Ancestral Clone Research Articles

Abstract 4007: Evolutionary history of transformation from chronic lymphocytic leukemia to Richter’s syndrome

Abstract Richter’s syndrome (RS) arising from chronic lymphocytic leukemia (CLL) is a striking example of an aggressive malignant histology that emerges from indolent cancer. RS is a major barrier to disease control in CLL and is associated with poor clinical outcomes and limited survival. The genetic basis of RS is poorly understood, and its relationship to the antecedent CLL remains incompletely characterized. To study RS, we performed whole-exome sequencing (WES) on samples collected from 52 patients with RS of diffuse large B cell lymphoma (DLBCL) histology. For this genomic characterization, samples from 42 patients were analyzed as ‘trios’ (matched germline, CLL and RS tissue DNA) and those from 10 as ‘duos’ (matched CLL and RS DNA). Beyond addressing contamination of CLL DNA in the germline sample, we developed methods for discriminating between the RS and CLL clones which often coexist in the same samples. The discovery cohort revealed that RS and CLL were clonally-related in 45/52 (87%) cases based on WES analysis, with a distinct RS clone emerging from a CLL subclone. The remaining 13% were determined to be clonally unrelated. RS clones presented ~3x higher rates of additional mutations than the ancestral CLL clones from which they developed. We identified novel RS somatic driver mutations (in IRF2BP2, SRSF1, B2M, DNMT3A and others), a high rate of copy number variations with recurrent deletions (e.g., del(17p) [TP53], del(13q14.3), del(7q36), and del(15q13.11) [MGA], del(9p21.3) [CDKN2A/B], del(16q12.2)), focal amplifications (amp(7q21.2) [CDK6], amp(8q24.2) [RECQL4, MYC], amp(13q31.2) [ERCC5], and frequent whole genome duplication. To further investigate RS and CLL clonal evolution, we performed single-cell RNA-sequencing on biopsies at the time of RS diagnosis in 5 individuals with clonally related transformation. Using our novel tool, CNVSingle, we inferred allele specific single-cell copy number alterations, yielding cluster-specific copy number profiles that matched the WES results of individual subclones of the RS and CLL populations. This enabled mapping genetic clones to specific expression patterns. Finally, we devised and tested a methodology that uses cfDNA for early detection of emerging Richter’s disease and have successfully identified Richter‘s tumor DNA in the blood several months prior to the clinical diagnosis. Our study thus defines drivers, distinct molecular subtypes and evolutionary path to RS and suggests strategies for its improved detection. Citation Format: Erin M. Parry, Ignaty Leshchiner, Romain Guièze, Connor Johnson, Eugen Tausch, Sameer Parikh, Camilla Lemvigh, Conor Messer, Daniel Rosebrock, Filippo Utro, Chaya Levovitz, Kahn Rhrissorrakrai, Matthew Davids, Raquel A. Jacobs, Kara Slowik, Julien Broseus, Shanye Yin, Shuqiang Li, Geoff Fell, Ziao Lin, Binyamin A. Knisbacher, Neil Ruthen, Dimitri Livitz, Christof Schneider, Jialin Ma, Julian Hess, Laura Z. Rassenti, Thomas J. Kipps, Nitin Jain, William Wierda, Florence Cymbalista, Neil E. Kay, Kenneth J. Livak, Brian P. Danysh, Chip Stewart, Donna Neuberg, Jennifer R. Brown, Laxmi Parida, Stephan Stilgenbauer, Gad Getz, Catherine J. Wu. Evolutionary history of transformation from chronic lymphocytic leukemia to Richter’s syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4007.

Pediatric T-ALL type-1 and type-2 relapses develop along distinct pathways of clonal evolution

The mechanisms underlying T-ALL relapse remain essentially unknown. Multilevel-omics in 38 matched pairs of initial and relapsed T-ALL revealed 18 (47%) type-1 (defined by being derived from the major ancestral clone) and 20 (53%) type-2 relapses (derived from a minor ancestral clone). In both types of relapse, we observed known and novel drivers of multidrug resistance including MDR1 and MVP, NT5C2 and JAK-STAT activators. Patients with type-1 relapses were specifically characterized by IL7R upregulation. In remarkable contrast, type-2 relapses demonstrated (1) enrichment of constitutional cancer predisposition gene mutations, (2) divergent genetic and epigenetic remodeling, and (3) enrichment of somatic hypermutator phenotypes, related to BLM, BUB1B/PMS2 and TP53 mutations. T-ALLs that later progressed to type-2 relapses exhibited a complex subclonal architecture, unexpectedly, already at the time of initial diagnosis. Deconvolution analysis of ATAC-Seq profiles showed that T-ALLs later developing into type-1 relapses resembled a predominant immature thymic T-cell population, whereas T-ALLs developing into type-2 relapses resembled a mixture of normal T-cell precursors. In sum, our analyses revealed fundamentally different mechanisms driving either type-1 or type-2 T-ALL relapse and indicate that differential capacities of disease evolution are already inherent to the molecular setup of the initial leukemia.

Spatiotemporal dynamics of clonal selection and diversification in normal endometrial epithelium

It has become evident that somatic mutations in cancer-associated genes accumulate in the normal endometrium, but spatiotemporal understanding of the evolution and expansion of mutant clones is limited. To elucidate the timing and mechanism of the clonal expansion of somatic mutations in cancer-associated genes in the normal endometrium, we sequence 1311 endometrial glands from 37 women. By collecting endometrial glands from different parts of the endometrium, we show that multiple glands with the same somatic mutations occupy substantial areas of the endometrium. We demonstrate that “rhizome structures”, in which the basal glands run horizontally along the muscular layer and multiple vertical glands rise from the basal gland, originate from the same ancestral clone. Moreover, mutant clones detected in the vertical glands diversify by acquiring additional mutations. These results suggest that clonal expansions through the rhizome structures are involved in the mechanism by which mutant clones extend their territories. Furthermore, we show clonal expansions and copy neutral loss-of-heterozygosity events occur early in life, suggesting such events can be tolerated many years in the normal endometrium. Our results of the evolutionary dynamics of mutant clones in the human endometrium will lead to a better understanding of the mechanisms of endometrial regeneration during the menstrual cycle and the development of therapies for the prevention and treatment of endometrium-related diseases.

