Audil, Hadiyah Y.

Introduction: Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is a rare form of large granular lymphocytic leukemia. The clinical characteristics and outcomes of patients (pts) with CLPD-NK in the literature are limited. We aim to describe the patient characteristics, laboratory features, and clinical outcomes of CLPD-NK by reporting on, to our knowledge, the largest single-center cohort of CLPD-NK in the United States. Methods: We retrospectively identified Mayo Clinic pts with CLPD-NK, defined per the WHO 5 th edition and 2022 International Consensus Classification. We conducted chart review to collect clinical and laboratory data. Overall survival (OS) was plotted using Kaplan-Meier. Time to first treatment (TTFT) was displayed using cumulative incidence methodology accounting for competing risk of death. Univariable Cox regression models investigated associations between patient characteristics and outcomes (OS and TTFT). Results: We identified 45 pts who were diagnosed with CLPD-NK between 1997 and 2022. The median age at diagnosis was 69 years (range: 25-90) and 32 pts (71%) were male. At the time of diagnosis, 10 pts (22%) had a concomitant autoimmune disorder and 9 (20%) had laboratory evidence of a serologic abnormality (monoclonal gammopathy, rheumatoid factor, etc.). In addition, 10 pts (22%) had a prior diagnosis of a hematologic malignancy and 12 (27%) had a prior diagnosis of a solid tumor. Clinically, 4 pts (9%) had B symptoms, 15 (33%) had splenomegaly, and 13 (29%) were transfusion dependent. At diagnosis, median hemoglobin (hgb) was 10.2 g/dL, with 25 pts (56%) showing a hgb <11g/dL and 8 (18%) demonstrating a hgb <8 g/dL. The median platelet count was 202x10^9/L, with 12 pts (27%) demonstrating a platelet count <150x10^9/L. The median white blood cell count was 5.8x10^9/L. The median absolute neutrophil count (ANC) was 1.2x10^9/L, with 29 pts (64%) demonstrating an ANC <1.5x10^9/L. The median absolute lymphocyte count (ALC) was 3.8x10^9/L, with 20 pts (44%) demonstrating an ALC >4x10^9/L. The median absolute abnormal NK cell count per flow cytometry was 2.2x10^9/L. Flow cytometry revealed killer cell immunoglobulin-like receptor (KIR) restriction in all pts, with 27 (61%) lacking KIR expression and 17 (39%) demonstrating KIR restriction in CD158a, CD158b, and/or CD158e. CD7 expression was present in 42 pts (93%; 31% dim/partial), CD8 expression was present in 22 (49%; 42% d/p), CD56 expression was present in 17 (39%; 23% d/p), CD57 expression was present in 26 (59%; 46% d/p), CD94 expression was present in 33 (77%; 7% d/p), and NKG2A expression was present in 21 (50%; 5% d/p). Bone marrow examination (n=30) revealed that the median number of NK infiltrate was 11% (range: 5-40%) of cellularity, with 20 demonstrating an intrasinusoidal infiltrative pattern. The median follow-up was 41 months (mo), and 23 pts required at least one line of treatment. Treatment indications included anemia (39%), neutropenia (4%), bi/pancytopenia (39%), lymphocytosis (4%), or other (13%). The median TTFT was 32 mo (95% CI 2-93 mo). Hgb <11g/dL was associated with a shorter TTFT (9.7 vs 187 mo); however, neutropenia and thrombocytopenia were not associated with TTFT. For first-line treatment, in addition to frequent steroid use, 16 pts (70%) received a methotrexate-based regimen, 6 (26%) received a cyclophosphamide-based regimen, and 1 (4%) received a cyclosporine-based regimen. The median TTFT was not reached. Of those who required systemic treatment, 9 pts required a second-line therapy. The median OS from diagnosis was 162 mo (95% CI: 84-not estimable, NE), with 33 pts alive at last follow-up (Figure 1a). Increasing age (HR 1.2, 95% CI 1.1-1.3, p=0.002) and lack of CD94 expression (HR 5.6, 95% 1.3-25.0, p=0.02) were associated with worse OS. In the 23 pts who required treatment, the median OS from treatment initiation was 84 mo (95% CI: 52-NE) (Figure 1b). Conclusions: In this large cohort of 45 pts, the majority had neutropenia and anemia, and approximately half required systemic treatment. Estimated median OS from diagnosis exceeded a decade for the entire cohort and was seven years after treatment initiation. Anemia was associated with a shorter TTFT, while increasing age and lack of CD94 expression were associated with worse OS. This rare disease warrants additional investigations to understand optimal management and improve outcomes, especially for those requiring treatment.

