Abstract: These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

Abstract: Background: Ibrutinib (IBR) and venetoclax (VEN) combination is a highly effective therapy for patients (pts) with CLL. The ideal treatment duration for the combination therapy is not known. Several ongoing trials are evaluating 1 year (yr), 2 yr, or an MRD-guided strategy for the duration of combination therapy. We previously reported results of the first-line cohort of a phase II trial of combined IBR and VEN for pts with CLL (Jain, NEJM 2019). Here we report updated data for these pts with focus on MRD. Methods: Pts with previously untreated CLL meeting IWCLL treatment criteria were enrolled. Pts received IBR 420 mg daily for 3 cycles followed by addition of VEN (weekly dose-escalation to 400mg daily). Combined therapy was given for 24 cycles (28 days/cycle). Pts with bone marrow (BM) undetectable MRD (U-MRD) (flow cytometry; sensitivity 10-4) at 24 cycles of combined therapy discontinued both VEN and IBR; MRD+ pts continued IBR. Response assessments were performed using BM and CT (2008 IWCLL criteria) after cycle 3 of IBR monotherapy, and then after cycles 3, 6, 9, 12, 18, and 24 of the combination. U-MRD was defined as & lt;0.01%; low MRD+ 0.01% to & lt;1%; high MRD+ ≥1%. Progression-free survival (PFS) was assessed as the time from the start of study drug to CLL progression, Richter transformation, or death from any cause. Overall survival (OS) was assessed as the time from the start of study drug to death from any cause. Results: A total of 80 pts were enrolled. The median age was 65 yrs. Baseline characteristics are shown in Table 1. The median follow-up was 33.8 months. Five pts came off study during IBR monotherapy; 75 pts initiated VEN. Serial BM MRD assessments are shown in Figure 1 (this includes all 80 pts as denominator, including the 5 pts who never started VEN in an intent-to-treat analysis). After 12 cycles of the combination, 45/80 (56%) achieved BM U-MRD remission; 24/80 (30%) were BM MRD-positive (low MRD+, n=19; high MRD+, n=5); 11/80 (14%) were off study prior to cycle 12 assessment. After 24 cycles of the combination, 53/80 (66%) achieved BM U-MRD remission; 14/80 (17%) were BM MRD-positive (low MRD+, n=13; high MRD+, n=1); 13/80 (17%) were off study prior to cycle 24 assessment. Overall, 60/80 (75%) achieved BM U-MRD at any time. For the 24 pts who were BM MRD+ at the end of cycle 12, with continued combination treatment, 12/24 (50%) achieved BM U-MRD at the end of cycle 24 [low MRD+, 10/19 (53%); high MRD+, 2/5 (40%)]. None of the pretreatment characteristics (age, gender, ALC, HGB, PLT, FISH abnormalities, IGHV status, CD38, ZAP-70, TP53-m, NOTCH1-m, SF3B1-m) were statistically associated with achieving BM U-MRD at 6 cycles, 12 cycles, 24 cycles, or MRD at any time. PFS and OS are shown in Figure 2. PFS by FISH and IGHV status is shown in Figure 3. No pt had CLL progression. Richter's transformation developed in 2 pts (during VEN dose escalation, n=1; during cycle 24 of combination, n=1); both pts are alive in remission after receiving allo-SCT. Three pts died. Two pts came off study during the cycle 1 of IBR monotherapy and died 6 mos and 27 mos later due to infection issues; 1 pt died in U-MRD remission during cycle 19 of the combination therapy when found unresponsive at home, had CPR, and CT scan done in hospital showed b/l pneumonia. This was deemed possibly ibrutinib related. Of the 53 pts who were BM U-MRD at the end of cycle 24 of the combination, 41 pts had a subsequent blood MRD assessment done in follow-up (others are too early) with a median time off study drugs of 11.6 mos; 5 pts had recurrence of blood MRD (range, 0.01-0.05%) without any clinical disease progression. There were 14 pts who were BM MRD+ at the end of cycle 24 of the combination (low MRD+, n=13; high MRD+, n=1). The only pt with high-MRD+ at end of cycle 24 was noted to have Richter transformation at that time (described above). The remaining 13 pts (all low MRD+ in BM, range 0.01-0.56%) are on IBR monotherapy; 4/9 who had a subsequent blood MRD assessment achieved blood U-MRD. Conclusions: Combined IBR and VEN is an effective time-limited oral regimen for pts with CLL. We report 50% conversion of BM MRD+ to U-MRD from cycle 12 to cycle 24 with ongoing combination therapy. Whether this would translate into improved PFS remains to be determined. Given improved