Detection of acquired TERT amplification in addition to predisposing p53 and Rb pathways alterations in EGFR-mutant lung adenocarcinomas transformed into small-cell lung cancers

IntroductionAmong the different mechanisms of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) reported in EGFR-mutated lung adenocarcinoma (EGFR-LUAD) patients, histological transformation into small cell carcinoma (SCLC) occurs in 3–14% of resistant cases, regardless of the generation of EGFR-TKI. In recent studies, bi-allelic inactivation of TP53 and RB1 has been identified in a vast majority of transformed SCLCs. However, the molecular mechanisms driving this histologic transformation remain largely unknown, mainly due to the rarity of samples. Patients and methodsOut of an initial cohort of 64 patients, tumor tissues of adequate quality and quantity for whole exome sequencing (WES) analysis were available for nine tumors for six patients: paired pre- and post-SCLC transformation samples for three Patients and post-SCLC transformation samples for three other patients. ResultsMutational analyses showed concurrent TP53 mutations and Rb pathway alterations in five of the six patients analyzed, confirming their suggested role as predisposing genetic alterations to SCLC transformation. In addition, TERT amplification was detected in four of the six patients and found to be an event acquired during SCLC transformation. Clonal history evolution analyses from the paired LUAD/SCLC samples showed different evolution patterns. In two patients, a large proportion of mutations were present in the most recent common ancestor cell of the initial LUAD and the transformed SCLC clones, whereas in the third patient, few clonal mutations were common between the LUAD and SCLC samples and the ancestor clone that lead to SCLC was present at low frequency in the initial LUAD. ConclusionDespite varied clinical presentations and clonal history evolution patterns, in addition to p53 and Rb pathways alterations, TERT amplification emerged as another common genetic mechanism of EGFR-LUAD to SCLC transformation in our cohort, and could represent a candidate therapeutic target in this subset of SCLC tumors.

Life histories of myeloproliferative neoplasms inferred from phylogenies.

Mutations in cancer-associated genes drive tumour outgrowth, but our knowledge of the timing of driver mutations and subsequent clonal dynamics is limited1-3. Here, using whole-genome sequencing of 1,013 clonal haematopoietic colonies from 12 patients with myeloproliferative neoplasms, we identified 580,133 somatic mutations to reconstruct haematopoietic phylogenies and determine clonal histories. Driver mutations were estimated to occur early in life, including the in utero period. JAK2V617F was estimated to have been acquired by 33 weeks of gestation to 10.8 years of age in 5 patients in whom JAK2V617F was the first event. DNMT3A mutations were acquired by 8 weeks of gestation to 7.6 years of age in 4 patients, and a PPM1D mutation was acquired by 5.8 years of age. Additional genomic events occurred before or following JAK2V617F acquisition and as independent clonal expansions. Sequential driver mutation acquisition was separated by decades across life, often outcompeting ancestral clones. The mean latency between JAK2V617F acquisition and diagnosis was 30 years (range 11-54 years). Estimated historical rates of clonal expansion varied substantially (3% to 190% per year), increased with additional driver mutations, and predicted latency to diagnosis. Our study suggests that early driver mutation acquisition and life-long growth and evolution underlie adult myeloproliferative neoplasms, raising opportunities for earlier intervention and a new model for cancer development.

Genetic and Phenotypic Diversities in Experimental Populations of Diploid Inter-Lineage Hybrids in the Human Pathogenic Cryptococcus.

To better understand the potential factors contributing to genome instability and phenotypic diversity, we conducted mutation accumulation (MA) experiments for 120 days for 7 diploid cryptococcal hybrids under fluconazole (10 MA lines each) and non-fluconazole conditions (10 MA lines each). The genomic DNA content, loss of heterozygosity (LOH) rate, growth ability, and fluconazole susceptibility were determined for all 140 evolved cultures. Compared to that of their ancestral clones, the evolved clones showed: (i) genomic DNA content changes ranging from ~22% less to ~27% more, and (ii) reduced, similar, and increased phenotypic values for each tested trait, with most evolved clones displaying increased growth at 40 °C and increased fluconazole resistance. Aside from the ancestral multi-locus genotypes (MLGs) and heterozygosity patterns (MHPs), 77 unique MLGs and 70 unique MPHs were identified among the 140 evolved cultures at day 120. The average LOH rates of the MA lines in the absence and presence of fluconazole were similar at 1.27 × 10−4 and 1.38 × 10−4 LOH events per MA line per mitotic division, respectively. While LOH rates varied among MA lines from different ancestors, there was no apparent correlation between the genetic divergence of the parental haploid genomes within ancestral clones and LOH rates. Together, our results suggest that hybrids between diverse lineages of the human pathogenic Cryptococcus can generate significant genotypic and phenotypic diversities during asexual reproduction.

Multi-ancestral origin of intestinal tumors: Impact on growth, progression, and drug efficacy.

BackgroundData are steadily accruing that demonstrate that intestinal tumors are frequently derived from multiple founding cells, resulting in tumors comprised of distinct ancestral clones that might cooperate or alternatively compete, thereby potentially impacting different phases of the disease process.AimWe sought to determine whether tumors with a multi‐ancestral architecture involving at least two distinct clones show increased tumor number, growth, progression, or resistance to drug intervention.MethodsMice carrying the Min allele of Apc were generated that were mosaic with only a subset of cells in the intestinal epithelium expressing an activated form of PI3K, a key regulatory kinase affecting several important cellular processes. These cells were identifiable as they fluoresced green, whereas all other cells fluoresced red.ResultsCell lineage tracing revealed that many intestinal tumors from our mouse model were derived from at least two founding cells, those expressing the activated PI3K (green) and those which did not (red). Heterotypic tumors with a multi‐ancestral architecture as evidenced by a mixture of green and red cells exhibited increased tumor growth and invasiveness. Clonal architecture also had an impact on tumor response to low‐dose aspirin. Aspirin treatment resulted in a greater reduction of heterotypic tumors derived from multiple founding cells as compared to tumors derived from a single founding cell.ConclusionThese data indicate that genetically distinct tumor‐founding cells can contribute to early intratumoral heterogeneity. The coevolution of the founding cells and their progeny enhances colon tumor progression and impacts the response to aspirin. These findings are important to a more complete understanding of tumorigenesis with consequences for several distinct models of tumor evolution. They also have practical implications to the clinic. Mouse models with heterogenous tumors are likely better for predicting drug efficacy as compared to models in which the tumors are highly homogeneous. Moreover, understanding how interactions among different populations in a single heterotypic tumor with a multi‐ancestral architecture impact response to a single agent and combination therapies are necessary to fully develop personalized medicine.

