Peripheral Blood Flow Cytometry Research Articles

Low frequency of Vγ9Vδ2 T cells predicts poor survival in newly diagnosed acute myeloid leukemia

AbstractIn several tumor subtypes, an increased infiltration of Vγ9Vδ2 T cells has been shown to have the highest prognostic value compared with other immune subsets. In acute myeloid leukemia (AML), similar findings have been based solely on the inference of transcriptomic data and have not been assessed with respect to confounding factors. This study aimed at determining, by immunophenotypic analysis (flow or mass cytometry) of peripheral blood from patients with AML at diagnosis, the prognostic impact of Vγ9Vδ2 T-cell frequency. This was adjusted for potential confounders (age at diagnosis, disease status, European LeukemiaNet classification, leukocytosis, and allogeneic hematopoietic stem cell transplantation as a time-dependent covariate). The cohort was composed of 198 patients with newly diagnosed (ND) AML. By univariate analysis, patients with lower Vγ9Vδ2 T cells at diagnosis had significantly lower 5-year overall and relapse-free survivals. These results were confirmed in multivariate analysis (hazard ratio [HR], 1.55 [95% confidence interval (CI), 1.04-2.30]; P = .030 and HR, 1.64 [95% CI, 1.06-2.53]; P = .025). Immunophenotypic alterations observed in patients with lower Vγ9Vδ2 T cells included a loss of some cytotoxic Vγ9Vδ2 T-cell subsets and a decreased expression of butyrophilin 3A on the surface of blasts. Samples expanded regardless of their Vγ9Vδ2 T-cell levels and displayed similar effector functions in vitro. This study confirms the prognostic value of elevated Vγ9Vδ2 T cells among lymphocytes in patients with ND AML. These results provide a strong rationale to consider consolidation protocols aiming at enhancing Vγ9Vδ2 T-cell responses.

Inflammatory activity evaluation in patients with axial spondyloarthritis using MRI relaxometry and mucosal-associated invariant T cells.

Currently, there is a lack of an objective quantitative measure to comprehensively evaluate the inflammatory activity of axSpA, which poses certain challenges in accurately assessing the disease activity. To explore the value of combined-parameter models of sacroiliacjoints (SIJs) MRI relaxometry and peripheral blood Mucosal-associated invariant T (MAIT) cells in evaluating the inflammatory activity of axial spondyloarthritis (axSpA). This retrospective clinical study included 88 axSpA patients (median age 31.0 (22.0, 41.8) years, 21.6% females) and 20 controls (median age 28.0 (20.5, 49.5) years, 40.0% females). The axSpA group was classified into active subgroup (n=50) and inactive subgroup (n=38) based on ASDAS-CRP. All participants underwent SIJs MRI examination including T1 and T2* mapping, and peripheral blood flow cytometry analysis of MAIT cells (defined as CD3+Vα7.2+CD161+) and their activation markers (CD69). The T1 and T2* values, as were the percentages of MAIT cells and CD69+MAIT cells were compared between different groups. Combined-parameter models were established using logistic regression, and ROC curves were employed to evaluate the diagnostic efficacy. The T1 values of SIJs and %CD69+MAIT cells in the axSpA group and its subgroup were higher than the control group (p<0.05), while %MAIT cells were lower than the control group (p<0.05). The T1 values and %CD69+MAIT cells correlated positively, while %MAIT cells correlated negatively, with the ASDAS-CRP (r=0.555, 0.524, -0.357, p<0.001). Between the control and axSpA groups, and between the inactive and active subgroups, the combined-parameter model T1 mapping+%CD69+MAIT cells has the best efficacy (AUC=0.959, 0.879, sensibility=88.6, 70%, specificity=95.0, 94.7%, respectively). The combined-parameter model T1 mapping+%CD69+MAIT cells allows a more accurate evaluation of the level of inflammatory activity.

Prevalence and clinicopathological features of incidentally detected TRBC1-dim populations in peripheral blood flow cytometry

Prevalence and clinicopathological features of incidentally detected TRBC1-dim populations in peripheral blood flow cytometry

Prognostic relevance of circulating lymphoma cells at diagnosis in newly diagnosed follicular lymphoma patients.

Follicular lymphoma (FL) is the most common indolent B-cell non-Hodgkin lymphoma. Circulating lymphoma (CL) cells can be seen at diagnosis in some FL patients, however, previous studies evaluating this have shown mixed results. Therefore, we sought to evaluate the impact of CL at diagnosis on outcomes in patients with newly diagnosed FL using data from a single center. Patients were divided into CL+ and CL- based on immunophenotyping via peripheral blood (PB) flow cytometry. CL was defined as detectable clonally restricted B-cells that matched the actual or expected B-cell immunophenotype of FL. The primary endpoint was progression-free survival (PFS) after first-line treatment and secondary endpoints included overall response rate (ORR), overall survival (OS), diagnosis to treatment interval (DTI), progression of disease within 2years of diagnosis (POD24), and cumulative incidence of transformation between the two groups. Among the 541 patients with FL, 204 had PB flow cytometry performed at diagnosis, and after excluding patients not meeting the eligibility criteria, 147 cases remained with 24 (16%) CL+ at diagnosis. Patients in the CL+ group were younger (53 vs. 58years, p=0.02), had more extranodal involvement (83% vs. 44%, p<0.01), follicular lymphoma international prognostic index 3-5 (55% vs. 31%, p=0.01), and a higher proportion received first-line immunochemotherapy (75% vs. 43%, p=0.01) compared to the CL-group. The median PFS was not significantly different between CL+ (6.27years, 95% CI=3.61-NR) and CL- (6.61years, 95% CI=5.10-9.82) cohorts regardless of the first-line treatment or level of absolute PB CL cells. There was no significant difference in ORR, median OS, DTI, POD24, and cumulative incidence of transformation between the two groups. In our study, we found that the presence of CL cells at diagnosis in FL in the contemporary era did not impact outcomes and survival.

Optimizing Autologous Stem Cell Transplantation in Multiple Myeloma: The Impact of Intensive Chemomobilization

