CD19-directed chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of relapsed/refractory B-cell non-Hodgkin lymphoma (B-NHL) and recently showed effects in autoimmune diseases, such as systemic lupus erythematosus (SLE). Despite high levels of inflammation, toxicity seemed to differ between patients with SLE and B-NHL. We therefore compared the CAR T-cell kinetics and treatment-related side effects to better define the toxicity profiles. In contrast with the similar CAR T-cell expansion, patients with SLE revealed a lower incidence and severity of cytokine-release syndrome, immune effector cell-associated neurotoxicity syndrome, and immune effector cell-associated hematotoxicity. Although the neutrophil nadir was lower in patients with SLE after therapy, the platelet counts remained close to normal and hematotoxicity was shorter in SLE than B-NHL. The reduced hematotoxicity correlated with lower acute-phase inflammation, better hematologic reserve before CAR T-cell therapy, and distinct serum cytokine profiles. Interestingly, CAR T-cell persistence was consistently shorter, and the reconstitution of conventional T and B cells was faster in SLE. In both cohorts, B-cell reconstitution correlated with functional CD4+ T-cell recovery, indicating a general biologic process of hematopoietic and immune system regeneration. In summary, similar lymphodepletion and CAR T-cell pharmacokinetics led to distinct toxicity, demonstrating that CAR T-cell therapy had a favorable side-effect profile in SLE, including faster recovery of the adaptive immune system.

Clonal hematopoiesis of indeterminate potential (CHIP) is associated with increased mortality and malignancy risk, yet the determinants of clonal expansion remain poorly understood. We performed sequencing at a depth of coverage of >4000× for CHIP mutations in 6976 postmenopausal women from the Women's Health Initiative (WHI) at 2 time points: the WHI baseline examination and ∼16 years later at the Long Life Study (LLS) visit. Among 3685 CH mutations detected at baseline (variant allele fraction [VAF] of ≥0.5%), 24% were not detected at LLS, 26% were micro-CH at LLS (0.5% ≤ VAF < 2%), and 50% were CHIP (VAF ≥ 2%). We confirmed that clonal expansion is highly dependent on initial clone size and CHIP driver gene, with SF3B1 and JAK2 mutations exhibiting the fastest growth rate. We identified germ line variants in TERT, IL6R, TCL1A, and MSI2 that modulate clonal expansion rate. Measured baseline leukocyte telomere length showed differential effects on incident CHIP risk, with shorter baseline leukocyte telomere length predisposing to incident PPM1D mutations and longer baseline leukocyte telomere length favoring incident DNMT3A mutations. We discovered that the IL6R missense variant p.Asp358Ala specifically impairs TET2 clonal expansion, supported by direct measurements of soluble interleukin-6 receptor and interleukin-6. Faster clonal growth rate was associated with increased risk of cytopenia, leukemia, and all-cause mortality. Notably, CHIP clonal expansion rate mediated 34.4% and 43.7% of the clonal hematopoiesis risk score's predictive value for leukemia and all-cause mortality, respectively. These findings reveal key biological determinants of CHIP progression and suggest that incorporating growth rate measurements could enhance risk stratification.

Patients with follicular lymphoma who experience disease progression within 24 months of diagnosis (POD24) have a lower survival. Positron emission tomography (PET) response and circulating tumor DNA (ctDNA) minimal residual disease (MRD) assessment at end of induction (EOI) may allow their early identification. A representative cohort of 141 patients from the RELEVANCE phase 3 trial with both available serum samples for ctDNA testing and PET images at randomization and at EOI (week 24) was investigated. Twelve percent were POD24. ctDNA was analyzed using a customized 130-kilobase capture panel, with phased variant (PV) enriched regions representing 39% of the panel. ctDNA was detected in 140 patients (99.3%) at baseline. To optimize specificity, only PVs, found in 124 patients (88%), were considered for ctDNA MRD assessment at EOI. Median progression-free survival (PFS) from EOI was not reached (NR) for the 112 patients with undetected ctDNA at EOI vs 17.7 months (95% confidence interval [CI], 1.4 to NR) for patients with positive ctDNA (MRD+) (P = .0038). Similarly, median PFS was NR for the 104 patients with undetected disease on PET at EOI vs 28.3 months (95% CI, 2.9 to NR; P = .0002) for patients with PET positivity. Both tests had a negative predictive value (NPV) of >90% for POD24. The positive predictive value was 58.3% for ctDNA MRD and 45% for PET but increased to 85.7% when both parameters were combined, without alteration of NPV. These data show that the combination of PET response and ctDNA MRD at EOI allows an early prediction of POD24, which may lead to a preemptive treatment decision. This trial was registered at www.clinicaltrials.gov as #NCT01650701.

