Standardization of flow cytometric detection of antigen expression.

Since response to antigen-based immunotherapy relies upon the level of tumor antigen expression we developed an antigen quantification assay using ABC values. Antigen quantification as a clinical assay requires methods for quality control and for interlaboratory and inter-cytometer platform standardization. A single lot of Cytotrol™ Lyophilized Control Cells (Beckman Coulter) used for all studies. The variability in antigen quantification across 4 different instrument platforms in 2 separate laboratories was evaluated. The effect of the antibody clone utilized, importance of custom 1:1 molar ratio (fluorophore to protein, F/P) verses off-the-shelf antibodies, and QuantiBrite PE calibration verses linearity calibration combined with a single point scale transformation with CD4 as reference were determined. Use of single lot control cells allowed validation of reproducibility between flow cytometer platforms and laboratories and allowed assessment of different antibody lots, cocktail preparation, and different antibody clones. Off the shelf antibody preparations provide reproducible estimates of antigen density, however custom 1:1 unimolar antibody preparations should be utilized for definitive measurement of antigen expression.Geometric Mean fluorescent Intensity (GeoMFI) was not comparable across instruments and inter-laboratory. The use of CD4 as the reference marker can minimize variability in ABC values. Comparable antigen quantification is vital in managing patients receiving antigen-based immunotherapy. If this assay is to be utilized in a clinical setting, quality control methods have to be instituted to assure reproducibility and allow validation across laboratories. We have demonstrated that use of a lyophilized cell control is highly valuable in achieveing these goals.

Rapid anti-myeloma activity by T cells expressing an anti-BCMA CAR with a human heavy-chain-only antigen-binding domain.

Multiple myeloma (MM) is a rarely curable malignancy of plasma cells. MM expresses B cell maturation antigen (BCMA). We developed a fully human anti-BCMA chimeric antigen receptor (CAR) with a heavy-chain-only antigen-recognition domain, a 4-1BB domain, and a CD3ζ domain. The CAR was designated FHVH33-CD8BBZ. We conducted the first-in-humans clinical trial of T cells expressing FHVH33-CD8BBZ (FHVH-T). Twenty-five patients with relapsed MM were treated. The stringent complete response rate (sCR) was 52%. Median progression-free survival (PFS) was 78 weeks. Of 24 evaluable patients, 6 (25%) had a maximum cytokine-release syndrome (CRS) grade of 3; no patients had CRS of greater than grade 3. Most anti-MM activity occurred within 2-4 weeks of FHVH-T infusion as shown by decreases in the rapidly changing MM markers serum free light chains, urine light chains, and bone marrow plasma cells. Blood CAR+ cell levels peaked during the time that MM elimination was occurring, between 7 and 15 days after FHVH-T infusion. C-C chemokine receptor type 7 (CCR7) expression on infusion CD4+ FHVH-T correlated with peak blood FHVH-T levels. Single-cell RNA sequencing revealed a shift toward more differentiated FHVH-T after infusion. Anti-CAR antibody responses were detected in 4 of 12 patients assessed. FHVH-T has powerful, rapid, and durable anti-MM activity.

COVID-19 in patients with classic and variant hairy cell leukemia.

Hairy cell leukemia (HCL), similar to its variant HCLv, is a B-cell malignancy associated with decreased humoral immunity. We prospectively monitored the largest cohort of patients with HCL/HCLv to date (n = 503) for COVID-19 by symptoms, antibody, and polymerase chain reaction (PCR) and/or antigen positivity. Fifty percent (253 of 503) of the patients with HCL/HCLv (238 HCL and 15 HCLv) had evidence of COVID-19, with 210 (83%) testing positive by PCR or rapid-antigen test. Of the 43 patients without positive tests, all had nucleocapsid antibodies indicating COVID-19 exposure, 7 recalled no symptoms, and 36 had mild symptoms. Of the 210 who tested positive, 23, 46, 129, and 12 cases occurred in 2020, 2021, 2022, and 2023, respectively. Among them, 175 began treatment for HCL/HCLv 0.4 to 429 (median, 66) months before, and 132 had their last dose of anti-CD20 monoclonal antibody 0.2 to 229 (median, 63) months before. Two patients died, including a young woman who began rituximab 2 months after first-line cladribine before vaccine availability. Nearly all patients with HCL/HCLv recovered uneventfully from COVID-19 including those without vaccination or those with significant immunosuppression and recent treatment. However, decreased normal B cells from HCL or treatment was associated with lower spike antibody levels as a response to COVID-19 (P = .0094) and longer recovery time (P = .0036). Thus, in a large cohort of patients with HCL/HCLv and in the first to determine relationships between COVID-19 outcome and immune markers, mortality was relatively low (∼1%), sequelae were uncommon, and recovery from COVID-19 was longer if normal B cells were low after recent treatment. The trials are registered at www.clinicaltrials.gov as #NCT01087333 and #NCT04362865.

