Clonal Hematopoiesis and COVID-19 Severity in Cancer Patients
Background: Advanced age, medical co-morbidities and a pro-inflammatory immunologic profile are associated with severe COVID-19. Acquired somatic mutations in hematopoietic stem cells (clonal hematopoiesis or CH) are common in elderly individuals. Previous studies have shown CH is associated with alterations in immunologic repertoire and function. Here, we investigate whether CH predisposes to severe COVID-19 or other infections. Methods: We performed a retrospective cohort study of 39,291 adult cancer patients with non-hematologic malignancies who had their tumor and blood sequenced using a panel of 468 known cancer driver genes. We identified individuals who tested positive for SARS-CoV-2 between March 8, 2020 and June 9, 2020. We determined the association between CH and severe COVID-19 (hypoxic event, hospitalization or death) using multivariable logistic regression models. We used an established aggregation schema for ICD-CM codes to identify distinct infection subtypes. The relationship between CH and risk of incident infections was determined using multivariable Cox regression. Results: In the study cohort, 436 patients were positive for SARS-CoV-2 and 165 developed severe COVID-19. COVID-19 patients with CH were more likely to develop severe disease compared to non-severe (OR=1.8; 95% CI: 1.1-2.9; p=0.01). CH was also more common among patients with severe COVID-19 compared to COVID-19 negative individuals (OR=1.6, 95% CI: 1.2-2.4, p=0.01). In 62,891 person-years of follow-up, 4,059 individuals developed an infection. CH was associated with risk of sepsis (HR=1.13; 95% CI 1.01-1.25, p=0.04) and bacterial enteritis (HR=1.8, 95% CI 1.1-2.8, p=0.01). Conclusions: CH is associated with severe COVID-19 and an increased risk of other infections in cancer patients. Disclosures Bolton: GRAIL: Research Funding. Jee:MDSeq Inc.: Patents & Royalties. Papaemmanuil:Celgene: Consultancy, Honoraria, Research Funding; Prime Oncology: Consultancy, Honoraria; Illumina: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; MSKCC: Patents & Royalties; Kyowa Hakko Kirin: Consultancy, Honoraria; Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Berger:Illumina: Research Funding; Grail: Research Funding; Roche: Consultancy. Levine:Prelude Therapeutics: Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy; Astellas: Consultancy; Morphosys: Consultancy; Novartis: Consultancy; Amgen: Honoraria; Gilead: Honoraria. Zehir:Illumina: Honoraria; Memorial Sloan Kettering Cancer Center: Current Employment.
Background Stably acquired mutations in hematopoietic cells represent substrates of selection that may lead to clonal hematopoiesis (CH), a common state in cancer patients that is associated with a heightened risk of leukemia development. Owing to technical and sample size limitations most CH studies have characterized gene mutations or mosaic chromosomal alterations (mCAs) individually. The relationship between acquired gene mutations and mCAs in CH and their joint roles in leukemia development have not been systematically investigated. Methods We developed a method to reliably map mCAs at low cell fractions from deep targeted sequencing data. We applied this method in a cohort of 32,442 solid tumor patients who have undergone prospective clinical sequencing (MSK-IMPACT). We characterized gene mutations in our patient cohort using an established variant calling procedure from our previous studies. Results We jointly characterized 383 mCA events (median aberrant cell fraction 32%, range 10%-90%) and 14,789 mutations across 457 genes. mCA was significantly associated with age (OR=1.8, P<0.001), male gender (OR=1.4, P=0.012), white race (OR=1.5, P=0.033) and prior receipt of external beam radiation therapy (OR=1.7, P=0.022). 217 (63%) mCAs co-occurred with at least one gene mutation, while 129 (37%) did not (OR=3.9, P<0.001). mCA was especially enriched in CH cases with high mutation number and VAF, detectable in 5.8% of subjects with ≥3 gene mutations and 4.8% of those with mutations at >20% VAF, compared to 1% of the general cohort. We identify co-mutational patterns characteristic of diverse mechanisms of clonal selection. We observe that mutations in DNMT3A, TET2, JAK2, MPL, EZH2, TP53 and ATM form recurrent double-hits with deletions or CNLOHs, resulting in either oncogene mutant dosage adjustment or inactivation of tumor suppressors. Notably, certain mCA events were highly directed events acting on previously acquired gene mutations in the corresponding loci. Of six events of 7qCNLOH, all six co-localized with an EZH2 (7q36.1) mutation (q<0.001). Of 12 cases with 9pCNLOH, 11 (92%, q <0.001) co-localized with a JAK2 V617F mutation. 4 out of 9 (44%, q <0.001) 1pCNLOH events co-localized with a MPL (1p34.2) mutation. In addition, we observe recurrent composite genotypes (4q24-/SRSF2, 7qCNLOH/ASXL1, 20q-/U2AF1) indicative of co-operating or epistatic interactions as well as loss of gatekeeper function (i.e. TP53) presenting with multiple chromosomal aneuploidies (5-, 7-, 3+). In total, these recurrent composite genotypes resembling known genetic interactions in leukemia genomes underlie 23% of all detected autosomal mCAs. During patient follow-up, the 3-year cumulative incidence of leukemias was significantly higher in patients with composite CH genotypes (14.6%, CI: 7-22%) as compared to patients with either mCA, gene mutation alone or no CH, of which all had a 3-year cumulative incidence of <1% (Figure 1). We performed a multivariable cause-specific Cox regression model and showed that mCA was independently predictive of subsequent leukemia diagnosis (HR=14, 95% CI: 6-33, P=1.2e-09) after adjusting for number of gene mutations and VAF in putative drivers. Conclusions Our joint characterization of gene mutations and mCAs in a large prospective sequencing cohort reveals a previously unrecognized layer of complexity in the evolutionary dynamics of clonal hematopoiesis that converges towards characteristic genotypes associated with distinct leukemia subtypes. This puts mCAs in the context of the continuous evolutionary process of oncogenesis that can often span years and sheds new lights on its patterns of acquisition and progression. We demonstrate that the integration of chromosomal aberrations provides additional resolution to risk stratification as well as interpretation of clinical phenotypes and that mCAs should be screened in conjunction with gene mutations to improve existing CH surveillance programs in cancer patients. Disclosures Bolton: GRAIL: Research Funding. Medina:Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Mantha:MJH Associates: Honoraria; Physicians Education Resource: Honoraria. Solit:Pfizer: Honoraria; Loxo Oncology: Honoraria; Lilly Oncology: Honoraria; Illumina: Honoraria; Vivideon Therapeutics: Honoraria. Diaz:Neophore: Consultancy, Current equity holder in private company; Merck: Consultancy; Johns