In retrospective studies, autologous stem cell transplantation (ASCT) conditioning with intravenous busulfan and melphalan (BUMEL) led to longer progression-free survival (PFS) than melphalan alone (MEL200). We compared long-term outcomes of BUMEL vs MEL200 in the context of intensified bortezomib, lenalidomide, and dexamethasone (VRD) induction and consolidation therapies. GEM12 was a phase 3 trial for patients with newly diagnosed multiple myeloma (NDMM) eligible for ASCT including 6 reinforced VRD cycles followed by ASCT conditioned with BUMEL or MEL200 and 2 VRD consolidation cycles. The primary end point was PFS. Subgroup analyses were based on International Staging System (ISS) stages and high-risk genetic abnormalities. Patients were randomized with an open-label 2 × 2 factorial design and 1:1:1:1 allocation ratio to ensure the balance between the GEM12 and the subsequent phase 3 GEM14 trial. Between 2013 and 2015, 458 patients were randomized (BUMEL, n = 230; MEL200, n = 228). The 10⁻⁶ MRD-negative rate was 63%, 68% for BUMEL vs 58% for MEL200 (odds ratio, 1.51; P = .035). The median PFS was 89 months for BUMEL and 73.1 for MEL200 (hazard ratio, 0.89 [95% confidence interval, 0.70-1.14]; P = .3). BUMEL showed benefit for patients with ISS stages II or III, t(14;16), and del(1p). For subcohorts ISS stages II or III treated with BUMEL and ISS I treated with MEL200 the median PFS was 96.5 months (95% confidence interval, 76 to not estimable). No safety concerns were observed. After a median follow-up of 8.4 years, GEM2012 demonstrated one of the longest PFS values reported in patients with NDMM, with significant differences favoring BUMEL in advanced ISS stages. The trial was registered at www.ClinicalTrials.gov as #NCT01916252 and at European Union Drug Regulating Authorities Clinical Trials as EudraCT 2012-005683-10.

Blood transfusions save millions of lives worldwide each year, yet formation of antibodies against nonself antigens remains a significant problem, particularly in patients who receive frequent transfusions. We designed and tested the Universal Blood Donor Typing (UBDT_PC1) array for automated high-throughput simultaneous typing of human erythrocyte antigens (HEAs), platelet antigens (HPAs), leukocyte antigens (HLAs), and neutrophil antigens to support selection of blood products matched beyond ABO/Rh. Typing samples from 6946 study participants of European, African, Admixed American, South Asian, and East Asian ancestry at 2 different laboratories showed a genotype reproducibility of ≥99% for 17 244 variants, translating to 99.98%, 99.90%, and 99.93% concordance across 338 372 HEA, 53 270 HPA, and 107 094 HLA genotypes, respectively. Compared with previous clinical typing data, concordance was 99.9% and 99.6% for 245 874 HEA and 3726 HPA comparisons, respectively. HLA types were 99.1% concordant with clinical typing across 8130 comparisons, with imputation accuracy higher in Europeans vs non-Europeans. Seven variant RHD alleles, a GYPB deletion underlying the U- phenotype, and 14 high-frequency antigen-negative types were also detected. Beyond blood typing, hereditary hemochromatosis-associated HFE variants were identified in 276 participants. We found that the UBDT_PC1 array can reliably type a wide range of blood cell antigens across diverse ancestries. Reproducibility and accuracy were retained when transfusion-relevant targets from the UBDT_PC1 array were incorporated into the UKBB_v2.2 genome-wide typing array. The results represent the potential for significant advancement toward improved patient care by reducing harm in transfusion recipients through extended matching.

