Analysis Of Clonal Architecture Research Articles

Deciphering Potential Molecular Signatures to Differentiate Acute Myeloid Leukemia (AML) with BCR::ABL1 from Chronic Myeloid Leukemia (CML) in Blast Crisis.

Acute myeloid leukemia (AML) with BCR::ABL1 has recently been recognized as a distinct subtype in international classifications. Distinguishing it from myeloid blast crisis chronic myeloid leukemia (BC-CML) without evidence of a chronic phase (CP), remains challenging. We aimed to better characterize this entity by integrating clonal architecture analysis, mutational landscape assessment, and gene expression profiling. We analyzed a large retrospective cohort study including CML and AML patients. Two AML patients harboring a BCR::ABL1 fusion were included in the study. We identified BCR::ABL1 fusion as a primary event in one patient and a secondary one in the other. AML-specific variants were identified in both. Real-time RT-PCR experiments demonstrated that CD25 mRNA is overexpressed in advanced-phase CML compared to AML. Unsupervised principal component analysis showed that AML harboring a BCR::ABL1 fusion was clustered within AML. An AML vs. myeloid BC-CML differential expression signature was highlighted, and while ID4 (inhibitor of DNA binding 4) mRNA appears undetectable in most myeloid BC-CML samples, low levels are detected in AML samples. Therefore, CD25 and ID4 mRNA expression might differentiate AML with BCR::ABL1 from BC-CML and assign it to the AML group. A method for identifying this new WHO entity is then proposed. Finally, the hypothesis of AML with BCR::ABL1 arising from driver mutations on a BCR::ABL1 background behaving as a clonal hematopoiesis mutation is discussed. Validation of our data in larger cohorts and basic research are needed to better understand the molecular and cellular aspects of AML with a BCR::ABL1 entity.

Abstract A41: Single Cell Multiomic Analysis Reveals Association of TP53-mut Loss of Heterozygosity with Primitive Phenotype in Acute Myeloid Leukemia

Abstract TP53 mutations in acute myeloid leukemia (AML) are associated with copy number abnormalities (CNA), structural variants and high risk of relapse (Döhner et al., 2017; Giacomelli et al., 2018; Bernard et al. 2020). In spite of relatively high remission rates obtained by targeted therapies, TP53 mutant (TP53-mut) clones persist, invariably resulting in relapse (Short et al., 2021; Takahashi et al., 2016). Delineating the clonal architecture and the immunophenotypes of TP53-mut clones during AML therapy may provide a better understanding of the role of TP53-mutations in AML biology. Recent progress in sequencing technologies allows the integration of genotyping and phenotyping at the single cell level. Here, we took advantage of MissionBio Tapestri’s newest platform: single cell DNA + protein for simultaneous genotyping and phenotyping with 45 surface oligo-conjugated antibodies in 10 paired samples from 5 patients with TP53-mut AML before and after therapy. Samples with at least 70% viability were stained with the TotalSeq™-D Human Heme Oncology Cocktail, V1.0. Following surface marker staining, single-cell suspension, encapsulation, and barcoding were performed according to manufacturer’s instruction. For scDNA library preparation, we utilized a validated custom panel (Morita et al. 2020) consisting of 279 amplicons covering recurrent mutations in 37 genes in AML. We sequenced a total of 44,550 cells from 10 samples. We confirmed mutations reported by MD Anderson molecular diagnostic laboratory in the genes covered by the scDNA custom panel. The clonal architecture analysis distinguished between TP53-mut clones with or without loss of heterozygosity (LOH) of the normal TP53 allele. Our data show a primitive immunophenotype in TP53-mut with LOH (LOH+) clones in comparison to TP53-mut LOH- clones. We see 2.4 LOG2FC increase in CD34 and 1.8 LOG2FC increase in CD117 (p<0.001) in TP53-mut LOH+ clones in comparison to TP53-mut LOH- clones. Clonal evolution analysis shows that TP53-mut LOH+ clones are significantly more resistant to therapy than TP53-mut LOH-, consistent with previous publications. On the other hand, TP53-mut LOH- clones showed significantly higher levels of CD2, CD16, CD5, CD3, and CD8 among other lineage markers (LOG2FC= 1.1, 1.2, 1.4, 1.1, and 1.1 respectively; p.value <0.0001) compared to TP53-mut LOH+. This data indicates that TP53-mut LOH- cells can express lymphoid phenotypic markers. Single cell cytokine analysis (IsoPlexis) reveals profound lack of secreted cytokines in T-cells from TP53-mut AML. Further data from bulk-RNA sequencing, ddPCR, and CyTOF will be presented that validates a lymphoid phenotype of TP53-mut LOH-. In summary, we utilize a scDNA+protein multiomic approach to dissect clonal architecture and provide a link between genotype-phenotype in TP53-mut AML. We show that while TP53-mut LOH+ clones are exclusively primitive, TP53-mut LOH- clones retain the capacity to exist outside primitive immunophenotype and might lack differentiation block. Citation Format: Edward Ayoub, Vakul Mohanty, Yuki Nishida, Tallie Patsilevas, Mahesh Basyal, Russell Pourebrahim, Muharrem Muftuoglu, Ken Chen, Ghayas C. Issa, Michael Andreeff. Single Cell Multiomic Analysis Reveals Association of TP53-mut Loss of Heterozygosity with Primitive Phenotype in Acute Myeloid Leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A41.

Molecular and Clinical Spectra of Protein Phosphatase, Mg2+/Mn2+-Dependent 1D (PPM1D)-Mutant Myeloid Neoplasms