The genomic architectures of tumour-adjacent tissues, plasma and saliva reveal evolutionary underpinnings of relapse in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is characterised by a dismal prognosis; nonetheless, limited studies have unveiled the mechanisms underlying HNSCC relapse. Next-generation sequencing was performed to identify the somatic mutations in 188 matched samples, including primary tumours, tumour-adjacent tissues (TATs), pre- and post-operative plasma, saliva and peripheral blood lymphocytes (PBLs) from 27 patients. The evolutionary relationship between TATs and tumours were analysed. The dynamic changes of tumour- and TAT-specific mutations in liquid biopsies were monitored together with survival analysis. Alterations were detected in 27 out of 27 and 19 out of 26 tumours and TATs, respectively. TP53 was the most prevalently mutated gene in TATs. Some TATs shared mutations with primary tumours, while some other TATs were evolutionarily unrelated to tumours. Notably, TP53 mutations in TATs are stringently associated with premalignant transformation and are indicative of worse survival (hazard ratio = 14.01). TAT-specific mutations were also detected in pre- and/or post-operative liquid biopsies and were indicative of disease relapse. TATs might undergo the processes of premalignant transformation, tumorigenesis and eventually relapse by either inheriting tumorigenic mutations from ancestral clones where the tumour originated or gaining private mutations independent of primary tumours. Detection of tumour- and/or TAT-specific genetic alterations in post-operative biopsies shows profound potential in prognostic use.

NPM1 Mutated AML Can Relapse with Wildtype NPM1 and Evolve with Novel Mutations: Implications for Pathobiology and MRD Monitoring

Introduction: Minimal residual disease (MRD) monitoring by qPCR is feasible in AML with mutated NPM1 (NPM1 mut ) . NPM1 mut leukemia is associated with a favorable outcome and MRD negativity is associated with an excellent prognosis. Cytogenetic and molecular analysis have proven the concept of clonal evolution in relapsed or refractory AML. Data is conflicting on NPM1 mut AML relapsing with wildtype NPM1 (NPM1 negative). In this study we analyzed 105 relapsed NPM1 mut AML patients including 15/105 patients that relapsed with NPM1 negative AML.Aims: To analyze in patients with NPM1 mut AML the frequency and mutational landscape of NPM1 negative AML at relapse and provide insight into clonal evolution.Patients and Methods: Between 2006 und 2017 we investigated a total of 105 intensively treated patients with NPM1 mut AML who attained a complete molecular remission (CMR) and finally relapsed. MRD was monitored by qPCR at 1394 consecutive time points (median=12 per patient, range 3-42). 15/105 patients experienced a NPM1 negative relapse. Unique molecular identifier based sequencing of 63 genes associated with hematological malignancies was performed for patients with NPM1 negative relapse in paired samples at diagnosis and at clinical relapse. The median coverage was 4,900x. Digital error suppression was applied to follow mutations in up to 1/500 molecules (VAF 0.2%).Results: For 15 patients with an NPM1 negative relapse the median time to relapse was 39 months (range 4-66). In contrast for 90/105 patients with an NPM1 mut relapse the median time to relapse was significantly shorter with 14 months (range 3-77, p=0.005). We analyzed the overall survival (OS) following relapse. For patients with NPM1 negative relapse OS at 2 years was 48% [95% CI, 12-77] versus 68% [52-79] for patients with NPM1 mut relapse (n.s.).For 61/90 patients with NPM1 mut relapse the diagnostic sample was available for analysis with the 63 gene panel. The mutational landscape of these patients revealed recurrent co-mutations in 14 different genes in 54/61 patients, with the published pattern: DNMT3A, TET2, FLT3- ITD, NRAS, SRSF2, IDH1, IDH2 and FLT3- TKD were affected in more than 10% of cases. A median of 2 genes were co-mutated per patient, no co-occurring mutation was found in only 7/61 patients (11%).For 12/15 cases with NPM1 negative relapse both diagnostic and relapse samples were available for 63 gene panel sequencing. At diagnosis we identified a total of 33 co-mutations in addition to NPM1 in a total of 12 different genes in 12/12 patients (DNMT3A (n=9 ), TET2 (6), IDH1 (4), SRSF2 (3), PTPN11 (2), IDH2 (1), KRAS (1), NRAS (1), EZH2, (1), STAG2 (1), ASXL1 (1) and FLT3- TKD (1)). All patients had at least one co-mutation with a median of 2 co-mutations per patient at diagnosis (range 1-6). At relapse we identified a total of 37 co-mutations in 13 different genes in 11/12 patients with a median of 2 mutations per patient (range 0-6). Eleven mutations in 9 genes (TET2, STAG2, PTPN11, SRSF2, KRAS, DNMT3A, EZH2, FLT3- TKD and NRAS) ) were lost at relapse and 15 mutations in 10 genes (TP53, RUNX1, NRAS, NF1, IDH2, SRSF2, WT1, IDH1, EZH2, TET2) were gained at relapse. We used digital error correction to reassess these mutations in the diagnostic sample. With a detection limit of up to 0.2% VAF (1/500 molecules) none of these mutations was detectable at diagnosis. DNMT3A was the most prevalent mutation (9/12 at diagnosis, 7/12 at relapse) and compared to the cohort with NPM1 mut relapse the presence of a DNMT3A mutation was associated with NPM1 negative relapse (p=0.024).One patient (8%) relapsed with the respective ancestral clone from diagnosis (w/o NPM1), 9 patients (75%) showed a relapse with the known ancestral clone plus evolution, and 2 patients (17%) relapsed with a completely unrelated clone.Conclusion: We identified 14% of NPM 1mut AML patients who relapsed with an NPM1 negative AML following intensive therapy. NPM1 negative relapse occurs significantly later than NPM1 mut relapse and is associated with the ancestral clone in the majority of patients. NPM1 negativity at relapse evades MRD assessment, however mutations in the ancestral clone should be detectable by NGS with digital error correction. This could be included in an extended MRD monitoring strategy, and performed in addition to qPCR for NPM1 at the routine time points. DisclosuresHöllein:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

In Depth Molecular Characterization of AML with RUNX1-RUNX1T1 at Diagnosis and Relapse

