IGHV allele similarity clustering improves genotype inference from adaptive immune receptor repertoire sequencing data.

In adaptive immune receptor repertoire analysis, determining the germline variable (V) allele associated with each T- and B-cell receptor sequence is a crucial step. This process is highly impacted by allele annotations. Aligning sequences, assigning them to specific germline alleles, and inferring individual genotypes are challenging when the repertoire is highly mutated, or sequence reads do not cover the whole V region. Here, we propose an alternative naming scheme for the V alleles, as well as a novel method to infer individual genotypes. We demonstrate the strengths of the two by comparing their outcomes to other genotype inference methods. We validate the genotype approach with independent genomic long-read data. The naming scheme is compatible with current annotation tools and pipelines. Analysis results can be converted from the proposed naming scheme to the nomenclature determined by the International Union of Immunological Societies (IUIS). Both the naming scheme and the genotype procedure are implemented in a freely available R package (PIgLET https://bitbucket.org/yaarilab/piglet). To allow researchers to further explore the approach on real data and to adapt it for their uses, we also created an interactive website (https://yaarilab.github.io/IGHV_reference_book).

Point-of-care anti-CD19 chimeric antigen receptor T-cell therapy for relapsed/refractory follicular lymphoma.

Patients with relapsed/refractory follicular lymphoma (R/R-FL) often require multiple treatment lines. We performed a phase 1b/2 single-center clinical trial of autologous point-of-care anti-CD19 chimeric antigen receptor (CAR) T-cells in R/R-FL patients treated patients with ≥ 2 treatment lines. All 26 patients enrolled received CAR T-cell infusion at a median of 11 days after leukapheresis. Seventy-seven percent of patients had POD24. At enrollment, disease stage was III-IV in 85% of the patients, 77% had high-risk FLIPI score, and 77% had progressive disease. Grade III-IV cytokine release and immune effector cell-associated neurotoxicity syndromes occurred in 12% and 16% of the patients, respectively. Overall response rate at 1-month was 88%. The median follow-up was 15.4 months. One-year overall and progression-free survival were 100% and 63%, respectively. In conclusion, point-of-care CAR T-cell, manufactured within 11 days, induced a high response rate with an acceptable safety profile in patients with high-risk R/R-FL.

B cell M-CLL clones retain selection against replacement mutations in their immunoglobulin gene framework regions.

Introduction: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, accounting for 30-40% of all adult leukemias. The dynamics of B-lymphocyte CLL clones with mutated immunoglobulin heavy chain variable region (IgHV) genes in their tumor (M-CLL) can be studied using mutational lineage trees. Methods: Here, we used lineage tree-based analyses of somatic hypermutation (SHM) and selection in M-CLL clones, comparing the dominant (presumably malignant) clones of 15 CLL patients to their non-dominant (presumably normal) B cell clones, and to those of healthy control repertoires. This type of analysis, which was never previously published in CLL, yielded the following novel insights. Results: CLL dominant clones undergo - or retain - more replacement mutations that alter amino acid properties such as charge or hydropathy. Although, as expected, CLL dominant clones undergo weaker selection for replacement mutations in the complementarity determining regions (CDRs) and against replacement mutations in the framework regions (FWRs) than non-dominant clones in the same patients or normal B cell clones in healthy controls, they surprisingly retain some of the latter selection in their FWRs. Finally, using machine learning, we show that even the non-dominant clones in CLL patients differ from healthy control clones in various features, most notably their expression of higher fractions of transition mutations. Discussion: Overall, CLL seems to be characterized by significant loosening - but not a complete loss - of the selection forces operating on B cell clones, and possibly also by changes in SHM mechanisms.

Cellular and humoral response to the fourth BNT162b2 mRNA COVID-19 vaccine dose in patients with CLL.