MRD response from cycle 12 to cycle 24, we have amended the trial to allow another 12 cycles of the combination for pts who are BM MRD+ at cycle 24. Ongoing studies will further help define the role and the optimal duration of this combination in CLL. Disclosures Jain: ADC Therapeutics: Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Pfizer: Research Funding; BMS: Research Funding; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Aprea Therapeutics: Research Funding; Precision Bioscienes: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; BeiGene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Thompson:Adaptive Biotechnologies: Consultancy, Research Funding; Pharmacyclics: Research Funding; AbbVie: Research Funding; Genentech: Consultancy; Janssen-Cilag: Honoraria. Burger:Pharmacyclics, an AbbVie company: Consultancy, Research Funding, Speakers Bureau; Gilead Sciences: Consultancy, Research Funding; Beigene: Research Funding, Speakers Bureau; AstraZeneca: Consultancy; TG Therapeutics: Research Funding, Speakers Bureau; Janssen Pharmaceuticals: Consultancy, Speakers Bureau. Borthakur:Incyte: Research Funding; BioLine Rx: Consultancy; Nkarta Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy; FTC Therapeutics: Consultancy; BioLine Rx: Research Funding; Novartis: Research Funding; AstraZeneca: Research Funding; Abbvie: Research Funding; Jannsen: Research Funding; BioTherix: Consultancy; PTC Therapeutics: Consultancy; Argenx: Consultancy; PTC Therapeutics: Research Funding; BMS: Research Funding; Xbiotech USA: Research Funding; Polaris: Research Funding; Curio Science LLC: Consultancy; Oncoceutics: Research Funding; Cyclacel: Research Funding; GSK: Research Funding. Sasaki:Pfizer Japan: Consultancy; Otsuka: Honoraria; Daiichi Sankyo: Consultancy; Novartis: Consultancy, Research Funding. Fowler:TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kadia:Novartis: Honoraria; BMS: Honoraria, Research Funding; Cellenkos: Research Funding; Genentech: Honoraria, Research Funding; Incyte: Research Funding; Pulmotec: Research Funding; Ascentage: Research Funding; Amgen: Research Funding; Astra Zeneca: Research Funding; Celgene: Research Funding; Cyclacel: Research Funding; JAZZ: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Astellas: Research Funding; Abbvie: Honoraria, Research Funding. Konopleva:Sanofi: Research Funding; Forty-Seven: Consultancy, Research Funding; Eli Lilly: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Kisoji: Consultancy; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Ascentage: Research Funding; Rafael Pharmaceutical: Research Funding; AbbVie: Consultancy, Research Funding; AstraZeneca: Research Funding; Cellectis: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Ablynx: Research Funding; Calithera: Research Funding; Agios: Research Funding. Alvarado:Sun Pharma: Research Funding; BerGenBio ASA: Research Funding; MEI Pharma: Research Funding; Tolero Pharmaceuticals: Research Funding; Astex Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding; Daiichi-Sankyo: Research Funding; FibroGen: Research Funding. Yilmaz:Pfizer: Research Funding; Daicho Sankyo: Research Funding; Pint Pharma: Honoraria. DiNardo:Syros: Honoraria; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Novartis: Consultancy; Notable Labs: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Jazz: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; MedImmune: Honoraria; ImmuneOnc: Honoraria; Calithera: Research Funding. Bose:Celgene Corporation: Honoraria, Research Funding; Kartos Therapeutics: Honoraria, Research Funding; Promedior, Inc.: Research Funding; Pfizer, Inc.: Research Funding; Constellation Pharmaceuticals: Research Funding; NS Pharma: Research Funding; Astellas Pharmaceuticals: Research Funding; CTI BioPharma: Honoraria, Research Funding; Incyte Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau; Blueprint Medicines Corporation: Honoraria, Research Funding. Pemmaraju:Plexxikon: Research Funding; AbbVie: Honoraria, Research Funding; Blueprint Medicines: Honoraria; Incyte Corporation: Honoraria; Stemline Therapeutics: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; MustangBio: Honoraria; Samus Therapeutics: Research Funding; DAVA Oncology: Honoraria; Cellectis: Research Funding; Affymetrix: Other: Grant Support, Research Funding; Pacylex Pharmaceuticals: Consultancy; Celgene: Honoraria; Daiichi Sankyo: Research