Chimeric Antigen Receptor T Cells as Salvage Therapy for Post-Chimeric Antigen Receptor T Cell Failure.

Outcomes for post-chimeric antigen receptor (CAR) T cell therapy (CART) relapse are poor. The utilization of a unique CAR T cell construct for post-CART failure is increasing, but this approach is not well described. In this study, with CART-A the first unique CAR T cell construct received and CART-B the second, the primary objective was to characterize outcomes following CART-B. Secondary objectives included evaluating safety and toxicity with sequential CART infusions; investigating the impact of potential factors, such as antigen modulation and interval therapy, on CART-B response; and characterizing long-term outcomes in patients receiving multiple CARTs. This was a retrospective review (NCT03827343) of children and young adults with B cell acute lymphoblastic leukemia (B-ALL) undergoing CART therapy who received at least 2 unique CART constructs, excluding interim CART reinfusions of the same product. Of 135 patients, 61 (45.1%) received 2 unique CART constructs, including 13 who received >2 CARTs over time. Patients included in this analysis received 14 distinct CARTs targeting CD19 and/or CD22. The median age at CART-A was 12.6 years (range, 3.3 to 30.4 years). The median time from CART-A to CART-B was 302 days (range, 53 to 1183 days). CART-B targeted a different antigen than CART-A in 48 patients (78.7%), owing primarily to loss of CART-A antigen target. The rate of complete remission (CR) was lower with CART-B (65.5%; 40 of 61) than with CART-A (88.5%; 54 of 61; P = .0043); 35 of 40 (87.5%) CART-B responders had CART-B targeting a different antigen than CART-A. Among the 21 patients with a partial response or nonresponse to CART-B, 8 (38.1%) received CART-B with the same antigen target as CART-A. Of 40 patients with CART-B complete response (CR), 29 (72.5%) relapsed. For the 21 patients with evaluable data, the relapse immunophenotype was antigennegative in 3 (14.3%), antigendim in 7 (33.3%), antigenpositive in 10 (47.6%), and lineage switch in 1 (4.8%). The median relapse-free survival following CART-B CR was 9.4 months (95% confidence interval [CI], 6.1 to 13.2 months), and overall survival was 15.0 months (95% CI, 13.0 to 22.7 months). Given the limited salvage options for post-CART relapse, identifying optimizing strategies for CART-B is critical. We raise awareness about the emerging use of CART for post-CART failure and highlight clinical implications accompanying this paradigm shift.

Daratumumab induces cell-mediated cytotoxicity of primary effusion lymphoma and is active against refractory disease.

Primary effusion lymphoma (PEL), an aggressive non-Hodgkin lymphoma caused by Kaposi sarcoma-associated herpesvirus (KSHV), lacks standard therapy and has a median survival of 10-22 months with combination chemotherapy. PEL is a tumor of plasmablast-like B cells generally expressing CD38, the target of daratumumab (Dara). Initially, we assessed PEL cells from eight patients and established that each expressed high levels of CD38 by flow cytometry. PEL cell lines were also evaluated and most had high CD38 expression. We then assessed Dara's effects on complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of PEL cell lines as well as its clinical benefits on two patients with PEL. Despite high CD38 expression, Dara did not induce CDC of PEL cell lines, due in part to high levels of the complement-inhibitory proteins, CD55 and CD59. However, Dara induced significant and dose-dependent increases in ADCC, particularly in those lines with high CD38 levels. Two FDA-approved drugs, all trans-retinoic acid (ATRA) and pomalidomide (Pom), significantly increased surface CD38 levels in low-CD38 expressing PEL cell lines, resulting in increased Dara-induced ADCC. Two patients with refractory PEL were treated with Dara alone or in combination with Pom. One patient with leptomeningeal PEL had a complete response to Dara and Pom combination treatment. Others had improvement in performance status and resolution of malignant ascites with Dara alone. Together, these data support the use of Dara monotherapy or in combination with ATRA or Pom as a potential therapeutic option for PEL.