We identified a patient with a recently described syndrome, monoclonal gammopathy of thrombotic significance (MGTS; Blood. 2023 Apr 6;141(14):1772-1776), and three additional patients with high 4Ts scores who were consistently negative in the PF4-polyanion HIT ELISA (Lifecodes PF4 IgG, Immucor). The MGTS patient, with a monoclonal IgG kappa, had recurrent thrombosis, and transient mild thrombocytopenia (nadir in the low 100s) over a period of years, and was noted to have a HIT ELISA optical density (OD) of 0.117. The three HIT patients experienced thrombocytopenia and thrombosis with ELISA ODs of 0.37, 0.18 and 0.20. All patients were positive in platelet-activation (functional) testing demonstrating PF4 dependence and inhibited with both high concentrations of heparin (100U/mL) and the FcγRIIa blocking monoclonal antibody (mAb) IV.3, consistent with the presence of anti-PF4 antibodies in the patient samples ( data not shown). Because the serologic profile of these patients suggested a novel HIT subtype characterized by negative HIT ELISA results, we undertook a functional platelet assay screen of 500 consecutive HIT ELISA-negative patients tested in our diagnostic laboratory. Research studies were approved by the Institutional Review Board of Mayo Clinic. Of the 500 patients tested, 20 (4.0%) stimulated platelet activation in the PF4-dependent P-selectin Expression Assay (PEA ≥19%; data not shown). To exclude non-specific activation that can be induced by HLA antibodies, additional testing was performed to evaluate whether platelet activation was PF4-dependent using buffer or PF4-treated platelets. Three patients demonstrated PF4-dependent activation that was inhibited by high heparin concentrations and mAb IV.3, as would be expected for HIT antibodies (ELISA-negative HIT, EN-HIT1-3; Figure 1). On retrospective review, all three patients thus identified had clinical histories and courses consistent with high clinical probability for HIT. To develop tools to study this new class of antibodies, monoclonal HIT antibodies were generated in mice after PF4/heparin immunizations and screened using a novel approach that included simultaneous testing of hybridoma supernatants in the PEA and PF4-polyanion ELISA. This strategy was designed to enabled us to identify mAbs that activated platelets (in the PEA), but that did not bind antigenic targets in the HIT ELISA. A novel mAb 431.4 that was developed using this approach, and the well-characterized HIT-like mAb KKO were evaluated for their abilities to both bind PF4-polyanion targets in ELISA, and to activate PF4-treated platelets in the PEA (Figure 2). In significant contrast to KKO, mAb 431.4 did not bind PF4/polyanion complexes, however, like KKO, strongly activated platelets in a PF4-dependent manner. Activation induced by both KKO and mAb 431.4 was inhibited by high heparin concentrations, as would be expected for HIT antibodies. In vivo murine studies using this novel mAb are pending at this time. In summary, the non-canonical diagnostic profile of HIT ELISA-negative but platelet activation-positive antibodies were found at a relatively high incidence in HIT-suspected patients (0.6%; 3 of 500 ELISA-negative patients). These findings have important diagnostic/management implications and suggest a re-evaluation of ASH's HIT management guidelines as noted in recommendations 2.7 and 2.8 of that document (“In patients with an intermediate (Recommendation 2.7) or high (Recommendation 2.8)-probability 4Ts score and a negative immunoassay, the ASH guideline panel recommends discontinuation of the non-heparin anticoagulant and resumption of heparin, if indicated ”; Blood Adv (2018) 2 (22): 3360-3392). Figure 1. A subset of ELISA-negative HIT-suspected patients harbor anti-PF4 platelet-activating antibodies. Three ELISA-negative (EN) HIT patient samples from a screen of 500 samples demonstrate PF4-dependent platelet activation and inhibition with high heparin concentration and mAb IV.3 incubation. PBS, phosphate buffered saline; IC, isotype control; HDH- high dose heparin (100U/mL). Figure 2. Murine mAb 431.4 serologically mimics pathogenic ELISA-negative HIT/MGTS antibodies. ELISA and PEA testing was performed using the “HIT-like” mAb KKO, novel mAb 431.4, or an isotype control (IC) antibody. PVS-polyvinyl sulfonate (polyanion used in the Immucor IgG ELISA assay).