Introduction: Minimal residual disease (MRD) monitoring by qPCR is feasible in AML with RUNX1-RUNX1T1 fusion. The absence of RUNX1-RUNX1T1 transcripts is considered as complete molecular remission (CMR). Risk stratification according to MRD is possible and initial studies allocating MRD positive patients to allogeneic stem cell transplantation have been undertaken (Zhu et al., Blood 2013). However, despite CMR about 10% to 30% of patients relapse. In our cohort 90/130 AML patients with RUNX1-RUNX1T1 fusion reached CMR. We analyzed all 17/90 that relapsed following CMR.Aims: To analyze the frequency, the clinical parameters and the mutational landscape of RUNX1-RUNX1T1 positive AML relapsing after CMR and to gain insights into the clonal evolution.Patients and Methods: Between 2005 und 2017 we investigated a total of 130 intensively treated AML patients with RUNX1 - RUNX1T1 fusion at diagnosis and at least two follow up samples. We applied quantitative real-time PCR to detect RUNX1-RUNX1T1 / ABL1 ratios with a sensitivity of 0.001%. We identified 17 patients with relapse following CMR and used unique molecular identifier (UMI) based gene panel sequencing to analyze the clonal architecture and evolution of this cohort in comparison to paired results at diagnosis. UMIs were used for digital error suppression to detect minimal variant allele fractions (VAFs) of up to 0.2%.Results: CMR was reached in 90/130 pts (69%) after a median of 5 months (range 5-45 months). Median event free survival (EFS) of patients with CMR was not reached (EFS at 2 years 82%). However, 17/90 (19%) relapsed during follow up with a median time to relapse of 13 months after CMR (range 4 to 38 months). The ratio of RUNX1-RUNX1T1 / ABL1 was 2.1 fold higher at diagnosis than at relapse with a median of 80 (range 4-508) versus 40 (range 2-501) respectively (n.s.).For all 17 cases who relapsed following CMR the diagnostic and relapse samples were subject to sequencing of 63 genes known to be mutated in hematologic neoplasms. At diagnosis we identified a total of 27 somatic mutations in 11 different genes co-occurring with RUNX1-RUNX1T1 in 14/17 patients: ASXL1 (n=6 ), ASXL2 (5), KIT (5), NRAS (2), CSF3R (2), FLT3- TKD (2), FLT3- ITD (1), IDH2 (1), KRAS (1), TET2 (1), RAD21 (1), SRSF2 (1). The median number of mutations per patient was 2 (range 0-5). At relapse we identified a total of 13 mutations in 7 different genes in 8/17 patients. 18 mutations in 9 genes (ASXL2, ASXL1, KIT, NRAS, CSF3R, FLT3-TKD, KRAS, RAD21, SRSF2) were lost at relapse and 4 mutations in 3 genes (KIT, PHF6, TET2)were gained at relapse. The median number of mutations per patient at relapse was significantly reduced: Fourteen patients (82%) had at least one mutation at diagnosis and only 9 patients (53%) at relapse (p=0.026). The median variant allele fraction (VAF) of co-occurring mutations at diagnosis was 24% (range 1-24) and 42% (range 3-67) at relapse (1.7 fold, n.s.). Changes in VAF did not reveal an evident general principle, however in some cases the gene with the highest VAF at diagnosis disappeared at relapse suggestive of the selection of a subclone.When analyzed for clonal evolution 13/17 patients (76%) showed a clonal change at relapse: nine of 17 patients (53%) relapsed with an ancestral clone, 3 (17%) with an ancestral clone with evolution and one (6%) with the initial clone with evolution. Four of 17 patients (24%) relapsed with the initial clone.As control we sequenced the diagnostic sample in 42/90 patients who did not relapse following CMR. A total of 30 mutations were found in 9 genes in 21/42 patients (median=0.5): ASXL2 (25%), TET2 (11%), KIT (10%), NRAS (10%), FLT3- ITD (6%), CBL (5%), ASXL1 (5%), FLT3- TKD (2%), IDH2 (2%). When compared to the 17 patients with relapse following CMR, the presence of an ASXL1 (p=0.0172) mutation at diagnosis and the total number of mutations at diagnosis (median=2 versus median=0.5; p=0.0024) were significantly associated with relapse.Conclusion: We analyzed the clonal evolution of RUNX1-RUNX1T1 positive AML following CMR. We observed losses and gains of mutations at relapse, but the majority of cases showed a reduction of numbers of mutations. This underscores that RUNX1-RUNX1T1 fusion is the key driver aberration and stable over time. However mutations in ASXL1 and the number of co-mutations at diagnosis is significantly associated with relapse following CMR. This should be considered when tailoring treatment strategies. DisclosuresHöllein:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Genotyping of Classical Hodgkin Lymphoma on the Liquid Biopsy