Protein phosphatase Mg2+/Mn2+-dependent 1D (PPM1D) gene (17q23) cooperates with TP53 in regulating DNA damage response (DDR). PPM1DMT are frequently encountered in clonal hematopoiesis of indeterminate potential (CHIP) and in therapy-related myeloid neoplasms (t-MN). Most typically lesions lead to C-terminal loss leading to truncation with paradoxical gain-of-function amplification of PPM1D effects in terms of inactivation of TP53 and are thus functionally synergistic with TP53 deficiency. Here, we investigated the clonal architecture and clinical implications of PPM1DMT in the context of TP53MT MN. A cohort of 4479 patients (pts) screened for the presence of PPM1DMT and TP53MT was analyzed in terms of clinical phenotype, cytogenetics, clonal hierarchy and outcomes (see Table). Overall, 67 (1.5%) pts harbored PPM1DMT and 367 (8%) TP53MT. PPM1DMT (n=70) were truncating, of which 57% were frameshift and 43% were stop codons, distributed across exon 6. No biallelic PPM1DMT case was found. When we studied PPM1D expression in wild type, mRNA levels were elevated in AML (59%; P<.0001, but not in MDS), with high risk AML having higher expression PPM1D levels compared to favorable cases (59% vs. 46%, P=.08). PPM1DMT cases were older than those with TP53MT [median 74yrs (range, 39-93) vs. 71yrs (14-95), P=.02]. PPM1DMT were significantly enriched within pts with CH (including CHIP and CCUS) compared to TP53MT (27 vs. 1%, P=.0001) while TP53MT were more frequently encountered in MN (71 vs. 31%, P=.0001). Review of the medical records of PPM1DMT and TP53MT cases revealed history of malignancy was ascertained in 69% of PPM1DMTvs. 30% in TP53MT; P<.0001, of which 30% had hematological malignancies (HM) and 7.5% were breast cancer (BC) survivors. Of note is that 9% PPMD1MT cases had autoimmune disorders. Overall, 37% received only chemotherapy, 20% radiotherapy only, 22% both chemo/radiotherapy and 13% immunosuppression. A total of 14% of TP53MT carriers had a prior history of HM and 5% had BC history. When compared to PPM1DMT, a higher fraction (58%; P=.005) received chemotherapy, whereas similar rates were noted for radiotherapy (14%), chemo/radiotherapy combination (17%) and immunosuppression (11%). PPM1DMT were also more commonly found in t-MN than TP53MT (42 vs. 28%, P=.05). Overall, 37% of PPM1DMT showed a normal karyotype (NK) compared to 14% of TP53MT (P=.0003). Indeed, up to 61% of TP53MT had complex karyotypes (CK vs. 15% in PPM1DMT, P=.0001). Most common genes associated with PPM1DMTvs.TP53MT were DNMT3A (21 vs. 10%; P=.05), TET2 (19 vs. 11%; P=.2), ASXL1 (15 vs. 10%, P=.4), KRAS/NRAS (1 vs 7%, P=.06) and NF1 (1 vs. 7%, P=.03). Analysis of clonal architecture showed that PPM1DMT were either dominant / codominant (46%; n=33) including PPM1DMT as a sole lesion(n=19); in these cases TP53MT was the most common 2nd/codominant hit (25%) followed by DNMT3A (12.5%, P=.04) and RUNX1 (12.5%, P=.04). Cases with dominant PPM1DMT were encountered more in t-MN (46 vs. 36%, P=.3) and CH (30 vs. 24%, P=.4) compared to subclonal PPM1DMT (n=34, 51%), which were subclonal to TET2MT (21%) followed by JAK2 (12%), DNMT3A (9%, P=.02) and TP53 (9%, P=.02). Notably, all deaths in PPM1DMT CH cases were not due to progression to MN. No significant difference was found in OS between ancestral and subclonal PPM1DMT carriers. PPM1DMT were more likely encountered in older pts (OR=3.2; P=.001), MN-LR (OR=2.8, P<.0001), CH (OR=12.6, P<.0001), t-MN (OR=4.8, P<.0001), NK (OR=1.9, P=.01) and DNMT3AMT carriers (OR=1.86, P=.04) and less likely to occur in pts with MN-HR (OR=0.38, P=.002; see Figure) Survival analysis showed no significant difference between PPM1DMT and PPM1DWT and between CH and non-CH PPM1DMT cases while there was a significant difference between PPM1DMTvs. TP53MT (102 vs.12mo., P<.0001).Of note is that 12 pts had both PPM1DMT and TP53MT (50% t-MN, of which 25% had a prior history of lymphoma). In such cases, PPM1DMT were mostly ancestral compared to TP53MT (67 vs. 33%; P<.0001). Configurations of PPM1D in the clonal hierarchy and pairing with secondary or primary hits are consistent with their role in MN evolving from CH. Association with CH implies that some of the t-MN may be derived from pre-existing CH a theory which we are currently being testing in serial samples. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genomic Analysis Reveals Heterogeneity Between Lesions in Synchronous Primary Right-Sided and Left-Sided Colon Cancer.

Background: The synchronous primary right-sided and left-sided colon cancer (sRL-CC) is a peculiar subtype of colorectal cancer. However, the genomic landscape of sRL-CC remains elusive.Methods: Twenty-eight paired tumor samples and their corresponding normal mucosa samples from 14 patients were collected from the Second Affiliated Hospital of Harbin Medical University from 2011 to 2018. The clinical–pathological data were obtained, and whole-exome sequencing was performed based on formalin-fixed and paraffin-embedded samples of these patients, and then, comprehensive bioinformatic analyses were conducted.Results: Both the lesions of sRL-CC presented dissimilar histological grade and differentiation. Based on sequencing data, few overlapping SNV signatures, onco-driver gene mutations, and SMGs were identified. Moreover, the paired lesions harbored a different distribution of copy number variants (CNVs) and loss of heterozygosity. The clonal architecture analysis demonstrated the polyclonal origin of sRL-CC and inter-cancerous heterogeneity between two lesions.Conclusion: Our work provides evidence that lesions of sRL-CC share few overlapping mutational signatures and CNVs, and may originate from different clones.

Paroxysmal Nocturnal Hemoglobinuria Results from Initial Clonal PIG-a Mosaicism

The pathogenesis of PNH involves alteration of at least one HSC clone, characterized by a somatic mutation in the PIG-A gene. The resulting phenotype appears to offer a growth advantage over other unaffected HSCs, yet the failure of PIG-A mutant clones to expand in animal models suggests that additional factors are necessary for the development of PNH. The association of PNH with aplastic anemia (AA) is evidence of selection pressure by immune attack, but secondary hits in additional genes may also be clonally acquired to render PNH clones competitive irrespective of immune pressure. With the advent of NGS, clonal architecture analysis has been revolutionized. Yet, despite increased resolution, NGS is hampered by the presence of normal/non-clonal cells at varying frequencies in the sequenced sample, often decreasing the detection threshold and precluding recognition of subclonal mosaicism.To study these issues in PNH, 133 patients with PNH (N=33), AA (N=33), and AA/PNH (N=67) were sequenced using deep targeted NGS covering all exons of the PIG-A gene. All patients had a detectable PNH clone by flow cytometry, with the mean WBC clone size of 0.22% in AA, 27.4% in AA/PNH, and 77.1% in PNH. 223 PIG-A mutations were identified in this cohort; 35% were frameshift mutations, 34% missense, 14% splice site, 13% nonsense, and 3% non-frameshift. In unpurified blood, PIG-A mutations were found in 27/33 (82%) of PNH patients, 36/67 (54%) of AA/PNH, and 2/33 (6%) of AA, with a clear association between PNH clone size and the ability to detect mutations by NGS (P<0.0001). In DNA extracted from cells flow sorted by FLAER negativity, detection rates were 26/29 (90%), with an average of nearly 3 mutations per patient, indicating that mechanisms such as microdeletions may affect as many as 10% of PNH or AA/PNH patients and that clonal mosaicism is common. Our results also show that indeed type III and II PNH cells are derived from 2 distinct clones, each with a distinct mutation.To examine disease pathogenesis, we studied 47 patients longitudinally using both PNH flow and deep sequencing. Serial testing illustrates possible modes of evolution: mutations can fluctuate in the contribution to clonal burden over time with the variant allelic frequency of some mutations increasing while others decrease. Linear regression of the number of mutations vs . time from diagnosis suggests that in AA/PNH (N=29), the number of mutant clones detected increases over time (P=.012) while in classic PNH (N=18) the trend is reversed, as the number of mutations detected decreases (P<.0001) and a dominant clone emerges to supply a majority of blood cells.Traditionally, PNH has been considered a monoclonal disease in which a PIG-A mutant HSC clone can outcompete normal hematopoiesis within the context of permissive conditions. Using deep NGS, our results clearly indicate that while a single clone ultimately may dominate, PNH begins as oligoclonal process with the eventual emergence of the most competitive HSC clone(s) populating hematopoietic space. Initially, an environment that “favors” a state of GPI-anchor deficiency thereby allows multiple clones to evolve. Over time, the most “fit” PNH HSC clones dominate, resulting in monoclonality. In addition, our results confirm the presence of distinct mutations with corresponding phenotype through sequencing of sorted type II and type III PNH cells. Clonal drift may also be observed, likely through a newly acquired advantage of one clone within the available pool of GPI-deficient PIGA mutant clones. Our previous work indicated that acquisition of additional enabling mutations results in a more complex clonal architecture comparable to that seen in MDS, an intrinsic mechanism of the selection process that involves mutations in genes altered in MDS. Additional intrinsic hits also explain patient-specific kinetics of PNH evolution and apparent independence of established PNH from the effect of immunosuppression. Thus, while immune selection may provide initial conditions favorable for oligoclonal PNH evolution, once pressure is relieved, intrinsic factors can lead to PNH monoclonality. Alternative mechanisms could include loss of dominance by exhaustion, senescence, or telomere shortening.In sum, our study clearly demonstrates initial polyclonality early in the pathogenesis of PNH with selection of dominant clones during disease duration. DisclosuresMakishima:Yasuda Medical Foundation: Research Funding.