Funding; SagerStrong Foundation: Other: Grant Support; LFB Biotechnologies: Honoraria; Roche Diagnostics: Honoraria. Jabbour:BMS: Other: Advisory role, Research Funding; Pfizer: Other: Advisory role, Research Funding; Takeda: Other: Advisory role, Research Funding; AbbVie: Other: Advisory role, Research Funding; Adaptive Biotechnologies: Other: Advisory role, Research Funding; Genentech: Other: Advisory role, Research Funding; Amgen: Other: Advisory role, Research Funding. Kantarjian:Jazz Pharma: Research Funding; Daiichi-Sankyo: Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Immunogen: Research Funding; Astex: Research Funding; Cyclacel: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ariad: Research Funding; BMS: Research Funding; Pfizer: Honoraria, Research Funding; Takeda: Honoraria; Amgen: Honoraria, Research Funding; Novartis: Research Funding. OffLabel Disclosure: Combination of ibrutinib and venetoclax is not FDA approved

Abstract: Background: Ibrutinib (IBR) and venetoclax (VEN) combination is a highly effective therapy for patients (pts) with CLL (Jain, NEJM 2019; Wierda, ASH 2020; Kater, EHA 2021). We previously reported results of the first-line cohort of a phase II trial of combined IBR and VEN for high-risk pts with CLL (Jain, NEJM 2019; Jain, JAMA Oncology 2021). Here we report updated data for these pts with focus on MRD. Methods: Pts with previously untreated CLL meeting IWCLL treatment criteria were enrolled. All pts had at least one high-risk feature: del(17p), mutated TP53, del(11q), unmutated IGHV, or age ≥65 years (yrs). Pts received IBR 420 mg daily for 3 cycles followed by addition of VEN (weekly dose-escalation to 400mg daily). Combined therapy was given for 24 cycles (28 days/cycle). Pts with bone marrow (BM) undetectable MRD (U-MRD) (flow cytometry; sensitivity 10 -4) at 24 cycles of combined therapy discontinued both VEN and IBR; MRD+ pts continued IBR. A trial amendment allowed an additional 12 cycles of combined VEN and ibrutinib for pts who remained BM MRD+ after Cycle 24. Response assessments were performed using BM and CT imaging studies (2008 IWCLL criteria). U-MRD was defined as & lt;0.01%; low MRD+ 0.01% to & lt;1%; high MRD+ ≥1%. Progression-free survival (PFS) was assessed as the time from the start of study drug to CLL progression, Richter transformation, or death from any cause. Blood MRD was monitored every 6 months in pts off treatment or on ibrutinib monotherapy beyond 24 cycles of combined treatment. Results: A total of 80 pts were enrolled. Baseline characteristics are shown in Table 1. The median follow-up was 44.1 months. Five pts came off study during 1 st 3 cycles of IBR monotherapy; 75 pts initiated VEN. We previously reported that after 12 cycles of the combination, 45/80 (56%) achieved BM U-MRD remission; 24/80 (30%) were BM MRD-positive (low MRD+, n=19; high MRD+, n=5). After 24 cycles of the combination, 53/80 (66%) achieved BM U-MRD remission; 14/80 (17%) were BM MRD+ (low MRD+, n=13; high MRD+, n=1). Overall, 60/80 (75%) achieved BM U-MRD as the best response. Updated PFS is provided in Figure 1. Of the 53 pts who were BM U-MRD at the end of cycle 24 of the combination, 52 pts had a subsequent blood MRD assessment done in follow-up (1 missed due to COVID-19); 51/53 discontinued all therapy, 2 pts continued IBR per treatment physician discretion. With a median time of 18.4 months post Cycle 24, 8 pts had recurrence of blood MRD (defined as MRD ≥ 0.01% in 2 consecutive visits) in follow-up with 1 pt with CLL progression. The sole pt with CLL progression had mutated IGHV with del(11q) and NOTCH1 mutation. The pt had delayed achievement of BM U-MRD with the pt achieving U-MRD for the first time at the end of Cycle 24 of combined therapy. She was noted to have disease progression 22 months off therapy; BTK or PLCG2 mutation were not detected and the patient is currently in clinical remission on acalabrutinib. The time to MRD conversion for these 53 pts is shown in Figure 2. There were 14 pts who were BM MRD+ at the end of cycle 24 of the combination (low MRD+, n=13; high MRD+, n=1). The only pt with high-MRD+ at end of cycle 24 was noted to have Richter transformation at that time. The remaining 13 pts (all low MRD+ in BM, range 0.01-0.56%) continued IBR monotherapy. With a recent trial amendment, MRD+ pts after Cycle 24 could get 12 additional cycles of venetoclax; 9/13 pts have resumed VEN. 6/9 pts have achieved U-MRD remission. 