Ferrer, Alejandro

Interstitial lung diseases (ILDs) in patients with shortened telomeres have not been well characterized. We describe demographic, radiologic, histopathologic, and molecular features, and p16 expression in patients with telomeres ≤10th percentile (shortened telomeres) and compare them to patients with telomere length >10th percentile. Lung explants, wedge biopsies, and autopsy specimens of patients with telomere testing were reviewed independently by 3 pathologists using defined parameters. High-resolution computed tomography scans were reviewed by 3 radiologists. p16-positive fibroblast foci were quantified. A multidisciplinary diagnosis was recorded. Patients with shortened telomeres (N=26) were morphologically diagnosed as usual interstitial pneumonia (UIP) (N=11, 42.3%), chronic hypersensitivity pneumonitis (N=6, 23.1%), pleuroparenchymal fibroelastosis, fibrotic nonspecific interstitial pneumonia, desquamative interstitial pneumonia (N=1, 3.8%, each), and fibrotic interstitial lung disease (fILD), not otherwise specified (N=6, 23.1%). Patients with telomeres >10th percentile (N=18) showed morphologic features of UIP (N=9, 50%), chronic hypersensitivity pneumonitis (N=3, 16.7%), fibrotic nonspecific interstitial pneumonia (N=2, 11.1%), or fILD, not otherwise specified (N=4, 22.2%). Patients with shortened telomeres had more p16-positive foci (P=0.04). The number of p16-positive foci correlated with outcome (P=0.0067). Thirty-nine percent of patients with shortened telomeres harbored telomere-related gene variants. Among 17 patients with shortened telomeres and high-resolution computed tomography features consistent with or probable UIP, 8 (47.1%) patients showed morphologic features compatible with UIP; multidisciplinary diagnosis most commonly was idiopathic pulmonary fibrosis (N=7, 41.2%) and familial pulmonary fibrosis (N=5, 29%) in these patients. In conclusion, patients with shortened telomeres have a spectrum of fILDs. They often demonstrate atypical and discordant features on pathology and radiology leading to diagnostic challenges.

Introduction Clonal cytopenias of undetermined significance (CCUS) is defined by the presence of somatic driver mutations/copy number variations in hematopoietic cells in patients with low blood counts, in the absence of morphological evidence for myeloid neoplasms. Patients with CCUS are often symptomatic and can be transfusion dependent (TD), with high rates of progression to myeloid neoplasms, especially myelodysplastic syndromes (MDS). In addition, similar to MDS, there are reports of CCUS patients responding to hypomethylating agents. Due to the lack of a formal diagnosis/morphological dysplasia, CCUS patients are often denied therapies or clinical trial enrollment. We carried out this study to validate our hypothesis that CCUS shares similar clinical and survival characteristics with lower risk MDS (LR-MDS). Methods CCUS patients were prospectively identified from the clonal hematopoiesis clinic, whereas LR-MDS patients were retrospectively identified from our institutional database. LR-MDS was defined as very low, low and intermediate risk MDS based on the R-IPSS categorization. Baseline demographics, blood counts, bone marrow (BM) morphology, cytogenetics, and NGS results were abstracted. Transfusion dependency was defined as requiring at least one unit of red cell or platelets every 4 weeks. The Mann-Whitney-U and Fischer's exact test were used to compare quantitative