Hopkins University: Patents & Royalties; Jounce Therapeutics: Current equity holder in private company; Thrive Earlier Detection: Current equity holder in private company; Personal Genome Diagnostics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Berger:Illumina: Research Funding; Roche: Consultancy; Grail: Research Funding. Levine:Lilly: Consultancy, Honoraria; Janssen: Consultancy; Roche: Consultancy, Honoraria, Research Funding; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Prelude Therapeutics: Research Funding; Gilead: Honoraria; Amgen: Honoraria; Morphosys: Consultancy; Novartis: Consultancy; Astellas: Consultancy; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Zehir:Memorial Sloan Kettering Cancer Center: Current Employment; Illumina: Honoraria. Papaemmanuil:Celgene: Consultancy, Honoraria, Research Funding; Prime Oncology: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Illumina: Consultancy, Honoraria; Kyowa Hakko Kirin: Consultancy, Honoraria; Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; MSKCC: Patents & Royalties.
Abstract Background. DNA methylation is a key epigenetic process involved in development, aging, and cancer. Mutations in DNMT3A and TET2 in the hematopoietic stem cell compartment lead to increased self-renewal. In addition to mutations in ASXL1, collectively, these DTA mutations are recognized as an aging phenomenon, known as the most common Clonal hematopoiesis of Indeterminate Potential (CHIP) mutations and alone are not predictive of increased risk for hematopoietic malignancy. Recently, DNMT3A mutations in donor hematopoietic cells were suggested to be associated with enhanced T-cell activity in allografted patients. Additionally, role of DNMT3A mutations in creating a proinflammatory state in cardiovascular disease setting and associated elevation of T-cell markers in the myocardium have been recently explored (Sano S et al. Circ Res. 2018). Since an inflamed tumor microenvironment is associated with improved immune checkpoint inhibitors (CPI) activity, we sought to determine the impact of CHIP (a proinflammatory state) on response to CPI and CPI's effects on clonal dynamics. Additionally, while classical chemotherapy (CTX) can create selective external pressure providing survival advantage to mutant stem cells, the selective pressure of T-cell activating therapies on hematopoietic stem cells is unclear. Methods. To study the relationship between CHIP and CPI, we used paired peripheral-blood samples taken before and after treatment with CPI therapy in patients (pts) with melanoma (MEL; n= 32) and non-small cell lung cancer (NSCLC; n=109). Serial samples (or post CPI samples) were evaluable in 5 MEL pts and 6 NSCLC pts. Error-corrected sequencing of a targeted panel of genes recurrently mutated in clonal hematopoiesis (CH) was performed on peripheral blood genomic DNA. Statistical comparisons between baseline and serial sample VAFs were performed using two-sided fisher's exact test, with a p < 0.05 considered significant. Results. In both the MEL and NSCLC cohort, baseline samples were collected before extensive therapy exposure. 90% (29/32) of the MEL cohort had no CTX or targeted therapy prior to the baseline sample; 28% (9/32) had prior radiotherapy (RT). 10% (11/109) of the NSCLC cohort samples had prior CTX, but only 2 of these were treated for more than 1 month before sample collection. CH was frequent in these minimally pre-treated patient samples; 28.1% (9/32) and 37.6% (41/109) of the baseline MEL and NSCLC samples, respectively. As expected, DTA mutations were the most common events in these cohorts. Samples with CH were from patients of older age, but had normal hematological parameters with exception of increased RDW (p=0.022). Primary tumor responses in this cohort were defined as durable (receipt of ≥12 CPI cycles) or not durable (<12 cycles). DNMT3Amut patients (VAF ≥1%, n=5) had more durable responses, i.e. higher median number of CPI cycles (21 cycles, range:10-40) compared to non-DNMT3Amut pts (7 cycles, range:1-13; p= NS). Additionally, pts with larger DNMT3Amut clones (figure 1- MEL cohort) tended to receive higher numbers of CPI cycles. In the serial sample analysis, we observed that mutations in DNMT3A and TET2 increased in size with longer CPI exposures (Figure 2, MEL cohort); pts 2, 3 and 5 received 13, 15 and 18 CPI cycles respectively, while pt 4 with the most notable clonal expansion in DNMT3A received 40 CPI cycles. All serial samples in MEL cohort showed a statistically significant change in VAF from baseline. In the serial sample analysis of NSCLC pts, we observed that those with ≥ 3 months of CPI exposure demonstrated decreases in clone size for non-DTA gene mutations such as SRCAP, STK11 and TPM1 (Table 1), but increases or stability in DNMT3A and TET2 mutations (Table 1). However, this VAF increase in DNMT3A and TET2 mutations in NSCLC cohort was not statistically significant. Conclusions. In this small cohort of pts with MEL and NSCLC, the presence of DNMT3A/TET2 CH was associated with longer checkpoint inhibitor exposure and increased allelic frequency over time. These findings need further validation in larger cohorts and delineation of the relationship between DTA mutations such as DNMT3A and enhanced immune activity. Acknowledgement: Data and samples for this study were provided by the Data Bank and BioRepository (DBBR), which is funded by the National Cancer Institute (P30 CA016056) and is a Roswell Park Cancer Institute Cancer Center Support Grant shared resource. Figure 1 Figure 1. Disclosures Griffiths: Taiho Oncology: Consultancy, Honoraria; Alexion Pharmaceuticals: Consultancy, Research Funding; Novartis: Honoraria; Boston Biomedical: Consultancy; Astex Pharmaceuticals: Honoraria, Research Funding; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Apellis Pharmaceuticals: Research Funding; Genentech: Research Funding; Takeda Oncology: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Hassane: Tempus Labs, Inc: Current Employment. Guzman: SeqRx: Consultancy; BridgeMedicines: Consultancy; Cellectis: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Wang: Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Genentech: Membership on an entity's Board of Directors or advisory committees; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kura Oncology: Consultancy, Honoraria, Other: Advisory board, steering committee, Speakers Bureau; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Other: Advisory Board; Mana Therapeutics: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory board, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Advisory board; DAVA Oncology: Consultancy, Speakers Bureau; Rafael Pharmaceuticals: Other: Data safety monitoring committee; Gilead: Consultancy, Honoraria, Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory board; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory board; Genentech: Consultancy; MacroGenics: Consultancy.