Patterns of Genomic Associations Can Define Acute Myeloid Leukemia (AML) Phenotype

AML can develop after an anteccedent myeloid malignancy (secondary AML [sAML]) or arise de novo (pAML). Genome sequencing technologies have illuminated the complexity of genomic abnormalities that drive AML and contribute to its phenotype. Since identifying a single gene or co-mutated genes unlikely to yield an understanding on how these mutations define disease biology and phenotype, an unbiased approach is needed to study the relationship of those abnormalities to each other and to AML biology.Here, we study these associations using an unbiased approach analogous to Netflix or Amazon's recommender system in which customer who buys A, and B is likely to buy C (the occurrence of mutation A, B, and C is likely to be associated with pAML or sAML).We performed targeted mutational analysis of 468 patients with rigorously defined pAML and sAML. The association between mutations and disease phenotype was investigated by Apriori market basket analysis algorithm. Association rule is a machine learning method that can identify relationships between variables in a large dataset. Clonal architecture of driver vs. subclonal mutations was evaluated by using allele fractions of point mutations in samples with 2 or more mutations and statistically significant clonal heterogeneity.Of 468 patients (pts) were included in the final analysis, 247 had pAML and 221 sAML. The median age for the entire cohort was 64 years (range, 18-100) and 222 pts (47.4%) had normal karyotype (NK). Compared to pts with pAML, those with sAML were older (68 vs 60 years, P <.001), had a lower WBC at presentation (3.85 vs 13 ×109 g/l, P <.001), less likely to have NK cytogenetics (35.7 vs 57.9%, P <.001), and more likely to have unfavorable cytogenetics (36.7 vs 23.9%, P=.002). The most commonly mutated genes in pAML included: NPM1 (27%), DNMT3A (25%), TET2 (25%), FLT3 (ITD/TKD, 21%), and RUNX1 (10%) and in sAML: TET2 (17%), ASXL1 (16%), SRSF2 (11%), U2AF1 (11%), and DNMT3A (10%). Association rules identified mutations in NPM1, FLT3, and DNMT3A as commonly associated with each other predominantly in pAML. DNMT3A was associated with NPM1 in 30 pts (12%) with pAML (21% with NK), with FLT3 in 10% of pAML (17% of NK and 16% with unfavorable karyotype), and without NPM1 or FLT3 in 25 pts (10%). DNMT3A co-occurred with NPM1 and FLT3 in 16 pts (6%) of pAML (11% of NK). Among pts with DNMT3AWT/ NPM1WT/ FLT3WT (triple negative), RUNX1 was the most commonly mutated gene in 18 pts (7%) followed by TET2 13 pts (5%). Clonal architecture analysis showed that DNMT3A commonly occurred as a driver mutation while NPM1 +/- FLT3 were subclonal. In pts with triple negative disease, RUNX1 or TET2 were commonly found as driver mutations.Association rules identified the combination of ASXL1 and TET2 along with one of spliceosome mutations (SRSF2, U2AF1, or ZRSR2) as highly specific combination for the sAML phenotype (6% of pts with sAML). Among pts with sAML who does not have any of these mutations, TP53 occurred in 16 pts (7%), DNMT3A in 14 (6%) (Commonly with NPM1WT and FLT3WT), and NRAS in 11 pts (5%). In sAML, 22 pts had TP53, 18 of which (82%) were associated with unfavorable karyotype. Clonal architecture analysis showed that ASXL1 and TET2 were commonly co-mutated as driver clone along with the spliceosome mutations.Association rules can define combinations of genomic abnormalities that can define AML phenotype. This study also show that defining AML phenotype by one gene or two gene mutations undermine the complexity of genomic abnormalities in AML. Unbiased genomic combinations using the recommender system approach may help to understand the complexity of genomic information in AML. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Advani:Pfizer: Consultancy; Takeda/ Millenium: Research Funding. Gerds:CTI BioPharma: Consultancy; Incyte: Consultancy.

Mutational Pattern and Clonal Hierarchy in MDS with Monosomy 7

Background: Monosomy 7 (-7) is a typical abnormality in MDS with a frequency of ~ 10%. -7 either occurs as the sole abnormality (~4%), accompanied by one additional abnormality (~1%) or in a complex karyotype (~5%). A detailed molecular analysis of MDS with -7 as the sole abnormality (-7 sole) has not been performed to date.Aims: Evaluation of molecular mutations in MDS with -7 sole and analysis of the clonal hierarchy.Patient cohorts and methods: 100 patients (pts) with MDS at diagnosis harboring -7 sole in chromosome banding analysis (59 male, 41 female) with available cytomorphology and material for mutation screening were selected. Median age was 70 years (range: 21-89 years). 86 pts had de novo and 14 pts therapy-related MDS. Blast count was <5% in 29 MDS and ≥5% in 71 MDS pts. 97 pts were also analyzed by interphase FISH using a probe for the centromeric region of chromosome 7. Targeted sequencing of 29 genes commonly mutated in myeloid malignancies was performed in all 100 pts (Illumina, San Diego, CA). Variants of unknown significance were excluded from statistical analysis.Results: The most frequently mutated genes were: ASXL1 (42%), SETBP1 (31%), U2AF1 (29%), DNTM3A (27%), RUNX1 (17%), ETV6 (14%), EZH2 (14%), TET2 (12%), SRSF2 (10%), CBL (9%), PTPN11 (9%), NF1 (6%), CSF3R (4%), JAK2 (4%), TP53 (3%), KRAS (3%), NRAS (3%), and MPL (3%). Genes mutated in 2% or less of pts were: GATA2, IDH1, IDH2, KIT, CALR, ETNK1, NPM1, SF3B1, and WT1 . No mutations were observed in CSNK1A1 and GATA1 (figure 1).RUNX1 mutations were significantly more frequent in t-MDS as compared to de novo MDS (43% vs 13%, p=0.02), while ASXL1 mutations were more frequent in MDS with blast count <5% (60% vs 41%, p=0.04).A median of 2 mutations per case were observed (range 0-6), with 94 pts harboring at least 1 mutation. No association between age and the number of mutations was observed. However, mutations in ETV6, SETBP1, U2AF1 and PTPN11 were associated with a higher number of mutations, while this was not the case for other gene mutations. In pts with 0-2 mutations ETV6, SETBP1, U2AF1 and PTPN11 were mutated in 4%, 15%, 14%, and 2%, respectively, while the respective mutation frequencies were 27%, 49%, 47%, and 19% in pts with >2 mutations (for all comparisons p<0.01).The DNMT3A mutation frequency was low in ASXL1 mutated pts as compared to ASXL1 wild type (wt) pts (8% vs 42%; p<0.0001), while RUNX1 mutations were more frequent in ASXL1 mutated pts (28% vs 10%, p=0.03). U2AF1 and ETV6 mutations frequently co-occurred as 67% of ETV6 mutated pts also harboured a U2AF1 mutation (p=0.002) while only 21% of ETV6 wt pts showed U2AF1 mutations. In addition, the following mutation combinations were more frequent than expected U2AF1 / ETV6 (p=0.002), RUNX1 / EZH2 (p=0.04), EZH2 / TET2 (p=0.04), SETBP1 / U2AF1 (p=0.01), SETBP1 / PTPN11 (p=0.02), and U2AF1 / TP53 (p=0.02), while the following mutation combinations co-occurred less frequently than expected: EZH2 / SETPB1 (p=0.05), RUNX1 / SETBP1 (p=0.02), and SRSF2 / U2AF1 (p=0.02).For clonal architecture analysis the clone size of cells harboring -7 was related to the mutation loads of the 11 most frequently mutated genes. The proportion of cells harboring -7 determined by FISH analyses varied between 6% and 95% (median: 57%). The -7 clone size was significantly correlated to the mutation loads of DNMT3A (r=0.67, p<0.001), RUNX1 (r=0.65, p=0.003), PTPN11 (r=0.71, p=0.02), and NF1 (r=0.81, p=0.01) indicating that -7 and the respective mutation were present together in the same clone. The mutation loads of the following mutations were significantly correlated: ASXL1 / PTPN11 (r=0.95, p=0.01), ASXL1 / U2AF1 (r=0.58, p=0.04), ETV6 / SETBP1 (r=0.91, p=0.005), and ETV6 / U2AF1 (r=0.87, p=0.03). Estimated based on clone size and mutation load -7 was present in the main clone (MC) in 61% of cases and in the subclone (SC) in 39%. Most mutations occurred in the MC as well as in the SC at comparable frequencies (ASXL1, EZH2, RUNX1, SETBP1, SRSF2). Mutations in ETV6 and PTPN11 were present slightly more frequently in a SC only (64% and 60%), while mutations in CBL, DNMT3A, TET2 and U2AF1 were found rarely in a SC only (33%, 18%, 18% and 17%) (figure 2).Conclusions: A broad spectrum of molecular mutations is present in MDS with -7 sole with the highest mutation frequencies in ASXL1, SETBP1, U2AF1, DNMT3A and RUNX1 . No clear hierarchy of -7 and mutations was identified, however, in most cases if mutated DNMT3A, TET2 and U2AF1 are present in the main clone. [Display omitted] DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Walter:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Clonal Architecture of EGFR Mutation Predicts the Efficacy of EGFR-Tyrosine Kinase Inhibitors in Advanced NSCLC: A Prospective Multicenter Study (NCT03059641).

