Witzig, TE

Abstract Introduction: Large B-cell lymphoma (LBCL) harboring chromosomal rearrangements (double-hit, DH) or coexpression of MYC and BCL2 (double-expressor, DE) represents biologically aggressive subgroups with inferior clinical outcomes. Oncogene-driven DNA replication stress (ODRS) is a hallmark of MYC and BCL2 dysregulation; however, the DNA damage response (DDR) pathways that sustain replication and cell survival under ODRS remain incompletely defined in DLBCL. Identifying DDR dependencies induced by ODRS may reveal therapeutically actionable vulnerabilities in DH/DE-LBCL. Methods: We studied DH/DE-LBCL cell lines (OCILY1, SUDHL2, DOHH2), non-DH/DE-LBCL cell lines (HT, KARPAS422, HBL1), and benign retinal pigment epithelial (RPE) cells engineered to overexpress MYC and/or BCL2. ODRS and DNA damage were assessed by phosphorylation of RPA (S4/S8, S33) and H2AX histone (γH2AX), respectively, using immunofluorescence, immunohistochemistry, and western blot (WB). Activity of homologous recombination (HR), nonhomologous end-joining (NHEJ), and microhomology-mediated end joining (MMEJ) pathways were quantified with established reporter assays. POLΘ (gene: POLQ) expression, an MMEJ marker, was evaluated by qPCR and WB. DDR activity was assessed using genomic signatures from whole-exome sequencing data of 404 LBCL patients. In vitro sensitivity of LBCL cells to POLΘ and PARP inhibitors was assessed with publicly available small-molecule inhibitors using CellTiterGlo and clonogenic assays, and therapeutic efficacy was validated in a DH-LBCL xenograft model in NRG mice. Results: DH/DE-LBCL cells and primary tumors exhibited increased ODRS and persistent DNA damage compared with non-DH/DE-LBCL models, including accumulation of γH2AX in mitosis. Functional profiling revealed selective upregulation of MMEJ in MYC/BCL2-overexpressing cells, with minimal changes in HR or NHEJ activity. POLQ expression was significantly increased in DH/DE-LBCL cell lines and primary samples, and the ID6 signature (associated with MMEJ activity) was significantly enriched in DH-LBCL tumors compared to non-DH/DE-LBCL tumors. POLΘ inhibition demonstrated greater toxicity in DH/DE-LBCL and in MYC-overexpressing RPE cells compared to non-DH/DE-LBCL and wild-type RPEs. Combined inhibition of POLΘ and PARP resulted in marked synergy in vitro. The therapeutic efficacy of POLΘ inhibition, with the synthetic lethality of concurrent PARP inhibition, was validated in the DH-LBCL xenograft model. Concurrent POLΘ and PARP inhibition with small-molecule inhibitors induced an antitumor effect in vivo, with increased overall survival in mice compared with vehicle- or olaparib-single-agent-treated groups. Conclusions: ODRS in DH/DE-LBCL creates a selective dependence on MMEJ mediated by POLΘ. Therapeutic targeting of POLΘ, alone or in combination with PARP inhibition, exploits this vulnerability and represents a promising strategy for this high-risk LBCL subset. These findings provide a biological rationale for clinical translation of POLΘ-directed therapies in DH/DE-LBCL. Citation Format: Cecilia Ayala-Zambrano, Dongni Yi, Michelle Manske, Kimberly Gwin, Richar Kandasamy, Surendra Dasari, Anne Novak, Shuhei Asada, Xiaosheng Wu, Zhenkun Lou, Thomas Witzig, Jithma Abeykoon. c-MYC/BCL2 induced replication stress drives dependence on POLQ-mediated microhomology-mediated end joining in aggressive large B-cell lymphoma [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A029.