Abstract Introduction Multiple myeloma (MM) causes lytic bone lesions, osteopenia, and fractures, which increase the morbidity of MM patients. Results from small previous studies have indicated that fractures in MM have a negative effect on survival. Aims The aim of the study was to evaluate the impact of fractures on survival in MM patients diagnosed in Sweden in the years 1990-2013. Furthermore, to analyze the effect of bone fractures at MM diagnosis on subsequent survival. Methods Patients diagnosed with MM in 1990-2013 were identified from the Swedish Cancer Registry. Information on date of birth, diagnosis, and death were collected from the Registry of Total Population. Information on all fractures were retrieved from the Swedish Patient Registry. Cox regression model was used with fractures as time-dependent variables. The effect of fractures on survival was assessed for any fracture or a subtype of fracture (a specific bone fracture or ICD-coded pathologic fracture). Either first fracture or the first subtype of fracture was used in the analysis. The effect of a fracture at MM diagnosis (within 30 days before or 30 days after MM diagnosis) on survival was also estimated using a Cox regression model. All models were adjusted for age, sex, time of diagnosis, and previous fractures. Results A total of 14,008 patients were diagnosed with MM in the study period. A total of 4,141 (29.6%) patients developed a fracture including fractures that occurred within a year before MM diagnosis and thereafter. Hereof 2,893 (20.7%) patients developed a fracture after MM diagnosis. The risk of death was significantly increased for patients that developed a fracture after the time of MM diagnosis with a hazard ratio (HR) of 2.00 (95% confidence interval (CI) 1.91-2.10) for all fractures combined. The risk of death was significantly increased for patients that developed all subtypes of fractures after MM diagnosis except ankle fractures. The risk of death was significantly increased for patients that developed pathologic fractures (HR=2.17; 95% CI 2.03-2.32), vertebral fractures (HR=1.73; 95% CI 1.61-1.87), hip fractures (HR=1.99; 95% CI 1.82-2.18), femoral fractures (HR=2.62; 95% CI 2.32-2.98), humerus fractures (HR=2.57; 95% CI 2.32-2.86), forearm fractures (HR=1.24; 95% CI 1.05-1.46), and rib fractures (HR=1.52; 95% CI 1.31-1.77), but not for ankle fractures (HR 1.07; 95% CI 0.79-1.44). A total of 942 (6.7%) of all MM patients were diagnosed with a fracture within 30 days before or 30 days after MM diagnosis. The patients with a fracture at diagnosis were at a significantly increased risk of death compared to those without (HR 1.31; 95% CI 1.21-1.41; Figure) Conclusions Our large population-based study, including over 14,000 patients diagnosed with MM in Sweden in the years 1990-2013, showed that MM patients that developed a fracture after the time of diagnosis were at twofold increased risk of dying compared to MM patients without a fracture. Furthermore, MM patients with a fracture at diagnosis had a 30% higher risk of dying compared to patients without a fracture. Our results indicate that fractures in MM reflect a more advanced disease at diagnosis and stress the importance of managing MM bone disease in all MM patients. Figure. Figure. Disclosures Landgren: Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding.