Preinfusion factors impacting relapse immunophenotype following CD19 CAR T cells.

Relapse following chimeric antigen receptor (CAR) T-cell therapy directed against CD19 for relapsed/refractory B-acute lymphoblastic leukemia (r/r B-ALL) remains a significant challenge. Three main patterns of relapse predominate: CD19 positive (CD19pos) relapse, CD19 negative (CD19neg) relapse, and lineage switch (LS). Development and validation of risk factors that predict relapse phenotype could help define potential pre- or post-CAR T-cell infusion interventions aimed at decreasing relapse. Our group sought to extensively characterize preinfusion risk factors associated with the development of each relapse pattern via a multicenter, retrospective review of children and young adults with r/r B-ALL treated with a murine-based CD19-CAR construct. Of 420 patients treated with CAR, 166 (39.5%) relapsed, including 83 (50%) CD19pos, 68 (41%) CD19neg, and 12 (7.2%) LS relapses. A greater cumulative number of prior complete remissions was associated with CD19pos relapses, whereas high preinfusion disease burden, prior blinatumomab nonresponse, older age, and 4-1BB CAR construct were associated with CD19neg relapses. The presence of a KMT2A rearrangement was the only preinfusion risk factor associated with LS. The median overall survival following a post-CAR relapse was 11.9 months (95% CI, 9-17) and was particularly dismal in patients experiencing an LS, with no long-term survivors following this pattern of relapse. Given the poor outcomes for those with post-CAR relapse, study of relapse prevention strategies, such as consolidative hematopoietic stem cell transplantation, is critical and warrants further investigation on prospective clinical trials.

Circulating CD22+/CD19-/CD24- progenitors and CD22+/CD19+/CD24- mature B cells: Diagnostic pitfalls for minimal residual disease detection in B-lymphoblastic leukemia.

BACKGROUND: Multiparametric flow cytometry (MFC) has become a powerful tool in minimal residual disease (MRD) detection in B-lymphoblastic leukemia/lymphoma (B-ALL). In the setting of targeted immunotherapy, B-ALL MRD detection often relies on alterative gating strategies, such as the utilization of CD22 and CD24. It is important to depict the full diversity of normal cell populations included in the alternative B-cell gating methods to avoid false-positive results. We describe two CD22-positive non-neoplastic cell populations in the peripheral blood (PB), including one progenitor population of uncertain lineage and one mature B-cell population, which are immunophenotypic mimics of B-ALL. METHODS: Using MFC, we investigated the prevalence and phenotypic profiles of both CD22-positive populations in 278 blood samples from 52 patients with B-ALL; these were obtained pre- and post-treatment with CD19 and/or CD22 CAR-T therapies. We further assessed whether these two populations in the blood were exclusively associated with B-ALL or recent anticancer therapies, by performing the same analysis on patients diagnosed with other hematological malignancies but in long-term MRD remission. RESULTS: The progenitor population and mature B-cell population were detected at low levels in PB of 61.5% and 44.2% of B-ALL patients, respectively. Both cell types showed distinctive and highly consistent antigen expression patterns that are reliably distinguishable from B-ALL. Furthermore, their presence is not restricted solely to B-ALL or recent therapy. CONCLUSIONS: Our findings aid in building a complete immunophenotypic profile of normal cell populations in PB, thereby preventing misdiagnosis of B-ALL MRD and inappropriate management.

A novel approach for characterization of KSHV-associated multicentric Castleman disease from effusions.