Most transplant-eligible multiple myeloma (MM) patients undergo autologous peripheral blood stem cell collection (PBSC) using G-CSF with on-demand plerixafor (G ± P). Chemomobilization (CM) can be used as a salvage regimen after G ± P failure or for debulking residual tumor burden ahead of autologous peripheral blood stem cell transplantation (ASCT). Prior studies utilizing cyclophosphamide-based CM have not shown long-term benefits. At our center, intensive CM (ICM) using a PACE- or HyperCVAD-based regimen has been used to mitigate "excessive" residual disease based on plasma cell (PC) burden or MM-related biomarkers. Given the lack of efficacy of non-ICM, we sought to determine the impact of ICM on event-free survival (EFS), defined as death, progressive disease, or unplanned treatment escalation. We performed a retrospective study of newly diagnosed MM patients who collected autologous PBSCs with the intent to proceed immediately to ASCT at our center between 7/2020 and 2/2023. Patients were excluded if they underwent a tandem autologous or sequential autologous-allogeneic transplant, had primary PC leukemia, received non-ICM treatment (i.e., cyclophosphamide and/or etoposide), or had previously failed G ± P mobilization. To appropriately evaluate the impact of ICM among those who potentially could have received it, we utilized a propensity score matching (PSM) approach whereby ICM patients were compared to a cohort of non-CM patients matched on pre-ASCT factors most strongly associated with the receipt of ICM. Of 451 patients identified, 61 (13.5%) received ICM (PACE-based, n = 45; hyper-CVAD-based, n = 16). Post-ICM/pre-ASCT, 11 patients (18%) required admission for neutropenic fever and/or infection. Among 51 evaluable patients, the overall response rate was 31%; however, 46 of 55 evaluable patients (84%) saw a reduction in M-spike and/or involved free light chains. Among those evaluated with longitudinal peripheral blood flow cytometry (n = 8), 5 patients (63%) cleared circulating blood PCs post-ICM. Compared to patients mobilized with non-CM, ICM patients collected a slightly greater median number of CD34+ cells (10.8 versus 10.2 × 10⁶/kg, P = .018). The median follow-up was 30.6 months post-ASCT. In a PSM multivariable analysis, ICM was associated with significantly improved EFS (hazard ratio [HR] 0.30, 95% CI 0.14 to 0.67, P = .003), but not improved OS (HR 0.38, 95% CI 0.10 to 1.44, P = .2). ICM was associated with longer post-ASCT inpatient duration (+4.1 days, 95% CI, 2.4 to 5.8, P < .001), more febrile days (+0.96 days, 95% CI 0.50 to 1.4, P < .001), impaired platelet engraftment (HR 0.23, 95% CI 0.06 to 0.87, P = .031), more bacteremia (OR 3.41, 95% CI 1.20 to 9.31, P = .018), and increased antibiotic usage (cefepime: +2.3 doses, 95% CI 0.39 to 4.1, P = .018; vancomycin: +1.0 doses, 95% CI 0.23 to 1.8, P = .012). ICM was independently associated with improved EFS in a matched analysis involving MM patients with excessive disease burden at pre-ASCT workup. This benefit came at the cost of longer inpatient duration, more febrile days, greater incidence of bacteremia, and increased antibiotic usage in the immediate post-ASCT setting. Our findings suggest that ICM could be considered for a subset of MM patients, but its use must be weighed carefully against additional toxicity.

Landscape and prognostic values of lymphocytes in patients with hepatocellular carcinoma undergoing transarterial embolization.

Transarterial embolization (TACE), the first-line treatment for hepatocellular carcinoma (HCC), does not always lead to promising outcomes in all patients. A better understanding of how the immune lymphocytes changes after TACE might be the key to improve the efficacy of TACE. However, there are few studies evaluating immune lymphocytes in TACE patients. Therefore, we aimed to evaluate the short- and long-term effects of TACE on lymphocyte subsets in patients with HCC to identify those that predict TACE prognosis. Peripheral blood samples were collected from 44 HCC patients at the following time points: one day before the initial TACE, three days after the initial TACE, and one month after the initial TACE and subjected to peripheral blood mononuclear cell isolation and flow cytometry. Dynamic changes in 75 lymphocyte subsets were recorded and their absolute counts were calculated. Tumor assessments were made every 4-6 weeks via computed tomography or magnetic resonance imaging. Our results revealed that almost all lymphocyte subsets fluctuated three days after TACE, but only Tfh and B cells decreased one month after TACE. Univariate and multivariate Cox regression showed that high levels of Th2 and conventional killer Vδ2 cells were associated with longer progressive-free survival (PFS) after TACE. Longer overall survival (OS) after TACE was associated with high levels of Th17 and viral infection-specific Vδ1 cells, and low levels of immature NK cells. In conclusion, TACE has a dynamic influence on the status of lymphocytes. Accordingly, several lymphocyte subsets can be used as prognostic markers for TACE.

Acalabrutinib, venetoclax, and obinutuzumab in relapsed/refractory CLL: final efficacy and ctDNA analysis of the CLL2-BAAG trial

AbstractThe phase 2 CLL2-BAAG trial tested the measurable residual disease (MRD)–guided triple combination of acalabrutinib, venetoclax, and obinutuzumab after optional bendamustine debulking in 45 patients with relapsed/refractory chronic lymphocytic leukemia (CLL). MRD was measured by flow cytometry (FCM; undetectable MRD <10–4) in peripheral blood (PB) and circulating tumor DNA (ctDNA) using digital droplet polymerase chain reaction of variable-diversity-joining (VDJ) rearrangements and CLL-related mutations in plasma. The median number of previous treatments was 1 (range, 1-4); 18 patients (40%) had received a Bruton tyrosine kinase inhibitor (BTKi) and/or venetoclax before inclusion, 14 of 44 (31.8%) had TP53 aberrations, and 34 (75.6%) had unmutated immunoglobulin heavy-chain variable region genes. With a median observation time of 36.3 months and all patients off-treatment for a median of 21.9 months, uMRD <10–4 in PB was achieved in 42 of the 45 patients (93.3%) at any time point, including 17 of 18 (94.4%) previously exposed to venetoclax/BTKi and 13 of 14 (92.9%) with TP53 aberrations. The estimated 3-year progression-free and overall survival rates were 85.0% and 93.8%, respectively. Overall, 585 paired FCM/ctDNA samples were analyzed and 18 MRD recurrences (5 with and 13 without clinical progression) occurred after the end of treatment. Twelve samples were first detected by ctDNA, 3 by FCM, and 3 synchronously. In conclusion, time-limited MRD-guided acalabrutinib, venetoclax, and obinutuzumab achieved deep remissions in almost all patients with relapsed/refractory CLL. The addition of ctDNA-based analyses to FCM MRD assessment seems to improve early detection of relapses. This trial was registered at www.clinicaltrials.gov as #NCT03787264.

Progressive multifocal leukoencephalopathy as the presenting feature in a patient with occult low-count monoclonal B-cell lymphocytosis.

Low Count Monoclonal B-Cell Lymphocytosis (LC-MBL) is a relatively poorly understood entity which has been suggested to be very common in asymptomatic adults and possibly related to infectious complications despite not progressing to CLL. We describe the first case of Progressive Multifocal Leukoencephalopathy (PML) presenting in a 72-year-old man with LC-MBL but no other immunocompromising conditions. A diagnosis of PML was confirmed with classic MRI findings in association with a high CSF John Cunningham polyomavirus (JCV) viral load (4.09' 105 copies/mL). An extensive search for underlying immunocompromising conditions only demonstrated LC-MBL representing approximately 4% of total leukocytes (0.2' 109/L). This is the first report of PML in association with LC-MBL. Careful review of peripheral blood flow cytometry results is necessary to identify this disorder. Further study of the epidemiology and infectious complications of LC-MBL are warranted.