Background Recent evidence suggests that age-related clonal hematopoiesis (CH) might represent the earliest precursor of myeloid neoplasms. Although the exact mechanism of clonal selection that shapes CH is still to be elucidated, both cell intrinsic and non-cell intrinsic effects of mutations, including the interplay between mutated cells and the bone marrow environment, are thought to play important roles, which are best studied using single-cell sequencing analysis of both mutations and gene expression. Methods We performed single-cell sequencing of hematopoietic stem and progenitors (HSPCs) from BM of the 16 patients with CH along with 16 control patients without CH identified by screening otherwise healthy individuals who received hip joint replacement, using a novel platform that enables simultaneous detection of gene mutations and expression based on the Fluidigm C1-HT system. Sequence data were analyzed with Seurat (Stuart et al Cell 2019) with integration of genotyping information. Cells were clustered and each cluster was assigned by marker-gene expressions for major cell-types in HSPCs, including hematopoietic stem cell (HSC)-like and erythroid progenitors. Cells were grouped by their genotypes and pathway analysis were performed. Results In total, we identified 35 subjects who had CH-related mutations, including those affecting DNMT3A, TET2, ASXL1, SF3B1, PPM1D, IDH1, GNB1 and TP53, of which 11 had more than one CH-related mutation. Most of these mutations showed a low variant allele frequency (VAF) ≤ 0.05. However, clones having double mutations of DNMT3A/TET2 or those having biallelic TET2 mutations tended to show a higher VAF as high as 0.4, suggesting an enhanced clonal advantage for clones having multiple mutations. Using our novel single-cell platform, we analyzed 3,767 cells from control patients without CH and 1,474 mutated cells and 7,234 wild-type (WT) cells from patients with CH. By targeting both genomic DNA and RNA, we successfully obtained a sufficient number of single-cell reads for genes whose expression was too low to evaluate by only targeting RNA, such as TET2 and DNMT3A. Although some clones having a high-VAF mutation caused a skewed clustering to be detected as a CH clone, many clones with low-VAF mutations did not make distinct clusters, indicating the importance of genotyping at a single cell level to identify and characterize mutated cells. Simultaneous detection of genotype and expression allowed us to see the effect of CH-mutations on cell phenotype and differentiation. For example, cells having compound TET2/DNMT3A mutations were significantly enriched in the erythroid cluster, while another clone with double TET2 mutations were more enriched in the HSC-like cluster, compared to cells from individuals without CH (WTcont). These are in line with the previous findings of TET2/DNMT3A double knockout mice or TET2 knockout mice, respectively. In another case with an IDH1 mutation, IDH1-mutated (MUTIDH1) cells less contributed to the HSC-like fraction, showing an enhancement of cell proliferation-signature, compared to WT (WTIDH1) cells in the same patient. Strikingly, compared to WTcont cells, WTIDH1 cells were significantly enriched in the HSC-like fraction and showed an enhanced expression of cytokine-related pathway genes, which was in line with a finding seen in mouse cells treated with 2-hydroxy-glutalate, an mutant IDH-related oncometabolite. Similarly, when compared to WTcont cells, WT cells from patients with DNMT3A- (WTDNMT3A) or TET2- (WTTET2) mutated CH significantly showed an enhanced cell proliferation. HSC-like WTTET2 cells also showed aberrant IFN-response signatures compared to corresponding WTcont cells, which was confirmed in competitive transplantation of Tet2 heterozygous knockout (hKO) and WT cells in a mouse model; HSPCs of WT competitors transplanted with Tet2-hKO cells showed a significant enhancement of IFN-response signatures compared to those transplanted with WT cells. Intriguingly, monocytes of Tet2-hKO donors showed aberrant expression of S100a8/a9, which might contribute to the non-cell intrinsic effect of Tet2-hKO cells. Conclusions In CH, not only mutated cells but also surrounding WT cells show an aberrant gene expression phenotype, suggesting the presence of non-cell autonomous phenotype or an altered bone marrow environment that favors the positive selection of CH-clones. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Ogawa:Eisai Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.
Abstract Background Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related phenomenon characterized by the presence of somatic mutations in peripheral blood (PMIDs: 25426837, 25426838). Although CHIP was originally defined in healthy older adults without cytopenias, it can be found in other contexts as well. For example, one recent report described four patients with therapy-related myeloid neoplasm (TMN) arising after treatment for other cancers, all of which were driven by TP53 mutations that could be found at very low levels in samples drawn years before the development of TMN (PMID: 25487151). However, there has not yet been a more systematic study of CHIP in this type of context. In this study, we sought to understand how CHIP behaves and influences outcomes in the context of autologous stem cell transplantation (ASCT), arguably the most extreme selective pressure that can be studied in the context of native hematopoiesis. We hypothesized that in patients with Non-Hodgkin Lymphoma (NHL) undergoing ASCT, the presence of CHIP at the time of transplantation would be associated with an increased risk of TMN and other adverse outcomes. Methods We analyzed exome sequencing data from 10 patients with TMN after ASCT (City of Hope Cancer Center, Duarte, CA), and performed targeted sequencing of 116 genes on banked, mobilized peripheral blood from an additional 401 patients with NHL who underwent ASCT (Dana Farber Cancer Institute, Boston, MA), to determine whether there is a clonal connection between CHIP at the time of ASCT and subsequent TMN, and to determine whether the presence of CHIP at the time of ASCT influences subsequent outcomes. Results In 7 of 10 TMN patients for whom we analyzed exome sequencing data, mutations present at the time of TMN were also detectable in the pre-ASCT sample. PPM1D, a key mediator of the DNA damage pathway, was mutated in 2 patients, as was TP53 (2 patients), TET2 (2 patients) and PRPF8 (1 patient). In our larger cohort of 401 unselected ASCT patients, CHIP was common (121 patients, 30.2%) and was associated with older age but not with other demographic or treatment-related factors. PPM1D was the most commonly mutated gene (54 mutations in 48 patients). In the ASCT cohort of 401 patients, 18 patients developed TMN. The presence of CHIP at the time of ASCT significantly increased this risk: the 10-year cumulative incidence of TMN, with death and allogeneic transplant as competing risks, was 12.4% for patients with CHIP, compared to 3.5% for patients without CHIP (P=0.002, Figure 1A). Moreover, the presence of CHIP at the time of ASCT conferred significant risks beyond TMN alone, as patients with CHIP had significantly inferior overall survival compared to patients without CHIP (10-year OS 30.6% versus 60.9%, P=0.0003, Figure 1B). This difference was driven primarily by late mortality and not by an increased risk of relapse or by the difference in rate of TMN. Although other variables were associated with OS in univariate analysis, multivariate analysis in a Cox proportional hazards model showed that only older age (60 or above), aggressive lymphoma, and presence of CHIP were significantly associated with survival. Conclusion We show that CHIP at the time of ASCT for NHL is common and is associated with an increased risk of TMN and decreased overall survival independent of the TMN risk. These results have substantial clinical and translational implications. They suggest the need to specifically study the connection between CHIP and lymphoma more deeply, which could be accomplished by assessing CHIP in patients with newly diagnosed lymphoma prior to the administration of any chemotherapy or mobilizing agents. They also suggest the need to consider alternative therapeutic approaches for patients with lymphoma and a high risk of TMN who are being considered for ASCT. Finally, they underscore the need to study clonal hematopoiesis in the context of treatment for other cancers to determine whether these results may be relevant to an even larger number of patients. Disclosures Lindsley: MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy. Mar:H3 Biomedicine: Other: Spouse's employment. LaCasce:Forty Seven: Consultancy; Seattle Genetics: Consultancy; Seattle Genetics: Consultancy. Koreth:LLS: Research Funding; amgen inc: Consultancy; takeda pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; kadmon corp: Membership on an entity's Board of Directors or advisory committees; prometheus labs inc: Research Funding; millennium pharmaceuticals: Research Funding. Ritz:Kiadis: Membership on an entity's Board of Directors or advisory committees. Soiffer:Kiadis: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.