A biopsy of lymphoid tissue is currently required to diagnose Kaposi sarcoma-associated herpesvirus (KSHV)-associated multicentric Castleman disease (KSHV-MCD). Patients showing clinical manifestations of KSHV-MCD but no pathological changes of KSHV-MCD are diagnosed as KSHV inflammatory cytokine syndrome. However, a lymph node biopsy is not always feasible to make the distinction. A pathognomonic feature of lymph nodes in KSHV-MCD is the expansion of KSHV-infected, lambda-restricted but polyclonal plasmablasts. To investigate whether these cells also reside in extra-nodal sites, effusion from 11 patients with KSHV-MCD and 19 with KSHV inflammatory cytokine syndrome was analysed by multiparametric flow cytometry. A distinct, lambda-restricted plasmablastic population (LRP) with highly consistent immunophenotype was detected in effusions in 8/11 patients with KSHV-MCD. The same population was also observed in 7/19 patients with KSHV inflammatory cytokine syndrome. The detection of LRP stratified KSHV inflammatory cytokine syndrome into two clinically distinct subgroups; those with detectable LRP closely resembled KSHV-MCD, showing similar KSHV viral load, comparable severity of thrombocytopenia and hypoalbuminaemia, and similar incidences of hepatosplenomegaly. Collectively, the detection of LRP by flow cytometry can serve as a valuable tool in diagnosing KSHV-MCD. KSHV inflammatory cytokine syndrome with LRP in effusions may represent a liquid-form of KSHV-MCD.

Efficacy of second CAR-T (CART2) infusion limited by poor CART expansion and antigen modulation.

Chimeric antigen receptor T-cells (CART) are active in relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), but relapse remains a substantial challenge. Reinfusion with the same CART product (CART2) in patients with suboptimal response or antigen positive relapse following first infusion (CART1) represents a potential treatment strategy, though early experiences suggest limited efficacy of CART2 with CD19 targeting. We report on our experience with CART2 across a host of novel CAR T-cell trials. This was a retrospective review of children and young adults with B-ALL who received reinfusion with an anti-CD19, anti-CD22, or anti-CD19/22 CART construct on one of 3 CAR T-cells trials at the National Cancer Institute (NCT01593696, NCT02315612, NCT0344839) between July 2012 and January 2021. All patients received lymphodepletion (LD) pre-CART (standard LD: 75 mg/m2 fludarabine, 900 mg/m2 cyclophosphamide; or intensified LD: 120 mg/m2 fludarabine, 1200 mg/m2 cyclophosphamide). Primary objectives were to describe response to and toxicity of CART2. Indication for CART2, impact of LD intensity, and CAR T-cell expansion and leukemia antigen expression between CART infusions was additionally evaluated. Eighteen patients proceeded to CART2 due to persistent (n=7) or relapsed antigen positive disease (n=11) following CART1. Seven of 18 (38.9%) demonstrated objective response (responders) to CART2: 5 achieved a minimal residual disease (MRD) negative CR, 1 had persistent MRD level disease, and 1 showed a partial remission, the latter with eradication of antigen positive disease and emergence of antigen negative B-ALL. Responders included four patients who had not achieved a CR with CART1. Limited cytokine release syndrome was seen following CART2. Peripheral blood CART1 expansion was higher than CART2 expansion (p=0.03). Emergence of antigen negative/dim B-ALL in 6 (33.3%) patients following CART2 contributed to lack of CR. Five of seven (71.4%) responders received intensified LD pre-CART2, which corresponded with higher CART2 expansion than in those receiving standard LD (p=0.029). Diminished CAR T-cell expansion and antigen downregulation/loss impeded robust responses to CART2. A subset of patients, however, may derive benefit from CART2 despite suboptimal response to CART1. Intensified LD may be one strategy to augment CART2 responses, though further study of factors associated with CART2 response, including serial monitoring of antigen expression, is warranted.

CD19/22 CAR T cells in children and young adults with B-ALL: phase 1 results and development of a novel bicistronic CAR.