Clonal architecture is fundamental for the understanding of cancer biology and therapy; however, multiregional sampling in advanced-stage cancers is not always applicable. This prospective clinical trial was to investigate whether paired tissue and circulating tumor DNA (ctDNA) could describe the clonal architecture of advanced non-small cell lung cancer (NSCLC) and its association with clinical outcome (NCT03059641). Paired tumor and plasma ctDNA samples were sequenced by target-capture deep sequencing of 1,021 genes. Clonal dominance analysis was performed on the basis of PyClone. Overall, 300 treatment-naïve patients with stage IIIB-IV NSCLC were recruited from 14 centers. Of the 94 patients with available ctDNA data for EGFR clonal architecture analysis, 72 (76.6%) showed EGFR as the dominant clone. The median progression-free survival was longer for these patients than for the 22 patients whose EGFR was nondominant clone [11 vs. 10 months; HR, 0.46; 95% confidence interval (CI), 0.24-0.88; P = 0.02]. The difference was more significant if both tissue and ctDNA defined EGFR as dominant clone (n = 43) versus those not (n = 8; 11 vs. 6 months; HR, 0.13; 95% CI, 0.04-0.50; P = 0.003). Moreover, multivariate Cox proportional HR analysis demonstrated EGFR clonal architecture as an independent prognostic indicator of the efficacy of EGFR-tyrosine kinase inhibitors (TKIs). Paired tissue and ctDNA could be analyzed for clonal architecture in advanced cancer. EGFR mutations do not always make up a dominant clone in advanced NSCLC, which was associated with the efficacy of EGFR-TKIs in NSCLC.

Double Genetic Hits and Subclonal Mosaicism in the Ras Signaling Pathway in Myeloid Neoplasia

The high prevalence of oncogenic mutations in the RAS family genes including their down or upstream partners (e.g., NRAS, KRAS, PTPN11, RIT1α) has strong implication in abnormal Ras signaling in the pathogenesis of myeloid neoplasia (MN). For many years RAS mutations have been considered non-targetable. However, recently multiple targeted agents have been introduced as possible future therapies. While common in JMML, RAS mutations are rarely seen in adult MN as the ancestral event. Instead they serve as subclonal hits. To that end, the clinical impact of RAS mutations has been controversial: whereas some studies did not find prognostic impact, others demonstrated that these mutations may confer a poor prognosis. Nevertheless, molecular analysis of core binding factor AML patients showed the coexistence of RAS clones harboring independent hits targeting the same pathway. We stipulate that occurrence of RAS mutations is not random and reasoned that studying cases with multiple RAS mutant subclones may reveal distinct molecular or etio-pathologic background attracting the emergence of RAS mutations as a way to augment MYC activity as a ubiquitous pathway of clonal progression. In a review of a molecular collection of data of a cohort of MN patients (n=1876) including MDS (n=692), MDS/MPN (n=282), AML (pAML, 710; sAML, 192) we found RAS mutations in 21% (403/1876) of the patients. Among them we encountered a subgroup of patients (9%; 38/403) harboring multiple RAS mutations. These multiple hits affected PTPN11, NRAS, KRAS and RIT1-α. Most of these multiple RAS mutants represented a subclonal mosaicism rather than biallelic subclones as indicated by single cell DNA sequencing. Three patients were assessed serially (2 patients/ 2 time points and 1 patient/ 5 time points). In one case NRAS (VAF-24%) and PTPN11 (VAF-28%) were acquired at the time of AML evolution. In an MDS/MPN-U, NRAS/KRAS were both detected at diagnosis with a VAF of 1% and 12% each. Both clones increased over 2 years' time span with NRAS ramping to 43% and reaching KRAS (50%). In the 3rd case, samples were collected over a period of 5 years with NRAS and PTPN11 being detected only at the later time point (VAF of 5% and 15%, respectively). In contrast to competing RAS subclones in adults, our historical cohort of JMML showed that at least one RAS mutation was found in 89% (82/92) of the patients as ancestral events and in mutually exclusive manner while biallelic RAS mutations occurred in 15% (12/82) of the cohort. In adult MN, the most common subclonal mosaicism was encountered in NRAS/KRAS (63%; 24/38), while less fraction of patients had competing subclonal mutations in NRAS (26%; 10/38) or in KRAS (8%; 3/38). Most of these mutations were found in canonical sites (NRAS: G12D/S/A/C, G13D/R/V, Q61A/H/R; KRAS: G12D/A/R, G13D/R/V, A146P/T). Patients carrying RAS mutations had significantly higher WBCs (20.5 vs 7.2, P&amp;lt;.001) consistent with the role of RAS in proliferation, possibly via augmentation of MYC. Isolated chromosome 7 abnormalities were more common among RAS mutant carriers who otherwise showed less complex karyotypes. RAS mutations were mostly enriched in MDS/MPN (26%) while absent in sAML suggesting the impact of these lesions on OS but not on PFS. Analysis of clonal architecture showed that accumulation of RAS subclones was most commonly related to the presence TET2 (17% vs. 8%) and EZH2 (6% vs. 1%) compared to WT. At the time of AML evolution, the presence of RAS mutation did not correlate with OS (18 vs. 13 mo., P=0.07). However, median OS was shorter in MDS and MDS/MPN carrying multiple RAS mutations compared to WT (10 vs. 30 mo., P=0.005) again supporting the contention that these clones may be markers of non-progression-related mortality. In sum, our study supports the notion that ancestral RAS hits are common in childhood MN with a hereditary component whereby the progression is related to the acquisition of secondary hits in biallelic configuration. In contrast in adult and elderly MN, specific genetic background predisposes to RAS mutant mosaicism such as ancestral TET2 and EZH2 lesions and MPN features reflecting the direction of selection pressure towards accumulation of multiple RAS pathway hits. Lack of the gene/dose effect on progression indicates, that in contrast to the compound heterozygous RAS childhood diseases, RAS mutant mosaicism reflects branching rather than linear evolution mode. Disclosures Sekeres: Pfizer: Consultancy; Takeda/Millenium: Consultancy; BMS: Consultancy. Carraway:Abbvie: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Takeda: Other: Independent Advisory Committe (IRC); Stemline: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; BMS: Consultancy, Other: Research support, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents &amp; Royalties: University of Illinois at Chicago. Patel:Alexion: Other: educational speaker. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Clonal Architecture Analysis of TET2 Identified Distinct Origins in Myelodysplastic Syndromes