Abstract BACKGROUND: Healthy adults with clonal hematopoiesis of indeterminate potential (CHIP) are at increased risk of death from vascular disease. CHIP-associated genetic mutations such as ASXL1, TET2 and DNMT3A have been linked to the pathogenesis of atherosclerosis (Jaiswal et al, 2017). MDS and CHIP share common genetics events; in fact, certain MDS cases are believed to arise from preexisting clonal hematopoiesis. A recent SEER study demonstrated that 60 months from diagnosis, patients (pts) with low-risk MDS are at higher risk of death from cardiovascular causes than from MDS itself (Brunner et al, 2017). As the relationship between vascular disease and clonal hematopoiesis is delineated, management of vascular disease in patients with MDS is becoming increasingly important. Here we performed a retrospective analysis of MDS pts from Roswell Park to evaluate whether MDS-related factors such as morphologic subtype, treatment, cytopenias, iron status, blast percentage and karyotype modify atherosclerosis risk when molecular events are taken into account. METHODS: Over the past decade, 850 pts were diagnosed with MDS were seen and treated at Roswell Park. For the purpose of this study, 192 pts with a confirmed diagnosis of MDS seen between 2012- 2017 were investigated for cardiac events, blood counts, iron or inflammatory status as ferritin, MDS-related morphology, molecular profile and treatment. Cases were comprehensively annotated for the presence of vascular events, (defined as imaging or procedure verified coronary artery disease, cerebrovascular accident, or peripheral vascular disease) by review of the medical record. Conventional karyotype information was present in 98% of the cohort and NGS based multi-gene sequencing results through FoundationOne Heme was evaluated in 30% of the cohort. Numeric variables were summarized using simple descriptive statistics. A logistic regression model was used to investigate the association between vascular events and a set of explanatory variables for multivariate analysis. RESULTS: The median age at diagnosis for this cohort is 69 (range: 21-91) years; 60% were male and 57% had MDS with excess blasts. By IPSS-R, 25% had very low/low risk MDS, 22% had intermediate risk MDS and 46% had high risk MDS. The most common karyotypic abnormalities were normal (42%), complex (17%), trisomy 8 (10%) and del 7/7q (10%). Of the 59 patients with molecular data, the most common recurrent somatic mutations were in ASXL1 (47%), TET2 (30%), SRSF2 (27%), RAS pathway (22%) SF3B1 (22%), RUNX1 (20%) and TP53 (17%). The overall incidence of vascular events in this cohort was 27%. Vascular disease was noted at similar frequencies for pts with low grade and high grade MDS (based on subtype of MDS), 23% vs. 30% (p=0.33) respectively. Women with MDS were significantly less likely to develop to vascular events than were men, 16% vs. 34% (p=0.007) and the mean age of pts with vascular disease was significantly higher, 71 vs. 65 years (p=0.005). Traditional risk factors such as hypertension and hyperlipidemia were more prevalent in MDS pts with vascular disease. Baseline hemoglobin, transfusion requirements and blast % levels were comparable in MDS patients with or without vascular disease. MDS pts with vascular disease had higher median ferritin levels, 432 vs. 301 ng/ml (p=0.093). When stratified by the IPSS-R, pts with very low risk MDS had a non-significant lower incidence of cardiac events, 14% vs. 25% (p=0.30), compared to other groups. Treatment with erythropoietin, lenalidomide or hypomethylating agents was not associated with vascular events. MDS pts with mutations in ASXL1 and SRSF2 had a higher incidence of vascular disease, 43% vs. 13% (p=0.01) and 38% vs 20% (p=0.18) respectively. On multivariable analysis, older age and male gender were most strongly predictive for vascular events. CONCLUSION: Here we retrospectively examined the potential factors associated with cardiovascular events in MDS pts treated at our institute over a 5-year time span. Male gender and older age were positively associated. Treatment regimen (erythropoietin injections or lenalidomide) and IPSS-R were not associated with an increased risk for vascular events. Factors independent of traditional cardiac risk factors, such as somatic mutations in ASXL1, iron overload and/or an inflammatory milieu i.e, ferritin, may contribute to atherosclerotic vascular disease in pts with MDS. Disclosures Baron: Pfizer Pharmaceuticals: Other: Previously served as a consult on the Advisory Boards (May 2017).. Wang:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Novartis: Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Jazz: Speakers Bureau; Jazz: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Griffiths:Pfizer, Inc.: Research Funding; Celgene, Inc: Honoraria, Research Funding; Astex/Otsuka Pharmaceuticals: Honoraria, Research Funding; Novartis, Inc.: Research Funding; Alexion Inc.: Honoraria, Research Funding. Thota:Incyte: Speakers Bureau.
