuclear stain and tissue autofluorescence to simulate eosin staining, we opted to combine fluorescent nuclear staining (through 4',6-diamidino-2-phenylindole) with actual eosin staining. We also output images derived from fluorescent nuclear staining and autofluorescence images for additional evaluation.

Phase 2 study of pembrolizumab for measurable residual disease in adults with acute lymphoblastic leukemia.

The presence of measurable residual disease (MRD) in acute lymphoblastic leukemia (ALL) confers a poor prognosis. CD19-targeted immunotherapy is effective against MRD but is logistically challenging, potentially toxic, and not applicable to T-cell ALL. We thus hypothesized that inhibition of PD-1 with pembrolizumab could also be effective for MRD, but without lineage restriction. The primary objective of this phase 2 study was to evaluate the efficacy of pembrolizumab in patients with ALL and MRD. Key eligibility criteria included adults with B- or T-cell ALL and MRD detectable by multiparameter flow cytometry or quantitative polymerase chain reaction from bone marrow aspirate (BMA) despite chemotherapy (plus ABL kinase inhibitor if Philadelphia chromosome positive). Pembrolizumab 200 mg IV was given every 3 weeks. Response was assessed by BMA using methods that previously detected MRD. The primary end point was complete MRD response rate. We stopped enrollment early; only 1 of 12 (8%) experienced a complete MRD response, which lasted 3 weeks. Interestingly, this patient had previously received hematopoietic cell transplantation and CD19-targeted chimeric antigen receptor-modified T-cell therapy and was the only patient to experience an immune-related adverse event from pembrolizumab (grade 3 Stevens-Johnson syndrome). Median overall survival from enrollment was 12.7 months. In summary, pembrolizumab had minimal activity against MRD but was generally well tolerated. These data can be compared with ongoing anti-PD-1 combination studies in ALL, and they further establish the role of trials specifically for patients with MRD. This trial was registered at www.clinicaltrials.gov as #NCT02767934.

Pembrolizumab with R-CHOP in previously untreated diffuse large B-cell lymphoma: potential for biomarker driven therapy.

Tumor programmed death-ligand 1 (PD-L1) expression in diffuse large B-cell lymphoma (DLBCL) is associated with inferior outcomes. The first-line immunologically-replete setting may be an opportune time for PD-1 inhibition. We evaluated pembrolizumab in combination with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) in untreated patients with DLBCL. Eligible patients were age 18 or older, had adequate organ function, and had DLBCL requiring full-course therapy. Patients received pembrolizumab 200 mg/cycle with R-CHOP, primarily to assess toxicity. Response assessment utilized standard criteria, and PD-L1 staining was performed at a validated central laboratory. Among 30 patients, toxicity was comparable to standard R-CHOP but with two grade ≥3 immune related adverse events (rash, pneumonitis). The overall and complete response rate was 90% and 77%. With 25·5 months of median follow-up, 2-year progression-free survival (PFS) is 83%. PD-L1 expression was associated with non-GCB subtype, and improved PFS and survival. Pembrolizumab can safely be added to R-CHOP, and is associated with a high CR rate and 2-year PFS. Improved PFS with PR-CHOP in PD-L1 expressing tumors contradicts historical data in R-CHOP treated patients, supporting evaluation of PD-L1 as a biomarker to identify DLBCL patients who may benefit from this first-line strategy.