Lasho, Terra L.

Abstract Background Histiocytic disorders are rare hematologic neoplasms characterized by their high clinical heterogeneity. Evidence of constitutive activation of the mitogen-activated protein kinase (MAPK) pathway in a number of these disorders has led to the increasing use of BRAF- and MEK-inhibitors as a therapeutic strategy. Response to these therapies is not universal and additional effective treatment options are required. Immune checkpoint inhibitors have proven effective for a wide array of malignancies. Tumor mutational burden (TMB), the total number of somatic pathogenic variants per coding region in a tumor genome, is a clinical biomarker associated with response to immunotherapy. We have previously reported preliminary findings of low TMB in a small cohort of patients with histiocytic neoplasms (Goyal G et al. Blood 2019). In this study, our aim was to confirm the findings in a larger clinical cohort using next generation sequencing studies. Methods A retrospective review of adult patients consecutively seen at Mayo Clinic from 2017 to April 2021 and diagnosis of a histiocytic neoplasm was performed. Electronic records were queried for demographic and laboratory data of all patients who consented to undergo tumor-tissue next-generation sequencing with the Tempus-xT ® or xO ® assay (Chicago, IL), throughout the course of their evaluation. TMB was reported as the number of nonsynonymous mutations per coding area of a tumor genome. TMB across groups was compared via an independent-samples T-test or via analysis of variance and post hoc subgroup testing via Turkey's Test as required. Results Seventy-three patients were included in the study. Individual diagnoses included: Erdheim-Chester disease (ECD) in 31.5% (n=23), including 3 patients with overlap features of ECD and other histiocytoses; Rosai Dorfman disease (RDD) in 28.8% (n=21); Langerhans cell histiocytosis in 21.9% (n=16), histiocytic sarcoma (HS) in 5.5% (n=4), and other histiocytic neoplasms in 12.3% (n=10) including adult xanthogranuloma, xanthoma disseminatum, undifferentiated histiocytoses, and one case of Langerhans cell sarcoma. Median tumor percentage on analyzed samples was 30% (range 10%-90%) and was ≤20% in 29 samples. TMB was <1.0 mutations per megabase (mpMb) in 42.5% (n=31) and >5.0 mpMb in only 6 cases. Median TMB was 1.58 (range 0 - 5.26) for ECD, 1.08 (range 0 - 6.3) for LCH, 0.2 (range 0 - 5.8) for RDD, 3.19 (range 1.67 - 6.8) for HS, and 1.6 (range 0 - 7.5) for other histiocytic neoplasms (Figure 1). No clear association between increasing TMB and number of systems with histiocytic infiltration was observed (Figure 2). A significant increase in TMB was observed among the 5 cases of sarcoma as compared to the rest of the cohort (p=0.014). When not considering sarcomas, no differences were observed in TMB between patients with ECD, LCH, RDD or other histiocytoses (F=0.775; p=0.512). Conclusion In this large cohort of histiocytic disorders, TMB was low compared to that historically seen in other neoplasms though this is likely influenced by tumor purity ≤20% in 29 cases. Only 6 tumors had TMB reports with mpMb above the threshold thought to be associated with an increased likelihood of response to PD-1/PDL1-targeted therapies (>5 mpMb). Our results suggest a low likelihood of response among histiocytic disorders using immune checkpoint inhibitors. Further exploration among malignant histiocytoses (HS and Langerhans cell sarcoma) is warranted given our findings of higher TMB compared with other histiocytic neoplasms. Figure 1 Figure 1. Disclosures Bennani: Kymera: Other: Advisory Board; Vividion: Other: Advisory Board; Kyowa Kirin: Other: Advisory Board; Daichii Sankyo Inc: Other: Advisory Board; Purdue Pharma: Other: Advisory Board; Verastem: Other: Advisory Board.