Abstract Castleman disease (CD) is a heterogeneous group of lymphoproliferative disorders anatomically classified by distribution (unicentric CD [UCD], oligocentric CD [oligoCD], or multicentric CD [MCD]). Human herpes virus 8–negative MCD is called idiopathic MCD (iMCD), which includes clinical subtypes with varying phenotypes and responses: TAFRO (thrombocytopenia, anasarca, fever, renal dysfunction and/or reticulin fibrosis, and organomegaly), IPL (idiopathic plasmacytic lymphadenopathy), and not otherwise specified. OligoCD has recently emerged as an intermediate form between UCD and MCD, with unclear clinical behavior, and IPL has not been validated in a Western cohort. We retrospectively analyzed 217 patients with CD evaluated at our institution between January 2004 and August 2024. Survival probabilities were compared using log-rank tests. Overall, 57% had UCD, 20% had oligoCD, and 23% had iMCD. Patients with oligoCD and iMCD more frequently exhibited systemic symptoms than those with UCD. Patients with oligoCD and iMCD had significantly shorter event-free survival (EFS) of 8.9 and 2.3 years, respectively, than those with UCD (not reached; P< .001 and P< .02). Among iMCD subtypes, iMCD-IPL demonstrated a longer EFS than iMCD-TAFRO (P = .02), with no deaths during the follow-up periods. The results validate oligoCD and iMCD-IPL as new subtypes in this Western cohort. The survival outcome in oligoCD was intermediate between UCD and iMCD, with only a subset of patients with oligoCD requiring systemic therapy. iMCD-IPL had a favorable survival outcome in this US cohort, similar to what has been reported in non-Western countries. Tailoring treatment strategies to disease subtypes and vigilant monitoring of oligoCD for progression may improve survival outcomes in CD.

8116 Background: POEMS syndrome, or osteosclerotic myeloma, is an uncommon plasma cell disorder associated with Peripheral neuropathy, Organomegaly, Endocrinopathy, M protein, and Skin changes, as well as bone lesions, Castlemans disease, and edema. The pathogenesis of POEMS is poorly understood, and its clinical course is distinct from Multiple Myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). While various cytogenetic abnormalities have been well described in MM and MGUS, we provide the first report of testing for these abnormalities in POEMS syndrome. Methods: Using a prospectively held dysproteinemia database, we reviewed all cases of POEMS seen at our institution, identifying 36 with metaphase cytogenetic testing results, and 9 with fluorescence in situ hybridization (FISH) testing results. Combined cytoplasmic immunoglobulin/FISH testing (cIg- FISH) was performed with fluorescent-tagged antibodies to kappa/lambda immunoglobulin and FISH probes corresponding to the following anomalies: trisomies 3, 7, 9, 11, 15 and 17; del13/del13q; 14q32 split/del14; and t(11;14). Results: Five patients had insufficient plasma cells for cIg-FISH, and 9 were adequate for testing. Monosomy 13 was seen in 5 of the 9 cIg-FISH samples of which 2 of these cases also had apparent monosomy 14. cIg-FISH in one patient revealed trisomy 3 and 7. No abnormalities were seen at 11q13 (CCND1), 17p13 (p53) or translocations involving 14q32 (IGH). Thrity-eight total karyotypes analyses were attempted, though two showed an insufficient number of suitable metaphases. Of the 36 karyotype analyses, 31 were normal, 2 displayed characteristic plasma cell anomalies (hyperdiploid with structural anomalies), 2 treated patients displayed del20q anomalies and 1 patient had a constitutional 13;14 translocation. Conclusion: We are the first to report del13 as a possibly common abnormality in POEMS syndrome, detected by cIg-FISH in 5 of 9 (55%) samples with sufficient plasma cells, similar to the rate observed in MM and MGUS. The lack of abnormalities at 14q32 (IgH locus) is in contrast to MM and MGUS. Abnormal karyotypes, consistent with dividing plasma cells, are rare in POEMS, observed in only 2 of 36 (5.5%) cases. These observations could possibly lend insight into all three disorders. No significant financial relationships to disclose.

Abstract The purpose of this study was to quantitate the number and labeling index of monoclonal plasma cells in the blood of patients with newly diagnosed multiple myeloma (MM) to learn if these values were independent prognostic factors for survival. Patients were candidates for this study if they had untreated myeloma requiring therapy, were evaluated at our institution between 1984 and 1993, and had a sample of blood analyzed with a sensitive immunofluorescence technique for monoclonal plasma cells and the blood B-cell labelling index (BLI). The % blood monoclonal plasma cells (%BPC) and the BLI were analyzed along with stage, marrow plasma cell LI, % marrow plasma cells, calcium, creatinine, albumin, beta-2-microglobulin, and C-reactive protein as univariate and multivariate factors for survival. Eighty percent of the 254 patients accrued to this study had monoclonal BPC detected. The median % BPC was 6% and 57% (144 of 254) of patients had a high number (> or = 4%). Patients with > or = 4% BPC had a median survival of 2.4 years vs 4.4 years for those with < 4% BPC (P < .001). The BLI was also prognostic (P = .008). In a multivariate analysis, the % BPC, age, albumin, stage, marrow plasma cell LI, and the BLI were independent factors for survival. The %BPC and the marrow plasma cell LI best separated the group into low, intermediate, and high risk myeloma with median survivals of 52, 35, and 26 months, respectively. Patients with high %BPC were less likely to have lytic bone disease from their MM (P = .002). The %BPC and the BLI are independent prognostic factors for survival and are useful in identifying patients as low, intermediate, and high risk. Clonal cells in the blood should be quantified in future clinical trials for myeloma.