and qualitative data in subgroups. Kaplan-Meier overall survival (OS) estimates were used for survival analysis and compared using the log-rank test. Results 186 patients were included in the study; 74 (40%) with CCUS and 112 (60%) with LR-MDS, median age 66 years, with 63% being male (Table 1). In the CCUS group, 93% had one or more mutations (67% >1 mutation) detected by NGS, while 7% had clonal cytogenetic abnormalities. Common mutations in CCUS included TET2 (30%), SRSF2 (20%), DNMT3A (13%) and ASXL1 (11%); with 28% of patients being red cell TD and 15% being platelet TD. On application of the R-IPSS stratification, 40%, 45%, 12% and 3% were in the very low, low, intermediate and high risk categories, respectively. LR-MDS subtypes included MDS-RS (50%), MDS-MLD (21%), MDS-EB (10%), MDS del5q (7%), and MDS-U (12%). SF3B1 mutations were seen in 67% (95% of MDS-RS), while TET2 and DNMT3A mutations were seen in 29% and 21%, respectively. 60% of LR-MDS patients had >1 mutation. 44% were red cell TD, while 10% were platelet TD. In comparison to patients with CCUS, LR-MDS patients were more likely to have higher white blood cell counts (p=0.002), higher neutrophil counts (p=0.009), higher platelet counts (p<0.001), be red cell TD (p=0.031), have BM ring sideroblasts (p<0.001), have higher BM blast% (p=0.004) and carry SF3B1 mutations (p<0.001); whereas CCUS patients were more likely to have higher monocyte counts (p=0.009) and more likely to carry IDH1 (p=0.005), ZRSR2 (p=0.005) and ATM (p=0.012) mutations. Importantly there were no differences with regards to R-IPSS cytogenetic risk groups, R-IPSS prognostic categories, platelet TD, and mutations involving TET2, DNMT3A, SRSF2 and ASXL1 and TP53. Notably signal pathway mutations and mutations involving tumor suppressor genes were infrequent (<5%) in both groups. The LR-MDS patients had a longer median follow up (53 vs 15 months) and at last follow up 72 (64%) and 11 (15%) deaths had been documented in the LR-MDS and CCUS groups respectively. 13 (18%) CCUS patients progressed to MDS (10) and AML (3) over a median of 15 months, while 9 (8%) LR-MDS patients progressed to higher grade MDS (1) and AML (8), respectively (median follow up 53 months). There was no difference in the median survival between CCUS (median OS not reached) versus LR-MDS (median OS 8.3 years) (p=0.372, Figure 1). Conclusion In spite of subtle phenotypic/molecular differences between LR-MDS and CCUS, both entities had similar prognostication, distribution of high risk mutations and survival outcomes. Within a short follow-up, 18% of CCUS patients progressed to MDS/AML, indicating that regardless of the absence of dysplasia these entities are a continuum of myeloid neoplasms. In order to improve access to treatments and clinical trial opportunities, we recommend that close consideration be given to consider CCUS as a MDS subtype. Disclosures Shah: Dren Bio: Consultancy.

Introduction Germline predisposition syndromes (GPS) are inherited disorders associated with germinal aberrations that increase the risk of malignancies. While aberrations in certain genes increase the risk for all types of malignancies (Tp53, ATM, CDKN2A, CHEK2), there is a growing list of genes associated specifically with hematological malignancies (GATA2, RUNX1, DDX41, ETV6, ANKRD26). At our institution, we have established a hematology GPS clinic to diagnose and manage GPS and with this report, detail our experience with 130 patients. Methods