AL amyloidosis is a disorder characterized by expansion of clonal plasma cells in the bone marrow and distant end organ damage mediated by misfolded immunoglobulin free light chains. There are currently limited data regarding the functional characteristics of AL amyloidosis plasma cells and their surrounding bone marrow microenvironment. We performed 5' single-cell RNA sequencing on newly diagnosed, treatment-naïve patients with AL amyloidosis and healthy subjects. We identified generalized suppression of normal bone marrow hematopoiesis with distinct expansion of monocytes and subsets of CD4+ T cells in patients with AL amyloidosis. We detected significant transcriptional changes broadly occurring among immune cells with increased tumor necrosis factor-α signaling and interferon response accompanied by increased inflammatory response in bone marrow plasma, as measured via quantitative proteomics with specific elevation of costimulatory molecule soluble CD276 (sB7-H3). A transcriptionally distinct population of nonmalignant plasma cells was disproportionately expanded in patients with AL amyloidosis and characterized by increased expression of CRIP1. Finally, clonal AL amyloidosis plasma cells were identified based on their unique variable-diversity-joining. rearrangement and showed increased expression of genes involved in proteostasis when compared with autologous, polyclonal plasma cells. Interpatient transcriptional heterogeneity was evident, with transcriptional states reflective of common genomic translocations easily identifiable. This study defines the transcriptional characteristics of AL amyloidosis plasma cells and their surrounding bone marrow microenvironment with identification of altered genes previously involved in the pathogenesis of other protein deposition disorders. Our data provide the rationale for functional validations of these genes in future studies.

TET2 is among the most commonly mutated genes in both clonal hematopoiesis and myeloid malignancies; thus, the ability to identify selective dependencies in TET2-deficient cells has broad translational significance. Here, we identify regulators of Tet2 knockout (KO) hematopoietic stem and progenitor cell (HSPC) expansion using an in vivo CRISPR-Cas9 KO screen, in which nucleotide barcoding enabled large-scale clonal tracing of Tet2-deficient HSPCs in a physiologic setting. Our screen identified candidate genes, including Ncoa4, that are selectively required for Tet2 KO clonal outgrowth compared with wild type. Ncoa4 targets ferritin for lysosomal degradation (ferritinophagy), maintaining intracellular iron homeostasis by releasing labile iron in response to cellular demands. In Tet2-deficient HSPCs, increased mitochondrial adenosine triphosphate production correlates with increased cellular iron requirements and, in turn, promotes Ncoa4-dependent ferritinophagy. Restricting iron availability reduces Tet2 KO stem cell numbers, revealing a dependency in TET2-mutated myeloid neoplasms.

Monoclonal antibodies targeting CD38 are a therapeutic mainstay in multiple myeloma (MM). Although they have contributed to improved outcomes, most patients still experience disease relapse, and little is known about tumor-intrinsic mechanisms of resistance to these drugs. Antigen escape has been implicated as a mechanism of tumor-cell evasion in immunotherapy. Yet, it is unknown whether MM cells can develop permanent resistance to anti-CD38 antibodies by acquiring genomic events leading to biallelic disruption of the CD38 gene locus. Here, we analyzed whole-genome and whole-exome sequencing data from patients 701 newly diagnosed MM, 67 patients at relapse with naivety to anti-CD38 antibodies, and 50 patients collected at relapse after anti-CD38 antibodies. We report a loss of CD38 in 10 of 50 patients (20%) after CD38 therapy, 3 of whom exhibited a loss of both copies. Two of these cases showed convergent evolution in which distinct subclones independently acquired similar advantageous variants. Functional studies on missense mutations involved in biallelic CD38 events revealed that 2 variants, L153H and C275Y, decreased binding affinity and antibody-dependent cellular cytotoxicity of the commercial antibodies daratumumab and isatuximab. However, a third mutation, R140G, conferred selective resistance to daratumumab, while retaining sensitivity to isatuximab. Clinically, patients with MM are often rechallenged with CD38 antibodies after disease progression and these data suggest that next-generation sequencing may play a role in subsequent treatment selection for a subset of patients.