INTRODUCTION. In classical Hodgkin lymphoma (cHL), the low representation (~5%) of Hodgkin-Reed-Sternberg cells (HRS) challenged tumor genotyping on the tissue biopsy. Cell free DNA (cfDNA) is shed into the blood by tumor cells and can be used as source of tumor DNA for the identification of somatic mutations, track clonal evolution of tumors and detect minimal residual disease during therapy. AIMS. The study aimed at: i) showing that cfDNA mirrors the genetics of HRS cells in cHL patients; ii) characterizing the mutational profile of a large cohort of newly diagnosed and chemorefractory cHL; iii) identifying molecular prognostic subtypes; iv) early detecting residual disease during therapy; and v) longitudinally tracking tumor clonal evolution under different treatment modalities. METHODS. The study included 80 newly diagnosed cHL and 32 chemorefractory cHL. The following biological material was analyzed: i) cfDNA from plasma collected at diagnosis, during ABVD courses, at refractory progression, before and during therapy with brentuximab or nivolumab; and ii) normal germline genomic DNA (gDNA) from granulocytes. For comparative purposes, paired tumor gDNA from microdissected HRS cells of 13 cases was also analyzed. A targeted resequencing panel optimized to include the coding exons and splice sites of 77 genes recurrently mutated in B-cell lymphomas was used for genotyping. Ultra-deep next-generation sequencing (NGS) of the gene panel was performed on NexSeq 500 (Illumina) using the CAPP-seq library preparation strategy (NimbleGen). RESULTS. In cHL patients, cfDNA surrogated gDNA from HRS cells, since it harbored 87.5% of the tumor confirmed mutations. Genes recurrently affected by non-synonymous somatic mutations in >20% of cHL included STAT6 (37.5%), TNFAIP3 (35%), and ITPKB (27.5%) (Fig. 1A). Mutations clustered in major pathways, including NF-κB, PI3K-AKT, cytokine and NOTCH signaling, and immune evasion. ITPKB mutations: i) were quite specific for cHL, being rare or absent in other lymphomas; ii) caused the subcellular delocalization of the protein in primary HRS cells of mutated patients; iii) correlated with clues of PI3K-AKT signaling activation both at gene expression and protein levels; and iv) consistent with the positioning of ITPKB downstream PI3K in the pathway, associate with resistance to PI3K inhibitors. Mutations of CD58, encoding a co-stimulatory molecule for T-cells, associated with short PFS independent of interim PET/CT results, pointing to immune escape genetic lesions as biomarkers of aggressive disease (Fig. 1B). Newly diagnosed and chemorefractory cHL shared a largely overlapping mutational landscape. TP53 mutations were not enriched in refractory cHL as instead commonly found in other types of refractory B-cell tumors. Conversely, more TET2 mutations were documented in refractory cHL, including newly acquired mutation, thus signaling towards aberrant DNA methylation programming as a mechanism of resistance in cHL with potential therapeutic implications. By longitudinal analysis, in patients relapsing under/after chemotherapy or brentuximab vedotin, pre-treatment/relapse tumor pairs branched through the acquisition of phase specific mutations from an ancestral clone, that always persisted (Fig. 1C). Conversely, in patients maintained under nivolumab, clones were cyclically suppressed and replaced by completely novel clones. We utilized the change in circulating tumor cfDNA load from baseline to interim timepoint to predict the best response to ABVD and to complement interim PET/CT in anticipating cure. A drop of 100-fold or 2-log drop in tumor cfDNA after 2 ABVD courses associated with an eventual complete response and cure. All cured patients that were inconsistently judged as interim PET/CT positive turned out to have a >2 log drop in tumor cfDNA. A drop of less than 2-log in tumor cfDNA after 2 ABVD courses associated with an eventual progression. All relapsed patients that were inconsistently judged as interim PET/CT negative turned out to have a <2 log drop in tumor cfDNA (Fig. 1D). CONCLUSIONS. Circulating tumor cfDNA allows to noninvasively detect tumor-specific mutations: i) identify prognostic subtypes; ii) early detect residual disease during therapy; and iii) longitudinally track tumor clonal evolution under different treatment modalities. [Display omitted] DisclosuresStathis:Celgene: Research Funding; Pfizer: Research Funding; Merck: Research Funding; Roche: Consultancy, Other: Advisory board; Amgen: Honoraria. Bertoni:Acerta Pharma: Research Funding; Bayer: Research Funding; Cellestia: Research Funding; Menarini: Research Funding; Piqur: Research Funding; Immunogen: Research Funding. Santoro:Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zucca:Bayer: Consultancy, Other: Advisory Role; Jannsen: Consultancy, Honoraria, Other: Advisory role; Takeda: Consultancy, Other: Advisory role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Celgene: Honoraria, Research Funding; Sandoz: Consultancy, Other: Advisory role; Bayer: Consultancy, Other: Advisory Role; Gilead Science: Consultancy, Other: Advisory role; Roche: Honoraria, Research Funding; Jannsen: Consultancy, Honoraria, Other: Advisory role; Roche: Advisory role, Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundipharma: Research Funding; Mundipharma: Research Funding; Gilead Science: Consultancy, Other: Advisory role; Takeda: Consultancy, Other: Advisory role; Sandoz: Consultancy, Other: Advisory role. Gaidano:Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Carlo-Stella:ADC Therapeutics: Research Funding.

Evolution in Myeloid Neoplasms Clonal Architecture Post Allogeneic Hematopoietic Cell Transplantation