We assessed the humoral and cellular response to the fourth BNT162b2 mRNA COVID-19 vaccine dose in patients with CLL. A total of 67 patients with CLL and 85 age matched controls tested for serologic response and pseudo-neutralization assay. We also tested the functional T-cell response by interferon gamma (IFNγ) to spike protein in 26 patients. Two weeks after the fourth vaccine antibody serologic response was evident in 37 (55.2%) patients with CLL, 20 /22 (91%) of treatment naïve, and 9/32 (28%) patients with ongoing therapy, compared with 100% serologic response in age matched controls. The antibody titer increased by 10-fold in patients with CLL, however, still 88-folds lower than age matched controls. Predictors of better chances of post fourth vaccination serologic response were previous positive serologies after second, third, and pre-fourth vaccination, neutralizing assay, and treatment naïve patients. T-cell response improved from 42.3% before the fourth vaccine to 84.6% 2 weeks afterwards. During the time period of 3 months after the fourth vaccination, 14 patients (21%) developed COVID-19 infection, all recovered uneventfully. Our data demonstrate that fourth SARS-CoV-2 vaccination improves serologic response in patients with CLL to a lesser extent than healthy controls and induces functional T-cell response.

Allogeneic Hematopoietic Cell Transplantation after Chimeric Antigen Receptor T Cell Therapy in Large B Cell Lymphoma.

Anti-CD19 chimeric antigen receptor T cell (CAR-T) therapy has transformed the care of patients with relapsed/refractory large B cell lymphoma (LBCL). However, approximately 60% of CAR-T recipients ultimately will experience disease recurrence or progression. Salvage therapies after CAR-T treatment failures are of limited efficacy and have a short duration of response. The objective of the present study was to evaluate the role of allogeneic hematopoietic cell transplantation (allo-HCT) after CAR-T therapy in LBCL patients. This was a multicenter observational study reporting the outcome of 39 adult LBCL patients who underwent allo-HCT following anti-CD19 CAR-T therapy. The median patient age was 47 years (range, 20 to 68 years). HLA-matched sibling, HLA-matched unrelated, and alternative donors were used in 36%, 36%, and 28% of transplantations, respectively. Conditioning regimens were primarily of low or intermediate intensity. Disease status at allo-HCT was complete response in 41%, partial response in 38%, and progressive disease in 21%. Allo-HCT was performed at a median of 127 days (range, 82 to 206 days) after CAR-T therapy. A high incidence of hepatic toxicity (28%), including sinusoidal obstruction syndrome (15.4%; 95% confidence interval; [CI], 6.2% to 28.5%), was observed. The 1-year cumulative incidence of grade II-IV and grade III-IV acute graft-versus-host disease (GVHD) was 38.5% (95% CI, 23.2% to 53.6%) and 15.4% (95% CI, 6.1% to 28.5%), respectively. The 2-year cumulative incidence of moderate-severe chronic GVHD was 11.1% (95% CI, 3.3% to 24.3%). Overall, 2-year nonrelapse mortality and relapse/progression incidence were 26% (95% CI, 13% to 41%) and 43% (95% CI, 27% to 59%), respectively. With a median follow-up of 32 months, the 2-year overall survival (OS) and progression-free survival (PFS) were 45% (95% CI, 31% to 66%) and 31% (95% CI, 19% to 50%), respectively. In multivariable analyses, pre-HCT elevated lactate dehydrogenase level and transformed lymphoma were predictive of OS and PFS, respectively. Our data suggest that allo-HCT after anti-CD19 CAR-T treatment failure is feasible with a relatively promising efficacy but possibly high toxicity rate.

Outcomes of first therapy after CD19-CAR-T treatment failure in large B-cell lymphoma.