Developing anti-factor VIII (FVIII) inhibitory antibodies (inhibitors) are a significant complication of FVIII protein replacement therapy in hemophilia A. Our previous study demonstrated that follicular helper T (TFH) cells play a critical role in FVIII inhibitor development. Follicular regulatory T (TFR) cells are a subset of forkhead box protein P3 positive (Foxp3+) T cells identified in the germinal center that can modulate TFH cell activation of B cells and antibody development. Here, we report that FVIII immunization significantly increases the TFR cells in the spleens of FVIII inhibitor-producing FVIIInull mice compared with saline-treated controls and non-inhibitor-producing animals. The TFH/TFR ratio significantly increased in FVIII inhibitor-producing mice. The emergence of TFR cells correlated with titers of FVIII inhibitors in FVIII-immunized mice. Using TFR-deficient Foxp3Cre+Bcl6fl/fl (Bcl6FC) mice, we found that the loss of TFR cells led to significantly decreased FVIII inhibitors compared with wild-type (WT) mice on FVIII immunization (24 ± 16 and 131 ± 114 Bethesda unit (BU)/mL, respectively) but not total anti-FVIII IgG levels and that TFR cells regulated IgG subclass switching and FVIII-specific B-cell responses. Interestingly, on FVIII immunization, mice with phosphatase and tensin (Pten) deficiency in Foxp3+ cells (Foxp3Cre+Ptenfl/fl), a model with augmented TFR cells, developed markedly lower FVIII inhibitor titers (8.1 ± 8.6 BU/mL) than WT controls. When CD4Cre+Bcl6fl/fl mice, a TFH- and TFR-deficient model, were immunized with FVIII, none of the animals developed FVIII inhibitors. In conclusion, FVIII immunization induces TFR cell activation and expansion. TFR cells have a dual function in regulating the development of FVIII inhibitors, and the TFH/TFR pathway is pivotal in FVIII inhibitor development in mice.

Although allogeneic hematopoietic stem cell transplantation is a leading treatment approach for myeloid malignancies, challenges in its immune biology and in treatment approaches remain. In the past decade, major advances in the knowledge of mechanisms of graft-versus-host disease (GVHD) has allowed development of new treatments both for GVHD prophylaxis and treatment. However, although successes did occur, failure did as well. Reasons for failure can be linked either to incomplete understanding of the pathophysiology of GVHD, or, in some cases, to errors in the design of clinical trials. Better GVHD prophylaxes and disease control have likely led to decreased nonrelapse mortality (NRM). However, although NRM rates have decreased, rates of relapse of the original malignancy have not significantly improved. Our current understanding of the biology of the graft-versus-leukemia (GVL) effect still lags behind that of GVHD, and treatment approaches to manipulate the GVL effect remain limited. The reasons for such a lag are numerous, but improved knowledge of the biology of hematological malignancies open the gate to new developments, providing that we can better understand the interplay between the immune system with leukemic clones. From a therapeutical perspective, much attention has been paid to the results from randomized clinical trials and from a biological perspective on recent discoveries, especially in the human setting. The objective of this perspective is to analyze what are the current challenges in the biology and treatment of GVHD and GVL and to provide a personal view on how some biological and therapeutic issues could be approached.

Disease response and progression assessment in multiple myeloma is based on various measurements of monoclonal protein (serum and urine protein electrophoresis, serum free light chain, and/or quantitative immunoglobulins). Currently, the International Myeloma Working Group consensus response criteria require 2 sequential assessments of any 1 marker made at any time before confirmation of disease progression and the institution of any new therapy. However, this can be cumbersome in clinical trials. Herein, we hypothesized that if 2 markers meet the progression criteria simultaneously, a repeat of either will not be necessary for confirmation. We retrospectively studied all sequential patients with myeloma enrolled in clinical trials at Mayo Clinic. We identified 583 episodes of confirmed progression in our study. Among the 583 progression episodes, nearly 70% (sensitivity of the simultaneous criteria) met the 2 simultaneous variable criteria at the first testing, indicating progression. Conversely, among 413 patients who met progression criteria by 2 simultaneous values, 98% (specificity of the simultaneous criteria) of patients subsequently had confirmed progression by sequential values. In summary, for patients with 2 disease burden markers meeting the simultaneous progression criteria, sequential assessment of either 1 for confirmation may not be necessary to determine disease progression.