Relapse-related mortality remains one of the main causes of death in patients (pts) with myeloid neoplasms (MN) who undergo allogeneic hematopoietic cell transplantation (allo-HCT). Molecular mutations and cytogenetic abnormalities present in hematopoietic cells are important determinants of relapse risk, with MRD predictive of relapse after allo-HCT. We studied the clonal hierarchy of MN at diagnosis compared to the relapsed state, to determine the effects of the ancestral hit and subclones on the impact on post-transplant recurrence.DNA from marrow/PB samples of MN pts was subjected to analysis at the time of diagnosis, post, and at the time of relapse to evaluate clonal expansion or evolution using a targeted multi-amplicon deep NGS panel of all ORFs of the top 60 most commonly mutated genes in MDS.We identified 43 pts with MDS and MDS/MPN who underwent allo-HCT from 2000 to 2016, whom were assessed serially in non-relapsed MN (NR-MN) and relapsed (R-MN). Median age at diagnosis was 54 years (y, range, 22-69) and 54y (range, 22-70) at time of HCT; 56% were male. NR-MN vs. R-MN pts median IPSS and IPSS-R scores at the time of diagnosis, 1.0 vs. 1.25 (P=0.5) and 4.0 vs. 4.0 (P=0.6), respectively. 53% underwent MAC and 58% had a PBSC. Pre-HCT treatments included HMA (42%), ESA (29%), 7+3 (10%), IMiD (10%), and HU (10%). Serial cases had a median follow up of 4 y (range 0.7 - 14.8) from diagnosis (Dx) and 2.3 (0.1-10.7) y from HCT. Rates before HCT of CR (30% vs. 30%), progressive disease (13% vs. 5%), stable disease (25% vs 25%), and disease progression (35% vs. 40%) (P=0.89). Median follow up post allo-HCT was 1.6y vs. 2.5y (P= 0.26). The incidence of acute GVHD in NR-MN vs. R-MN was 43% vs. 65% (P= 0.269) and 39% vs. 15% for chronic GVHD (P= 0.156), respectively. No difference in median blast percentage pre allo-HCT 4% vs 3.5% (P=0.5722).In NR-MN pts (N=23) after transplant, their pre-HCT characteristics showed 45% (10/23) had abnormal cytogenetics, with the most common abnormalities being del17p, del7q, and/or monosomy 7. SNP analysis showed pathogenic lesions in 43% of assessable cases (6/14), with most frequent abnormalities being loss of 14q11.2 (N=3) and aberrations (loss/UPD) of chr 7 (N=3). Furthermore, 17/21 pts had ≥1 mutation in decreasing frequency TET2 (N=4) , BCOR/BCORL1 (3) , SRSF2 (3), STAG2 (3), ASXL1 (3) , TP53 (2). Post-HCT NGS analyses are ongoing.In R-MN pts (n=20) after transplant, their pre-HCT characteristics showed 65% had abnormal cytogenetics at diagnosis, with the most common chromosomal aberrations involving monosomy 7/7q, trisomy 8, del5q, and del20q. Pre-HCT, 14/20 pts had ≥1 mutation in decreasing frequency TP53 (N=4) , SF3B1 (3), U2AF1 (3) , DNMT3A (2) , NRAS (2), and ASXL1 (2) . NGS at disease diagnosis, post-HCT, and at relapse was performed for 9 pts. In 5 pts relapse of the original ancestral hit (PHF6, SF3B1, NRAS, TP53, and DNMT3A) evolution of additional mutations (ASXL1, KRAS, EZH2) (4 featured in Figure 1). Clinical management of relapsed pts included a 2nd allo-HCT in 7 pts (35%), of which 2 are alive, and DLI in 6 (30%), of which 3 are alive.Logistic regression analysis demonstrated that mutations in DNMT3A and TP53 were non-significantly associated with higher relapse rates (OR= 3.27; P=0.1403). Univariable analysis for time to relapse demonstrated a significant association with earlier relapse with chromosome 11 aberrations (HR=6.4; P=0.005), but DNMT3A and TP53 together (HR=2.285; P=0.1071), TP53 alone (HR=2.257; P=0.1576), and complex cytogenetics (HR=2.15; P=0.1733) with relapse did not reach statistically significance, due to limited sample size.In conclusion, serial analysis of R-MN pre and post-HCT indicated recurrences of the original ancestral mutation, but often included acquisition of new mutations. Retraction but not elimination of the initial ancestral clone in R-MN pts demonstrated the relevance of an earlier ontogenetic stage. The initial hit could potentially drive the phenotype and resultant characteristics of the disease of subsequent lesions or vice versa. We plan to apply ultra-deep sequencing followed by an in-house bio-analytical pipeline to exclude false positive low frequency hits thereby determining the true MRD for the mutation identified in the original MN pre allo-HCT. Deeper NGS of MN pts in the pre & post allo-HCT setting will expand understanding of primary clonal aberrations and their subsequent re-emergence or regression with disease evolution. [Display omitted] DisclosuresGerds:CTI BioPharma: Consultancy; Incyte: Consultancy. Advani:Pfizer: Consultancy; Takeda/ Millenium: Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Majhail:Sanofi: Honoraria; Anthem, Inc.: Consultancy.

The genomic architechtures of tumor-adjacent tissues, plasma and saliva reveal evolutionary underpinnings of relapse in head and neck squamous cell carcinoma.

e18046 Background: Head and neck squamous cell carcinoma (HNSCC) is characterized by dismal prognosis, nonetheless limited studies have unveiled the mechanisms underlying HNSCC relapse. Methods: Next-generation sequencing 1 5 was performed to identify somatic mutations in 188 matched samples including primary tumors, tumor adjacent tissues (TATs), pre- and post-operative plasma, saliva and peripheral blood lymphocytes (PBLs) from 27 patients. Evolutionary relationship between TATs and tumors were analyzed. The dynamic changes of tumor- and TAT-specific mutations in liquid biopsies were monitored together with survival analysis. Results: Alterations were detected in 27/27 and 19/26 tumors and TATs respectively. TP53 was the most prevalent genes mutated in TATs. Some TATs shared mutations with primary tumors, while some other TATs were evolutionarily unrelated to tumors. Notably, TP53 mutations in TATs are stringently associated with premalignant transformation and indicative of worse survival (HR=14.01). TAT-specific mutations were also detected in pre- and/or post-operative liquid biopsies, and indicative of disease relapse. Conclusions: TATs might undergo the processes of premalignant transformation, tumorigenesis, and eventually relapse by either inheriting tumorigenic mutations from ancestral clones where tumor originated or gaining private mutations independent to primary tumor. Detection of tumor- and/or TAT-specific genetic alterations in post-operative biopsies shows profound potential in prognostic use.

Tumor evolutionary trajectories during the acquisition of invasiveness in early stage lung adenocarcinoma

The evolutionary trajectories of early lung adenocarcinoma (LUAD) have not been fully elucidated. We hypothesize that genomic analysis between pre-invasive and invasive components will facilitate the description of LUAD evolutionary patterns. We micro-dissect malignant pulmonary nodules (MPNs) into paired pre-invasive and invasive components for panel-genomic sequencing and recognize three evolutionary trajectories. Evolutionary mode 1 (EM1) demonstrates none of the common driver events between paired components, but another two modes, EM2A and EM2B, exhibit critical private alterations restricted to pre-invasive and invasive components, respectively. When ancestral clones harbor EGFR mutations, truncal mutation abundance significantly decrease after the acquisition of invasiveness, which may be associated with the intratumoral accumulation of infiltrated B cells. Harboring EGFR mutations is critical to the selective pressure and further impacts the prognosis. Our findings extend the understanding of evolutionary trajectories during invasiveness acquisition in early LUAD.

Comparison of Deep Whole Exome Versus Targeted Gene Sequencing for Assessment of Persistent Molecular Disease in Acute Myeloid Leukemia Samples

