Mikhail Roshal

Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells

Classical Hodgkin lymphoma (cHL) is characterized by sparsely distributed Hodgkin and Reed-Sternberg (HRS) cells amid reactive host background, complicating the acquisition of neoplastic DNA without extensive background contamination. We overcame this limitation by using flow-sorted HRS and intratumor T cells and optimized low-input exome sequencing of 10 patient samples to reveal alterations in genes involved in antigen presentation, chromosome integrity, transcriptional regulation, and ubiquitination. β-2-microglobulin (B2M) is the most commonly altered gene in HRS cells, with 7 of 10 cases having inactivating mutations that lead to loss of major histocompatibility complex class I (MHC-I) expression. Enforced wild-type B2M expression in a cHL cell line restored MHC-I expression. In an extended cohort of 145 patients, the absence of B2M protein in the HRS cells was associated with lower stage of disease, younger age at diagnosis, and better overall and progression-free survival. B2M-deficient cases encompassed most of the nodular sclerosis subtype cases and only a minority of mixed cellularity cases, suggesting that B2M deficiency determines the tumor microenvironment and may define a major subset of cHL that has more uniform clinical and morphologic features. In addition, we report previously unknown genetic alterations that may render selected patients sensitive to specific targeted therapies.

Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia

Background CD19‐specific chimeric antigen receptor (CAR) T cells induce high rates of initial response among patients with relapsed B‐cell acute lymphoblastic leukemia (ALL) and long‐term remissions in a subgroup of patients. Methods We conducted a phase 1 trial involving adults with relapsed B‐cell ALL who received an infusion of autologous T cells expressing the 19‐28z CAR at the Memorial Sloan Kettering Cancer Center (MSKCC). Safety and long‐term outcomes were assessed, as were their associations with demographic, clinical, and disease characteristics. Results A total of 53 adults received 19‐28z CAR T cells that were manufactured at MSKCC. After infusion, severe cytokine release syndrome occurred in 14 of 53 patients (26%; 95% confidence interval [CI], 15 to 40); 1 patient died. Complete remission was observed in 83% of the patients. At a median follow‐up of 29 months (range, 1 to 65), the median event‐free survival was 6.1 months (95% CI, 5.0 to 11.5), and the median overall survival was 12.9 months (95% CI, 8.7 to 23.4). Patients with a low disease burden (<5% bone marrow blasts) before treatment had markedly enhanced remission duration and survival, with a median event‐free survival of 10.6 months (95% CI, 5.9 to not reached) and a median overall survival of 20.1 months (95% CI, 8.7 to not reached). Patients with a higher burden of disease (≥5% bone marrow blasts or extramedullary disease) had a greater incidence of the cytokine release syndrome and neurotoxic events and shorter long‐term survival than did patients with a low disease burden. Conclusions In the entire cohort, the median overall survival was 12.9 months. Among patients with a low disease burden, the median overall survival was 20.1 months and was accompanied by a markedly lower incidence of the cytokine release syndrome and neurotoxic events after 19‐28z CAR T‐cell infusion than was observed among patients with a higher disease burden. (Funded by the Commonwealth Foundation for Cancer Research and others; ClinicalTrials.gov number, NCT01044069.)

Enasidenib induces acute myeloid leukemia cell differentiation to promote clinical response

Recurrent mutations at R140 and R172 in isocitrate dehydrogenase 2 (IDH2) occur in many cancers, including ∼12% of acute myeloid leukemia (AML). In preclinical models these mutations cause accumulation of the oncogenic metabolite R-2-hydroxyglutarate (2-HG) and induce hematopoietic differentiation block. Single-agent enasidenib (AG-221/CC-90007), a selective mutant IDH2 (mIDH2) inhibitor, produced an overall response rate of 40.3% in relapsed/refractory AML (rrAML) patients with mIDH2 in a phase 1 trial. However, its mechanism of action and biomarkers associated with response remain unclear. Here, we measured 2-HG, mIDH2 allele burden, and co-occurring somatic mutations in sequential patient samples from the clinical trial and correlated these with clinical response. Furthermore, we used flow cytometry to assess inhibition of mIDH2 on hematopoietic differentiation. We observed potent 2-HG suppression in both R140 and R172 mIDH2 AML subtypes, with different kinetics, which preceded clinical response. Suppression of 2-HG alone did not predict response, because most nonresponding patients also exhibited 2-HG suppression. Complete remission (CR) with persistence of mIDH2 and normalization of hematopoietic stem and progenitor compartments with emergence of functional mIDH2 neutrophils were observed. In a subset of CR patients, mIDH2 allele burden was reduced and remained undetectable with response. Co-occurring mutations in NRAS and other MAPK pathway effectors were enriched in nonresponding patients, consistent with RAS signaling contributing to primary therapeutic resistance. Together, these data support differentiation as the main mechanism of enasidenib efficacy in relapsed/refractory AML patients and provide insight into resistance mechanisms to inform future mechanism-based combination treatment studies.