GPS were investigated in pediatric and adult patients with one or more first degree relatives with hematological/visceral malignancies or in those with antecedent thrombocytopenia (ANKRD26, RUNX1, ETV6), or with specific syndromic features (short telomere syndromes/STS, GATA2 haploinsufficiency, Fanconi anemia/FA, Shwachman-Diamond syndrome/SDS). Depending on the phenotype, specific functional assays such as flow-FISH for telomere length assessment and chromosomal breakage assays were ordered. After informed consent and genetic counselling, germline testing was carried out on peripheral blood mononuclear cell, skin fibroblast, or hair follicle-derived DNA. A custom-designed marrow failure NGS panel (200 genes) was used in most cases and interrogation of variants, in silico studies, and functional assays were carried out as previously described (Mangaonkar et al MC Proc 2019). Copy number variations were identified by aCGH. At the time of progression/worsening cytopenias, bone marrow/lymph node biopsies and NGS (next generation sequencing) were carried out where indicated. Results 130 patients with germline predisposition have been identified to date. The spectrum of disorders seen include STS 29 (22%), FA 17 (13%), GATA2 16 (12%), Diamond Blackfan anemia/DBA 13 (10%), RUNX1-FPD 12 (9%), ATM deletions/mutations 11 (8%), ANKRD26 6 (5%), SDS 5 (4%), DDX41 4 (3%), MPL 3 (2%), CHEK2, MECOM, Tp53 mutations 2 (2%) each, and CBL, CEPBA, ELANE, NF1, CDKN2A, CSF3R, ETV6, and GATA1 mutations, 1 (1%) each. Evidence for clonal evolution (CCUS) and hematological malignancies were seen in 51 (39%) patients, involving all the aforementioned genes/syndromes with the exception of DBA, CBL, ETV6, MPL, CSF3R, and GATA1. Seven (64%) of 11 patients with germline ATM deletions/mutations developed lymphoid malignancies; homozygous ATM (Follicular NHL-1, Burkitt lymphoma-1, T-ALL-1, T-LPD-1) and heterozygous ATM (T-PLL-1, DLBCL-1, CLL-1). Clonal evolution occurred in 11 (69%) of 16 GATA2 haploinsufficient patients (CCUS-2, MDS-3, CMML-1, AML-5) and in 7 (58%) of 12 RUNX1-FPD patients (CCUS-1, MDS-1, MDS/MPN-3, AML-2). Five of 29 (17%) STS patients had clonal progression (CCUS-2, MDS-2, AML-1), and 5 (29%) of 17 FA patients progressed to MDS-2 or AML-3. JMML was seen in one patient with a germline NF1 mutation, while 1 (20%) of 5 SDS patients progressed to AML. NGS data at progression was available in 24 (55%) of 44 myeloid/CCUS progressions, with somatic truncating ASXL1 mutations being most frequent (29%), followed by RAS pathway mutations (15%). AML/MDS progressions in STS, FA, and SDS were universally associated with complex/monosomal karyotypes, translating to refractory disease. Seventeen (39%) of 44 patients with myeloid predisposition underwent allogenic HCT (GATA2-7, FA-3, RUNX1-FPD-3, STS-2, NF1-1, Tp53-1), with 10 (59%) being alive at last follow up (Table 1). Conclusion We demonstrate the spectrum of germline aberrations associated with predisposition to hematological malignancies and outline the phenotypic heterogeneity of clonal transformation. The advent of NGS allows identification of clonal progression earlier than morphological changes, with mutations in ASXL1 and RAS pathway genes being commonly implicated. This study supports the universal development of dedicated germline predisposition clinics. Disclosures Pruthi: CSL Behring: Honoraria; Genentech Inc.: Honoraria; Bayer Healthcare: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria.