Persistence or recurrence of large B-cell lymphoma after CD19-CAR-T is common, yet data guiding management are limited. We describe outcomes and features following CAR-T treatment failure. Of 305 adults who received CD19-CAR-T, 182 experienced disease recurrence or progression (1-year cumulative incidence 63% [95%CI: 57-69]). Of 52 post-CAR-T biopsies evaluated by flow cytometry, 49 (94%) expressed CD19. Subsequent anti-cancer treatment was administered in 135/182 (74%) patients with CAR-T treatment failure. Median OS from the first post-CAR-T treatment was 8 months (95%CI 5.6-11.0). Polatuzumab-, standard chemotherapy-, and lenalidomide-based treatments were the most common approaches after CAR-T. No complete responses (CRs) were observed with conventional chemotherapy, while CR rates exceeding 30% were seen following polatuzumab- or lenalidomide-based therapies. Factors associated with poor OS among patients treated post-CAR-T were pre-CAR-T bulky disease (HR 2.27 [1.10-4.72]), lack of response to CAR-T (2.33 [1.02-5.29]), age >65 years (HR 2.65 [1.49-4.73]) and elevated LDH at post-CAR-T treatment (HR 2.95 [1.61-5.38]). The presence of ≥2 of these factors was associated with inferior OS compared to ≤1 (56% vs. 19%). In this largest analysis to date of patients who progressed or relapsed after CD19-CAR-T, survival is poor, though novel agents such as polatuzumab and lenalidomide may have hold promise.

IgTreeZ, A Toolkit for Immunoglobulin Gene Lineage Tree-Based Analysis, Reveals CDR3s Are Crucial for Selection Analysis.

Somatic hypermutation (SHM) is an important diversification mechanism that plays a part in the creation of immune memory. Immunoglobulin (Ig) variable region gene lineage trees were used over the last four decades to model SHM and the selection mechanisms operating on B cell clones. We hereby present IgTreeZ (Immunoglobulin Tree analyZer), a python-based tool that analyses many aspects of Ig gene lineage trees and their repertoires. Using simulations, we show that IgTreeZ can be reliably used for mutation and selection analyses. We used IgTreeZ on empirical data, found evidence for different mutation patterns in different B cell subpopulations, and gained insights into antigen-driven selection in corona virus disease 19 (COVID-19) patients. Most importantly, we show that including the CDR3 regions in selection analyses - which is only possible if these analyses are lineage tree-based - is crucial for obtaining correct results. Overall, we present a comprehensive lineage tree analysis tool that can reveal new biological insights into B cell repertoire dynamics.

Point-of-care CAR T-cell therapy as salvage strategy for out-of-specification tisagenlecleucel.

Tisagenlecleucel (tisa-cel) is an anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for patients with relapsed/refractory large B-cell lymphoma. Outcomes of patients with out-of-commercial specification (OOS) CAR T products are not well characterized. We therefore assessed 37 adult patients who underwent leukapheresis for tisa-cel therapy in a single center. In nine (24%) patients, manufactured tisa-cel was considered OOS. Three of them (33%) received tisa-cel after institutional review board approval; 2/9 (22%) did not receive tisa-cel due to disease progression; and 4/9 (44%) received academic point-of-care (POC) CAR T-cell as salvage therapy, at a median of 35 days following OOS notification. Three of those four patients achieved a complete response. In univariate analysis, risk factors for OOS were ≥ 4 prior therapies or previous bendamustine exposure. In conclusion, we report high OOS incidence of 24% in real-life setting. Forty-four percent of those patients received POC CAR T-cell as salvage therapy.

Point-of-care anti-CD19 CAR T-cells for treatment of relapsed and refractory aggressive B-cell lymphoma.

Anti CD19 chimeric antigen receptor (CAR) T-cell therapy has transformed the care of relapsed and refractory aggressive B-cell lymphoma. However, financial toxicity and manufacturing time represent barriers to its widespread implementation. Study applicability, toxicity, and efficacy of a locally produced autologous CD19-directed CAR T-cell product were studied. We performed a phase 1b/2 clinical trial with a point-of-care (POC) CAR T-cell product that contains a CD28 costimulatory domain. Adult patients with aggressive B-cell lymphoma or transformed low-grade lymphoma who received at least 2 prior regimens were eligible. A total of 73 patients, with a median age of 49 years, met inclusion criteria. CAR T-cell production time from apheresis was 10 days (interquartile range 10-11), negating the need for bridging chemotherapy. Overall and complete response rates were 62.5% and 37.5%. Median progression-free and overall survival were 3.7 and 12.1 months, respectively. Overall and progression-free survival at 12 months were 52.1% (confidence interval [CI]: 40.8%-66.5%) and 40% (CI: 30%-53.7%), respectively. Patients who achieved response had longer progression-free and overall survival. Grade 3-4 cytokine release syndrome was observed in 9.5% of the patients, and immune effector cell-associated neurotoxicity syndrome grade 3-4 in 21.9%. No deaths occurred due to CAR T-cell toxicity. Fifteen patients (20%) underwent allogeneic stem cell transplantation at a median time of 60 days after CAR T-cell therapy; 8 were alive at last follow-up. Of the 6 patients who underwent the transplantation in complete response 2 deceased because of toxicity. POC CAR T-cells are a feasible therapeutic option in aggressive B-cell lymphoma. They provide good efficacy while minimizing production time and the need for bridging therapy.