Immaturity associated antigens are lost during induction for T cell lymphoblastic leukemia: implications for minimal residual disease detection.

Induction chemotherapy for acute leukemia often leads to antigenic shifts in residual abnormal blast populations. Studies in precursor B cell ALL (B‐ALL) have demonstrated that chemotherapy commonly results in the loss of antigens associated with immaturity, limiting their utility for minimal residual disease (MRD) detection. Little information is available about the stability of these antigens in precursor T cell ALL (T‐ALL) though it is presumed that CD99 and terminal deoxynucleotidyl transferase (TdT) are highly informative based on limited studies.

Mutations in a gene encoding a midbody kelch protein in familial and sporadic classical Hodgkin lymphoma lead to binucleated cells

Classical Hodgkin lymphoma (cHL) is a malignancy of B-cell origin in which the neoplastic cells, known as “Reed-Sternberg” (RS) cells, are characteristically binucleated. Here we describe a family where multiple individuals developing cHL have inherited a reciprocal translocation between chromosomes 2 and 3. The translocation disrupts KLHDC8B, an uncharacterized gene from a region (3p21.31) previously implicated in lymphoma and related malignancies, resulting in its loss of expression. We tested KLHDC8B as a candidate gene for cHL and found that a 5′-UTR polymorphism responsible for decreasing its translational expression is associated with cHL in probands from other families with cHL and segregates with disease in those pedigrees. In one of three informative sporadic cases of cHL, we detected loss of heterozygosity (LOH) for KLHDC8B in RS cells, but not reactive T lymphocytes, purified from a malignant lymph node. KLHDC8B encodes a protein predicted to contain seven kelch repeat domains. KLHDC8B is expressed during mitosis, where it localizes to the midbody structure connecting cells about to separate during cytokinesis, and it is degraded after cell division. Depletion of KLHDC8B through RNA interference leads to an increase in binucleated cells, implicating its reduced expression in the formation of cHLs signature RS cell.

Rational design of novel red-shifted BRET pairs: Platforms for real-time single-chain protease biosensors

Bioluminescence resonance energy transfer (BRET) systems to date have been dominated by use of blue‐green Renilla luciferase (Rluc) as the energy donor. Although effective in many cases, the expense and unfavorable biochemical attributes of the substrate (phenylcoelenterazine) limit utility of Rluc‐based BRET systems. Herein we report a series of novel BRET pairs based on luciferases that utilize D‐luciferin, resulting in red‐shifted photonic outputs, favorable biochemical attributes, and increased efficacy. We developed a modified Förster equation to predict optimal BRET luciferase donor‐fluorophore pairs and identified tdTomato as the optimal red fluorophore acceptor for click beetle green luciferase (CBG). A prototypical single‐chain protease biosensor, capable of reporting on executioner caspase activity in live cells and in real‐time, was generated by inserting a DEVD linker between CBG and tdTomato and validated in vitro with recombinant caspases and in cellulo with apoptosis‐sensitive and ‐resistant cell lines. High signal‐to‐noise ratios (∼33) and Z′ factors (0.85) were observed in live cell longitudinal studies, sufficient for high‐throughput screening. Thus, we illustrate a general methodology for the rational design of new BRET systems and provide a novel single‐chain BRET protease biosensor that is long lived, red‐shifted, and utilizes D‐luciferin.

Optimization of Plasma Fluorogenic Thrombin-Generation Assays

We optimized fluorogenic thrombin-generation assays with regard to sample volume, calibration, analytic corrections, and activation reagents. Lower sample volumes (40 vs 80 microL) were associated with better recovery of thrombin activity, lower interference due to absorbance of light, and higher total thrombin generation (area under the curve), even using internal standards to calibrate plasma samples. With lower sample volumes, there was no advantage to internal calibration of samples without obvious interference (hemolysis). Previously developed corrections for measured vs expected fluorescence units, residual thrombin-alpha(2)-macroglobulin activity, and hemolysis improved the analytic accuracy of the assay. An optimized assay with a 40-microL sample volume, analytic corrections, and a corn trypsin inhibitor to block contact activation showed that 0.6 pmol/L tissue factor activator was better than 5 pmol/L at differentiating healthy subjects from patients with sepsis while demonstrating good reproducibility (area under the curve, 4% within-run and 7% between-run coefficient of variation).