Background Allogeneic stem cell transplantation (HSCT) involves the transfer of healthy donor hematopoietic cells, including hematopoietic stem cells (HSCs) and mature immune effector cells, to recipients with high-risk hematologic malignancies. The success of HSCT is fundamentally dependent on engraftment of normal donor-derived hematopoiesis. Rarely, donor-derived cells undergo transformation to myeloid leukemia as a late complication of HSCT (donor cell leukemia, DCL), a process linked in some cases to pre-existing mutations in donor hematopoietic stem cells (HSCs). In healthy older adults, HSC mutations can cause clonal hematopoiesis of indeterminate potential (CHIP), which is associated with increased risk of hematologic malignancy and an increased risk of all-cause mortality. Among donors over age 40, the frequency of donor CHIP can be estimated to be 5 to 20-fold higher than the frequency of donor cell leukemia, suggesting that non-leukemic endpoints of donor derived clonality may be more prevalent and consequential than previously recognized. Methods We performed targeted next generation sequencing in patients who developed peripheral cytopenias or myeloid neoplasms in the context of 100% donor cell chimerism after allogeneic HSCT. We also prospectively sequenced potential donors who had abnormal CBC parameters, including WBC differential and RBC indices. To determine whether the mutations detected in recipients were of donor derivation, and to assess clonal evolution over time, we performed ultra-deep genotyping at 30,000-100,000X or droplet digital PCR (ddPCR) in the donor stem cell product and serial post-SCT samples. Results We identified four allogeneic SCT recipients with 100% chimerism who met inclusion criteria, two with late-onset hematologic malignancy (latency 23 and 10 years) and two with progressive cytopenias but no evidence of hematologic malignancy on bone marrow examination (latency 18 and 13 months). In each of these patients, sequencing of bone marrow or peripheral blood specimens revealed at least one somatic mutation (SF3B1 K666Q; U2AF1 S34F; DNMT3A T868N; DNMT3A Q356X) that could be used to assess the possibility of donor derivation. In all patients, we identified at least one mutation present in both the diagnostic post-HSCT recipient sample and the pre-HSCT banked donor stem cell product. Figure 1 shows post-HSCT clonal evolution from one case. In another case, we evaluated contemporaneous samples from the donor and recipient obtained at the time of diagnosis of DCL in the recipient (23 years after HSCT). We identified two key findings: 1) a shared SF3B1 K666 mutation in donor and recipient, and 2) divergent clonal evolution, with ASXL1 C789X in the donor, and ASXL1 G642fs, SETBP1 D868N, and SUZ12 D605E in the recipient (Figure 2). In this context, the recipient had a morphologic diagnosis of MDS/MPN overlap, while the donor had abnormal RBC indices but no cytopenias. Since CHIP is associated with subtle abnormalities in hematologic parameters even in the absence of overt cytopenias, we evaluated potential donors with non-exclusionary CBC abnormalities, such as abnormal white blood cell differential or elevated MCV. In two cases, we prospectively identified DNMT3A mutations (R736Cand R882H, variant allele fractions 11.5% and 2%, respectively), consistent with donor CHIP. In one case, the donor with CHIP was used for transplantation. At 90 days after HSCT, donor cell chimerism was 99%; sequencing of recipient peripheral blood revealed the donor-derived DNMT3A mutation and the recipient remained persistently anemic, macrocytic, and thrombocytopenic. Conclusion We identified healthy stem cell donors with clonal hematopoiesis, marked by mutations in canonical genetic drivers of myeloid malignancies. In each instance, the aberrant clone engrafted in the transplant recipient, underwent selective expansion, and caused development of abnormal hematopoietic function. Our findings suggest that donor-derived clonal hematopoiesis may be common, especially in grafts from older donors, and that donor CHIP may confer an unrecognized and increased risk of leukemic and non-leukemic endpoints. We further demonstrate that donor CHIP can be identified prospectively, thus highlighting an emerging challenge in HSCT that may influence donor selection strategies as the clinical significance of donor CHIP is systematically elucidated. Disclosures Ritz: Kiadis: Membership on an entity's Board of Directors or advisory committees. Soiffer:Juno: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Lindsley:MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy.
Clonal hematopoiesis (CH), defined by expansion of clones in the hematopoietic system, has been linked to internal (aging) and external (smoking and oncologic therapy) factors in patients with advanced solid tumors. The effects of inherited variants, however, have yielded inconclusive results in few studies so far. While comparison of CH prevalence between monozygotic and dizygotic pairs failed to detect genetic predisposition to CH, others have shown associations with germline variants near TERT and TET2, and a potential link between pathogenic CHEK2 germline variants and CH has been proposed. Here, utilizing prospective tumor-blood paired sequencing data from a large institutional clinical cohort, we link inherited pathogenic variants in cancer predisposition genes to CH and show interactions between germline and somatic mutations in DNA damage repair (DDR) genes. We analyzed sequencing data from 32,748 cancer patients who have undergone MSK-IMPACT testing using paired tumor and blood samples to study the associations between CH and rare pathogenic and likely-pathogenic (P/LP) germline variants. P/LP germline mutations were identified in the blood using an in-house developed random forest algorithm, trained using known P/LP mutations. Following joint anonymization of P/LP germline mutations and CH variants, linear regression models by gene were used to test for associations, accounting for known CH confounders. In our cohort, 9,697 patients (29.6%) harbored at least one CH mutation, while P/LP germline variants were detected in 1,793 of these CH patients. P/LP germline variants in CHEK2 (OR=1.29, p=4.49x10-2) were found to be significantly associated with CH in a pan-cancer analysis. Interestingly, germline P/LP and CH co-mutations in CHEK2 were very rare; we identified only one patient with CH and germline mutations in CHEK2 out of 161 patients with germline CHEK2 mutations, compared to 304 patients with CH CHEK2 mutation when CHEK2 is wild-type in the germline. In contrast, ATM and TP53 showed increased number of patients with co-mutations in P/LP germline and CH mutations in the same gene, with 14 out of 84 patients with germline mutations in ATM compared to 254 when not mutated (OR=7.05, p=2.08x10-7) , and 3 out of 16 compared to 387 for TP53 (OR=10.94, p=5.44x10-3). Further, we found mutual exclusivity between CHEK2 P/LP germline variants and CH mutations in PPM1D (OR=0.18, p=1.79x10-2). This