Remission durability following single-antigen targeted chimeric antigen receptor (CAR) T-cells is limited by antigen modulation, which may be overcome with combinatorial targeting. Building upon our experiences targeting CD19 and CD22 in B-cell acute lymphoblastic leukemia (B-ALL), we report on our phase 1 dose-escalation study of a novel murine stem cell virus (MSCV)-CD19/CD22-4-1BB bivalent CAR T-cell (CD19.22.BBζ) for children and young adults (CAYA) with B-cell malignancies. Primary objectives included toxicity and dose finding. Secondary objectives included response rates and relapse-free survival (RFS). Biologic correlatives included laboratory investigations, CAR T-cell expansion and cytokine profiling. Twenty patients, ages 5.4 to 34.6 years, with B-ALL received CD19.22.BBζ. The complete response (CR) rate was 60% (12 of 20) in the full cohort and 71.4% (10 of 14) in CAR-naïve patients. Ten (50%) developed cytokine release syndrome (CRS), with 3 (15%) having ≥ grade 3 CRS and only 1 experiencing neurotoxicity (grade 3). The 6- and 12-month RFS in those achieving CR was 80.8% (95% confidence interval [CI]: 42.4%-94.9%) and 57.7% (95% CI: 22.1%-81.9%), respectively. Limited CAR T-cell expansion and persistence of MSCV-CD19.22.BBζ compared with EF1α-CD22.BBζ prompted laboratory investigations comparing EF1α vs MSCV promoters, which did not reveal major differences. Limited CD22 targeting with CD19.22.BBζ, as evaluated by ex vivo cytokine secretion and leukemia eradication in humanized mice, led to development of a novel bicistronic CD19.28ζ/CD22.BBζ construct with enhanced cytokine production against CD22. With demonstrated safety and efficacy of CD19.22.BBζ in a heavily pretreated CAYA B-ALL cohort, further optimization of combinatorial antigen targeting serves to overcome identified limitations (www.clinicaltrials.gov #NCT03448393).

Acute and delayed cytopenias following CAR T-cell therapy: an investigation of risk factors and mechanisms.

Prolonged myelosuppression after chimeric antigen receptor (CAR) T-cell therapy is common and poorly understood. A retrospective analysis of 43 patients was conducted to investigate factors contributing to CAR T-cell-related cytopenias. Thirty-five patients were evaluable for analysis of delayed cytopenias occurring after initial hematologic recovery. Time to hematologic recovery (TTHR) was defined as number of days after CAR T-cell infusion for recovery to hemoglobin ≥8.0 g/dL, platelets ≥50.0 k/µL, and neutrophil count ≥1.0 k/µL without transfusions or growth factors for 7 days. Baseline percent bone marrow (BM) malignancy involvement correlated with TTHR (p = .0047). Patients with grades 3-4 cytokine-release syndrome (CRS) had longer TTHR than those with grades 0-2 CRS (p = .0479). Patients who developed prolonged or delayed cytopenias after anti-BCMA CAR T cells had a higher percentage of BM aspirate CAR+ cells at 2 months (n = 10; p = .0159).

Phase II Trial of Inotuzumab Ozogamicin in Children and Adolescents With Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia: Children's Oncology Group Protocol AALL1621.

PURPOSE: Children's Oncology Group trial AALL1621 was conducted to prospectively determine the safety and efficacy of inotuzumab ozogamicin (InO) in pediatric and adolescent patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). PATIENTS AND METHODS: This single-arm phase II trial enrolled patients age 1-21 years with R/R CD22-positive B-ALL. In cycle 1, InO dosing was 0.8 mg/m2 intravenously on day 1 and 0.5 mg/m2 on days 8 and 15 of a 28-day cycle with response evaluation at day 28. Using a two-stage design, the trial was continuously monitored for dose-limiting toxicities and sinusoidal obstruction syndrome (SOS). CD22 expression was retrospectively evaluated by central flow cytometry. RESULTS: Forty-eight patients were evaluable for response and toxicity; 19 had complete response (CR) and nine CR with incomplete count recovery (CRi) after cycle 1 (CR/CRi rate: 58.3%; two-sided 90% CI, 46.5 to 69.3). Twenty-seven of 28 patients with CR or CRi had minimal residual disease measured by flow cytometry; 18 (66.7%) had minimal residual disease < 0.01%. Seven of 28 patients (25%) with CR or CRi had delayed count recovery past day 42 in cycle 1. Three (6.3%) patients had grade 3 ALT elevation and one patient had grade 3 hyperbilirubinemia in cycle 1. Of 21 patients undergoing hematopoietic stem-cell transplantation after InO, 6 (28.6%) developed grade 3 SOS. Partial CD22 expression and lower CD22 site density were associated with lower likelihood of response to InO. CONCLUSION: InO is effective and well tolerated in heavily pretreated children and adolescents with R/R CD22-positive B-ALL. SOS after hematopoietic stem-cell transplantation and prolonged cytopenias were notable. CD22 modulation was identified as a mechanism of resistance. Expanded study of InO combined with chemotherapy is underway.

Blinatumomab Nonresponse and High-Disease Burden Are Associated With Inferior Outcomes After CD19-CAR for B-ALL.