AbstractGain of 1q22 at diagnosis portends poorer outcomes in multiple myeloma (MM), but the prognostic significance of acquired 1q22 gain is unknown. We identified 63 MM patients seen at Mayo Clinic from 1/2004 to 12/2019 without 1q22 gain at diagnosis who acquired it during follow up and compared them to 63 control patients who did not acquire 1q22 gain with similar follow up. We also compared outcomes in the acquired 1q22 gain group with outcomes in 126 patients with 1q22 gain present at diagnosis. The incidence of acquired 1q22 gain was 6.1% (median follow‐up 6.8 years); median time to acquisition was 5.0 years (range: 0.7–11.5 years). Abnormalities on baseline fluorescence in situ hybridization (FISH) included trisomies (54%) and monosomy 13 (39%); 16 (25%) had high‐risk (HR) translocations or del(17p). Median progression‐free survival with front line therapy was 29.5 months in patients with acquired 1q22 gain, versus 31.4 months in control patients (p = .34) and 31.2 months in patients with de novo 1q22 gain (p = .04). Median overall survival (OS) from diagnosis was 10.9 years in patients with acquired 1q22 gain, versus 13.0 years in control patients (p = .03) and 6.3 years in patients with de novo 1q22 gain (p = .01). Presence of HR FISH at baseline increased risk of 1q22 gain acquisition. We demonstrate that acquisition of 1q22 gain is a significant molecular event in MM, associated with reduced OS. Among HR patients for whom this clonal evolution is determined, a risk‐adapted approach and/or clinical trial should be considered.

Background: Within the heterogeneous genomic landscape of multiple myeloma (MM), clonal evolution including the acquisition of risk-defining mutations and chromosomal abnormalities is a recurrent event and can be detected by fluorescence in situ hybridization (FISH). The effects of acquired abnormalities on clinical outcomes have not been well defined. We previously reported that patients who acquired 17p deletion [del(17p)] during the course of their disease had a significantly reduced overall survival (OS) by 38 months compared to patients who did not acquire del17p (Lakshman et al 2019). Similarly, while de novo gain of the long arm of chromosome 1 (1q22 gain) is a known high-risk aberration associated with significantly shorter OS and progression-free survival (PFS) in MM, the prognostic significance of acquired 1q22 gain has not been described. The primary objective of this study was to analyze factors predictive for acquired 1q22 gain and determine its impact on survival. Methods: We identified MM patients from the Mayo Clinic Dysproteinemia Database who had at least one follow up FISH performed ≥6 months from diagnosis. The clinical characteristics, concomitant cytogenetic abnormalities, first line treatments administered, and OS were compared between patients with acquired 1q22 gain and patients who did not acquire this abnormality. The Mayo Clinic IRB approved this study. Results: A total of 1041 MM patients met the inclusion criteria. Of these, 63 patients (6.1%) had acquired 1q22 gain, defined as being negative for 1q22 gain on initial FISH at diagnosis and having this abnormality detected on subsequent FISH. Median age at diagnosis was 59 years and 56% were male. Median time to acquisition of 1q22 gain was 60 months (range 8-140 months). We identified one control patient for each case who was diagnosed within one year of the case and had a subsequent FISH performed at a similar duration from diagnosis. Patients with acquired 1q22 gain had similar baseline characteristics except for a higher proportion of high-risk (HR) FISH at diagnosis [t(4;14), t(14;16), t(14;20), and del(17p13)] when compared to controls (27% HR FISH versus 6% HR FISH; P<0.01). 1q22 gain was concomitantly present with trisomies in 33 patients (54%), monosomy 13 in 24 patients (39%), t(4;14) in 8 patients (13%), and del(17p13) in 7 patients (12%). All patients received treatment prior to acquisition of 1q22 gain. Of the 63 patients, first line induction therapy consisted of proteasome inhibitor (PI) with steroid in 43%, immunomodulatory drugs (IMiD) with steroid in 40%, and PI + IMiD with steroid in 17% of patients. 