FOXO1 repression contributes to block of plasma cell differentiation in classical Hodgkin Lymphoma

The survival of classical Hodgkin lymphoma (cHL) cells depends on activation of NF-κB, JAK/STAT, and IRF4. Whereas these factors typically induce the master regulator of plasma cell (PC) differentiation PRDM1/BLIMP-1, levels of PRDM1 remain low in cHL. FOXO1, playing a critical role in normal B-cell development, acts as a tumor suppressor in cHL, but has never been associated with induction of PC differentiation. Here we show that FOXO1 directly upregulates the full-length isoform PRDM1α in cHL cell lines. We also observed a positive correlation between FOXO1 and PRDM1 expression levels in primary Hodgkin-Reed-Sternberg cells. Further, we show that PRDM1α acts as a tumor suppressor in cHL at least partially by blocking MYC. Here we provide a link between FOXO1 repression and PRDM1α downregulation in cHL and identify PRDM1α as a tumor suppressor in cHL. The data support a potential role for FOXO transcription factors in normal PC differentiation.

Multicolor Flow Cytometry and Multigene Next-Generation Sequencing Are Complementary and Highly Predictive for Relapse in Acute Myeloid Leukemia after Allogeneic Transplantation

Minimal residual disease (MRD) in acute myeloid leukemia (AML) is typically measured using multiparameter flow cytometry (MFC). Detection of leukemia mutations using multigene next-generation sequencing (NGS) can potentially be used to measure residual disease. We used a targeted 28-gene NGS panel to detect mutations and different-from-normal 10-color MFC to measure MRD in AML patients before allogeneic hematopoietic stem cell transplantation (HCT). Residual disease was defined when any abnormal blast population was detected using MFC and when any leukemia allele was detected with a variant allele frequency (VAF)  ≥ 5% using NGS. We tracked the clearance of leukemia alleles between AML diagnosis and immediately before HCT and found that mutations in DNMT3A, TET2, and JAK2 were less likely to be cleared than NPM1, IDH 1/2, and FLT3-ITD. Despite varying sensitivities, the concordance rate of residual disease detection before HCT using the 2 assays was 44 of 62 (71%) evaluable cases. Discordance could be explained by residual mutations in DNMT3A and TET2 that were not detected by MFC and presence of residual leukemia mutations with VAF below the established thresholds for mutation calling. Presence of flow MRD and residual mutations immediately before HCT using the 2 assays was associated with relapse risk (MFC: hazard ratio,  4.62; 95% confidence interval [CI], 1.32 to 16.09; P = .016 and NGS: hazard ratio,  4.35; 95% CI, 1.63 to 11.6; P = .003) and survival (MFC: hazard ratio,  2.44; 95% CI, 1 to 5.97; P = .05 and NGS: hazard ratio, 2.1; 95% CI, .97 to 4.55; P = .059) after HCT. Residual disease detected concurrently by MFC and NGS conferred the highest relapse risk compared with patients who were either negative by both assays or had discordant status (overall, P = .008). Although MFC is universally applicable, a multigene NGS approach to measuring residual disease in AML provides additional information on differential clearance of disease alleles and can assess clonal architecture before transplantation.

Single-Tube 10-Fluorochrome Analysis for Efficient Flow Cytometric Evaluation of Minimal Residual Disease in Plasma Cell Myeloma

OBJECTIVES Widespread adoption of recent recommendations for minimal residual disease (MRD) detection in myeloma has partly been impeded by a paucity of studies detailing multiparameter flow cytometry (MPF) assay validation. In response, we have validated a novel and efficient single-tube 10-color assay for MRD detection that incorporates the recently recommended plasma cell markers. METHODS Aspirate samples from 53 patients with plasma cell disorder were analyzed using a novel single-tube 10-color method. The limit of detection, precision of measurement, and linearity of measurement of our new assay were determined using serial dilution experiments. The stability of the new antibody cocktail and the viability/specificity of stained samples were evaluated using serial time course measurements. RESULTS There was a high degree of quantitative agreement between our new 10-color method and an established eight-color method. Four positive samples detected by the 10-color assay were below or at the limit of detection of the eight-color assay, confirming its high sensitivity. In two cases, subsequent revision of the International Myeloma Working Group Uniform Response Criteria was necessary. CONCLUSION Adoption of our validated 10-color assay would enable clinical laboratories to satisfy current MRD recommendations without significantly increasing the demands on current workflow practices.