PURPOSE: CD19-targeted chimeric antigen receptor T cells (CD19-CAR) and blinatumomab effectively induce remission in relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) but are also associated with CD19 antigen modulation. There are limited data regarding the impact of prior blinatumomab exposure on subsequent CD19-CAR outcomes. PATIENTS AND METHODS: We conducted a multicenter, retrospective review of children and young adults with relapsed or refractory ALL who received CD19-CAR between 2012 and 2019. Primary objectives addressed 6-month relapse-free survival (RFS) and event-free survival (EFS), stratified by blinatumomab use. Secondary objectives included comparison of longer-term survival outcomes, complete remission rates, CD19 modulation, and identification of factors associated with EFS. RESULTS: Of 420 patients (median age, 12.7 years; interquartile range, 7.1-17.5) treated with commercial tisagenlecleucel or one of three investigational CD19-CAR constructs, 77 (18.3%) received prior blinatumomab. Blinatumomab-exposed patients more frequently harbored KMT2A rearrangements and underwent a prior stem-cell transplant than blinatumomab-naïve patients. Among patients evaluable for CD19-CAR response (n = 412), blinatumomab nonresponders had lower complete remission rates to CD19-CAR (20 of 31, 64.5%) than blinatumomab responders (39 of 42, 92.9%) or blinatumomab-naive patients (317 of 339, 93.5%), P < .0001. Following CD19-CAR, blinatumomab nonresponders had worse 6-month EFS (27.3%; 95% CI, 13.6 to 43.0) compared with blinatumomab responders (66.9%; 95% CI, 50.6 to 78.9; P < .0001) or blinatumomab-naïve patients (72.6%; 95% CI, 67.5 to 77; P < .0001) and worse RFS. High-disease burden independently associated with inferior EFS. CD19-dim or partial expression (preinfusion) was more frequently seen in blinatumomab-exposed patients (13.3% v 6.5%; P = .06) and associated with lower EFS and RFS. CONCLUSION: With the largest series to date in pediatric CD19-CAR, and, to our knowledge, the first to study the impact of sequential CD19 targeting, we demonstrate that blinatumomab nonresponse and high-disease burden were independently associated with worse RFS and EFS, identifying important indicators of long-term outcomes following CD19-CAR.

Characterization of extramedullary disease in B-ALL and response to CAR T-cell therapy.

Chimeric antigen receptor (CAR) T cells effectively eradicate medullary B-cell acute lymphoblastic leukemia (B-ALL) and can traffic to and clear central nervous system (CNS) involvement. CAR T-cell activity in non-CNS extramedullary disease (EMD) has not been well characterized. We systematically evaluated CAR T-cell kinetics, associated toxicities, and efficacy in B-ALL non-CNS EMD. We conducted a retrospective review of B-ALL patients with non-CNS EMD who were screened for/enrolled on one of three CAR trials (CD19, CD22, and CD19/22) at our institution. Non-CNS EMD was identified according to histology or radiographic imaging at extramedullary sites excluding the cerebrospinal fluid and CNS parenchyma. Of ∼180 patients with relapsed/refractory B-ALL screened across multiple early-phase trials over an 8-year period, 38 (21.1%) presented with isolated non-CNS EMD (n = 5) or combined medullary/non-CNS EMD (n = 33) on 18-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) imaging. A subset receiving CAR T cells (18 infusions) obtained FDG PET/CT scans preinfusion and postinfusion to monitor response. At best response, 72.2% (13 of 18) of patients showed a medullary minimal residual disease-negative complete remission and complete (n = 7) or partial (n = 6) non-CNS EMD response. Non-CNS EMD responses to CAR T cells were delayed (n = 3), and residual non-CNS EMD was substantial; rarely, discrepant outcomes (marrow response without EMD response) were observed (n = 2). Unique CAR-associated toxicities at non-CNS EMD sites were seen in select patients. CAR T cells are active in B-ALL non-CNS EMD. Still, non-CNS EMD response to CAR T cells may be delayed and suboptimal, particularly with multifocal disease. Serial FDG PET/CT scans are necessary for identifying and monitoring non-CNS EMD.

Long term follow-up of a phase II study of cladribine with concurrent rituximab with hairy cell leukemia variant.