54 patients (85%) received upfront stem cell transplant (SCT) (median 5.9 months to SCT), compared to 50 patients (79%) in the control group who received SCT. The median follow up of all 126 patients was 6.8 years. There was a statistically significant reduction in median OS from diagnosis in patients with acquired 1q22 gain compared to the control group (10.8 years versus 13.0 years; P = 0.02; Figure 1). Predictors of acquisition of 1q22 gain were identified using a case-control method. Age ≥70 at diagnosis and presence of HR FISH at baseline appeared to increase the risk of acquiring 1q22 gain. Conclusion: Acquisition of 1q22 gain is a relatively uncommon occurrence, but notably reduced OS by 2.2 years compared to patients who did not acquire 1q22 during the course of their disease (P = 0.02). Older age and the presence of HR FISH at diagnosis increased the risk of acquisition of 1q22 gain. The presence of high-risk translocations at baseline suggests that acquisition of 1q22 gain occurs in the context of more aggressive disease biology. Disclosures Kapoor: Cellectar: Consultancy; Celgene: Honoraria; Janssen: Research Funding; Sanofi: Consultancy, Research Funding; Amgen: Research Funding; Takeda: Honoraria, Research Funding; GlaxoSmithKline: Research Funding. Dispenzieri:Alnylam: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; Takeda: Research Funding; Intellia: Research Funding; Janssen: Research Funding. Gertz:Prothena: Consultancy; Celgene: Consultancy; Johnson and Johnson: Speakers Bureau; DAVA oncology: Speakers Bureau; Advisory Board for Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Advisory Board for Proclara: Membership on an entity's Board of Directors or advisory committees; i3Health: Consultancy; Springer Publishing: Patents & Royalties; Amyloidosis Foundation: Research Funding; International Waldenstrom Foundation: Research Funding; NCI SPORE MM: Research Funding; Ionis/Akcea: Consultancy; Physicians Education Resource: Consultancy; Medscape: Consultancy, Speakers Bureau; Amgen: Consultancy; Appellis: Consultancy; Annexon: Consultancy; Spectrum: Consultancy, Research Funding; Janssen: Consultancy; Sanofi: Consultancy; Data Safety Monitoring board from Abbvie: Membership on an entity's Board of Directors or advisory committees; Alnylam: Consultancy. Dingli:Apellis: Consultancy; Rigel: Consultancy; Millenium: Consultancy; Bristol Myers Squibb: Research Funding; Karyopharm Therapeutics: Research Funding; Sanofi-Genzyme: Consultancy; Alexion: Consultancy; Janssen: Consultancy. Lin:Novartis: Consultancy; Celgene: Consultancy, Research Funding; Bluebird Bio: Consultancy, Research Funding; Juno: Consultancy; Merck: Research Funding; Takeda: Research Funding; Gamida Cells: Consultancy; Sorrento: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vineti: Consultancy; Legend BioTech: Consultancy; Janssen: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding. Kumar:Cellectar: Other; Genecentrix: Consultancy; Dr. Reddy's Laboratories: Honoraria; AbbVie: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; BMS: Consultancy, Research Funding; Sanofi: Research Funding; Karyopharm: Consultancy; MedImmune: Research Funding; Takeda: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Novartis: Research Funding; Kite Pharma: Consultancy, Research Funding; Oncopeptides: Consultancy, Other: Independent Review Committee; IRC member; Genentech/Roche: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Celgene/BMS: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Janssen Oncology: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Adaptive Biotechnologies: Consultancy; Amgen: Consultancy, Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments, Research Funding; Merck: Consultancy, Research Funding; Carsgen: Other, Research Funding; Tenebio: Other, Research Funding.