2 pts had Richter transformation and 3 pts have died (Jain, JAMA Oncology 2021). Conclusions: We report long term follow-up of combined IBR and VEN in first-line CLL. Remissions were durable with some pts having recurrence of blood MRD in follow-up, which may be an early indicator of relapse. In a small subset of the pts with BM MRD+ disease at 24 cycles of combined therapy, additional VEN appears to lead to U-MRD remission in majority of the pts. Whether this will lead to improved long-term PFS remains to be determined. Figure 1 Figure 1. Disclosures Jain: TG Therapeutics: Honoraria; Beigene: Honoraria; Janssen: Honoraria; Fate Therapeutics: Research Funding; Aprea Therapeutics: Research Funding; Precision Biosciences: Honoraria, Research Funding; Incyte: Research Funding; Adaptive Biotechnologies: Honoraria, Research Funding; Cellectis: Honoraria, Research Funding; ADC Therapeutics: Honoraria, Research Funding; Servier: Honoraria, Research Funding; Pfizer: Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Pharmacyclics: Research Funding. Thompson: AbbVie: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding; Amgen: Other: Institution: Honoraria, Research Grant/Funding; Genentech: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding; Adaptive Biotechnologies: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding, Expert Testimony; Pharmacyclics: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding; Janssen: Consultancy, Honoraria; Gilead: Other: Institution: Advisory/Consultancy, Honoraria. Ferrajoli: BeiGene: Other: Advisory Board, Research Funding; Janssen: Other: Advisory Board ; AstraZeneca: Other: Advisory Board, Research Funding. Burger: Novartis: Other: Travel/Accommodations/Expenses, Speakers Bureau; TG Therapeutics: Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel/Accommodations/Expenses, Speakers Bureau; Beigene: Research Funding, Speakers Bureau; Pharmacyclics LLC: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Gilead: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; AstraZeneca: Consultancy. Borthakur: GSK: Consultancy; ArgenX: Membership on an entity's Board of Directors or advisory committees; University of Texas MD Anderson Cancer Center: Current Employment; Protagonist: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding; Ryvu: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees. Takahashi: Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Celgene/BMS: Consultancy; GSK: Consultancy. Sasaki: Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding. Kadia: Cellonkos: Other; Aglos: Consultancy; Dalichi Sankyo: Consultancy; AbbVie: Consultancy, Other: Grant/research support; BMS: Other: Grant/research support; Amgen: Other: Grant/research support; Cure: Speakers Bureau; Jazz: Consultancy; Genentech: Consultancy, Other: Grant/research support; Liberum: Consultancy; Novartis: Consultancy; Pfizer: Consultancy, Other; Pulmotech: Other; Sanofi-Aventis: Consultancy; AstraZeneca: Other; Astellas: Other; Genfleet: Other; Ascentage: Other. Konopleva: Sanofi: Other: grant support, Research Funding; Cellectis: Other: grant support; Calithera: Other: grant support, Research Funding; KisoJi: Research Funding; Agios: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Ablynx: Other: grant support, Research Funding; Stemline Therapeutics: Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; AstraZeneca: Other: grant support, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Forty Seven: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights. Alvarado: BerGenBio: Research Funding; Jazz Pharmaceuticals: Research Funding; Astex Pharmaceuticals: Research Funding; Sun Pharma: Consultancy, Research Funding; MEI Pharma: Research Funding; FibroGen: Research Funding; Daiichi-Sankyo: Research Funding; CytomX Therapeutics: Consultancy. Yilmaz: Pfizer: Research Funding; Daiichi-Sankyo: Research Funding. DiNardo: Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Takeda: Honoraria; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding; Forma: Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; Foghorn: Honoraria, Research Funding. Bose: Kartos Therapeutics: Honoraria, Research Funding; Sierra Oncology: Honoraria; Novartis: Honoraria; Constellation Pharmaceuticals: Research Funding; NS Pharma: Research Funding; Celgene Corporation: Honoraria, Research