Next-generation sequencing (NGS) has deepened our understanding of myelodysplastic syndromes (MDS). Genetic mutations of TET2 were common in MDS and were also detected in asymptomatic elder persons, which were defined as aged related clonal haematopoiesis (ARCH) and considered as a myelodysplastic syndrome precursor state. In this study, we analyzed the mutation profiles of TET2 in 770 newly-diagnosed MDS subjects. 73 mutations were found in 67 of 770(8.7%) subjects. 14.9% were frame shifts, 29.9% were nonsense, 44.8% were missense and 10.7% harbored 2 TET2 mutations. TET2MT subjects were older than TET2WT subjects (P=0.024) and the variant allele frequency (VAF) of TET2 was positively correlated with age (r=0.318, P=0.009). Clonal architecture was determined using a copy number-adjusted VAF difference between 2 mutation events in 49 TET2MT subjects with 2 or more mutations. 19 (38.3%) were TET2ancestral and 30(61.2%) were TET2subclonal. TET2ancestral subjects were significantly older than TET2WT subjects (P=0.013) while no difference was found between TET2subclonal subjects and TET2WT subjects (P=0.509). The frequency of cytosine-to-thymine (C→T) transition was significantly higher in TET2ancestralsubjects compared with TET2subclonal subjects (P=0.029), which was considered to be a somatic mutational signature of aging, indicating that MDS driven by TET2ancestral was likely derived from TET2MT ARCH. The most common upstream mutations in TET2subclonal subjects were U2AF1 (16.7%) and ASXL1 (13.3%), and the most common downstream mutations in TET2ancestral subjects were ASXL1 (21.1%) and RUNX1 (15.8%). TET2 mutations rarely existed alone in TET2ancestral subjects (14.3%) and were always accompanied by another mutations like U2AF1, SF3B1 and ASXL1, suggesting that the acquisition of another mutation led to the progress of ARCH to MDS in TET2ancestral subjects. TET2MT had no impact on survivals in overall cohort (P=0.218) while predicted poorer survivals in IPSS-R lower risk group (P=0.004). Additional ASXL1, U2AF1 and RUNX1 mutations in TET2MT subjects indicated poorer prognosis compared with TET2WT subjects (P=0.005; P=0.04; P&amp;lt;0.001) .There was no significant difference in OS of TET2MT subjects with different clonal architecture (P=0.76). Subjects with TET2 VAF≥30% had poorer survivals compared with TET2WT subjects (P=0.057). In conclusion, TET2MT MDS subjects with different clonal architectures may have different origins. TET2ancestral subjects may be derived from ARCH and progressed to MDS after secondary hits. The effects of TET2 mutation on the prognosis depended on the accompanying mutations and clonal burdens. Disclosures No relevant conflicts of interest to declare.

Clonal Architecture of &lt;i&gt;EGFR&lt;/i&gt; Mutation Predicts the Efficacy of EGFR-Tyrosine Kinase Inhibitors in Advanced NSCLC: A Prospective Multicenter Study

Background: Although EGFR mutations in lung cancer are identified most often as trunk mutations in the early stage, cancer evolution often follows a branched trajectory. Whether EGFR would always be the dominant clone in the advanced stage is unknown. Methods: This prospective multicenter clinical trial recruited treatment naive patients with stage IIIB-IV non–small cell lung cancer from 14 centers in China. Paired tumor and plasma ctDNA samples were sequenced by target-capture deep sequencing of 1021 genes. Clonal dominance analysis was performed on the basis of PyClone. Findings: Overall, 300 patients were recruited and 132 patients treated with first line targeted therapy were followed. The median follow-up time was 10 months (IQR 6–13). Of the 94 patients with available ctDNA data for EGFR clonal architecture analysis, 72 (76.6%) showed EGFR as the dominant clone. The mPFS was longer for these patients than for the 22 patients who had EGFR non-dominant clone (11 months vs 10 months, hazard ratio [HR] 0.46, 95% CI 0.24–0.88, p = 0.02). The difference was more significant if patients who were defined as having EGFR dominant clone by both tumor tissue and plasma (n = 43) versus those not (n = 8) (11 vs 6 months, HR = 0.13, 95% CI 0.04–0.50, p = 0.003). Moreover, multivariate cox proportional hazard ratio analysis demonstrated EGFR clonal architecture as an independent prognostic indicator of the efficacy of EGFR-tyrosine kinase inhibitors. Interpretation: In advanced NSCLC,EGFR mutations do not always make up a dominant clone. Clonal architecture of EGFR in the pretreatment ctDNA is associated with the efficacy of EGFR-TKIs in NSCLC. Trial Registration: The trial has been registered with the number NCT03059641. Funding Statement: Geneplus-Beijing Co. Ltd. Declaration of Interests: RC, PL, LC and XY were employees of Geneplus-Beijing Co. Ltd. All other authors declare no conflict of interests. Ethics Approval Statement: This study was performed under a protocol (KS1707) approved by the Institutional Review Board of Shanghai Chest hospital (Shanghai, China).

Spontaneous Remission of Secondary Acute Lymphoblastic Leukemia Associated with Lenalidomide Therapy for Multiple Myeloma

Introduction: Multiple myeloma (MM) is an incurable malignancy of B-cell lineage. Introduction of immunomodulatory drugs such as lenalidomide has significantly improved overall survival of patients with MM. Lenalidomide maintenance is currently the standard of care for maintaining remission in MM. However, second primary malignancies are known to arise in patients on lenalidomide though their pathogenesis is not known. We report a patient who developed B-lymphoblastic leukemia (B-ALL) while on lenalidomide, which went into spontaneous remission after stopping lenalidomide. Whole exome sequencing (WES) was performed to examine the mutational landscape and clonal evolution of the different malignant clones. We also aimed to postulate a mechanism for lenalidomide-induced ALL. Case history and methods: A 59-year-old female with history of rheumatoid arthritis was diagnosed with IgG-κ MM and treated with 8 cycles of bortezomib, Lenalidomide and dexamethasone followed by lenalidomide-maintenance (10 mg/day). At 36 months after initiation of treatment for MM, she developed lymphopenia and her bone marrow (BM) biopsy showed 38% leukemic B lymphoblasts. Lenalidomide was discontinued and a follow-up BM biopsy done 2 months later showed spontaneous complete remission of ALL, which was confirmed at 8 months. She remains in complete remission of ALL and MM without any specific treatment at 12 months after the diagnosis of ALL. BM aspirate samples at diagnosis of MM, on lenalidomide-maintenance with MM in remission, at diagnosis of ALL, and after stopping lenalidomide with MM and ALL in remission were used to perform WES with 2x150 bp reads and 100x coverage utilizing the Illumina Hiseq. The raw reads were mapped to reference genome (hg19) and compared with peripheral blood T-lymphocytes as germ line control. Variants were annotated using dbSNP, CLINVAR and COSMIC through Mutect. Clonal evolution was analyzed by SciClone. The study was approved by Institutional Review Board. Results: The burden of somatic mutations was significantly higher at diagnosis of MM when compared to the three other time points. Analysis of clonal architecture revealed the distinct clustering of mutations at specific time points (Figure). Most mutations detected at diagnosis of MM (e.g. NOTCH2, BTG1, BCLAF1) disappeared after treatment for MM. Similarly, most mutations detected only at diagnosis of ALL (e.g.PIK3CD, CDK16) became undetectable at spontaneous remission. Interestingly, clones with mutations of IGSF3 (immunoglobulin superfamily) and CXXC4 (Wnt signaling pathway) were detectable while the patient was on lenalidomide and at diagnosis of ALL but disappeared after stopping lenalidomide, which suggests that these clones gained pro-survival advantage from lenalidomide. Only a few mutations (GSDMC, NBPF20, ANAPC1) persisted in both MM and ALL stages. GSDMC is a gasdermin family member which may modulate function of MYC. NBPF20 has been described in relapsed pediatric ALL. ANAPC1 is a cell cycle gene whose transcription is regulated by Ikaros. Loss of repressor function of Ikaros was recently reported to deregulate ANAPC1 expression and cause mitotic progression of ALL in vitro. As lenalidomide is known to induce degradation of Ikaros, we hypothesize that lenalidomide may create a favorable selection pressure for B-cell clones harboring mutations in Ikaros-dependent genes. Conclusions: Clonal evolution analysis suggests that MM and ALL arose from different B-cell sub-clones, which was consistent with previous observation. However, there are a few shared mutations between MM and pre-B ALL, which may be responsible for leukemogenesis in our case. Lenalidomide may affect intracellular protein interactions to induce selection of rare B-cell clones evolving into secondary ALL. Also, our case demonstrated that simply stopping lenalidomide may lead to spontaneous and durable regression of ALL. Transcriptomic and proteomic analysis including Ikaros expression is required to further understand the mechanism of appearance of ALL and its regression after stopping lenalidomide. Figure Disclosures Shlomchik: BlueSphere Bio: Other: Founder and Equity Interest.