Hairy cell leukemia variant (HCLv) responds poorly to purine analogue monotherapy. Rituximab concurrent with cladribine (CDAR) improves response rates, but long-term outcomes are unknown. We report final results of a phase 2 study of CDAR for patients with HCLv. Twenty patients with 0 to 1 prior courses of cladribine and/or rituximab, including 8 who were previously untreated, received cladribine 0.15 mg/kg on days 1 to 5 with 8 weekly rituximab doses of 375 mg/m2 beginning day 1. Patients received a second rituximab course ≥6 months after cladribine, if and when minimal residual disease (MRD) was detected in blood. The complete remission (CR) rate from CDAR was 95% (95% confidence interval, 75-100). Sixteen (80%) of 20 patients (95% confidence interval, 56-94) became MRD negative according to bone marrow at 6 months. The median duration of MRD-negative CR was 70.1 months, and 7 of 16 are still MRD negative up to 120 months. With a median follow-up of 69.7 months, 11 patients received delayed rituximab, and the 5-year progression-free survival (PFS) and overall survival (OS) were 63.3% and 73.9%, respectively. Five patients with TP53 mutations had shorter PFS (median, 36.4 months vs unreached; P = .0024) and OS (median, 52.4 months vs unreached; P = .032). MRD-negative CR at 6 months was significantly associated with longer PFS (unreached vs 17.4 months; P < .0001) and OS (unreached vs 38.2 months; P < .0001). Lack of MRD in blood at 6 months was also predictive of longer PFS and OS (P < .0001). After progression following CDAR, median OS was 29.7 months. CDAR is effective in HCLv, with better outcomes in patients who achieve MRD-negative CR. This trial is registered at www.clinicaltrials.gov as #NCT00923013.

Carfilzomib, Lenalidomide, and Dexamethasone Followed by Lenalidomide Maintenance for Prevention of Symptomatic Multiple Myeloma in Patients With High-risk Smoldering Myeloma: A Phase 2 Nonrandomized Controlled Trial.

IMPORTANCE: High-risk smoldering myeloma has a 5-year risk of progression to symptomatic multiple myeloma of approximately 75%. Treatment with lenalidomide decreases the risk of progression; however, novel triplet regimens are superior, and earlier disease may be more treatment sensitive. OBJECTIVE: To evaluate the use of carfilzomib, lenalidomide, and dexamethasone (KRd) with lenalidomide maintenance therapy as early intervention in high-risk smoldering myeloma and to determine the rates of minimal residual disease (MRD)-negative complete response (CR). DESIGN, SETTING, AND PARTICIPANTS: In this single-arm, single-center, phase 2 nonrandomized controlled trial, responses were evaluated at every cycle during KRd treatment and every 3 cycles subsequently. Bone marrow biopsies and imaging were performed by cycle 8 and then annually. The study enrolled patients from May 29, 2012, to July 23, 2020, at the National Institutes of Health Clinical Center, a highly specialized tertiary cancer center. Patient key eligibility criteria included a diagnosis of high-risk smoldering myeloma based on the Mayo Clinic, Spanish, and/or Rajkumar, Mateos, and Landgren criteria. INTERVENTIONS: Patients received eight 4-week cycles of intravenous carfilzomib 36 mg/m2 (first 2 doses, 20 mg/m2), dexamethasone (20 mg, cycles 1-4; 10 mg, cycles 5-8 twice weekly), and lenalidomide 25 mg (days 1-21) followed by twenty-four 28-day cycles of maintenance lenalidomide 10 mg (days 1-21). Stem cell harvest and storage were optional. MAIN OUTCOMES AND MEASURES: The primary outcome was the MRD-negative CR rate. Key secondary outcomes included duration of MRD-negative CR and progression to multiple myeloma. RESULTS: A total of 54 patients (median age, 59 years [range, 40-79 years]; 30 men [55.6%]; and 2 Asian [3.7%], 15 Black [27.8%], 1 Hispanic [1.9%], and 36 White [66.7%] patients) were enrolled, with a median potential follow-up time of 31.9 months (range, 6.7-102.9 months). The MRD-negative CR rate was 70.4% (95% CI, 56.4%-82.0%), with a median sustained duration of 5.5 years (95% CI, 3.7 years to not estimable). The 8-year probability of being free from progression to multiple myeloma was 91.2% (95% CI, 67.4%-97.9%), and no deaths occurred. Nonhematologic grade 3 adverse events occurred in 21 patients (38.9%) and included thromboembolism, rash, and lung infection, with no grade 4 events. CONCLUSIONS AND RELEVANCE: Results of this phase 2 nonrandomized controlled trial suggest that treatment of high-risk smoldering myeloma with novel triplet regimens, such as KRd and lenalidomide maintenance therapy, may alter the natural history of smoldering myeloma by significantly delaying development of end-organ disease. Randomized clinical trials are needed to confirm this favorable benefit-to-risk profile. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01572480.