Abstract 3619: Post CAR-T lymphocytosis as a surrogate of BCMA CAR-T expansion, response, and prognosis in multiple myeloma

Abstract Introduction: Expansion of chimeric receptor antibody (CAR)-T cells after infusion has been described as important marker in multiple myeloma (MM); however, the phenomenon of overt lymphocytosis has not been reported. We describe the kinetics of absolute lymphocyte count (ALC) after BCMA CAR-T in MM as a marker of depth of response and progression-free survival (PFS). Methods: 158 patients (pts) were included (93 pts ciltacabtagene autoleucel (cilta-cel) and 65 pts idecabtagene vicleucel (ide-cel) from three institutions. Baseline characteristics, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), ALC on day -5 through 14, and outcomes were collected. A cohort of 84 pts who received CD19 CAR-T for non-Hodgkin lymphoma (NHL) as comparator. Peripheral blood (PB) flow cytometry was performed on days 7, 14 and 21 in 4 patients. Results: Early ALC increase after BCMA CAR-T was frequent, with a median max ALC 1.3 × 103/uL (IQR 1-4.4) from a median baseline of 0. However, when compared to ide-cel, cilta-cel had a higher median Max ALC (2 × 103/uL (1 - 4.4) vs 0.8 (0.7 - 2.8), p &amp;lt; 0.001), longer time-to-max ALC (11 vs 12 days, p = 0.003), and higher absolute ALC increase (2.1 × 103/uL vs 0.7 × 103/uL, p &amp;lt;0.001), while there were no differences in ALC before lymphodepleting conditioning or day 0. Higher max ALC was associated with CRS (OR 4.8, CI 1.3-17.2, p 0.01). Max ALC and absolute increase in ALC were significantly associated with deeper response (VGPR vs PR or less), improved PFS and duration of response (DoR) in univariable analysis. Max ALC &amp;gt;1 × 103/uL (HR 0.4, 0.2-0.7, p &amp;lt;0.001) and non-paraskeletal EMD (HR 2.1, 1.2-.4, p &amp;lt;0.01) remained associated with PFS/DoR in multivariable model after adjusting for CAR-T Product, high-risk cytogenetics, and number of previous lines. PB flow cytometry in 3 responder patients (max ALC range 2-14 × 103) showed expansion of CD3+ BCMA CAR-T from day 7 (BCMA CAR-T &amp;lt;0.2% of CD3+ cells) to day 14 (BCMA CAR-T range 54-88.6%), while a non-responder (max ALC 0.4 × 103) patient had no expansion despite presence of CD3+ BCMA CAR-T at day 7, 14 and 21. BCMA CAR-T had higher max ALC when compared to CD19 CAR-T (median 1.3 × 103 vs 0.4 × 103 (0.2-0.7), p &amp;lt; 0.001), while there were no differences in baseline ALC between BCMA and CD19 CAR-T. Median max ALC did not differ between response status after CD19 CAR-T (0.4 × 103 (0.2-0.7) vs 0.6 (0.3-0.7), p = 0.3). Discussion: Post-CAR-T lymphocytosis is common after BCMA CAR-T therapy for MM and is associated with response, PFS, and DoR after adjusting for other prognostic variables. A cut-off of max ALC &amp;gt; 1 × 103 identified patients with superior PFS, while max ALC &amp;lt;0.5 × 103 represents high-rates of poor response and early progression. ALC expansion correlated with BCMA CAR-T expansion as assessed by flowcytometry, supporting ALC assessment as an accessible surrogate that is clinically applicable and accessible. Citation Format: Mateo Mejia Saldarriaga, Darren Pan, Caitlin Unkenholz, Tarek Mouhieddine, Josh Fein, Jorge Monge, Cara Rosenbaum, Roger Pearse, David Jayabalan, Christian Gordillo, Hei Ton Chan, Markus Mapara, Giorgio Inghirami, Suzanne Lentzsch, Ren Reshef, Adriana Rossi, Samir Parekh, Sundar Jagannath, Shambavi Richards, Ruben Niesvizky, Mark Bustoros. Post CAR-T lymphocytosis as a surrogate of BCMA CAR-T expansion, response, and prognosis in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3619.

Nodal Low-Grade B-Cell Lymphoma Co-Expressing CD5 and CD10 but Not CD23, IRTA1, or Cyclin D1: The Diagnostic Challenge of a Splenic Marginal Zone Lymphoma.

The diagnosis of lymphoma is based on histopathological and immunophenotypical features. CD5 and CD10 are traditionally considered a T-cell antigen and a germinal center B-cell antigen, respectively. It is very unusual for a low-grade B-cell lymphoma (BCL) to co-express CD5 and CD10. Although the biologic basis or clinical significance of such co-expression is unclear, this rare event may pose a significant diagnostic challenge. Here, we report a case of a 63-year-old male presenting with bilateral cervical lymphadenopathy and lymphocytosis. Histologically, the nodal tumor was largely diffuse with neoplastic small atypical lymphocytes co-expressing CD5, CD10, and CD20, but not CD23 or cyclin D1. The leukemic cells in the peripheral blood exhibited hairy projections. Taking together the marked splenomegaly, involvement of lymph nodes, bone marrow, and peripheral blood, a final diagnosis of splenic marginal zone lymphoma (SMZL) was reached. The patient was alive with partial response for 10 months after immunochemotherapy. The dual expression of CD5 and CD10 is extremely unusual for low-grade BCL and may lead to an erroneous diagnosis. Integrating the findings into peripheral blood smear tests, flow cytometry, histopathology, imaging, and clinical features is mandatory to exclude other lymphoma types and to reach a correct diagnosis, particularly for a case with nodal presentation.

A Rare Case of Hairy Cell Leukemia with COVID Infection: A Case Report

Abstract Hairy cell leukemia (HCL) is a distinct subset of chronic lymphoid leukemia, presents with unique features, including the characteristic "hairy" projections seen in neoplastic B cells. We reported a case on hairy cell leukemia (HCL), emphasizing the challenges posed by this rare hematological malignancy, especially in the context of concurrent health crises such as the COVID-19 pandemic. This malignancy, though uncommon, necessitates comprehensive diagnostic methodologies, such as peripheral blood studies, flow cytometry, and bone marrow biopsy. Treatment strategies for HCL involve tailored approaches based on symptomatic presentations. Purine analogs, cladribine and pentostatin, stand as first-line interventions, showcasing efficacy in inducing and sustaining remission. However, the prolonged immunosuppression resulting from these treatments warrants vigilant monitoring for potential infectious complications. Emerging therapies, such as the BRAF inhibitor vemurafenib, provide additional options for refractory or progressive cases. The post-treatment phase demands meticulous follow-up, with an emphasis on regular blood work assessments by an oncology nurse and primary care physician. Our case further highlights the need for heightened awareness among healthcare professionals, particularly in the post-COVID-19 phase, where persistent symptoms led to the discovery of an underlying hematological disorder. Keywords: Hairy cell leukemia, Evan syndrome, Covid-19.

Cytogenetic, Clinical, Hematologic, Demographic, Immunohistochemical, and Flow Cytometry Characteristics of Patients with Plasma Cell Neoplasm in Five Years: A First Report from Iran.