Funding; Blueprint Medicines: Honoraria, Research Funding; Pfizer: Research Funding; Promedior: Research Funding; Astellas: Research Funding; Incyte Corporation: Honoraria, Research Funding; BMS: Honoraria, Research Funding; CTI BioPharma: Honoraria, Research Funding. Pemmaraju: Blueprint Medicines: Consultancy; LFB Biotechnologies: Consultancy; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Roche Diagnostics: Consultancy; MustangBio: Consultancy, Other; Affymetrix: Consultancy, Research Funding; Samus: Other, Research Funding; ImmunoGen, Inc: Consultancy; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; Aptitude Health: Consultancy; Plexxicon: Other, Research Funding; Springer Science + Business Media: Other; Protagonist Therapeutics, Inc.: Consultancy; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; Clearview Healthcare Partners: Consultancy; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; CareDx, Inc.: Consultancy; Sager Strong Foundation: Other; Daiichi Sankyo, Inc.: Other, Research Funding; Incyte: Consultancy; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Bristol-Myers Squibb Co.: Consultancy; DAVA Oncology: Consultancy; Pacylex Pharmaceuticals: Consultancy; Celgene Corporation: Consultancy; Cellectis S.A. ADR: Other, Research Funding. Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Wang: Stemline Therapeutics: Honoraria. Kantarjian: Taiho Pharmaceutical Canada: Honoraria; Precision Biosciences: Honoraria; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Jazz: Research Funding; BMS: Research Funding; AbbVie: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; NOVA Research: Honoraria; KAHR Medical Ltd: Honoraria; Ipsen Pharmaceuticals: Honoraria; Astra Zeneca: Honoraria; Astellas Health: Honoraria; Aptitude Health: Honoraria; Amgen: Honoraria, Research Funding; Ascentage: Research Funding. Wierda: Juno Therapeutics: Research Funding; AstraZeneca: Research Funding; Xencor: Research Funding; Janssen: Research Funding; Loxo Oncology, Inc.: Research Funding; Cyclacel: Research Funding; Oncternal Therapeutics, Inc.: Research Funding; Miragen: Research Funding; KITE Pharma: Research Funding; Sunesis: Research Funding; Gilead Sciences: Research Funding; Acerta Pharma Inc.: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Karyopharm: Research Funding; Genentech: Research Funding; GSK/Novartis: Research Funding; Genzyme Corporation: Consultancy; AbbVie: Research Funding. OffLabel Disclosure: The combination of ibrutinib and venetoclax is not FDA approved

Abstract: Acute myeloid leukemia (AML) with KMT2A ( MLL ) rearrangement is known for monocytic or myelomonocytic differentiation, but the full immunophenotypic spectrum and dynamic changes of the immunophenotype in this genetically defined disease have not been systematically studied. Methods We reviewed the immunophenotype, karyotype, and mutations at the time of initial diagnosis and relapse of adults with AML with KMT2A rearrangement in our institution between 2007 and 2020. Results We identified 102 patients: 44 men and 58 women with a median age of 52 years (range, 18–87). Forty‐three patients were considered to be therapy‐related. Twenty‐four out of 64 patients relapsed from complete remission after induction therapy, 34 had persistent/progressive disease, and 58 patients died with a median overall survival of 17 months. We detected five immunophenotypes: immature monocytic (38%); myelomonocytic (22%); myeloblastic (22%); mature monocytic (10%); and acute promyelocytic (APL)‐like (8%). By chromosomal breakpoints, we presumed 11 different partners; t(9;11) (p22;q23)/ MLLT3‐KMT2A was the most common rearrangement ( n  = 56, 55%), followed by t(6;11) (q27;q23)/ AFDN‐KMT2A ( n  = 13,13%). Patients with t(6;11) (q27;q23)/ AFDN‐KMT2A preferentially showed a myeloblastic phenotype ( p  = 0.026). Mutations were detected in 39/64 (61%) cases, and RAS pathway ( NRAS/KRAS/PTPN11 ) was involved in 26/64 (41%) cases. None of the APL‐like cases had mutations detected. At the time of disease relapse, 10/24 (42%) showed major immunophenotypic change, and 7/10 cases gained additional cytogenetic and/or molecular alterations. Conclusion The immunophenotype of AML with KMT2A rearrangement is more diverse than previously recognized, with a substantial subset showing no evidence of monocytic differentiation. Major immunophenotype change is common at the time of relapse.