Abstract Introduction: Mutations involving isocitrate dehydrogenase 1/2 (IDH) are known oncogenic drivers in hematological malignancies, conferring neomorphic enzymatic activity to IDH 1/2, resulting in the oncometabolite, 2-hydroxyglutarae (2-HG). 2-HG in turn suppresses TET activity, making IDH and TET2 mutations synthetically lethal and almost mutually exclusive. The frequency of IDH mutations in CMML is <10% and their prognostic impact remains unclear. We carried out this study in a large database of molecularly annotated CMML patients to better define the clinical profile and prognostic impact of these mutations. Methods: After IRB approval, CMML patients from the Mayo Clinic, Minnesota and the Moffitt Cancer Center (MCC), Tampa, Florida, were included in the study. All patients had bone marrow (BM) biopsies with cytogenetics and molecular genetics done either at diagnosis, or at first referral. Clinical and mutational data were abstracted and retrospectively analyzed. Overall survival (OS) was calculated from date of CMML diagnosis to date of death/last follow, while AML-free survival (AML-FS) was calculated from date of CMML diagnosis to date of leukemic transformation (LT). Patients that had undergone allogeneic HCT were excluded from the study (n=3). Statistical analysis was carried out using the Blue Sky software. Results: Six hundred and forty four patients were included in the study (Mayo Clinic-357, MCC- 287), median age 71 years (range, 20-95 years), 67.8% being male. Forty-three (6.7%) patients had IDH mutations, 35 (82%) IDH2 and 8 (18%) IDH1; of which, 34 (97%) involved the IDH2R140 hotspot and 5 (62.5%) involved the IDH1R132 hotspot, respectively. The median variant allele fractions (VAF) for IDH1 mutations was 41% (range, 8-46%) and for IDH2 mutations was 46% (range, 7-70%). There were no significant demographic or clinical differences between IDH mutant and wild type CMML patients, with the exception that IDH mutant CMML patients were less likely to be thrombocytopenic (p=0.006), were less likely to have TET2 co-mutations (14% vs 53.2%; p<0.001) and were more likely to have SRSF2 co-mutations (69.8% VS 40.3%; p<0.001). Importantly there were no differences in proliferative or dysplastic subtypes (p=0.3), cytogenetic (p=0.12) and molecular risk stratifications (p=045). There were also no significant demographic or clinical differences between IDH1 vs IDH2 mutant CMML patients. Six (14%) IDH mutant CMML patients had TET2 co-mutations; 5 (83%) with IDH2R140Q (median VAF-28%;all male) and 1 (17%) with IDH1R132H (VAF-44%;female) (Figure 1). Five (11%) IDH2 mutant patients were treated with enasidenib (IDH2 inhibitor), none with a durable response, while none of the IDH1 mutant patients received targeted therapy. At last follow up (median 18 months), 337 (52%) deaths and 119 (18.5%) LT have been documented, with IDH mutant patients having a higher LT rate (30.2% vs 17.6%, p=0.04) compared to wildtype patients. The median OS of the entire cohort was 35 months, with no difference in OS between IDH mutant and wild type patients (34.5 vs 35 months, p=0.12), with IDH1 mutant patients having a shorter OS in comparison to IDH2 mutant patients (31 vs 37 months; p=0.005- Figure 1). IDH mutant CMML patients also had a shorter AML-FS in comparison to wild type patients (36.6 vs 210 months, p=0.005), with there being no differential impact on AML-FS of IDH1 vs IDH2 mutations (p=0.26, Figure 1). Conclusions: IDH mutations are infrequent in CMML (7%), with IDH2 mutations being more common than IDH1 mutations (80 vs 20%). IDH mutations co-occur very infrequently with TET2 mutations (14%), with IDH mutant patients being less likely to have thrombocytopenia and more likely to have SRSF2 co-mutations. IDH mutations negatively impacting AML-FS without a significant impact on OS. Prospective clinical trials testing the safety and efficacy of IDH1/2 inhibitors in CMML are much needed. Figure 1 Figure 1. Disclosures Komrokji: AbbVie: Consultancy; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Speakers Bureau; Acceleron: Consultancy; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Geron: Consultancy. Al-Kali: Novartis: Research Funding; Astex: Other: Research support to institution. Padron: BMS: Research Funding; Stemline: Honoraria; Taiho: Honoraria; Kura: Research Funding; Incyte: Research Funding; Blueprint: Honoraria. Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding.