The aggregation of clonal plasma cells causes plasma cell neoplasms, which vary in severity and clinical outcomes. The present research focused on the epidemiological, clinical, immunologic, and cytogenetic characteristics of plasma cell neoplasms. In this five-year retrospective cross-sectional study, demographic information such as age and sex, calcium elevation, renal insufficiency, anemia, and bone lesion (CRAB) characteristics, as well as laboratory data including bone marrow and peripheral blood film results, immunohistochemistry, flow cytometry, and cytogenetic study outcomes were collected at Shiraz University of Medical Sciences, Shiraz, Iran. The collected data were analyzed using SPSS Statistics software (version 20.0). Descriptive statistics were reported as numbers, percentages, and mean±SD. 417 newly diagnosed plasma cell neoplasm patients were confirmed by bone marrow or other tissue biopsy tests. 279 patients were men (66.9%). The most prevalent age group was 60-64 years old (18.46%). Plasma cell myeloma (PCM) affected 355 (85.13%) patients, while monoclonal gammopathy of undetermined significance (MGUS) affected 6 (1.43%) patients. Solitary plasmacytoma was seen in 56 (13.42%) patients. At the time of diagnosis, 119 (33.52%) of 355 PCM patients were asymptomatic, whereas 236 (66.47%) patients had at least one CRAB symptom, 55 (15.49%) had two or more, and 14 (3.94%) had three or more. There were 7 (1.97%) cases of amyloidosis. Cytogenetic abnormalities were found in 51.28% (40/78) of the patients. Twenty-one individuals (52.5%) were hyperdiploid with multiple trisomy, while 19 (47.50%) were not. When diagnosed, Iranian PCM patients might have more advanced disease. PCM was more prevalent in young adults, and hyperdiploid was the most common cytogenetic finding in this investigation.

Flow Cytometry in Mature T- and NK-cell Neoplasms: A Retrospective Descriptive Study from a Tertiary Care Cancer Centre in Kerala, India

Introduction: Mature T- and NK-cell Neoplasms (MTNKN) constitute around 12% of all non-Hodgkin lymphomas. They have a variable clinical course, ranging from indolent to highly aggressive tumours. Flow cytometry immunophenotyping is crucial for the diagnosis, staging, and classification of MTNKN. Aim: To determine the frequency, morphologic, and immunophenotypic profile of various subtypes of MTNKN diagnosed by flow cytometry. Materials and Methods: This was a retrospective descriptive study conducted at the Department of Pathology, Regional Cancer Centre, Thiruvananthapuram, Kerala, India. All cases of MTNKN diagnosed by flow cytometry in peripheral blood, bone marrow aspirates, or body fluids from January 1, 2010 to June 30, 2020 (10.5 years duration) at the cancer centre were studied. The morphology of tumour cells and immunophenotype by flow cytometry were analysed. Clinical parameters (lymphadenopathy, hepatosplenomegaly, skin lesions, effusions, B symptoms) and follow-up details, including Progression-free Survival (PFS) and Overall Survival (OS), were also noted. Descriptive statistics for continuous variables and frequencies and percentages for categorical variables were obtained. The Kaplan-Meier method for survival plots was used. Results: Mature T- and NK-cell neoplasms constituted 83 cases. The median age of patients was 56 years. The majority of patients were males (n=49). Adult T-cell Leukaemia/Lymphoma (ATLL) (n=50) constituted the most common subtype, followed by Mycosis fungoides/Sezary syndrome (MF/SS) (n=14), T-cell Large Granular Lymphocytic Leukaemia (T-LGLL) (n=7), T-cell Prolymphocytic Leukaemia (T-PLL) (n=4), Aggressive NK-cell Leukaemia (ANKL) (n=3), Hepatosplenic T-cell Lymphoma (HSTL) (n=3), and Anaplastic Large Cell Lymphoma (ALCL) (n=2). OS and PFS at three years were 22.3% and 16.6%, respectively. Conclusion: Mature T- and NK-cell neoplasms presenting as leukaemia is rare. ATLL was the most common subtype of MTNKN. Flower cells, Sezary cells, and prolymphocytes are useful morphological clues for diagnosis. Immunophenotyping by flow cytometry, along with clinicopathologic correlation, is crucial for the diagnosis and subclassification of MTNKN. All subtypes except T-LGLL show inferior PFS and OS.

Updated Results of the Phase I BALLI-01 Trial of UCART22 Process 2 (P2), an Anti-CD22 Allogeneic CAR-T Cell Product Manufactured By Cellectis Biologics, in Patients with Relapsed or Refractory (R/R) CD22+ B-Cell Acute Lymphoblastic Leukemia (B-ALL)

Introduction: UCART22 is a genetically modified allogeneic T-cell product manufactured from healthy donor cells. Donor-derived T-cells are transduced using a lentiviral vector to express the anti-CD22 chimeric antigen receptor (CAR) and are further modified using Cellectis' TALEN ® technology to disrupt the T-cell receptor alpha constant ( TRAC) and CD52 genes to minimize risk of graft-vs-host disease (GvHD) and allow use of an anti-CD52 antibody for lymphodepletion (LD). Preliminary results from patients treated with UCART22 manufactured by a CMO (Process 1 (P1)) showed that UCART22 was well-tolerated and meaningful responses were achieved at the highest dose level (DL 3; 5 x 10 6 cells/kg). The fludarabine, cyclophosphamide, and alemtuzumab (FCA) LD regimen was also shown to extend host lymphocyte suppression and improve UCART22 expansion versus fludarabine and cyclophosphamide (FC) alone (Boissel N, et al. EHA 2023). We now report updated results from the BALLI-01 study that includes the first patients treated with UCART22 Process 2 (P2) manufactured by Cellectis Biologics. Methods: The primary endpoints are safety, tolerability, and determining the MTD/RP2D of UCART22. Additional endpoints are anti-leukemic activity and expansion of UCART22. Eligibility criteria include age 15‒70y, B-ALL blast CD22 expression ≥ 70%, and ≥ 2 prior treatment regimens. After FCA (F 30 mg/m 2 × 3d, C 0.5g/m 2 × 3d, A 20 mg/d × 3d) LD regimen, pts received a single infusion of UCART22-P2. In vitro comparability assays suggested that UCART22 P2 was more potent than UCART22 P1, so dose escalation with UCART22 P2 started at DL2 (1 x 10 6 cells/kg) compared to the highest studied dose DL3 (5 x 10 6 cells/kg) with UCART22 P1. Results: As of 01 July 2023, 3 pts were enrolled into the first UCART22 P2 cohort at DL2. Pt 1 is a 17yo female with B-ALL with a hypodiploid karyotype and a germline TP53 mutation whose disease had previously failed to respond to multiagent chemotherapy, blinatumomab (blina), inotuzumab (ino), venetoclax (ven), allogeneic hematopoietic stem cell transplantation (HSCT), and autologous CD19 CAR T-cell therapy (CAR19) x2. Pt 2 is a 68yo female with Ph-negative B-ALL who relapsed with CD19-low disease after multiagent chemotherapy, ino, and blina. Pt 3 is a 27yo male with B-ALL with an ABL2 fusion who had failed multiagent chemotherapy, ino, blina, TKIs, and an experimental autologous CAR19. UCART22 P2 administered after the FCA LD regimen was well tolerated. No DLTs or ICANS were observed. Cytokine release syndrome (CRS) occurred in 2/3 (67%) pts, with one G1 that resolved without treatment and one G2 that resolved after tocilizumab x1. There was one G5 sepsis SAE at D40 considered related to UCART22 P2 and FCA LD in Pt 1. Responses were assessed beginning on D28. Up to FC/FCA-DL3 with 18 pts treated with UCART22 P1, 1 CR, 4 CRi, and 2 morphologic leukemia-free states (MLFS) were observed, with 3 responses occurring out of 6 (50%) patients treated with FCA-DL3 (previously reported, EHA 2023). For UCART22 P2, FCA-DL2, 2/3 pts (67%) responded: Pt 2 achieved an MRD neg CR lasting over 84 days after UCART22 infusion; Pt 1 achieved an MRD negative MLFS up to D40. Pt 3 was refractory to treatment, however this pt received bridging therapy with dasatinib for his ABL2 fusion, and on Day -1, only 47% of the leukemic cells expressed CD22 (down from 88% at screening). UCART22 P2 expansion was observed by flow cytometry in peripheral blood with peak of 79 cells/μl in Pt 1 and 225 cells/μl in Pt 2, both at D11, with predominantly CD8 cells expanding. Inflammatory markers such as ferritin, IFN-γ, TNF-α and IL-6 levels increased more than 3-fold, correlating with UCART22 P2 expansion and CRS. Summary : In vitro comparability studies indicated that UCART22 P2 was more potent than UCART22 P1, and this was suggested clinically, as there was a 67% response rate at DL2 with UCART22 P2 compared to 50% at DL3 with UCART22 P1. Use of UCART22 P2 did not lead to any grade ≥3 CRS, and no DLTs or ICANS was observed. UCART22 P2 expansion was seen in the two responders, which closely correlated with CRS and changes in inflammatory markers. These data support the safety and preliminary efficacy of UCART22 P2 in this poor-risk R/R B-ALL population. The study continues to enroll pts treated with UCART22 P2 at dose level 2i (2.5 x 10 6 cells/kg), and updated data will be presented.