Abstract In this study, we aimed to characterize the molecular mechanisms of two very rare subtypes of AML: acute megakaryoblastic leukemia (AMKL) and acute panmyelosis with myelofibrosis (APMF). Our goal is to describe the clinical-pathologic features of APMF and explore the genomic landscape AMKL to further understand these rare malignancies. We identified 35 patients diagnosed with AMKL (n=27) and APMF (n=8) at Mayo Clinic between 1995 and 2015 by retrospective chart review. For 10 of the AMKL patients, tissue was available for whole-exome sequencing and was analyzed in comparison to a published AML reference population. The median overall survival was shorter in patients with AMKL (3.9 months, 95% CI 1.7-7.4) than in patients with APMF (14.3 months, 95% CI 0.7-NE, p = 0.026,). For both AMKL and APMF, patients who underwent allogeneic hematopoietic stem cell transplantation had better overall survival (14.3 months, 95% CI 1.9-NE) compared to those not undergoing transplantation (4.6 months, 95% CI 1.5-7.4, p = 0.003). Patients with AMKL presented more frequently with splenomegaly, with higher peripheral blood blast counts, and with higher serum lactate dehydrogenase concentrations. 9 out of 10 AMKL patients with available tissue for sequencing demonstrated variants in 22 of 35 AML driver genes, with JAK2 V617F as the most common mutation. Although our small study numbers preclude firm conclusions, it does reflect the rarity of these AML subtypes. Furthermore, inferior survival in AMKL (compared to APMF) was reflected in its proliferative disease features such as splenomegaly, high blast counts, and high lactate dehydrogenase. Interestingly, the presence of JAK2 V617F variants in 40% of the interrogated samples was higher than expected. Although JAK2 is a known molecular driver in myeloproliferative neoplasms, it is actually very rare in de novo AML (~1%). Despite improved survival with allogeneic stem cell transplant, we can conclude that treatment outcomes for AMKL and APMF with standard-of-care therapy were sobering and that patients with these rare AML subtypes should undergo molecular evaluation to qualify them for clinical trials with novel targeted therapies. Citation Format: Marissa Li, Moritz Binder, Basma Basha, Ben Zhang, Alejandro Ferrer, Yuanhan Liu, Mrinal Patnaik, David Viswanatha, Naseema Gangat. Acute megakaryoblastic leukemia is associated with poor overall survival and enriched in JAK2 variants [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B03.

ABSTRACT Inactivating mutations of the ENPP1 gene are associated with generalized arterial calcification of infancy (GACI) and less often autosomal-recessive hypophosphatemic rickets type 2 (ARHR2). We aimed to investigate the spectrum of phenotypes in a family with monoallelic and biallelic mutations of ENPP1 after identification through whole exome sequencing of a 54-year-old female with biallelic mutation of ENPP1, c.323G > T; p.Cys108Phe and c.1441C > T; p.Arg481Trp. Including the proband, 2 subjects had biallelic mutations, 5 had monoallelic mutations, and 2 had no mutation of ENPP1. The maternal mutation, a known pathogenic variant associated with GACI, was found in 3 subjects with monoallelic mutations, while the paternal mutation, which was not previously reported, was present in 2 subjects with monoallelic mutations. Both subjects with biallelic mutations had bowing of bilateral femurs, periarticular mineral deposition, normocalcemic primary hyperparathyroidism with multigland parathyroidectomy, increased carotid intima-media thickness, and enthesopathy was also noted in one subject. Intact FGF23 was elevated in both subjects with biallelic mutations, while C-terminal FGF23 was only elevated in one and PPi was reduced in one. Subjects with monoallelic mutations did not have periarticular calcifications or bone deformities. To conclude, patients with biallelic GACI causing mutations can survive well into adulthood, and despite the same biallelic ENPP1 pathogenic variants, clinical and biochemical manifestations can significantly differ, and include enthesopathy and primary hyperparathyroidism, which have not been previously described. Although carriers of monoallelic ENPP1 variants appear unaffected by classic disease manifestations, there may be subtle biochemical and clinical findings that warrant further investigation. © 2019 American Society for Bone and Mineral Research.