Imaging of Hematological Patients in the Era of COVID-19.

The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulted in changes in management and imaging routines for patients with hematological malignancies. Treating physicians had to familiarize themselves with a new disease, with distinct imaging manifestations, sometimes overlapping with other infections prevalent in this patient population. In some aspects, infected hematological patients might exhibit a different disease course, and routine imaging in asymptomatic hematological patients may result in unexpected COVID-19 findings, implying covert infection, that should be further explored. Furthermore, some complications of hematological diseases and treatments may present with findings similar to COVID-19 manifestations, and treating physicians must consider both possibilities in the differential diagnosis. In this review, we aimed to present the influence the COVID-19 pandemic had on hematological malignancy imaging.

Targeting the actin nucleation promoting factor WASp provides a therapeutic approach for hematopoietic malignancies.

Cancer cells depend on actin cytoskeleton rearrangement to carry out hallmark malignant functions including activation, proliferation, migration and invasiveness. Wiskott-Aldrich Syndrome protein (WASp) is an actin nucleation-promoting factor and is a key regulator of actin polymerization in hematopoietic cells. The involvement of WASp in malignancies is incompletely understood. Since WASp is exclusively expressed in hematopoietic cells, we performed in silico screening to identify small molecule compounds (SMCs) that bind WASp and promote its degradation. We describe here one such identified molecule; this WASp-targeting SMC inhibits key WASp-dependent actin processes in several types of hematopoietic malignancies in vitro and in vivo without affecting naïve healthy cells. This small molecule demonstrates limited toxicity and immunogenic effects, and thus, might serve as an effective strategy to treat specific hematopoietic malignancies in a safe and precisely targeted manner.

Outcomes Related to FDG-PET-CT Response in Patients With Hodgkin Lymphoma Treated With Brentuximab-Vedotin at Relapse or Consolidation.

BACKGROUND: Brentuximab-vedotin (BV) monotherapy has shown high efficacy in heavily pre-treated patients with relapsed or refractory Hodgkin lymphoma (HL) after high-dose chemotherapy or autologous stem cell transplantation (ASCT). We retrospectively analyzed the outcomes of treatment with BV of HL patients and examined the predictive ability of PET-CT for response in this setting. PATIENTS AND METHODS: Records of 49 HL patients (median age, 39 years, 55% male) treated with BV for relapse (71.4%) or consolidation (28.6%) post-ASCT were analyzed. Patients who did not reach complete response (CR) on PET/CT after 4 cycles (non-responders) discontinued BV and received the next treatment line. Overall survival (OS) and progression-free survival (PFS) were compared between responders and non-responders. RESULTS: After a median follow-up of 19.1 months, all consolidation patients were alive and none progressed. Median OS in 23 relapsed patients that did not achieve CR after 4 cycles and continued to the next treatment was 55.0 months, while all those in CR (n = 24) were alive (P = .0120). No statistically significant differences in OS were observed between responders and non-responders with relapsed HL (P = .1072). Median PFS evaluated after 4 BV cycles was significantly longer in responders compared to non-responders (47.9 vs. 1.5 months, P < .0001). Neuropathy and neutropenia were the main toxicities observed. CONCLUSIONS: HL patients treated with BV for relapse or consolidation who achieved CR by PET-CT after 4 cycles showed improved PFS and OS compared to non-responders. Non-responders treated for relapsed HL who proceeded to the next treatment line demonstrated comparable OS to responders.