relationship was replicated by comparing CHEK2 germline mutations and somatic PPM1D mutations in the solid tumor samples within the same cohort and same pattern of mutual exclusivity also persisted in comparisons of other DDR pathway genes (i.e. ATM vs PPM1D and TP53 vs PPM1D). When studying specific cancer types, a significant association between CHEK2 P/LP germline variants and CH in breast cancer patients was also identified (OR=2.33, p=2.64x10-3). Furthermore, trends between germline mutations in ATM and CH rates in breast cancer (OR=1.84, p=9.82x10-2) and lung adenocarcinoma (OR=2.22, p=8.91x10-2) patients were observed. Our results support the already reported link between rare pathogenic germline mutations in CHEK2 with CH in cancer patients, with stronger association among breast cancer patients (Comen E. et al, 2019). Moreover, this study suggests a close relationship between inherited variants and CH mutations within the DDR genes in solid tumor patients and points out to interesting mutual exclusivity patterns between these same genes. Intriguingly, hematopoietic stem cells have been largely related to proficient DDR systems in order to regulate HSC maintenance and tissue homeostasis in the hematopoietic system. All in all, associations identified in this study might translate into enhanced clinical surveillance for CH and associated comorbidities cancer patients harboring these germline mutations. Disclosures Bolton: GRAIL: Research Funding. Papaemmanuil:Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Illumina: Consultancy, Honoraria; Prime Oncology: Consultancy, Honoraria; Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Kyowa Hakko Kirin: Consultancy, Honoraria; MSKCC: Patents & Royalties. Levine:Prelude Therapeutics: Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Astellas: Consultancy; Morphosys: Consultancy; Novartis: Consultancy; Amgen: Honoraria; Gilead: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Berger:Illumina: Research Funding; Grail: Research Funding; Roche: Consultancy. Zehir:Illumina: Honoraria; Memorial Sloan Kettering Cancer Center: Current Employment.
Introduction The World Trade Center (WTC) disaster exposed first responders to high levels of aerosolized carcinogens (Lioy et. al. Env. Health Perspect 2002). Clonal hematopoiesis is associated with exposure to smoking and genotoxic stimuli (Jaiswal et. al. NEJM 2014; Genovese et. al. NEJM 2015). We sought to determine its incidence in WTC-exposed first responders. We also assessed the effect of WTC particulate matter (WTC-PM) on genome integrity in vitro, and in murine studies. Methods Deep targeted sequencing was performed on blood collected from 481 first responders (429 WTC-exposed firefighters, 52 WTC-exposed emergency medical service workers) and 52 non-exposed first responders. Samples were analyzed for 237 genes mutated in hematologic malignancies and interpreted using reference databases. Non synonymous somatic mutations were annotated and analyzed. Results In the WTC-exposed cohort, 57 individuals with 66 somatic mutations of expected pathogenic potential were identified (overall prevalence 11.9%). In the non-exposed cohort, only one pathogenic mutation was found in the IDH2 gene (overall prevalence 1.9%). There was a strong association between increasing age and prevalence of mutations in the WTC-exposed cohort (Fig 1A). DNMT3A (16/66), TET2 (7/66), SF3B1 and SRSF2 (3/66 each) were the most common genes identified in the WTC-exposed cohort (Fig 1B). Median VAF was 12% and missense mutations were most frequent alteration. Aging, smoking, DNA repair and alkylating agent exposure related mutational signatures were observed with a cytosine to thymine (C→T) transition being most common. Next, we assessed the effect of WTC-PM on genome integrity and replication in vitro. WTC-PM that was collected in the first three days after 9/11 was used in concentrations mimicking exposure levels. Lymphocytes exposed to WTC-PM demonstrated a significant increase in phosphorylated H2AX foci accumulation, suggesting a DNA damage response (Fig 2). Since common fragile sites (CFSs) detect basal levels of stress in the cell, and activate DNA damage response (DDR), we profiled DNA replication dynamics at CFS-FRA16D at very high resolution using the single molecule analysis of replicated DNA (SMARD) assay. Treatment with WTC-PM significantly altered replication at two common fragile sites (regions 1 and 2 of FRA16D, Fig 3A) with replication pausing being observed at multiple sites (Fig 3B-I, white rectangles). Striking increase in replication initiation was seen, characterized as dormant origins activated to rescue replication pausing (Fig 3E, J). These alterations were accompanied by a corresponding increase in replication speed, conditions that lead to DNA replication errors and mutagenesis (Fig 3F, K). Next, we treated mice with WTC-PM via the oropharyngeal route to mimic first responder exposures, and then harvested and analyzed their bone marrow compartments. Significant expansion of hematopoietic stem cells (Kit+, Sca1+, Lineage-ve, KSL) was seen in WTC-PM treated mice (Fig 4A,B). Whole genome sequencing of sorted stem cells showed a significant increase in non-synonymous SNPs, deletions and indels in the WTC-PM treated samples when compared to control (Fig 4C-E). These genomic alterations were found to occur at low VAF throughout the whole genome, demonstrating widespread genotoxic effects of WTC-PM on hematopoietic stem cells in vivo (Fig 4F). Discussion We report a high burden of mutations in 11.9% (57/481) WTC-exposed first responders compared to the non-exposed cohort (1.9%, 1/52). The frequency of the somatic mutations was many fold higher than in previous studies (Jaiswal et. al. NEJM 2014; Genovese et. al. NEJM, 2015). In the 50-59 year age group, 10% of WTC-exposed individuals carried somatic mutations, compared to the frequency of 2.5% reported by Jaiswal et. al. for the same age group. Despite deeper sequencing performed in our study, the median VAF in our study was 12%, indicating that the difference in technique did not bias our study towards increased detection of small, subclinical clones when compared to previous studies. Furthermore, we demonstrate that WTC-PM can perturb DNA replication and increased genomic instability in vivo, potentially leading to higher burden of clonal hematopoiesis in WTC-exposed first responders. These results demonstrate adverse environmental exposures can be associated with a high rate of clonal hematopoiesis. Disclosures Landgren: Sanofi: Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Theradex: Other: IDMC; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Fletcher:Genoptix/Neogenomics: Employment. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Celgene: Research Funding; Deerfield: Research Funding. Steidl:GlaxoSmithKline: Research Funding; Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Will:Novartis Pharmaceuticals: Research Funding. Verma:Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding.