Single cell analysis of clonal architecture in acute myeloid leukaemia

We used single cell Q-PCR on a micro-fluidic platform (Fluidigm) to analyse clonal, genetic architecture and phylogeny in acute myeloid leukaemia (AML) using selected mutations. Ten cases of NPM1c mutant AML were screened for 111 mutations that are recurrent in AML and cancer. Clonal architectures were relatively simple with one to six sub-clones and were branching in some, but not all, patients. NPM1 mutations were secondary or sub-clonal to other driver mutations (DNM3TA, TET2, WT1 and IDH2) in all cases. In three of the ten cases, single cell analysis of enriched CD34+/CD33− cells revealed a putative pre-leukaemic sub-clone, undetectable in the bulk CD33+ population that had one or more driver mutations but lacked NPM1c. Cells from all cases were transplanted into NSG mice and in most (8/10), more than one sub-clone (#2-5 sub-clones) transplanted. However, the dominant regenerating sub-clone in 9/10 cases was NPM1+ and this sub-clone was either dominant or minor in the diagnostic sample from which it was derived. This study provides further evidence, at the single cell level, for genetic variegation in sub-clones and stem cells in acute leukaemia and demonstrates both a preferential order of mutation accrual and parallel evolution of sub-clones.

PHF6 Somatic Mutations and Their Functional Role in the Pathophysiology of Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Recurrent somatic nonsense PHF6 mutations have been reported in patients with T-acute lymphocytic leukemia, AML and chronic myeloid leukemia in blast crisis. Germ line (GL) PHF6 mutations are responsible for Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation and dysmorphic features. PHF6 is a highly conserved 41kDa protein with ubiquitous expression in hematopoietic cells, including CD34+ cells.We screened patients (N=1166) with myeloid neoplasms by targeted multi-amplicon deep NGS targeting all ORFs of PHF6 to determine the prevalence and distribution and molecular context of PHF6 gene alterations. In total, we identified and verified 52 cases with somatic PHF6 mutations, 32 of which were frameshift or nonsense mutations and with a strong male predominance (76%). Mutations were distributed almost equally between 2 DNA binding domains. Previously, PHF6 has been included in other screening panels (Haferlach et al. 2014 and Papaemmanuil 2013) with somatic mutations found in 24/944 and 21/738 MDS cases, respectively. SNP-array karyotyping showed that microdeletions involving the PHF6 locus were present in about 1.2% of myeloid neoplasms, but affected only female patients.The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (P=.018). The most commonly associated somatic mutations included RUNX1 (P=.001) and IDH1 (P=.008) but not IDH2 (P>.1). There was no impact on overall survival with respect to PHF6 mutant status in total or within individual risk groups (low risk (RA,RARS) vs. high-risk (RAEB1/2). Concomitant PHF6 and RUNX1 mutations were associated with particularly poor prognosis. RUNX1 mutational status correlated with PHF6 expression levels and PHF6 expression inversely correlated with RUNX1 mRNA levels. Subsequent analysis of clonal architecture using VAF calculations and serial samples for these cases suggested that PHF6 may function as a founder driver gene in 18% of cases. PHF6 variant allelic frequency (VAF) varied between disease subtypes, with the highest clonal burden found in AML patients (P<.01). Within MDS patients we also found lower expression of PHF6 mRNA in CD34+ cells in MDS overall vs. controls (P<.01), as well as lower expression of PHF6 in advanced myeloid neoplasms (P<.05). Lower expression (defined as mean+1SD of controls) was found in 12% and 23% of patients with lower- or higher- risk MDS, respectively.Recent studies have proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at G2/M phase and DNA damage. Following shRNA knockdown, hematopoietic cell lines showed only moderately accelerated growth and increased response to growth factors, while EPO-dependent UT7, did not result in growth factor autonomy. To delineate the possible pathophysiological pathway involving PHF6, we compared transcriptional expression profiles of 5 different cell lines with shPHF6 to WT counterparts. We then studied the consequences of PHF6 knockdown on transcriptional profiles. We have found 1020 transcripts differentially expressed (with at least 1.5x change up/down) in the context of shPHF6 knock down. Concordant results among all 5 cell lines resulted in 354 genes that were upregulated and 766 that were down-regulated. Analyses with primary patient data derived from low PHF6 expressors and mutant cases found a concordance of 71 upregulated genes and 80 genes that were downregulated.The most significant functional group of transcripts that was found to be modulated was a family belonging to ribosomal biogenesis pathway (pFDR<1x10-6). Mass spec fingerprinting found protein-protein interaction partners that were found to be dysregulated on a transcriptional level. This finding of protein interaction/transcriptional dependence might suggest feedback mechanisms on a transcriptional level.In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms, frequently associated with RUNX1/IDH1 mutations. Our functional in vitro studies along with recently published reports suggest an association of PHF6 deficiency with transcriptional regulation and thereby provide a basis for a phenotype conveyed by ancestral lesions, consistent with its role as a tumor suppressor gene. DisclosuresSekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Makishima:The Yasuda Medical Foundation: Research Funding.