Background There is growing evidence that genomics-based assessment of persistent molecular disease (PMD) may be a useful risk stratification tool for patients with acute myeloid leukemia (AML). However, there is no consensus about the optimal approach for detection and monitoring of leukemia-associated mutations (LAMs) for PMD testing. One approach is whole exome sequencing (WES), which provides a comprehensive assessment of the clonal architecture by identifying LAMs across the exome at diagnosis, which can then be measured in follow-up samples. Another approach is to use highly sensitive targeted gene sequencing (TGS) to detect persistent LAMs even at very low levels, although detection is limited to genes interrogated by the panel. Although both of approaches have been shown to predict outcome in retrospective studies, there are substantial differences in the number of LAMs identified and the limit of detection, and a head-to-head comparison has not yet been reported. Here, we use a defined cohort enrolled in a prospective clinical trial to compare PMD results from deep WES to error-corrected TGS. Methods Cohort: Patients were age 18-60 with de novo AML classified as intermediate risk by European Leukemia Net criteria, who achieved a morphologic remission after undergoing standard induction therapy. All patients were enrolled in a prospective clinical trial (NCT02756962). Sequencing: Deep WES was performed using DNA from normal tissue (buccal swab or skin) and pre- and post-induction (~day 30) bone marrow (BM) samples to achieve an average coverage depth of ~600x. LAMs identified via paired tumor/normal analysis of the pre-induction sample were queried in the post-induction sample for assessment of PMD. PMD testing by TGS was performed using error-corrected sequencing of 40 genes recurrently mutated AML genes to an average error-corrected coverage depth of ~4500x. Definitions: Based on previously published work, we used two separate variant allele frequency (VAF) cutoffs to define PMD. For WES, PMD+ was defined as ≥1 LAM with a VAF &amp;gt;2.5% (Klco JAMA 2015). For TGS, PMD+ was defined as having ≥1 LAM with a VAF &amp;gt;0.5% (Duncavage NEJM 2018). LAMs were sub-classified per Table 1. TGS mutations not identified as LAMs by WES at diagnosis were excluded from the primary analysis. Results 31 patients were studied. LAMs are summarized in Table 1. 20 patients (65%) were PMD+ by WES after induction, and 22 patients (71%) were PMD+ by TGS. The concordance between WES and TGS was 81% (25/31) (Table 2). Two patients were PMD+ by WES only, due to the persistence of LAMs in the exome space, but not in the targeted panel. Four patients were PMD+ by TGS only, due to the presence of recurrent mutations at VAFs below the detection limit of WES (range: 0.59 - 1.90%). Two patients were PMD+ by both assays, but due to different mutations. All other patients who were PMD+ by both assays had at least one overlapping mutation. Analysis of the mutations that persisted after therapy in both assays showed that 26% of patients (8/31) were PMD+ by TGS because of mutations in DNMT3A, TET2, or ASXL1 (i.e., "DTA" mutations). All of these patients were also PMD+ by WES, with 7 of the 8 patients having ≥1 additional non-DTA mutation (median: 3, range: 1 - 7). In an exploratory analysis, 22 additional mutations in 13 patients were identified by TGS that were not detected by WES on the diagnostic sample. This included two patients who were PMD-, but who had new mutations in TET2 and DNMT3A, respectively, likely representing selection for ancestral clones that were unrelated to the AML founding clone. Conclusion Concordance between WES and TGS-based PMD assessment was high. Discordant results were generally driven by non-recurrent mutations detected by WES, and low-level mutations detected by the high coverage, error-corrected TGS. Although isolated DTA mutations were common on TGS, WES analysis showed additional LAMs accompanied these variants in most cases, indicating the persistence of an ancestral leukemic clone that may provide useful prognostic information. We also observed new, low-level mutations that emerged after therapy in 42% of patients, some of which were not part of the leukemic clone identified at diagnosis. This indicates that use of highly sensitive PMD approaches may be challenging without pre-induction mutation testing, which is required to understand the relevance of markers of persistent molecular disease. Disclosures Jacoby: AbbVie: Research Funding; Takeda: Consultancy; Jazz Pharmaceuticals: Research Funding.

Genetic Determinants and Evolutionary History of Richter's Syndrome

Genetic Determinants and Evolutionary History of Richter's Syndrome

Genomic Analysis of Congenital Myeloid Sarcoma Identifies Significant Bone Marrow Involvement in the Absence of Morphologic Blasts

Myeloid sarcoma is a tumor mass consisting of blasts at extramedullary sites, most frequently the skin and soft tissues. Skin involvement, also termed leukemia cutis, is a well-described entity in congenital leukemia and is present in approximately 50% of patients diagnosed within the first 4 weeks of life. Most cases of congenital leukemia cutis harbor KMT2A rearrangements, as do up to 80% of cases of infantile leukemia, and are typically accompanied by bone marrow involvement. Here we describe a rare CIC-rearrangement as the driver mutation for a case of congenital cutaneous myeloid sarcoma in a newborn female who was noted at birth to have a violaceous, nodular rash. Skin biopsy of the nodular rash shortly after birth with immunohistochemical staining was consistent with myeloid sarcoma. Bilateral bone marrow aspirates and biopsies were also performed, but failed to reveal an aberrant blast population by morphology and flow cytometry. Cytogenetics of the cutaneous myeloid sarcoma was significant for t(10;19)(q23;q13.2) and fluorescence in situ hybridization performed on the tumor confirmed a CIC-rearrangement. CIC-rearranged sarcomas are a new entity of undifferentiated small round cell sarcoma characterized by fusion events involving the CIC gene but are not a known to be driver mutations in myeloid neoplasms. Despite the absence of a blast population in the bone marrow, 75% of analyzed metaphases carried the t(10;19)(q23;q13.2). To further explore the underlying genomic events, whole genome, exome, and transcriptome sequencing was performed on both sarcoma and bone marrow specimens. Sequencing revealed an in-frame CIC-NUTM2A fusion gene present in both the skin and the bone marrow which has been previously described in a single case of undifferentiated soft tissue sarcoma. There were no large-scale chromosomal losses or gains and no copy neutral loss of heterozygosity events. Two exonic single nucleotide variations (SNVs) were detected, both of which were limited to the skin sarcoma and not predicted to be pathogenic. In vitro and in vivo modeling demonstrated that the CIC-NUTM2A fusion protein was highly leukemogenic. Colony forming assays performed using transduced murine bone marrow revealed that the CIC-NUTM2A fusion conferred self-renewal in contrast to CIC, NUTM2A, and the reciprocal NUTM2A-CIC product which failed to serially replate (p&amp;lt;0.001). Transplantation of CIC-NUTM2A modified murine bone marrow cells resulted in a fully penetrant myeloid leukemia with a median survival of 21 days in primary transplants and 16 days in secondary transplants. Transcriptome analysis of these tumors revealed a distinct gene expression profile when compared to several classic myeloid associated fusion genes including AML1-ETO and MLL-AF6. To further understand the acquisition of a malignant phenotype by morphology between the bone marrow and the sarcoma, targeted deep sequencing was performed for all tier 1, 2 and 3 mutations identified by whole genome and exome sequencing. A shared ancestral clone was identified in both specimens along with three subclones specific to the sarcoma. Four SNVs acquired in the sarcoma were found to be present in regulatory regions of genes that were also differentially expressed between the sarcoma and the bone marrow including C6orf120, SMURF1, TJAP1, and PID1. PID1 was found to be downregulated in the malignant sarcoma specimen and has been previously shown to be a regulator of the AKT/PI3K pathway. Low PID1 expression has been associated with poor outcomes in other malignancies, including pediatric glioblastoma. A genome wide CRISPR screen of our CIC-NUTM2A positive murine leukemia cells revealed an enrichment for PID1 deficient cells as well as PTEN confirming the cooperativity between CIC-NUTM2A and the AKT/PI3K pathway (p=0.009 and p=0.005 respectively). In conclusion, we describe a rare fusion gene, CIC-NUTM2A, which leads to an aggressive myeloid malignancy in both humans and mice. Targeted deep sequencing demonstrated the clonal evolution from the bone marrow and acquisition of a cooperating mutation targeting the AKT/PI3K pathway in a subset of extramedullary cells that led to morphologic transformation. A thorough interrogation of the bone marrow in patients with myeloid sarcoma is warranted even in the absence of morphologic and immunophenotypic blasts by flow cytometry to optimally track residual disease during treatment. Disclosures No relevant conflicts of interest to declare.