Abstract We review the clinical manifestations and long-term outlook of patients with chronic natural killer (NK) cell lymphocytosis. After reviewing more than 1,500 peripheral blood lymphoid flow cytometry reports and molecular genetics data from patients with suspected large granular lymphocyte (LGL) proliferation, we identified 10 patients (median age at diagnosis, 60 years; range, 35 to 76 years; male:female ratio, 3:2) with persistent (greater than 6 months) increase in phenotypically determined NK cells (CD3-CD16+). Southern blot analysis performed on 9 patients showed no clonal T-cell receptor gene rearrangements. Disease duration was measured from time of initial recognition of LGL or NK cell excess (greater than 40% of the lymphocyte fraction). Clinical data from these 10 patients were compared with those from 68 patients with T-cell LGL (T-LGL) leukemia. Currently, all patients are alive (median disease duration, 5 years; range, 0.8 to 8 years). Associated disease manifestations included pure red blood cell aplasia, recurrent neutropenia, recurrent neutropenic sepsis, and vasculitic syndromes, all of which were responsive to immunosuppressive therapy. No patient had palpable lymphadenopathy or splenomegaly. Compared with the patients with T-LGL leukemia, patients with chronic NK cell leukemia has similar lymphocyte counts, associated conditions, treatment responses, and survival but had less neutropenia and anemia.

Abstract The plasma cell labeling index (PCLI) and serum beta 2-microglobulin (beta 2M) are independent prognostic factors in multiple myeloma (MM). Recently, levels of thymidine kinase (TK) and C-reactive protein (CRP) have been shown to have prognostic value. We studied 107 patients with newly diagnosed myeloma to determine whether TK and CRP values added prognostic information not already available using the PCLI and beta 2M. Univariate survival analysis showed prognostic significance for the PCLI, TK, beta 2M, age, serum albumin, and CRP. Multivariate analysis showed that only PCLI and beta 2M have independent prognostic significance. The survival curves were better separated using the PCLI and beta 2M than with other combinations of variables. Among nine patients under age 65 with low PCLI and low beta 2M, eight were alive almost 6 years after starting chemotherapy. These good-risk patients could not be identified by standard clinical features. Although creatinine and calcium were normal, other features such as bone lesions, osteoporosis, fracture, and anemia were present and stage distribution was similar to other patients in the study. In conclusion, PCLI and beta 2M measured at diagnosis are independent prognostic factors. They must be considered when interpreting the results of clinical trials and should be helpful in counseling patients and in designing new trials. When the PCLI and beta 2M values are known, the TK and CRP values do not add useful additional prognostic information.

Abstract A 73-year-old woman with metastatic transitional cell carcinoma of the bladder developed vaginal bleeding a few days after undergoing radical cystectomy. She had no other signs of mucocutaneous bleeding. Coagulation studies revealed a markedly prolonged thrombin time (greater than 600 seconds), a slightly prolonged reptilase time (20 seconds), and mildly elevated fibrinogen (4.39 g/L), and fibrin D-dimer (200 to 500 ng/mL) levels. Treatment of the patient's plasma in vitro with protamine or barium sulfate normalized the thrombin time. The anticoagulant activity corresponded to 0.15 heparin U/mL when measured by a thrombin time assay using normal plasma as substrate and standardized with porcine heparin. The anticoagulant was quantitatively bound to and subsequently eluted with 1 mol/L NaCl from quaternary aminoethyl (QAE) Sephadex, and then isolated by affinity chromatography on immobilized antithrombin III. The isolated anticoagulant was shown to be sensitive to heparinase digestion. Therefore, the inhibitor has functional and chemical properties similar to those of high-affinity heparin. Thus far, this is the only anticoagulant of this type isolated from the plasma of a patient bearing a tumor other than plasma cell myeloma.