Background: Cytogenetic abnormalities detected by fluorescence in situ hybridization (FISH) are known to differentially impact prognosis in patients with chronic lymphocytic leukemia (CLL). Deletion6q23 (del6q23) has been reported in ~2-3% of CLL patients at diagnosis; however, its prognostic significance remains indeterminate. Methods: We identified previously untreated CLL patients from the Mayo Clinic CLL Database seen between 1/1995 - 2/2020 who had FISH performed. The clinical characteristics, concomitant cytogenetic abnormalities, time to first therapy (TTFT), and overall survival (OS) were compared between patients with del6q23 to those without this abnormality. TTFT was analyzed from FISH date until treatment or last known untreated date, accounting for competing risk of death. OS was analyzed using Kaplan-Meier methods from FISH date. In a matched (CLL-IPI risk score and sex) analysis, Cox regression analysis was used to determine the association between TTFT/OS and presence of del6q23. The Mayo Clinic IRB approved this study. Results: A total of 3101 CLL patients were identified; their median age at diagnosis was 64 years and 66% were male. Of these patients, 42 (1.3%) had evidence of del6q23 by FISH. Compared to patients without del6q23, patients with del6q23 carried a more adverse clinical and cytogenetic profile at the time of diagnosis including advanced Rai stage (22% Rai III/IV vs. 9% Rai III/IV; P = 0.002), unmutated IGHV genes (91% vs. 43%; P < 0.001), and high/very high risk CLL-IPI (50% vs. 27%; P < 0.001). Del6q23 was the sole abnormality in 13 patients (31%). Of the remaining patients, del6q23 was concomitantly present with del13q in 8 patients (19%), and with either del11q or del17p in 21 patients (50%). Of the 42 patients with del6q23, 29 received first line therapy: chemoimmunotherapy in 16 patients, BTK inhibitor in 7 patients, and an anti-CD20 monoclonal antibody alone in 6 patients. The median follow-up of the entire cohort was 7.3 years. The median TTFT in patients with del6q23 was 0.7 years while the 5-year OS was 76%. Patients with del6q23 demonstrated significantly shorter TTFT and reduced OS when compared to patients without del6q23 (Figures 1A and 1B). The shorter TTFT was also evident for patients with del6q23 compared to patients without del6q23 divided according to the Dohner risk category (Figure 2A), although there was no difference in OS between these groups of patients (Figure 2B). Because del6q23 patients were enriched for other unfavorable prognostic markers, we identified four control patients without del6q23, matched by CLL-IPI risk score and sex, to each treatment-naïve patient with del6q23. Patients with del6q23 showed a borderline statistically significant shorter TTFT (median 0.7 years versus 3.9 years; P = 0.07) and a nonsignificant association with OS (median 10.9 years versus 10.3 years; P = 0.28). Conclusion: Del6q23 by FISH in previously untreated CLL represents an uncommon abnormality. When compared to patients without del6q23, patients with del6q23 tend to have more adverse clinical and cytogenetic profiles at baseline, which likely confer a worse prognosis as reflected by shorter TTFT compared to patients without this abnormality. Figure Disclosures Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding. Wang:Novartis: Research Funding; Innocare: Research Funding; Incyte: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Kay:Sunesis: Research Funding; Abbvie: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; MEI Pharma: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees. Kenderian:Juno: Research Funding; Gilead: Research Funding; Lentigen: Research Funding; MorphoSys: Research Funding; Sunesis: Research Funding; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Tolero: Research Funding; BMS: Research Funding. Parikh:Ascentage Pharma: Research Funding; Merck: Research Funding; AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; TG Therapeutics: Research Funding; Pharmacyclics: Honoraria, Research Funding; GlaxoSmithKline: Honoraria; Janssen: Honoraria, Research Funding; MorphoSys: Research Funding; Genentech: Honoraria; Verastem Oncology: Honoraria.