Heritable epigenetic diversity for conservation and utilization of epigenetic germplasm resources of clonal East African Highland banana (EAHB) accessions

Key messageGenetically identical East African Highland banana (EAHB) clones are epigenetically diverse with heritable epialleles that can contribute to morphological diversity.Heritable epigenetic variation can contribute to agronomic traits in crops and should be considered in germplasm conservation. Despite the genetic uniformity arising from a genetic bottleneck of one ancestral clone, followed by subsequent vegetative propagation, East African Highland bananas (EAHBs) display significant phenotypic diversity potentially arising from somatic mutations, heritable epialleles and/or genotype-by-environment interactions. Here, we use DNA methylation profiling across EAHB accessions representing most of the primary EAHB genepool to demonstrate that the genetically uniform EAHB genepool harbours significant epigenetic diversity. By analysing 724 polymorphic DNA methylation sites by methylation-sensitive AFLP across 90 EAHB cultivars, we could differentiate the EAHB varieties according to their regions (Kenya and Uganda). In contrast, there was minimal association of DNA methylation variation with the five morphological groups that are used to classify EAHBs. We further analysed DNA methylation patterns in parent–offspring cohort, which were maintained in offspring generated by sexual (seed) and asexual (vegetative) propagation, with higher levels of altered DNA methylation observed in vegetatively generated offspring. Our results indicate that the phenotypic diversity of near-isogenic EAHBs is mirrored by considerable DNA methylation variation, which is transmitted between generations by both vegetative reproduction and seed reproduction. Genetically uniform vegetatively propagated crops such as EAHBs harbour considerable heritable epigenetic variation, where heritable epialleles could arise in offspring and contribute to functional traits. This study provides a basis for developing strategies for conservation of epigenetic resources and for integration of epimarkers into crop breeding programmes.

A dynamic mutational landscape associated with an inter-regionally diverse immune response in malignant rhabdoid tumour.

Malignant rhabdoid tumour (MRT) is a childhood neoplasm of high malignancy characterised by biallelic mutation and/or loss of the epigenetic master regulator SMARCB1, accompanied by no or few other oncogenic drivers. In spite of their generally low mutational burden, an intratumoural T-cell response has been reported in a subset of MRTs, indicating that immune checkpoint inhibition may be considered a viable therapy option for some patients. We assess here the evolution over time and space of predicted neoantigens and indicators of immune checkpoint status in two MRT patients who progressed under treatment. Both patients showed an accumulation of novel clonal and subclonal mutations, including predicted neoantigens, in metastases compared to their inferred ancestral clones in the primary tumours. The first patient had peritoneal metastases from an MRT of the liver. Clonal deconvolution revealed polyclonal seeding from the primary tumour to a single metastatic site, followed by a local subclonal burst of mutations. The second patient had a renal MRT with multiple pulmonary metastases, each of which could be traced back to a single genetically unique founder cell, with formation of novel subclones in two metastases. Both patients showed a regionally heterogeneous landscape of predicted neoantigens and of tumour-infiltrating lymphocytes expressing CD8 and PD1. In both patients, some tumour regions fulfilled established criteria for PD-L1 positivity (> 1% of tumour cells), while others did not. This suggests that even in a tumour type like MRT, with a single driver mutation, there can be heterogeneity in neoantigen repertoire, immune response, and biomarkers for checkpoint blockade among sampled locations. This must be taken into account when assessing progressed MRT patients for checkpoint inhibition therapy. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Clonal lineage from normal endometrium to ovarian clear cell carcinoma through ovarian endometriosis.

Clear cell carcinoma of the ovary is thought to arise from endometriosis. In addition, retrograde menstruation of shed endometrium is considered the origin of endometriosis. However, little evidence supports cellular continuity from uterine endometrium to clear cell carcinoma through endometriosis at the genomic level. Here, we performed multiregional whole‐exome sequencing to clarify clonal relationships among uterine endometrium, ovarian endometriosis and ovarian clear cell carcinoma in a 56‐year‐old patient. Many somatic mutations including cancer‐associated gene mutations in ARID1A, ATM, CDH4, NRAS and PIK3CA were shared among epithelium samples from uterine endometrium, endometriotic lesions distant from and adjacent to the carcinoma, and the carcinoma. The mutant allele frequencies of shared mutations increased from uterine endometrium to distant endometriosis, adjacent endometriosis, and carcinoma. Although a splice site mutation of ARID1A was shared among the four epithelium samples, a frameshift insertion in ARID1A was shared by adjacent endometriosis and carcinoma samples, suggesting that the biallelic mutations triggered malignant transformation. Somatic copy number alterations, including loss of heterozygosity events at PIK3CA and ATM, were identified only in adjacent endometriosis and carcinoma, suggesting that mutant allele‐specific imbalance is another key factor driving malignant transformation. By reconstructing a clonal evolution tree based on the somatic mutations, we showed that the epithelium samples were derived from a single ancestral clone. Although the study was limited to a single patient, the results from this illustrative case could suggest the possibility that epithelial cells of ovarian endometriosis and clear cell carcinoma were descendants of uterine endometrial epithelium.