BCOR and BCORL1 mutations in Myelodysplastic Syndromes (MDS): Clonal Architecture and Impact on Outcomes

BackgroundSeveral recurrent somatic mutations have been identified in MDS and these mutations play an important role in disease pathophysiology and outcome. BCOR and BCORL1 are located on chromosome X and interact with histone deacetylases and other cell functions. The BCOR gene is mutated (BCORMUT) in 4-6% of MDS patients (pts) and is associated with poor outcome. BCORL1 mutations (BCORL1MUT ) are present in <1% of MDS pts with an unknown impact on OS. We investigated the clonal architecture of BCOR and BCORL1 in MDS and the impact of these mutations on clinical outcome.MethodsWe sequenced DNA samples from pts with MDS and related myeloid malignancies (MDS/myeloproliferative neoplasms (MPN) and secondary AML) for the presence of BCOR and BCORL1 mutations and 58 other genes that have been described as recurrently mutated in myeloid malignancies. The Revised International Prognostic Scoring System (IPSS-R) was calculated as descried previously. Overall survival (OS) was measured from the time of diagnosis to time of death or last follow up. Variant allele frequencies (VAFs) adjusted by zygosity were used to define architecture of driver clones.ResultsOf 621 included MDS pts, 29 (5%) had BCOR mutations and 13 (2%) had BCORL1 mutations. Patients with BCOR mutations were younger (median age 63 vs. 68 years, p= .04), and had a lower platelet counts at diagnosis (63 vs. 93 109/L, p = .01) compared to BCORWT pts. Cytogenetic risk categories per IPSS-R for BCORMUT was similar to BCORWT: very good 3% vs. 2%, good 65% vs. 62%, intermediate 10% vs. 17%, poor 13% vs. 7% and very poor 6% vs. 9%, respectively, p = .62). Risk categories per IPSS-R were also similar for BCORMUT compared toBCORWT:very low 10 vs. 16%, low 31 vs. 40%, intermediate 24 vs. 18%, high 17 vs. 14%, and very high 13 vs. 10%, respectively, p = .69). BCOR mutations were missense in 9 pts (31%), frameshift insertion/deletion in 10 (34%), stopgain in 8 (28%), and nonsense in 2 (6%). BCOR is commonly co-mutated with ASXL1 (p=.008), RUNX1 (p= .0001), NF1 (p =.002), ETV6 (p =.026), BCORL1 (p =.0001), MECOM (p =.021), RAD21 (p =.021), and CEBPA (p = .0001). Clonal architecture analysis identified BCOR mutations as ancestral, subclonal, and mosaic in 41%, 21% and 38% of cases respectively. The median OS for BCORMUT pts was 24.5 months compared to 17.9 months for BCORWT, p = .23). The impact of BCOR mutations on OS was neutral even after adjustment for age and IPSS-R risk categories.Pts with BCORL1MUT had lower WBC counts (median 2.7 vs. 4 109/L p = .02), and lower platelets counts at diagnosis (72 vs. 91 109/L, p = .02) compared to BCORL1WT pts. In cytogenetic analyses, BCORL1MUT was associated with a higher incidence of very good cytogenetics per IPSS-R (15% vs. 2%, p =.001). Further, BCORL1MUT were more likely to be classified in the intermediate risk group per IPSS-R (46 vs. 18%, p = .01) and less likely to be classified in the low risk group (7 vs. 40%, p = .01) compared to BCORL1WT. BCORL1 mutations were missense in 7 patients (53%), frameshift insertion/deletion in 3 (23%), stopgain in 1 (7%), and nonsense in 2 (15%). BCORL1 is commonly co-mutated with TET2 (p =.001), BCOR (p <.001) DHX29 (p = .001), C7orf55 (p =.02), and FLT3 (p =.03). Clonal architecture analysis identified BCORL1 mutations as ancestral, subclonal, and mosaic in 61%, 23% and 15% of patients respectively. The median OS for BCORL1MUTpts was longer compared to BCORL1WT, but was not significantly different (43.8 vs. 19.9, respectively, p = .16) even after adjustment for age and IPSS-R risk categories.ConclusionBCOR and BCORL1 mutations occur with low frequency in MDS. These mutations can be ancestral or subclonal. The impact of BCOR mutations on OS was neutral even after adjustment for age and IPSS-R risk scores. Although the median OS for pts with BCORL1MUT was longer compared to BCORL1WT,it was not statistically significant. A larger pt population with BCOL1 may show a positive impact of these mutations on OS. DisclosuresMukherjee:Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding. Advani:Blinatumomab: Research Funding; Pfizer Inc.: Consultancy, Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

The immune landscape of renal cell carcinoma and its association with intratumoral clonality.

605 Background: Clear cell renal cell carcinoma (ccRCC) is among the most highly infiltrated tumors despite a relatively low mutation burden. Despite success in treating subsets of these patients with immunotherapy, the high immune-infiltration of ccRCC remains a conundrum that has not been sufficiently addressed. Methods: We utilized an original RNAseq-based aggregate immune score (Senbabaoglu Y, bioRxiv, 2015) to compare immune infiltration levels across 20 tumor types profiled in The Cancer Genome Atlas (TCGA) and validated our results with an independent cohort of ccRCC patients (Sato Y, Nat Genet, 2013). We also evaluated the immunogenic effect of intratumoral heterogeneity (ITH) through an analysis of clonal architecture in ccRCC tumors from both of the aforementioned data sets using the SciClone (Miller C, PLoS Comput Biol, 2014) algorithm. Results: We identified ccRCC as an immunogenic outlier that is unique from other highly infiltrated tumors in its exceptional overexpression of antigen presentation machinery compared to normal tissue. In a focused decomposition of immune cell levels within the 415 ccRCC tumors from TCGA, three unique clusters of tumors emerged primarily separated by varying degrees of T cell infiltration; termed (1) T-cell-enriched, (2) heterogeneous, and (3) non-infiltrated groups. These clusters were validated in an independent ccRCC cohort of 101 patients. A comparison of the degree of clonality with immune infiltration levels revealed that tumors with less ITH (i.e. fewer subclones) had significantly higher immune infiltration in both the TCGA and the validation cohort . Conclusions: Our findings provide novel insight into the immune landscape of ccRCC tumors and explore a potential mechanism for the immunogenicity of these tumors. Such information can potentially be used to aid in predicting therapeutic response to immunomodulatory agents.

Transition of Chronic Myeloid Leukemia to Chronic Myelomonocytic Leukemia As a Tool to Observe Development of Chronic Myelomonocytic Leukemia

Introduction. Development of abnormal Philadelphia (Ph) negative clones following treatment of chronic myeloid leukemia (CML) patients with imatinib has been observed in 3 to 9% of patients. Here we report on a 77 year old male diagnosed with CML that responded to imatinib treatment and subsequently developed chronic myelomonocytic leukemia (CMML). He achieved major cytogenetic response within 3 months but this response coincided with the emergence of monocytosis diagnosed as CMML. Five months after starting imatinib treatment the patient succumbed to CMML. We analyzed five sequential samples to determine whether a chronological order of mutations defined the emergence of CMML and to characterize the clonal evolution of the CMML population.Materials and Method. Five samples (diagnostic and four follow up samples) were available for analysis. CMML mutations were identified by whole exome sequencing (WES) in CD14+ cells following the onset of CMML, using CD3+ cells as constitutional control. Mutations were validated by Sequenom MassARRAY and Sanger sequencing and quantified by pyrosequencing. Deep WES was performed on the diagnostic sample to determine whether the mutations were present at CML diagnosis. To determine the clonal architecture of the emerging CMML, colony formation assays were performed on the diagnostic and the next two follow-up samples (Samples 1-3). More than 100 colonies per sample were plucked for DNA and RNA isolation. The DNA from these colonies were tested for the presence of the confirmed CMML mutations and the RNA was used for detection of BCR-ABL1 transcript using a Taqman real time assay.Results. Four mutations were identified by Sequenom and WES throughout the patient's time course [KRASG12R, MSLNP462H, NTRK3V443I and EZH2I669M ]. Sequenom did not identify these at diagnosis while deep WES did. Clones derived from colony formation assay revealed three distinct clones present in all samples analysed. Clone 1 had only KRASG12R, clone 2 had KRASG12R, MSLNP462H, and NTRK3V443I, and clone 3 had all four mutations. All clones containing any of these four mutations were BCR/ABL1 negative. Analysis of clonal architecture indicated that KRASG12R was acquired first and EZH2I669M last, while MSLNP462H and NTRK3V443I were acquired in between. These CMML clones increased proportionately as clinical CML metamorphosed into clinical CMML after initiation of imatinib therapy. Consistent with the colony data, pyrosequencing revealed that the ratio between the mutants remained largely stable throughout the follow up period.Conclusion. This case illustrates how targeted therapy impacts clonal competition in a heterogeneous MPN. While the CML clone was dominant in the absence of imatinib, it was quickly outcompeted by the CMML clones upon initiation of imatinib therapy. The clonal architecture analysis, in combination with in vivo kinetics data, suggest that the KRASG12R mutation alone was able to produce a CMML phenotype as clones with just KRASG12R remained at a relatively stable ratio during follow up. Unexpectedly, acquisition of additional mutations, including EZH2I669M as the last mutational event identified in this patient, did not increase clonal competitiveness, at least in the peripheral blood. These data show that clonal evolution may not invariably increase clonal fitness, suggesting that factors other than Darwinian pressures contribute to clonal diversity in myeloproliferative neoplasms. [Display omitted] DisclosuresDeininger:Gilead: Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