A Multicenter Study Analyzing Survival and Prognostic Factors in Patients with Leukemic Phase Follicular Lymphoma

Introduction Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma. Leukemic-phase FL (LP-FL) is a rare entity comprising 1-3% of FL patients (pts). Controversy exists about prognosis of LP-FL with some studies suggesting worse outcome. No clear cut-point of circulating cells has been used to define LP-FL; however, a level &amp;gt; 5x103^3 cells/uL is defined in GELF criteria for high burden disease. No unified treatment recommendation is present for these pts with some opting to treat as indolent FL and some choosing a more aggressive approach. Furthermore, limited case series (n=7 to 37) on LP-FL are available, hence we aimed to better understand prognosis and treatment strategies associated with better outcome in LP-FL. Methods Pts with a pathology proven diagnosis of FL in lymph node and/or bone marrow and a positive flow cytometry in peripheral blood for clonal B cells (CBs), at diagnosis or at relapse, were included in the study. Data was collected from 5 academic institution in the US. We report time-to-event outcomes including median progression-free survival (mPFS) and overall survival (mOS) estimated by Kaplan-Meier method (only pts who received therapy included) . Uni and multivariable analyses were performed using Cox regression. CBs were divided into 4 groups: (&amp;lt;1, 1-4.99, 5-9.99, &amp;gt;10) 10^3/uL. Pts treatment was grouped in 4 categories: anti-CD20 monoclonal antibody (CD20mAb), CD20mAb+Bendamustine, CD20mAb+ CHOP, and other. Pts with CD10 negative clones on flow cytometry were excluded as they might represent different lymphoma type. Results Sixty-one pts (period 1998 to 2022) were identified in participating institutions with LP-FL, 10 of which had CD10- flow cytometry and excluded from study. Of 51 remaining pts, median age was 56 years (y). Nine (17.6%) pts were Hispanic and 44 (86.2%) pts were white. Most pts (37%) presented asymptomatic lymphadenopathy, 22% with B symptoms, 14% with incidental elevated WBC, 12% with organ compromise and 12% due to other reasons. Overall, B symptoms were present in 20 (39%) pts. Hepato/splenomegaly was present in 22 (43%) pts, high tumor burden (GELF criteria) in 12 (23%) pts, and most pts (n=42; 82%) presented ECOG PS 0-1. We less frequently observed anemia (Hg &amp;lt;12g/dL, n= 10; 20%) and elevated LDH (n=17; 33%). High risk FLIPI score (≥3) was present in 28 (55%) pts. Median count of CBs of 1.62 x 10^3/uL. CBs were &amp;lt;5 10^3/uL in 35 (69%) pts, CBs=5-10 10^3/uL in 6 (12%) pts and CBs &amp;gt;10 10^3/uL in 10 (20%) pts. Median absolute lymphocyte count was 3.0 x10^3/uL. Cytogenetic data was available in tissue biopsies in 13 (25%) pts and all carried t(14;18). Treatment was as follows: 24 (47%) pts with bendamustine + CD20mAb , 15 (29%) pts with CD20mAb + CHOP, 7 (14%) pts were treated with CD20mAb, 3 (6%) pts underwent active surveillance, and 2 (4%) with other treatments. Overall response rate (ORR) was 87%. Of those 31 (65%) pts had complete response (CR) and 11 (23%) pts had partial response (PR). With a median follow up of 5.5 y the mPFS was 3.6 y and mOS was 9.6 y (Figure 1). Relapse/ progression of disease occurred in 26 (54%) pts and CBc was detect in 12/13 pts in which flow cytometry was done. Transformation on relapse occurred in 7 pts. In pts experiencing disease relapse/progression, CAR T-cell was given to 6 pts with an ORR of 83.3% (CR: in 4 and PR in 1 pts); one pt died due to CAR-T grade 5 neurotoxicity. Death occurred in 13 pts with 6 (46% of all deaths) being lymphoma-related. Age≥60 and male sex were associated with worse PFS in univariable analysis (Table 1), with age only remaining significant in a multivariable analysis that also included sex and frontline therapy (HR 3.19, p=0.01 ). Notably, FLIPI score, CBs count category, and type of frontline therapy did not predict PFS. Progression of disease within 24 months (POD24) predicted OS in univariable analysis (HR= 3.93; p=0.029). Multivariable analysis for OS was not performed due to small number of deaths. Conclusion To our knowledge, this is the largest study of LP-FL. Median OS was around 10 years, a comparable outcome to historical nodal FL survival. Treatment type also did not predict PFS or OS. Circulating monoclonal cell levels did not impact survival . This is notable as the current GELF criteria includes circulating malignant cells ≥5 x10^3/uL as an indication for treatment while our date shows that no set cut-point may be needed. In addition, CAR T-cell appears to be an effective therapy with similar results as in nodal FL.