We studied 25 T-cell chronic lymphocytic leukemia (T-CLL) cases collected over a 15-year period. Immunophenotypic analysis was performed in each case; 12 cases were evaluated by cytogenetics, and gene rearrangement studies were performed in 14 cases. The median age was 57 years with a male predominance (M:F, 15:10). The median presenting lymphocyte count was 36.3 x 10(9)/L (range, 3.9 to 438 x 10(9)/L). Fourteen patients (56%) had shotty adenopathy and ten (40%) had mild-to-moderate splenomegaly at presentation; four (16%) had erythematous skin lesions. The lymphocytes were predominantly small; some cases had a minor component of medium-sized cells (< 10%). The nuclear: cytoplasmic ratios were uniformly high with round to oval nuclei; however, a wide spectrum of nuclear outlines could be found, ranging from minimally to markedly convoluted. Nucleoli were either absent or small and inconspicuous. These lymphocytes did not have the morphology of prolymphocytes and did not contain cytoplasmic granules. Bone marrow infiltration was generally in an interstitial pattern; the degree of involvement ranged from 15% to 90%. Immunophenotyping showed that the lymphocytes were mature T-cells with a predominant CD4+ immunophenotype. Three cases displayed a CD8+ immunophenotype. The patients were treated with a variety of chemotherapeutic regimens with only a minimal response observed in two of 20 patients. We conclude that T-CLL is an uncommon chronic lymphoproliferative disorder (CLPD) that can be morphologically similar to B-CLL, is distinct from T- prolymphocytic leukemia, and has an aggressive clinical course that is refractory to therapy. It may also be difficult to distinguish T-CLL from other T-CLPD, especially the leukemic phase of peripheral T-cell lymphoma and some cases of Sezary syndrome.

We review the clinical manifestations and long-term outlook of patients with chronic natural killer (NK) cell lymphocytosis. After reviewing more than 1,500 peripheral blood lymphoid flow cytometry reports and molecular genetics data from patients with suspected large granular lymphocyte (LGL) proliferation, we identified 10 patients (median age at diagnosis, 60 years; range, 35 to 76 years; male:female ratio, 3:2) with persistent (greater than 6 months) increase in phenotypically determined NK cells (CD3-CD16+). Southern blot analysis performed on 9 patients showed no clonal T-cell receptor gene rearrangements. Disease duration was measured from time of initial recognition of LGL or NK cell excess (greater than 40% of the lymphocyte fraction). Clinical data from these 10 patients were compared with those from 68 patients with T-cell LGL (T-LGL) leukemia. Currently, all patients are alive (median disease duration, 5 years; range, 0.8 to 8 years). Associated disease manifestations included pure red blood cell aplasia, recurrent neutropenia, recurrent neutropenic sepsis, and vasculitic syndromes, all of which were responsive to immunosuppressive therapy. No patient had palpable lymphadenopathy or splenomegaly. Compared with the patients with T-LGL leukemia, patients with chronic NK cell leukemia has similar lymphocyte counts, associated conditions, treatment responses, and survival but had less neutropenia and anemia.

The plasma cell labeling index (PCLI) and serum beta 2-microglobulin (beta 2M) are independent prognostic factors in multiple myeloma (MM). Recently, levels of thymidine kinase (TK) and C-reactive protein (CRP) have been shown to have prognostic value. We studied 107 patients with newly diagnosed myeloma to determine whether TK and CRP values added prognostic information not already available using the PCLI and beta 2M. Univariate survival analysis showed prognostic significance for the PCLI, TK, beta 2M, age, serum albumin, and CRP. Multivariate analysis showed that only PCLI and beta 2M have independent prognostic significance. The survival curves were better separated using the PCLI and beta 2M than with other combinations of variables. Among nine patients under age 65 with low PCLI and low beta 2M, eight were alive almost 6 years after starting chemotherapy. These good-risk patients could not be identified by standard clinical features. Although creatinine and calcium were normal, other features such as bone lesions, osteoporosis, fracture, and anemia were present and stage distribution was similar to other patients in the study. In conclusion, PCLI and beta 2M measured at diagnosis are independent prognostic factors. They must be considered when interpreting the results of clinical trials and should be helpful in counseling patients and in designing new trials. When the PCLI and beta 2M values are known, the TK and CRP values do not add useful additional prognostic information.