Progression of disease within 24 months of initial therapy (POD24) detected incidentally in imaging does not necessarily indicate worse outcome.

Progression of disease within 24 months of initial therapy (POD24) has previously been identified as a predictor of reduced overall survival (OS) for patients with follicular lymphoma (FL). Here we attempt to validate this finding in a retrospective cohort and understand whether the method by which progression is determined, clinically or radiographically, influences POD24 robustness. We reviewed records of 635 patients with FL and included 317 patients in our analysis. POD24 occurred in 21.5% of patients and it was evident that OS was significantly lower in the POD24 group. In multivariate analysis both POD24 and FLIPI were independently associated with inferior OS. POD24 that was detected by incidental routine imaging did not predict reduced OS as opposed to progression that was detected clinically. Although surveillance imaging is generally discouraged in FL, it still is a routine practice by many physicians, and therefore our findings are of significant clinical implications.

Recognizing severe fatigue and decline in quality of life in Hodgkin lymphoma survivors.

Hodgkin lymphoma (HL) is common in young adults and considered curable in most patients. Young HL survivors (HLS) are at risk of long-term adverse effects. Our study aimed to assess various fatigue and quality of life (QoL) complaints, and their correlations with treatment. Self-reported questionnaires assessing fatigue (MFI-20) and QoL-related issues (EORTC-QOL-C-30) were used to examine HLS aged 18-65 who completed first-line chemotherapy ± radiotherapy (RT) and were in complete remission for at least six months post-therapy. The cohort included 120 HLS (median age 32 years), assessed between 6 months and 15 years post-treatment. About 28% presented with severe fatigue and severely reduced QoL. Higher fatigue levels were associated with four cycles of the ABVD + RT. Young HLS experience high levels of persistent physical fatigue, emotional distress, and cognitive decline that are insufficiently investigated. Assessment of these complaints is essential and further investigation may provide tailored solutions for a better QoL for HLS.

Efficacy of Gemcitabine as Salvage Therapy for Relapsed and Refractory Aggressive Non-Hodgkin Lymphoma.

Gemcitabine-based salvage therapy is considered an effective treatment for relapsed and refractory Non-Hodgkin's lymphoma (NHL). We analyzed the outcome of 41 consecutive NHL patients treated with gemcitabine-based regimens between January 2007 and October 2015. Twenty-eight males and 13 females (median age 66.4 years) were included. The median follow-up from gemcitabine initiation was 7.3 months. Thirty patients (73%) had B-cell, and eleven (27%) had T-cell, lymphoma. All patients received a median of 2 prior regimens, of which at least 1 was anthracycline based. Twenty-eight patients (78%) received full-dose while 9 (22%) received reduced-dose regimens. The overall response rate was 37%, with 24% (n = 10) complete response, 12% (n = 5) partial response, and 63% (n = 22) progressive disease or stable disease. The median progression-free survival (PFS) was 47 days (range 12-1,318), the median overall survival (OS) was 1.9 years. Twenty patients (49%) died during follow-up. Grade 3-4 hematological toxicity was reported in 21 patients (51%). Relapsed vs. refractory disease, as well as a response to gemcitabine, predicted better PFS and OS. Use of a full-dose regimen predicted a better OS. Compared to previously published data, we observed less favorable outcomes. The administration of gemcitabine-based therapy as a salvage regimen for patients with relapsed or refractory NHL had limited success. Innovative therapies for these patients are an unmet need.

FDG PET-CT evaluation in neurolymphomatosis: imaging characteristics and clinical outcomes.