Post CAR-T Peripheral Lymphocytosis and Its Kinetics Are a Potential Biomarker for Response and Immune Mediated Toxicities

Introduction Although expansion of chimeric receptor antibody (CAR)-T cells after infusion has previously been described, overt lymphocytosis with atypical morphology has not been widely reported in multiple myeloma (MM). Here we describe the kinetics of absolute lymphocyte count (ALC), its relationship with immune mediated toxicities, and clinical outcomes. Methods Thirty-nine patients (pts) receiving BCMA CAR-T (25 pts ciltacabtagene autoleucel (cilta-cel) and 14 pts idecabtagene vicleucel (ide-cel)) from two institutions were retrospectively reviewed. Data on baseline characteristics, presence of extramedullary disease (EMD), conditioning regimen, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), severity of CRS/ICANS (according to American Society of Transplantation and Cellular Therapy (ASTCT) classification), ALC on day -5 through 14, time to best response (TBR), time to first response (TFR), depth of response, and outcomes were collected. Response was evaluated in pts with &amp;gt;3 months of follow up. Peripheral blood flow cytometry was performed in 3 cases at time of highest ALC. ALC data for 84 pts who received CD19 CAR-T (77 pts with axicabtagene ciloleucel and 7 pts with brexucabtagene autoleucel) for non-Hodgkin lymphoma (NHL) were included as a comparison. Maximum ALC (max-ALC) was defined as the highest ALC between day 0 and +15, baseline ALC was defined as the lowest ALC during day 0 through +3, and absolute ALC increase was defined as the difference of max-ALC and baseline ALC. Results Baseline characteristics of the cohort are described in Table A. Pts who received cilta-cel had earlier onset of CRS (median day +6 (IQR 2 - 8) vs +2 (IQR 1 - 2), p = 0.02), had higher max-ALC after CRS (median 1.7 cel/uL (IQR 0.8 - 5.3) vs 0.81 cel/uL (IQR 0.6 - 1.1), p = 0.02), and had a trend towards later max-ALC (median day +12 (IQR 11 - 13) vs +10 (IQR 9 - 13), p = 0.1). Baseline ALC did not differ by BCMA CAR-T product or number of previous lines of therapy. When compared to the NHL cohort, patients who received BCMA CAR-T had higher max-ALC (1.01 cel/uL (IQR 0.6 - 1.9) vs 0.4 cel/uL (IQR 0.2 - 0.7), p &amp;lt; 0.001) and this max-ALC occurred later (median day max-ALC +12 (IQR 10 - 13) vs day +10 (8 - 10), p &amp;lt; 0.01) Figure C. Higher max-ALC and absolute ALC increase were associated with depth of response and occurrence of CRS/ICANS. Pts who had a deeper response (VGPR/CR) had a higher median max-ALC (1.2 cel/uL (IQR 0.77 - 1.81) vs 0.5 cel/uL (IQR 0.2 - 0.7) p &amp;lt; 0.01) and absolute ALC increase (1.2 cel/uL (IQR 0.7 - 1.8) vs 0.6 cel/uL (IQR 0.2 - 0.7) vs, p = 0.01) compared to those with PR or worse Figure B. Similarly, pts who did not progress on follow-up had higher median max-ALC (0.58 cel/uL (IQR 0.4 - 0.8) vs 1.3 cel/uL (IQR 0.9 - 1.9), p &amp;lt; 0.001) and absolute ALC increase (1.3 cel/uL (0.9 - 1.8) vs 0.6 cel/uL (0.5 - 0.8), p &amp;lt; 0.01) compared to progressors. Pts who experienced CRS had a higher absolute ALC increase i(1.05 cel/uL (0.7 - 2.7) vs 0.27 cel/uL (0.2 - 0.5), p = 0.02), and pts who had ICANS had higher max ALC (3.2 cel/uL (0.8 - 6.4) vs 1.01 cel/uL (0.6 - 1.8), p = 0.2). These results were consistent across BCMA CAR-T product, and there were no differences in baseline ALC across CAR-T product, response, disease progression, or number of previous lines. Furthermore, higher max-ALC was associated with higher odds of having VGPR/CR (OR 32.3 (2.23 - 240), p = 0.04) in univariable logistic regression. Flow cytometry data on 3 pts showed expansion of CD8+ T Cells during peak ALC with increase of CD8:CD4 ratio. Multicolor flow cytometry using an antibody BCMA at time of peak ALC showed expansion of CD4+ and CD8+ CAR-T, and further immunophenotypic characterization (differentiation, maturation, exhaustion) is currently being performed. Conclusions Post-CAR-T lymphocytosis was a common event after BCMA CAR-T therapy for MM, which presented a higher max-ALC, and at a later day when compared to CD19 CAR-T for NHL. Furthermore, ALC dynamics differed by BCMA CAR-T product, with cilta-cel exhibiting later CRS with higher max-ALC peaking at a later day when compared to ide-cel. Furthermore, higher max-ALC and absolute ALC increase were associated with deeper response (VGPR/CR) and sustainable response on follow up. Our findings suggest that peripheral lymphocytosis after CAR-T is disease specific (NHL vs MM) and post CAR-T lymphocytosis in peripheral blood is a potential biomarker for clinical outcomes and toxicity.

Long-Term Outcomes in Chronic Lymphocytic Leukemia Treated with Ibrutinib: 10-Year Follow-up of a Phase 2 Study

Background: Targeted agents have replaced conventional chemotherapy in treatment of chronic lymphocytic leukemia (CLL) with substantially improved outcomes in CLL with TP53 alterations (del17p or TP53 mutations). Whereas fludarabine, cyclophosphamide, and rituximab chemoimmunotherapy (CIT) had a median progression-free survival (mPFS) of 11.2 months and median overall survival (mOS) of 33.1 months as frontline therapy in del17p CLL (Fisher et al. Blood 2016), we previously reported a 6-year PFS of 61% and OS of 79% in CLL with TP53 alterations receiving frontline continuous ibrutinib (Ahn et al. NEJM 2020). In a similar patient subset, the CLL14 study demonstrated a mPFS of 51.9 months and a 5-year OS of 60.0% using fixed-duration venetoclax and obinutuzumab (Al-Sawaf et al. EHA 2023; Al-Sawaf et al. Nat. Comm 2023). Although more effective than CIT, long-term outcomes in the era of targeted therapy are not yet fully defined. With a median follow-up of nearly 10 years, we analyzed efficacy, safety, and depth of response in CLL patients treated with long-term ibrutinib therapy in a phase 2 study (NCT01500733). Methods: Eligible patients had high-risk CLL, defined by TP53 alterations or age ≥65 years-old (N = 84 evaluable), and received ibrutinib 420 mg orally once daily until disease progression or intolerable side effects. Response assessments were performed annually with CT scans up to five years and subsequently with physical exam. Minimal residual disease (MRD) was performed annually with peripheral blood flow cytometry. Undetectable MRD (uMRD) was defined as &amp;lt;1 CLL cell per 10,000 leukocytes. For patients discontinuing the study, long-term outcomes were obtained when possible through a separate natural history study (NCT00923507). Results: As of December 31, 2022, a median follow-up of 113 months and 117 months (reverse Kaplan-Meier) was available for calculation of PFS and OS, respectively. The mPFS for all patients was 85.9 months (Table 1). mPFS was significantly shorter in those with TP53 alterations (67.3 months, N = 53) vs. those without (not reached, N = 31) and in relapsed disease (49 months, N = 32) vs. frontline treatment (108 months, N = 52). The OS at 117 months was 58.5% for all patients, 73.8% for treatment-naïve patients, and 54.1% in patients with TP53 alterations. Among patients with TP53 alterations receiving frontline ibrutinib (N = 34), the mPFS was 81 months with an OS of 69.7% at 117 months. Most patients (82.1%, 69/84) had discontinued ibrutinib at the time of analysis with 15 patients (17.9%) remaining on therapy. 37 (44.0%) patients discontinued ibrutinib for progressive disease including 6 cases with histologic transformation. 27 (32.1%) patients discontinued therapy related to treatment-emergent adverse events (AEs) including 8 cardiac AEs (four cases were grade ≥2 atrial fibrillation), 7 second cancers, 4 infections, 3 cases of dementia (ages 78, 80, and 85), 2 pleural effusions, and 3 other AEs. Five patients (5.9%) withdrew from the study with 4 switching to commercial supply of ibrutinib and one changing to CAR-T therapy. uMRD was observed in 15.4% (13/84) of all patients. The median time to uMRD was 5 years (range: 2 - 10 years). There was no enrichment in these patients for Rai stage ≥3-4, unmutated IGHV, TP53 alteration, prior treatment, or age. Most patients achieving uMRD also had a complete response (CR) to therapy (10/13) and remained progression-free at last follow-up (12/13). The one patient with progressive disease achieved uMRD and CR at 24 months, had detectable MRD at 36 months, and had nodal progression at 60 months. Among the progression-free patients, 8 had subsequent MRD assessments demonstrating sustained uMRD for 1-6 years including one patient who had been off any CLL-directed therapy for 4 years. Conclusions: These results further define the long-term prognosis of patients treated with BTK inhibitors for CLL, most notably with substantial improvements in the OS of patients with TP53 alterations over CIT. Additionally, these results may inform discussions of continuous BTK inhibitor therapy versus fixed-duration venetoclax combinations in frontline therapy of high-risk CLL. In a subset of ibrutinib-treated patients, depth of response increased over multiple years to the point of uMRD suggesting the possibility that such patients might safely discontinue therapy, following paradigms established in patients with chronic myelogenous leukemia.