Characterization of HLH-like manifestations as a CRS variant in patients receiving CD22 CAR T cells.

Chimeric antigen receptor (CAR) T-cell toxicities resembling hemophagocytic lymphohistiocytosis (HLH) occur in a subset of patients with cytokine release syndrome (CRS). As a variant of conventional CRS, a comprehensive characterization of CAR T-cell-associated HLH (carHLH) and investigations into associated risk factors are lacking. In the context of 59 patients infused with CD22 CAR T cells where a substantial proportion developed carHLH, we comprehensively describe the manifestations and timing of carHLH as a CRS variant and explore factors associated with this clinical profile. Among 52 subjects with CRS, 21 (40.4%) developed carHLH. Clinical features of carHLH included hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, coagulopathy, hepatic transaminitis, hyperbilirubinemia, severe neutropenia, elevated lactate dehydrogenase, and occasionally hemophagocytosis. Development of carHLH was associated with preinfusion natural killer(NK) cell lymphopenia and higher bone marrow T-cell:NK cell ratio, which was further amplified with CAR T-cell expansion. Following CRS, more robust CAR T-cell and CD8 T-cell expansion in concert with pronounced NK cell lymphopenia amplified preinfusion differences in those with carHLH without evidence for defects in NK cell mediated cytotoxicity. CarHLH was further characterized by persistent elevation of HLH-associated inflammatory cytokines, which contrasted with declining levels in those without carHLH. In the setting of CAR T-cell mediated expansion, clinical manifestations and immunophenotypic profiling in those with carHLH overlap with features of secondary HLH, prompting consideration of an alternative framework for identification and management of this toxicity profile to optimize outcomes following CAR T-cell infusion.

Long-Term Follow-Up of CD19-CAR T-Cell Therapy in Children and Young Adults With B-ALL.

PURPOSE: CD19 chimeric antigen receptor (CD19-CAR) T cells induce high response rates in children and young adults (CAYAs) with B-cell acute lymphoblastic leukemia (B-ALL), but relapse rates are high. The role for allogeneic hematopoietic stem-cell transplant (alloHSCT) following CD19-CAR T-cell therapy to improve long-term outcomes in CAYAs has not been examined. METHODS: We conducted a phase I trial of autologous CD19.28ζ-CAR T cells in CAYAs with relapsed or refractory B-ALL. Response and long-term clinical outcomes were assessed in relation to disease and treatment variables. RESULTS: Fifty CAYAs with B-ALL were treated (median age, 13.5 years; range, 4.3-30.4). Thirty-one (62.0%) patients achieved a complete remission (CR), 28 (90.3%) of whom were minimal residual disease-negative by flow cytometry. Utilization of fludarabine/cyclophosphamide-based lymphodepletion was associated with improved CR rates (29/42, 69%) compared with non-fludarabine/cyclophosphamide-based lymphodepletion (2/8, 25%; P = .041). With median follow-up of 4.8 years, median overall survival was 10.5 months (95% CI, 6.3 to 29.2 months). Twenty-one of 28 (75.0%) patients achieving a minimal residual disease-negative CR proceeded to alloHSCT. For those proceeding to alloHSCT, median overall survival was 70.2 months (95% CI, 10.4 months to not estimable). The cumulative incidence of relapse after alloHSCT was 9.5% (95% CI, 1.5 to 26.8) at 24 months; 5-year EFS following alloHSCT was 61.9% (95% CI, 38.1 to 78.8). CONCLUSION: We provide the longest follow-up in CAYAs with B-ALL after CD19-CAR T-cell therapy reported to date and demonstrate that sequential therapy with CD19.28ζ-CAR T cells followed by alloHSCT can mediate durable disease control in a sizable fraction of CAYAs with relapsed or refractory B-ALL (ClinicalTrials.gov identifier: NCT01593696).