Abstract: Introduction : The current World Health Organization (WHO) classification considers acute myeloid leukemia (AML) with PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 to be AML regardless of blast count. Myeloid neoplasms with KMT2A rearrangement present with high blast percentages and are classified as AML in the vast majority of cases; however, rare myeloid neoplasms with KMT2A rearrangement may present with & lt;20% blasts and these are currently considered to be myelodysplastic syndromes (MDS) or chronic myelomonocytic leukemia (CMML). We questioned whether these oligoblastic KMT2A rearranged neoplasms shared similar biology with AML, and if this genetic finding should be considered to be AML-defining. Methods : We searched the archives of three large institutions in the United States for untreated adult with oligoblastic ( & lt;20%) myeloid neoplasms with KMT2A rearrangement and untreated adult AML patients with KMT2A rearrangement and compared their clinical and laboratory characteristics. KMT2A rearrangement was detected by chromosomal analysis and/or fluorescence in-situ hybridization (FISH) using KMT2A breakapart probe. KMT2A rearrangement partners were assessed based on a conventional karyotype results and/or RNA based fusion assay. Results: Between 2010 and 2021 we identified 22 patients with MDS or CMML and KMT2A rearrangement and 88 patients with AML and KMT2A rearrangement. The clinical and laboratory information are summarized below. MDS/CMML with KMT2A-rearrangement showed a similar age and sex distribution to AML, but had more patients with preceding history of chemotherapy for unrelated malignancies (68% vs 37%, p=0.012). Aside from a low blast count in PB and BM, patients with MDS/CMML had a lower WBC and were less anemic and thrombocytopenic. MDS/CMML patients also showed a lower percentage of BM cells with KMT2A rearrangement by FISH (58% vs. 92%, p & lt;0.0001), and significantly less likely to have MLLT3 as the partner gene (23% vs. 58%, p=0.0005). With a similar mutation frequency among patients tested (42% versus 34%, p=0.22), MDS/CMML patients had significantly fewer mutations involving the RAS pathway (KRAS, NRAS, FLT3, PTPN11) than AML patients (p=0.0035). Seventeen of 22 MDS/CMML patients (77%) progressed to AML with a median time of progression being 4 months (range, 1-27 months). Of the 5 patients who did not progress to AML, 3 received allogeneic stem cell transplantation (SCT) and remain in complete remission, while 2 remain AML-free 3 and 16 months after the initial diagnosis. With a median follow-up of 12 months (range, & lt;1-117), 15 MDS/CMML patients and 58 AML patients died. There was no difference in use of SCT to treat MDS/CMML versus AML (p=0.47). MDS/CMML and AML patients displayed similar overall survival (OS) (p=0.9059, Figure 1). There was also no statistical difference in OS between MDS/CMML and AML when therapy-related (p=0.569) and de novo (p=0.962) neoplasms were analyzed separately. On multivariable analysis, therapy-related disease (HR 1.774 [1.058-2.975], p=0.03), SCT (HR 0.166 [0.000-0.291] , p & lt;0.0001), and platelet count (HR 0.992 [0.000-0.998], p=0.013) were independently associated with shorter OS. Neither AML versus MDS/CMML nor blast percentage (whether dichotomized as & lt;5% or & gt;=5% or as a continuous variable) were associated with shorter OS. Conclusion: Oligoblastic myeloid neoplasms with KMT2A rearrangement tend to progress rapidly to overt AML and demonstrate similar aggressive behavior to AML patients with KMT2A rearrangement, irrespective of therapy relatedness. Patients with KMT2A rearrangement and & lt;20% blasts are currently classified as MDS or CMML, and may be inadequately treated. Our findings suggest that all myeloid neoplasms with KMT2A (MLL) rearrangement should be classified as AML, irrespective of the blast count. Figure 1 Figure 1. Disclosures Ravandi: AbbVie: Honoraria, Research Funding; Prelude: Research Funding; Taiho: Honoraria, Research Funding; Xencor: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding. Issa: Novartis: Consultancy, Research Funding; Kura Oncology: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding. Pozdnyakova: Scopio Labs: Consultancy. Wang: Stemline Therapeutics: Honoraria.

Abstract: Fluorescence in situ hybridization (FISH) is a confirmatory test to establish a diagnosis of inv(16)/t(16;16) AML. However, incidental findings and their clinical diagnostic implication have not been systemically studied. We studied 1629 CBFB FISH cases performed in our institution, 262 (16.1%), 1234 (75.7%), and 133 (8.2%) were reported as positive, normal, and abnormal, respectively. The last included CBFB copy number changes (n = 120) and atypical findings such as 3′CBFB deletion (n = 11), 5′CBFB deletion (n = 1), and 5′CBFB gain (n = 1). Correlating with CBFB-MYH11 RT-PCR results, totally 271 CBFB rearrangement cases were identified, including five with discrepancies between FISH and RT-PCR due to new partner genes (n = 3), insertion (n = 1), or rare CBFB-MYH11 variant (n = 1) and eight with 3′CBFB deletion. All cases with atypical findings and/or discrepancies presented clinical diagnostic challenges. Correlating FISH signal patterns and karyotypes, additional chromosome 16 aberrations (AC16As) show impacts on the re-definition of a complex karyotype and prognostic prediction. The CBFB rearrangement but not all AC16As will be detected by NGS-based methods. Therefore, FISH testing is currently still needed to provide a quick and straightforward confirmatory inv(16)/t(16;16) AML diagnosis and additional information related to clinical management.