Introduction: Short telomere syndromes (STS) are accelerated aging syndromes affecting hematopoietic, pulmonary, hepatobiliary and/or immunological systems. Clinical assessment of age-appropriate telomere length (TL) is performed using flow cytometry & fluorescence in-situ hybridization (flowFISH). Screening for germline variants in STS-related genes is guided by flowFISH-determined centile categories of TL, with screening recommended for TL <1st centile or 1-10th centile in lymphocytes (L) or granulocytes (G). However, the utility of genetic testing for patients with TL >10th centile and integration of clinical phenotype with flowFISH data in predictive algorithms is currently unclear. Methods: FlowFISH testing was done at reference laboratories in Vancouver (Repeat Diagnostics; Canada) & Johns Hopkins University (JHU, USA). Salient clinical features were pre-determined as, personal history of premature hair greying (onset at age < 30 years), idiopathic pulmonary fibrosis (IPF) or IPF/emphysema overlap (in smokers), cryptogenic cirrhosis or NRH, unexplained cytopenias &/or immunodeficiency, & family history of the above (in >1 1st or 2nd degree relatives). Clinical likelihood score (CLS) was assigned as low (1), intermediate (int, 2) or high (>2), based on the number of aforementioned clinical features present prior to flowFISH testing. Genetic testing was performed using either an in-house or commercial bone marrow failure-specific next generation sequencing (NGS) panel or whole exome sequencing (WES), and data for known variants affecting telomerase or telomeric function (TERT, TERC, DKC1, TINF2, NHP2, NOP10, TCAB1, NAF1, & RTEL1) was recorded. Results: One hundred forty-nine patients at our institution underwent TL assessment at Repeat diagnostics (n=38) and JHU (n=111) laboratories, respectively. Median age was 56 (range: 7-79) years; 88 (59%) being males. Significant family history was present in 40 (27%) patients, while premature greying of hair was present in 13 (9%) patients. Organ-specific clinical features included unexplained cytopenias (n=89, 60%) IPF (n=71, 48%), cryptogenic cirrhosis or NRH (n=21, 14%), & unexplained immunodeficiency (n=14, 9%). CLS stratification included low (n=74, 50%), int (n=54, 36%), & high (n=21, 14%), with higher CLS significantly correlating with lower delta TL for L (p=0.0005) but not G (p=0.3). Genetic testing was performed in 51 (35%) patients (NGS-51, WES-1) among which 13 (26%) patients had a telomere-associated variant; 5 (10%) pathogenic (pv, all TERT). CLS alone was unable to predict likelihood of finding a telomere-associated variant (p=0.4). Based on age-appropriate centile categorization of L & G TL (information for both available in 134 patients), patients were stratified into six groups (table 1). TL <1st centile in L: This group was further divided into two groups; TL<1st centile in both L & G [A1, n=7, CLS low-3 (43%), int-2 (29%), & high-2 (29%)] and TL <1st centile in L and 1-50th centile in G (A2, n=2, CLS low & high) patients. Among the 4 (57%) patients who underwent genetic (NGS-3, WES-1) testing, 3 (75%) had TERTpv in A1 subgroup and 1 A2 subgroup patient had a VUS in TERT.TL <1st centile in G, 1-10th centile in L (n=18): This group included 9 (50%) low, 8 (44%) int and 1 (6) high CLS patients, of which only 1 of 8 NGS-tested patients had TERTpv.TL 1-10th centile in L or G: This group was divided into; 1-10th centile in both L & G [C1, n=28, CLS low-3 (11%), int-14 (50%), high-11(39%)] of whom 8 (22%) underwent NGS with no pathogenic variants but 3 VUS in RTEL1, NAF1 & PARN genes, and 1-10th centile in L, >1-90th centile in G [C2, n=36, CLS low-19 (53%), 16 (44%), 1 (3%)] of whom 8 (22%) underwent NGS with 1 TERTpv and 2 VUS in TINF2TL >10th centile in L & 1-90th centile in G (n=43, 32%): CLS stratification in this group included 27 (63%) low, 12 (28%) int, 4 (9%) high. NGS testing was done in 13 (30%) patients [CLS low-9(69%), int 2(15%), high 2 (15%)], of whom only 2 (15%) had VUS in TINF2 and TERT gene, but no pathogenic variants (figure 1). Conclusion: Our study demonstrates the importance of using a flowFISH assay based predictive algorithm to screen patients with suspected STS for telomere-related genetic alternations, in comparison to a clinical likelihood score. We also demonstrate a limited role for genetic testing in patients with lymphocyte TL >10th centile, regardless of the clinical likelihood score. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Quantitative activity through the TCR-proximal protein network specifies thymic positive versus negative selection.