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 >4,000x depth of coverage for CHIP mutations in 6,976 postmenopausal women from the Women's Health Initiative at two timepoints: the WHI baseline exam and approximately 16 years later at the Long Life Study (LLS) visit. Among 3,685 CH mutations detected at baseline (VAF ≥ 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 germline 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 (FL) who experience disease progression within 24 months of diagnosis (POD24) have a lower survival. Positron emission tomography (PET) response and circulating tumor DNA (ctDNA) residual disease (MRD) assessment at end of induction therapy (EOI) may allow their early identification. A representative cohort of 141 patients from the RELEVANCE phase III 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 PFS from EOI was not reached (NR) for the 112 patients with undetected ctDNA at EOI versus 17.7 months (95%CI : 1.4-NA) for patients ctDNA MRD (+) (p=0.0038). Similarly, median PFS was NR for the 104 patients with undetected disease on PET at EOI versus 28.3 months (95%CI : 2.9-NR, p=0.0002) for patients with PET(+). Both tests had a negative predictive value (NPV) above 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 for an early prediction of POD24 which may lead to a preemptive treatment decision.

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 BUMEL vs. MEL200 outcomes in newly diagnosed multiple myeloma (NDMM) patients receiving intensified bortezomib, lenalidomide and dexamethasone (VRD) induction and consolidation therapy. The GEM12 phase III trial enrolled 458 patients (from 2013 to 2015) who were randomized to BUMEL (n=230) or MEL200 (n=228) conditioning after induction with six reinforced VRD cycles and followed by two similar VRD consolidation cycles. The primary endpoint was PFS, including subgroup analyses by International Staging System (ISS) stages and high-risk genetic abnormalities. Randomization used 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 III GEM14 maintenance trial. After 2 years of maintenance, the global 10⁻⁶ MRD-negative rate was 63%, (68% BUMEL vs. 58% MEL200; OR 1.51, P= 0.035). The PFS was not significantly better in the BUMEL vs. MEL200, even though it was almost 16 months longer (median PFS 89 vs. 73.1 months; HR 0.89, 95%CI, 0.70-1.14, P= 0.3). BUMEL showed benefits in ISS stages 2/3, t(14;16), and del(1p). In a combined subgroup jointly considering patients with ISS2/3 treated with BUMEL and patients with ISS1 treated with MEL200 the median PFS was 96 months (95%CI, 76-NE). No safety concerns emerged. After a median follow-up of 8.4 years, GEM2012 reported one of the longest PFS durations in NDMM patients, with BUMEL significantly favoring advanced ISS stages. The trial is registered at ClinicalTrials.gov (NCT01916252) and EudraCT (2012-005683-10).

Blood transfusions save millions of lives worldwide each year, yet formation of antibodies against non-self antigens remains a significant problem, particularly in frequently transfused patients. We designed and tested the Universal Blood Donor Typing (UBDT_PC1) array for automated high-throughput simultaneous typing of human erythroid, platelet, leukocyte, and neutrophil antigens (HEA, HPA, HLA, and HNA, respectively) to support selection of blood products matched beyond ABO/Rh. Typing samples from 6946 donors of European, African, Admixed American, South Asian, and East Asian ancestry at two 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 to 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 versus 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 donors. 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 towards improved patient care by reducing harm in transfused patients 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 AL amyloidosis patients and healthy subjects. We identified generalized suppression of normal bone marrow hematopoiesis with distinct expansion of monocytes and subsets of CD4+ T cells in AL amyloidosis patients. We detected significant transcriptional changes broadly occurring among immune cells with increased TNF-a signaling and interferon response accompanied by increased inflammatory response in bone marrow plasma as measured via quantitative proteomics with specific elevation of co-stimulatory molecule soluble CD276 (sB7-H3). A transcriptionally distinct population of non-malignant plasma cells was disproportionately expanded in AL amyloidosis patients and characterized by increased expression of CRIP1. Finally, clonal AL amyloidosis plasma cells were identified based on their unique VDJ rearrangement and showed increased expression of genes involved in proteostasis when compared to autologous, polyclonal plasma cells. Inter-patient 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 physiological setting. Our screen identified candidate genes, including Ncoa4, that are selectively required for Tet2 KO clonal outgrowth compared to wild-type (WT). Ncoa4 targets ferritin for lysosomal degradation (ferritinophagy), maintaining intracellular iron homeostasis by releasing labile iron (Fe2+) in response to cellular demands. In Tet2-deficient HSPCs, increased mitochondrial ATP 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). While 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 701 newly diagnosed patients, 67 patients at relapse with naivety to anti-CD38 antibodies, and 50 patients collected at relapse following anti-CD38 antibodies. We report a loss of CD38 in 20% (10/50) of patients post-CD38 therapy, three of which exhibited a loss of both copies. Two of these cases showed convergent evolution where distinct subclones independently acquired similar advantageous variants. Functional studies on missense mutations involved in biallelic CD38 events revealed that two 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 following 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-FVIII inhibitory antibodies (inhibitors) is 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 Foxp3+ T cells recently 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 to 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 to wild-type (WT) mice upon FVIII immunization (24±16 and 131±114 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, upon FVIII immunization, mice with phosphatase 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 HCT 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 non relapse mortality (NRM). However, while 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 effect (GvL) still lag beyond 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 (MM) is based on various measurements of monoclonal protein (serum and urine protein electrophoresis, serum free light chain (FLC, and or quantitative immunoglobulins). Currently, the IMWG consensus response criteria require two sequential assessments of any one 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 two 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 two simultaneous variable criteria at the first testing, indicating progression. Conversely, among 413 patients who met progression criteria by two simultaneous values, 98% (specificity of the simultaneous criteria) of patients subsequently had confirmed progression by sequential values. In summary, for patients with two disease burden markers meeting the simultaneous progression criteria, sequential assessment either one for confirmation may not be necessary to determine disease progression.