Abstract: Background: Ibrutinib (IBR), a BTK inhibitor, and venetoclax (VEN), a BCL-2 inhibitor are approved for patients (pts) with CLL. The rationale for combining IBR and VEN includes: 1) preclinical models showing synergism, 2) non-overlapping toxicities; 3) non-overlapping mechanisms of action. We recently reported results of the first-line cohort of an investigator-initiated phase II trial of combined IBR and VEN for pts with CLL (Jain N et al. NEJM 2019). Here we report results of the 80 pts from the R/R CLL cohort of the trial (NCT02756897). Methods: Pts with R/R CLL meeting 2008 IWCLL treatment criteria were enrolled. Pts received IBR monotherapy (420 mg daily) for 3 cycles followed by addition of VEN (weekly dose-escalation to the 400mg daily target dose). Combined therapy was administered for 24 cycles. Pts with bone marrow (BM) undetectable MRD (U-MRD) (multi-color flow cytometry; sensitivity 10-4) at 24 cycles of combined therapy stopped both VEN and IBR; MRD+ pts could continue IBR. Response assessments were performed using blood, BM and CT imaging (2008 IWCLL criteria) at the following time-points (after cycle 3 of IBR monotherapy, and then after cycles 3, 6, 9, 12, 18, and 24 of combined therapy). Progression-free survival (PFS) was assessed as the time from the start of study drug to CLL progression, Richter transformation, or death. Overall survival (OS) was assessed as the time from the start of study drug to death. Results: A total of 80 pts were enrolled. The median age was 61.5 yrs (32-79). The baseline characteristics are shown in Table 1. Overall, 30 (38%) pts had a TP53 aberration. The median follow-up for all pts is 22.3 months. Of the 80 pts, 1 pt was later reclassified as splenic marginal zone lymphoma and is excluded from further analyses. Five pts came off study during IBR monotherapy phase (reasons listed below). 74 pts initiated VEN. Serial BM MRD responses are shown in Figure 1. One pt with a prior allo-SCT had no marrow disease at screening and is therefore excluded from serial MRD analyses. After 3 cycles of IBR monotherapy, none of the 73 pts achieved BM U-MRD. After addition of VEN, increasing proportions of pts achieved BM U-MRD remission. After 3 cycles of the combination, 7/68 (10%) achieved BM U-MRD remission. After 6 cycles of the combination, 15/67 (22%) achieved BM U-MRD remission. After 12 cycles of the combination, 29/60 (48%) achieved BM U-MRD remission. After 24 cycles of the combination, 16/24 (67%) achieved BM U-MRD remission. To assess the incremental benefit of the combined IBR and VEN beyond the first 12 cycles, we analyzed the MRD for pts who had received 24 cycles of combined therapy (n=24). Among these 24 pts, 13 were MRD+ at end of 12 cycles; 4/13 (31%) became U-MRD at end of 18 cycles of combination. Similarly, among these 24 pts, 9 were MRD+ at end of 18 cycles of combination; only 1/9 (11%) achieved U-MRD after 24 cycles of combination. PFS and OS are shown in Figure 2. Two pts had CLL progression after completing 24 cycles of combined therapy. 1 pt was in U-MRD remission at 24 cycles and stopped both IBR and VEN per study design; he relapsed 3 months later and responded to IBR monotherapy. The second pt was MRD+ at 24 cycles of combined therapy and continued IBR monotherapy after 2 yrs per study design; he relapsed few months later and is now in remission post CD19 CAR-T. One pt developed Hodgkin's transformation. Two pts died; both due to infectious complications during the IBR monotherapy. One pt developed severe cytopenias during the VEN dose escalation, was non responsive to multiple therapies for worsening cytopenias, then underwent an allogeneic stem cell transplant. One pt, with prior FCR therapy developed MDS. A total of 15 (19%) pts have come off trial; 5 pts came off study during IBR monotherapy (death from infection, n=2; skin rash, n=1; insurance issue, n=1; pt decision, n=1). Three pts came off trial during VEN ramp up (cytopenias, n=2; noncompliance, n=1). Four pts came off trial during the combination phase of IBR and VEN (arthralgia, n=1; Hodgkin's transformation, n=1; renal cancer, n=1; MDS, n=1). Three pts came off trial after completing 24 cycles of combined therapy (CLL progression, n=2; pt decision to continue VEN beyond 24 cycles, n=1). Grade 3-4 neutropenia occurred in 29% pts. Grade 3-4 thrombocytopenia occurred in 3% pts. Atrial fibrillation occurred in 7 (9%) pts. Conclusions: Combined VEN and IBR is an effective well-tolerated chemotherapy-free oral regimen for pts with R/R CLL. Disclosures Jain: Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding. Thompson:Gilead: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Pharmacyclics: Research Funding; Pfizer: Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Research Funding. Burger:Gilead Sciences: Research Funding; Pharmacyclics, an AbbVie company: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; Aptose Biosciences, Inc: Research Funding; BeiGene: Research Funding; AstraZeneca: Honoraria. Borthakur:Cyclacel: Research Funding; Eli Lilly and Co.: Research Funding; PTC Therapeutics: Consultancy; AbbVie: Research Funding; Xbiotech USA: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Research Funding; Agensys: Research Funding; Bayer Healthcare AG: Research Funding; AstraZeneca: Research Funding; BMS: Research Funding; Oncoceutics, Inc.: Research Funding; Tetralogic Pharmaceuticals: Research Funding; Merck: Research Funding; Arvinas: Research Funding; Polaris: Research Funding; Strategia Therapeutics: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; Cantargia AB: Research Funding; NKarta: Consultancy; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Eisai: Research Funding; Janssen: Research Funding; GSK: Research Funding; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding. Takahashi:Symbio Pharmaceuticals: Consultancy. Bose:Constellation: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Promedior: Research Funding; NS Pharma: Research Funding; CTI BioPharma: Research Funding; Incyte Corporation: Consultancy, Research Funding, Speakers Bureau; Blueprint Medicine Corporation: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Kartos: Consultancy, Research Funding. Fowler:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kadia:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Bioline RX: Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; AbbVie: Consultancy, Research Funding. Konopleva:Ablynx: Research Funding; Eli Lilly: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Agios: Research Funding; Astra Zeneca: Research Funding; Forty-Seven: Consultancy, Honoraria; Cellectis: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Ascentage: Research Funding; Genentech: Honoraria, Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Kisoji: Consultancy, Honoraria. Alvarado:Jazz Pharmaceuticals: Research Funding; Abbott: Honoraria. DiNardo:medimmune: Honoraria; agios: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; syros: Honoraria; daiichi sankyo: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; celgene: Consultancy, Honoraria; jazz: Honoraria. Pemmaraju:samus: Research Funding; abbvie: Consultancy, Honoraria, Research Funding; mustangbio: Consultancy, Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; novartis: Consultancy, Research Funding; plexxikon: Research Funding; Daiichi-Sankyo: Research Funding; sagerstrong: Research Funding; affymetrix: Research Funding; incyte: Consultancy, Research Funding; cellectis: Research Funding; celgene: Consultancy, Honoraria. Jabbour:Cyclacel LTD: Research Funding; Pfizer: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; BMS: Consultancy, Research Funding. Sasaki:Pfizer: Consultancy; Otsuka: Honoraria. Garg:Garglet LLC: Other: Owner; Enlitic inc.: Other: Advisor. Plunkett:Cyclacel Ltd: Research Funding. Kantarjian:Daiichi-Sankyo: Research Funding; Amgen: Honoraria, Research Funding; Takeda: Honoraria; Immunogen: Research Funding; AbbVie: Honoraria, Research Funding; Ariad: Research Funding; Astex: Research Funding; Cyclacel: Research Funding; Novartis: Research Funding; Jazz Pharma: Research Funding; Pfizer: Honoraria, Research Funding; Agios: Honoraria, Research Funding; BMS: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Wierda:Oncternal Therapeutics Inc.: Research Funding; Miragen: Research Funding; Genentech: Research Funding; Sunesis: Research Funding; Pharmacyclics LLC: Research Funding; KITE pharma: Research Funding; Acerta Pharma Inc: Research Funding; Juno Therapeutics: Research Funding; Gilead Sciences: Research Funding; Xencor: Research Funding; Janssen: Research Funding; Loxo Oncology Inc.: Research Funding; Cyclcel: Research Funding; AbbVie: Research Funding; GSK/Novartis: Research Funding. OffLabel Disclosure: Combination of ibrutinib and venetoclax is not FDA approved