Neurolymphomatosis (NL) often represents unidentified non-Hodgkin lymphoma relapses. Considering its severity, early detection and treatment are crucial. We outline one hospital's 18F-FDG-PET-CT imaging findings of NL, along with the patients' clinical characteristics. Clinical records and imaging findings of 19 NL patients, PET-CT diagnosed, were retrospectively reviewed. Patient data, FDG-PET-CT findings and the presence of coexisting diseases, especially CNS involvement, were documented. Available MRI and clinical data verified the findings. All cases had increased linear FDG uptake along anatomic nerve sites. CTs showed varying degrees of corresponding soft-tissue-thickening. Clinical correlations also contributed to the diagnosis. In 4/19 patients, lymphoma presented with NL, in 15/19 it appeared with disease recurrence/progression. In 9/19, clinical symptoms suggested neural involvement while 10/19 had nonspecific symptoms. Eleven died of lymphoma within 0.9 years of diagnosis despite directed-therapy. Eight, however, survived up to 7.82 years post-diagnosis. Whole-body FDG-PET-CT can assist in early NL diagnosis, possibly enhancing survival.

Ethnic variation in medical and lifestyle risk factors for B cell non-Hodgkin lymphoma: A case-control study among Israelis and Palestinians.

BACKGROUND: Risk factors for B-cell non-Hodgkin lymphoma (B-NHL) have not been assessed among Palestinian Arabs (PA) and Israeli Jews (IJ). METHODS: In a case-control study we investigated self-reported medical and lifestyle exposures, reporting odds ratios (ORs) and 95% confidence intervals [CIs], by ethnicity, for overall B-NHL and subtypes. RESULTS: We recruited 823 cases and 808 healthy controls. Among 307 PA/516 IJ B-NHL cases (mean age at diagnosis = 51 [±17] versus 60 [±15] years, respectively) subtype distributions differed, with diffuse large B-cell lymphoma (DLBCL) being prominent among PA (71%) compared to IJ (41%); follicular lymphoma (FL), was observed in 14% versus 28%, and marginal zone lymphoma, in 2% versus 14%, respectively. Overall B-NHL in both populations was associated with recreational sun exposure OR = 1.43 [CI:1.07-1.91], black hair-dye use OR = 1.70 [CI:1.00-2.87], hospitalization for infection OR = 1.68 [CI:1.34-2.11], and first-degree relative with hematopoietic cancer, OR = 1.69 [CI:1.16-2.48]. An inverse association was noted with alcohol use, OR = 0.46 [CI:0.34-0.62]. Subtype-specific exposures included smoking (FL, OR = 1.46 [CI:1.01-2.11]) and >monthly indoor pesticide use (DLBCL, OR = 2.01 [CI:1.35-3.00]). Associations observed for overall B-NHL in PA only included: gardening OR = 1.93 [CI:1.39-2.70]; history of herpes, mononucleosis, rubella, blood transfusion (OR>2.5, P<0.01 for all); while for IJ risk factors included growing fruits and vegetables, OR = 1.87 [CI:1.11-3.15]; and self-reported autoimmune diseases, OR = 1.99 [CI:1.34-2.95]. CONCLUSIONS: In these geographically proximate populations we found some unique risk factors for B-NHL. Heterogeneity in the observed associations by ethnicity could reflect differences in lifestyle, medical systems, and reporting patterns, while variations by histology infer specific etiologic factors for lymphoma subtypes.

Infections associated with bendamustine containing regimens in hematological patients: a retrospective multi-center study.

A multi-center retrospective analysis of a cohort of patients in Israel treated with any bendamustine containing regimen between 2010-2014 was performed in order to determine the incidence and predictors for infection. The Kaplan Meier Model, employing log rank analysis, was used to assess time-to-infection. The Cox Proportional Hazards model was used to analyze multivariate effects of risk and 234 patients were included in the analysis. One hundred and nine (46.6%) developed at least one infection and 33.76% had severe infections. Seventy-six (41.5%) developed bacterial infection, nine (3.8%) fungal infection and 26 (11.5%) had viral infections. Factors significantly associated with time to infection on multivariable analysis were: bendamustine-combinations [hazard ratio (HR) = 0.589 (95% CI = 0.374-0.926), p = 0.022], Hb level [HR = 0.791 (95% CI = 0.716-0.875), p < 0.0001] and ischemic heart disease [HR = 1.828 (95% CI = 1.165-2.868), p = 0.009]. Infections were associated with a higher mortality and hospitalization rate.