Modulation of the Btla-HVEM Axis to Enhance CAR T Cell Immunotherapy Against Cancer

Introduction: The efficacy of adoptive T cell immunotherapies against cancer, such as chimeric antigen receptor (CAR) T cells, is severely blunted by the immunosuppressive tumor microenvironment (TME). We sought to investigate the role of the TME in cancer resistance to effector T cells in order to define actionable targets to enhance CAR T cell immunotherapies. We initially used Hodgkin lymphoma (HL) as an ideal tumor model since it is characterized by a TME that is profoundly infiltrated by immunosuppressive cells, and then we expanded our findings to multiple cancer models. We first sought to identify the dominant interactions of immunosuppressive cellular compartments and effector T cells by analyzing single-cell RNA sequencing data on a total of 26 (4 exploratory + 22 validation) HL patient tumor biopsies. Using the CellPhoneDB algorithm, we inferred that the ligand B- and T-lymphocyte attenuator (BTLA) on effector T cells and the receptor Herpesvirus entry mediator (HVEM, TNFRSF14) on immunosuppressive cells (e.g., regulatory T cells, monocytes) strongly interact in the TME and promote T cell dysfunction ( Fig 1a). Akin to the canonical checkpoint PD1, BTLA recruits two potent tyrosine phosphatases, SHP-1 and SHP-2, to disable early T cell activation. Thus, we rationalized that BTLA expression on T cells might reduce their anti-tumor function. We hypothesized that deleting BTLA in CAR T cells would abolish BTLA-HVEM trans interactions at the immunological synapse and unleash the cytotoxic potential of CAR T cells. Methods and Results: We first generated BTLA KO anti-CD30 CAR T (CART30) cells against HL. To test their function in vivo, 15x10 6 HDLM-2 (CD30+HVEM+ HL) cells were subcutaneously implanted into NSG mice on day 0, and on day 62, 3x10 5 CAR30+ T cells were infused intravenously. Our results demonstrated that BTLA KO significantly enhances the function of anti-CD30 CAR T cells in HVEM+ HL ( Fig 1b), as assessed via tumor size (caliper) and CART30 expansion in the peripheral blood (flow cytometry). Additionally, we generated BTLA KO 4-1BBζ CART19 cells, which showed greater in vivo anti-tumor function in a subcutaneous tumor model of DLBCL (CD19+ HVEM+ OCI-Ly18). Serum collected from OCI-Ly18-bearing NSG mice infused with BTLA KO CART19 was enriched in effector cytokines (e.g., TNF, IFNγ, IL-2) as measured by Luminex. We then extended these findings into HVEM+ solid tumor models. BTLA KO improved tumor control in vitro in short-term killing experiments (Incucyte SX5) for both CAR and TCR T cells, respectively directed against prostate cancer (HER2+ PC-3) and melanoma (GP-100+ DM-6). Importantly, we demonstrated that BTLA KO in primary BALB/c-derived murine CAR T cells enhances tumor control in A20 murine lymphoma. In this model, BTLA KO tumor-infiltrating CAR T cells showed substantially reduced exhaustion in the TME relative to wild-type CAR T cells. Mechanistically, we showed that targeted mutations in BTLA intracellular tyrosine motifs effectively reduce the recruitment of SHP-1/2 and preserve the docking domain of the pro-stimulatory Grb2, thus maintaining high NFAT signaling and increased persistence in vivo. These effects were dependent on the expression of HVEM on tumor cells. Finally, we found that high BTLA RNA expression in tisagenlecleucel infusion products correlates with poor response to treatment in patients with DLBCL and FL (NCT02030834). Conclusion: Our results reveal a critical role of the BTLA-HVEM axis in inhibiting CAR T cell function, and demonstrate that CRISPR-Cas9 deletion of BTLA leads to enhanced anti-tumor efficacy in multiple models of cancer. The key mechanism is the reduction of SHP-1/2 recruitment and the consequent increase in CAR T cell activation. The results of this study will be translated into a first in human clinical trial of BTLA-deficient CAR T cells for relapsed or refractory cancer.

A Prognostic Model Integrating Circulating Clonal Plasma Cells into R-ISS and PET-CT Based Tumor Burden in Transplant-Ineligible Multiple Myeloma