T-cell large granular lymphocyte leukemia (T-LGLL) is a clonal proliferation of cytotoxic T lymphocytes (CTL). T-LGLL mainly manifest in elderly and is associated with autoimmune diseases including rheumatoid arthritis (RA), B cell dyscrasias, non-hematologic cancers and immunodeficiency (e.g., hypogammaglobulinemia). LGL manifestations often resemble reactive immune processes leading to the dilemmas that LGLs act like CTL expansion during viral infections (for example EBV associated infectious mononucleosis). While studying a cohort of 246 adult patients with T-LGLL seen at Cleveland Clinic over the past 10 years, we encountered 15 cases of overt T-LGLL following transplantation of solid organs (SOT; n=8) and hematopoietic stem cell transplantation (HSCT; n=7). Although early studies reported on the occurrence of LGL post-transplant, these studies focused on the analysis of oligoclonality skewed reactive CTL responses rather than frank T-LGLL. We aimed to characterize post-transplantation T-LGLL in SOT and HSCT simultaneously and compare them to a control group of 231 de novo T-LGLL (cases with no history of SOT or HSCT). To characterize an unambiguous "WHO-defined T-LGLL" we applied stringent and uniform criteria. All cases were diagnosed if 3 out of 4 criteria were fulfilled, including: 1) LGL count >500/µL in blood for more than 6 months; 2) abnormal CTLs expressing CD3, CD8 and CD57 by flow cytometry; 3) preferential usage of a TCR Vβ family by flow cytometry; 4) TCR gene rearrangement by PCR. In addition, targeted deep sequencing for STAT3 mutations was performed and charts of bone marrow biopsies were reviewed to exclude other possible conditions. Diagnosis was made 0.2-27 yrs post-transplantation (median: 4 yrs). At the time of T-LGLL diagnosis, relative lymphocytosis (15-91%), T lymphocytosis (49-99%) and elevated absolute LGL counts (>500 /µL; 93%) were also seen. Post-transplantation T-LGLL were significantly younger than de novo T-LGLL, (median age: 48 vs. 61 yr; P<.0001). Sixty% of post-transplantation T-LGLL patients were males. Fifteen% of patients had more cytogenetic abnormalities compared to de novo T-LGLL, had a lower absolute LGL count (median: 4.5 vs. 8.5 k/µL) and had less frequent neutropenia, thrombocytopenia and anemia (27 vs. 43%, 33 vs. 35% and 20% vs. 55%; P=.01). TCR Vb analysis identified clonal expansion of ≥1 of the Vb proteins in 60% (n=9) of the patients; the remaining 40% (n=6) of the cases had either a clonal process involving a Vb protein not tested in the panel (20%; n=3) or no clear expansion (20%; n=3). Signs of rejection were observed in 20% (n=3/15) and GvHD in 13% (n=2/15) of the patients. Post-transplantation, 27% of cases presented with neutropenia (absolute neutrophil count <1.5 x109/L; n=4), 33% with thrombocytopenia (platelet count <150 x109/L; n=5) and 25% with anemia (hemoglobin <10 g/dL; n=3). T-LGLL evolved in 10 patients (67%; 10/15) despite IST including cyclosporine (n=5), tacrolimus (n=4), mycophenolate mofetil (n=5), cyclophosphamide (n=1), anti-thymocyte globulin (n=1), and corticosteroids (n=6). Lymphadenopathy and splenomegaly were seen in 13% (n=2) and 33% (n=5) of the patients. Other conditions observed were MGUS (20%; n=3) and RA (7%; n=1). Conventional cytogenetic showed normal karyotype in 89% (n=11, tested individuals 13/15). Somatic STAT3 mutations were identified in 2 patients. Sixty% of cases (n=9) were seropositive for EBV when tested at different time points after transplant. Similarly, 53% (n=8) were seropositive for CMV, of which, 5 were positive post-transplantation and 3 pre-/post-transplantation. The complexity of T-LGLL expansion post-transplantation might be due to several mechanisms including active viral infections, latent oncogenic viral reactivation and graft allo-antigenic stimulation. However, in our cohort graft rejection or GvHD was encountered in a few patients (2 allo-HSCT recipients). Autoimmune conditions were present in 50% of SOT recipients (n=4/ 8, including RA, ulcerative colitis, systemic lupus erythematosus). Some of our patients also had low immunoglobulin levels. Overt EBV (post-transplant lymphoproliferative disorder) and CMV reactivation was diagnosed in only 27% (4/15) of the patients. In sum we report the long term follow up of a cohort of T-LGLL and emphasize the expansion of T-LGLL post-transplant highlighting the difficulty in assigning one unique origin of LGLL. Disclosures Hill: Genentech: Consultancy, Research Funding; Takeda: Research Funding; Celegene: Consultancy, Honoraria, Research Funding; Kite: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; TG therapeutics: Research Funding; AstraZeneca: Consultancy, Honoraria. Majhail:Atara Bio: Consultancy; Mallinckrodt: Honoraria; Nkarta: Consultancy; Anthem, Inc.: Consultancy; Incyte: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.