Abstract Background: Chronic myelomonocytic leukemia (CMML) is an aging disease that arises on the background of clonal hematopoiesis, commonly involving multiple TET2 mutations or TET2/SRSF2 co-mutations. In a prior study of 1084 CMML patients (Leukemia 2020; 34:1407-1421), we demonstrated a high prevalence of TET2 mutations (56%) in CMML, with approximately 30% of patients having >1 TET2 mutation. The cell-type and allele specific distribution of these mutations remains of great interest and are thus far poorly defined. To visualize the genotypic and immunophenotypic correlates, we performed single-cell DNA analysis and simultaneous protein profiling on 2 CMML patients with multiple TET2 mutations. Methods: After Mayo Clinic IRB approval, two CMML patients with dual TET2 mutations and available peripheral blood mononuclear cells were included in the study. Patient samples were labeled with TotalSeq TM -D Heme Oncology Cocktail (BioLegend, San Diego, CA) consisting of 45 unique oligo-conjugated cell surface markers. The samples were then washed and loaded into a Tapestri microfluidics cartridge, where single cells (3,500 cells/µL) were encapsulated, lysed and barcoded using the MissionBio Myeloid custom amplicon panel consisting of 312 amplicons in 45 genes relevant to myeloid disorders (MissionBio, San Francisco, CA). The individual barcoded DNA and protein for each cell was purified, recovered, and labeled with Illumina i5/i7 indices, quanitifed by Agilent Bioanalyzer and pooled for sequencing. The libraries were sequenced via Illumina NovaSeq by the Mayo Genomics Core, and data was recovered and analyzed using MissionBio Tapestri Insights v3.0.2. Results: Two dual TET2 mutated CMML patients were included in the study, both male, ages 79 and 83, and both with CMML-0. Case 1 had TET2 p.G1172Efs*53 (variant allele fraction/VAF-6%) and p.H1732Tfs*13 (VAF-13%) mutations, while case 2 had TET2 p.Y620Ffs* (VAF-85%), p.G898* (VAF-82%), and SRSF2 p.95H (VAF-45%) mutations, respectively. In both patients, TET2 mutations were found to be preferentially distributed in monocytes (CD11b+, CD14+, CD64+, CD13+, CD33+, HLA-DR+), with very few classically defined granulocyte (CD16+,CD13+.CD33+, HLA-DR neg), B (CD19+,CD22+)/T (CD2+,CD4+,CD5+,CD7+) lymphocytes, NK cells (CD16+/CD56+/HLA-DR neg) and plasma cells (CD38+/CD138+), demonstrating mutant TET2. Among monocytes, TET2 mutations were enriched in classical/M01 (CD11b+, CD14+,CD16-) and intermediate/M02 monocytes and were infrequent in the MO3 fraction, commensurate with monocyte repartitioning seen in CMML. Interestingly, a large number of single cells with TET2 mutations also demonstrated an aberrant surface phenotype, with universal expression of CD14+ and CD11b+, along with a mixture of other markers (CD163+, CD11c, CD4+, CD2+, and/or CD56+). With regards to allelic distribution, while several permutations and combination were seen involving heterozygosity and homozygosity for the TET2 mutations identified on bulk sequencing, data was corroborative for both TET2 mutations being located on trans alleles. Conclusions: In this study, we demonstrate that TET2 mutations in CMML are highly enriched in classical circulating monocytes and infrequently occur in classical granulocytes, B/T and NK cells; with multiple TET2 mutations most commonly co-existing on trans alleles. Analyses on additional samples along with copy number variation assessments are ongoing. Figure 1: Lollipop plot of TET2 exons with dual TET2 mutations labeled above and oxygenase domain labeled in green; below are number of cells identified with each genotype for Case 1 (A) and Case 2 (B). Mutations are labeled proximal (yellow) and distal (red) and alleles are in grey. The directionality of the arrows to different genotypes is based on decreasing cell number and genotype. Uniform manifold approximation and projection (UMAP) plot of Case 1 (C) and Case 2 (D) with cells clustered by immunophenotype and labeled by genotype (non TET-2 mutated purple, heterozygous TET2 in green, homozygous TET2 in yellow). Relative protein expression of T-cell phenotype (CD14-, CD3+, CD4+/-,CD8+/-, CD5+, CD7+, CD2+), or classical monocytes (CD14+/CD16-) for Case 1 (E & F) and Case 2 (G & H) overlayed on UMAP. Royal blue represents non TET2-mutated cells, and mutated cells are labeled in pastel blue, green and light orange by mutation type. Figure 1 Figure 1. Disclosures Durruthy-Durruthy: Mission Bio Inc.: Current Employment. Arribas-Layton: Mission Bio, Inc.: Current Employment. Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding.

AbstractGermline predisposition syndromes (GPS) result from constitutional aberrations in tumor suppressive and homeostatic genes, increasing risk for neoplasia in affected kindred. In this study, we present clinical and genomic data on 144 Mayo Clinic patients with GPS; 59 evaluated prospectively using an algorithm‐based diagnostic approach in the setting of a dedicated GPS/ inherited bone marrow failure syndrome (IBMFS) clinic. Seventy‐two (50%) patients had IBMFS (telomere biology disorders‐32,Fanconi anemia‐18, Diamond Blackfan Anemia – 11, congenital neutropenia–5, Schwachman‐Diamond Syndrome‐5 and Bloom Syndrome‐1), 27 (19%) had GPS with antecedent thrombocytopenia (RUNX1‐FPD‐15, ANKRD26‐6, ETV6‐2, GATA1‐1, MPL‐3), 28 (19%) had GPS without antecedent thrombocytopenia (GATA2 haploinsufficiency‐16, DDX41‐10, CBL‐1 and CEBPA‐1) and 17 (12%) had general cancer predisposition syndromes (ataxia telangiectasia‐7, heterozygous ATM variants‐3, CHEK2‐2, TP53‐2, CDK2NA‐1, NF1‐1 and Nijmegen Breakage Syndrome‐1). Homozygous and heterozygous ATM pathogenic variants were exclusively associated with lymphoproliferative disorders (LPD), while DDX41 GPS was associated with LPD and myeloid neoplasms. The use of somatic NGS‐testing identified clonal evolution in GPS patients, with ASXL1, RAS pathway genes, SRSF2 and TET2 being most frequently mutated. Fifty‐two (91%) of 59 prospectively identified GPS patients had a change in their management approach, including additional GPS‐related screening in 42 (71%), referral for allogenic HSCT workup and screening of related donors in 16 (27%), medication initiation and selection of specific conditioning regimens in 14 (24%), and genetic counseling with specific intent of fertility preservation and preconceptual counseling in 10 (17%) patients; highlighting the importance of dedicated GPS screening, detection and management programs for patients with hematological neoplasms.

Abstract The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. Significance: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression. This article is highlighted in the In This Issue feature, p. 1307