Introduction: Circulating clonal plasma cell (cCPC) is an easily obtained and non-invasive biomarker in multiple myeloma (MM), providing distinct prognostic information from the revised-international staging system (R-ISS). However, its prognostic relevance has been investigated primarily in transplant-eligible and clinical trial settings. Consequently, there is limited data on the impact of cCPC on outcomes and optimal cut-off values in transplant-ineligible (TIE) MM patients. Moreover, appropriate combinations to refine risk stratification, such as tumor burden measured by positron emission tomography-computed tomography (PET-CT), have yet to be fully explored. Methods: We performed a retrospective analysis on newly diagnosed TIE-MM patients with available peripheral blood (PB) flow cytometry (FCM) and PET-CT data from 2011 to 2021. Patients with primary plasma cell leukemia (n=8), defined as &amp;gt;5% monoclonal plasma cells in PB, were excluded from the study. Quantification of cCPC was performed using 6-color (2011-2016) or 8-color (2017-2021) FCM staining for CD19, CD56, CD38, CD138, cytoplasmic κ, and cytoplasmic λ to confirm monoclonality (Beckman Coulter, USA), which required at least 150000 mononuclear cell counts with the detectable limit at 0.01% (10 -4) based on previous reports (Gonsalves et al). Tumor burden was assessed by calculating total lesion glycolysis (TLG) using the following formula: the mean standardized uptake value (SUV) multiplied by the total tumor volume with an absolute SUV &amp;gt;2.5, using Metavol Software (Hokkaido University, Japan). Receiver operating curve analysis with the Youden index was employed to determine the optimal cutoff for predicting 2-year progression-free survival (PFS) and 5-year overall survival (OS). The predictive performance of R-ISS, cCPC, and TLG alone, along with their combinations, was compared using Harrell's concordance index (c-index). Results: 114 patients with TIE-MM were analyzed. The median age was 78 years (interquartile range [IQR] 73-83). The number of patients classified as R-ISS Ⅰ, Ⅱ, and Ⅲ was 12 (10.5%), 82 (71.9%), and 20 (17.5%), respectively. Most patients (85.1%) received proteasome inhibitor-based induction remission therapy. cCPCs were detectable in 90 patients (80.7%), with a median level of 6 ×10 -4 (IQR 1-22). Regarding tumor burden, 73 patients (64%) had at least one fluorodeoxyglucose avid lesion with a median TLG of 34.7 (IQR 0-268). cCPC levels were significantly correlated with CPC in bone marrow (R =0.24, P =0.01) but not with TLG (R =-0.01, P =0.88) and involved free light chain levels (R =0.07, P =0.41). The optimal cut-off values in predicting 2-year PFS and 5-year OS were 9 and 10 ×10 -4 for cCPC, and 325 and 343 for TLG, respectively. For simplicity, we classified &amp;gt;10 ×10 -4 for cCPC and &amp;gt;300 for TLG as high-risks. Notably, approximately two-thirds of the patients (62.3%) had at least one high-risk feature (Figure 1). Both high cCPC and TLG showed significant associations with worse OS and PFS (5-year OS rate: cCPC high vs. low, 40.2% vs. 69.3%; TLG high vs. low, 38% vs. 64.5%; 2-year PFS rate: cCPC high vs. low, 45.7% vs. 69.1%; TLG high vs. low, 46.4% vs. 64% all P &amp;lt; 0.05). These factors retained prognostic significance after adjustment for R-ISS on multivariate analysis. Finally, we constructed a novel predictive model integrating RISS, cCPC, and TLG. This model discriminated OS among patients with 0 (5-year OS rate, 80% [95% confidence interval 68.4%-93.6%]), 1 (50.5% [37.7%-67.7%]), and ≧2 factors (29% [13.3%-63.5%], P &amp;lt;0.001, Figure 2). The c-index was the highest (0.651) in this combined model compared to the single use of these risk factors (0.585, 0.594, and 0.569). Conclusions: We demonstrated the prognostic impact of cCPC, which is independently associated with R-ISS and TLG, on real-world TIE-MM patients. The new prediction model, integrated with these different clinical and biological features, may provide robust outcome discrimination.

Comparison of Venetoclax Based Treatments for Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia

Background: Venetoclax (Ven) is a BCL-2 inhibitor that produces deep responses with high rates of undetectable minimal residual disease (uMRD) for patients (pts) with chronic lymphocytic leukemia (CLL) (Al-Sawaf et al. 2020). Currently, ven is FDA approved as either monotherapy (mono) or in combination (combo) with rituximab (R) In patients with relapsed/refractory (R/R) CLL (Seymour et al. 2018). However, the optimal ven based treatment (tx) for pts with R/R CLL either as mono, or in combo with anti-CD20 monoclonal antibody (mab), or bruton tyrosine kinase inhibitor (BTKi) is uncertain. We performed a single institution retrospective analysis of pts with R/R CLL treated with ven based regimens evaluating efficacy of different ven based txs. Methods: 98 pts with R/R CLL treated between the years of 2012 and 2023 were evaluated. Ven based treatment consisted of ven mono or in combo with R, obinutuzumab (G), or ibrutinib (BTKi) with standard ven dose ramp up to a maximum dose of 400mg daily. R and G were administered per FDA standard dosing. Ibrutinib was administered as 420mg daily, as either fixed or continuous duration tx. Response assessment was performed using iwCLL criteria. Minimal residual disease (MRD) was assessed by multi-parametric peripheral blood flow cytometry with a sensitivity of 10 -4. Time-to-event analyses were performed with the Kaplan-Meier method, with log-rank test used to assess statistically significant differences between variables. Hazard ratios (HR) were calculated by use of Cox proportional modelling. Results: The pts had a median age of 68 years (range; 46-89). Rai stage at time of tx initiation with ven: I 14.2%; II 36.3%; III 8.8%; Stage IV 40.7%. The median number of previous txs was 4 (range; 1-14). Pts harbored the following cytogenetic abnormalities: del(13q) 50.5%; del(11q) 18.5%; trisomy 12 15.5%; normal 15.5%; del(17p)/TP53 mutation 24.7%; and complex in 33%. Seventy percent of pts harbored an unmutated immunoglobulin heavy chain. 25.5% of pts had genomic complexity, with ≥ 5 gene mutations. 43.9% of pts were treated with ven mono; 19.4% were treated with venR; 25.5% of pts were treated with venG; and 11.2% of pts were treated with ven + BTKi. The median number of months of ven based tx was 16.3 (range; 0.9-118.6). 14.3% of pts currently remain on ven. 60.8% of pts had progression after ven based tx; with 62.2% requiring additional tx. 15.3% of pts developed Richter's Transformation. The best overall response for the cohort was: CR 67%; PR 24.5%; SD 1.1%; and PD 7.4%. The best MRD response for the whole cohort was uMRD 76.8%; low MRD 19.5; and high MRD 3.6%. uMRD as best MRD response occurred in 66.7% of ven mono; 82.4% of venR; 86.4% of venG; and 70% of ven + BTKi treated pts. With a median follow-up of 52 months (mo), the median progression-free survival (PFS) for the whole cohort was 67.8 mo. Ven combo had superior PFS to ven mono; median PFS 76.6 vs 58.9 mo (p = 0.04; HR 0.5037; 95% CI 0.2583 - 0.9635). Among the combination treatment approaches, there was a trend for improved PFS for venG compared to ven mono; median PFS not reached (NR) vs 58.9 mo (p = 0.08; HR 0.4385; 95% CI 0.1608 - 1.0240). Similarly, there was also a trend to improved PFS for ven + BTKi compared to ven mono; median PFS NR vs 58.9 mo (p = 0.07; HR 0.2895; 95% CI 0.0462 - 0.9950). Both the presence of del17p/TP53 mutation; and the presence of MRD (+MRD) 12 months after stopping ven based tx were associated with inferior PFS. The median PFS for pts without del17p/TP53 mutation vs with del17p/TP53 mutation was 118 vs 39.7 mo (p &amp;lt; 0.0001; HR 0.2242; 95% CI 0.0533 - 0.8366). Median PFS for pts with uMRD vs +MRD 12 months after ven was NR vs 42.8 mo (p = 0.0001; HR 0.0689; 95% CI 0.0037 - 0.3810). Conclusions : In a high-risk population of pts with R/R CLL, we found that ven based tx leads to durable remissions, with high rates of uMRD. Combo approaches with the addition of either an anti-CD20mab or BTKi led to improved PFS. While venR is the current FDA approved combo regimen for pts with R/R CLL, we found that there was a trend for improved PFS with either venG or ven + BTKi compared to ven mono. Both the presence of a del17p/TP53 mutation and the presence of MRD 12 months after stopping ven based tx significantly adversely affected PFS. However, pts who maintained uMRD 12 months after completing ven, had exceptionally durable long-term remission. This has possible implications for employing MRD adapted txs for pts with CLL.