PHF6 - Somatic Mutations and Their Role in Pathophysiology of MDS and AML

Recently, rare somatic nonsense PHF6 mutations and deletions have been reported in patients with T-ALL, AML and blast crisis CML. Germ line PHF6 mutations have been described in Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation and somatic deformities. Patients with BFLS have been also reported to develop leukemia, suggesting PHF6 mutations may predispose to cancer. PHF6 is a highly conserved 41kDa protein showing ubiquitous expression in a variety of tissues, including bone marrow, CD34+ cells and leukocytes. The function and molecular pathogenesis in hematological disorders is unknown. PHF6 has been suggested to be a tumor suppressor gene (TSG) involved in the control of rRNA synthesis. Recent CHIPseq experiments showed that PHF6 binds upstream of the regulatory sequence of RUNX1.In an index case of a young adult female patient with proliferative CMML with dysmorphic features, we have identified remarkable GL mosaicism for PHF6 mutation (p.K44fs), confirmed by deep sequencing of bone marrow, CD3+ cells, spleen and skin tissue. Subsequently, we screened patients with myeloid neoplasms by targeted multi-amplicon sequencing to determine the prevalence and distribution of PHF6 gene alterations. Sequencing results from 1122 cases were analyzed (778 by targeted deep sequencing and 344 by whole exome sequencing). In total, we identified 45 cases with PHF6 mutations, 32 of which were frameshift or nonsense mutations. Previously, PHF6 have been included in screening panels by Haferlach et al., (Leukemia 2014) and Papaemmanuil et al., (Blood 2013) and somatic mutations were found in 24/944 and 21/738 cases of MDS, respectively. The somatic nature of these defects was confirmed by analysis of non-clonal CD3+ lymphocytes, Thus the incidence of PHF6 mutations ranges from 4.3% in current study to 2.8% and 2.5% reported by others and are most frequently observed among patients with secondary AML (33%), suggesting that PHF6 mutations are not uncommon driver events in myeloid neoplasia. Gender distribution showed a strong male predominance (76%), indicating that retention of a single copy of PHF6 may be protective. There was no significant sex difference in the transcriptional expression of PHF6 itself. The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (P =.08). The most commonly associated somatic mutations were in RUNX1 (P =.001) and IDH2 (p=.008). Concomitant PHF6 and RUNX1 mutations are associated with a poor prognosis in AML, and occur predominantly in males. There was no association observed between low expressors of PHF6 and RUNX1 mutations or RUNX1 expression levels. Conversely, RUNX1 mutant cases without somatic PHF6 mutations were not observed to have low transcriptional PHF6 levels. Subsequent analysis of clonal architecture using variant allelic frequency calculations and serial sampling suggested that mutated PHF6 may function as a founder driver gene in proportion of cases, while RUNX1 mutations are acquired as secondary events.Recent studies proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at the G2/M phase and an increase in DNA damage. To delineate a possible pathophysiological pathway involving PHF6 we compared transcriptional expression profiles of low expressors to those with normal levels of PHF6. The most notably deregulated group of genes were clustered to a functionally related group of ribosomal RNA proteins (p<.00001). To better understand functional properties of PHF6 we conducted PHF6 specific immuno-precipitation followed by mass spectrometric fingerprinting on K562 cells to identify protein partners. We have identified a novel association of PHF6 in RNA degradation/stability and ribosomal proteins, including MOV and PABPC families.In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms and arefrequently associated with RUNX1/IDH2 mutations. Our functional in vitro studies, along with recently published reports, suggest an association of PHF6 deficiency with transcriptional regulation and thereby provide a basis for a transcriptional repressor phenotype conveyed by ancestral lesions, consistent with a role for PHF6 as a TSG. DisclosuresLevine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees.

Determinants of Phenotypic Commitment and Clonal Progression--Conclusions from the Study of Clonal Architecture in CMML

CMML is heterogeneous clinically (a varying degree of dysplastic or proliferative clinical features) and in terms of its molecular pathogenesis. Analysis of the spectrum of genomic lesions in CMML may contribute to understanding of the pathogenesis and help identify certain mutations as diagnostic biomarkers. Apart of the mere presence of a somatic lesion, phenotypic features may be shaped by initial hits. Conversely sub-clonal events may determine the phenotype or progression. Finally, initial hits may predetermine e.g., mutator phenotype, or differentiation block and therefore selecting for specific sub-clonal hits.We have selected a large CMML cohort to establish generalizable pathogenetic and clinical associations to account for molecular heterogeneity and serial samples have to be analyzed to assess clonal dynamics and hierarchy. The study group consisted of 242 patients, including 150 CMML cases (96 CMML-1, 27 CMML-2, 27 post CMML sAML) and JMML (N =92); 15 patients were studied serially. We also used comparison cohorts of M4/M5 AML (N =64) and advanced (N= 231) and low risk MDS (N= 199) serving as risk adjusted match for CMML subtypes. The CMML entity was further sub-classified based on clinical parameters and pathomorphologic features, 57% dysplastic (MD-CMML) and 43% proliferative form (MP-CMML). Analysis was performed using WES (paired germ line/tumor samples) and multiamplicon NGS targeting top 60 most commonly affected genes. For clonal architecture analysis, cross-sectional variant allelic frequency (VAF) concept based analysis was performed including assessment of affected genes by ranking of the corresponding clonal burden rather than the absolute cellular frequency. The results of this analysis were confirmed in serial samples to identify expanding, declining and stable subclones.Comparison of mutational spectra between the disease entity show profound differences in morphologically similar entities as particularly evident in comparison of CMML to JMML (TET2, ASXL1) or to lesser degree low risk MDS and CMML1 while progression in advanced cases was often associated with similar spectrum of additional subclonal events. However, the differences were more striking when clonal hierarchy was assessed to identify dominant/codominant and subclonal mutational events. We found that top 4 dominant/codominant clonal events, included TET2 (56%), SRSF2 (42%), ASXL1 (46%), DNMT3A (45% of patients), while in MDS corresponding frequency of these dominant events was TET2 (15%) SRSF2 (8%), ASXL1 (11%) and DNMT3A (8%), with most common ancestral events ranked SF3B1, TET2, ASXL1 etc. The clinical importance of these dominant events in CMML is highlighted by their impact on survival in KM analysis (p=.018).Our analysis also demonstrated that for certain founding events not pathognomonic for CMML either codominant or subsequent subclonal events determine the phenotypic features (1st generation) or progression (2nd generation). For instance initial TET2 in CMML was followed often by SRSF2 or in conjunction, RAS pathway mutations while MDS was driven by TET2, SF3B1, TP53, and many other events. Progression in our cohort was driven in both CMML and MDS by ASXL1, RUNX1, NPM1. When other common mutations were categorized by their role in individual patients 27% EZH2, 20% of CBL and 22% of SETBP1 were dominant. Serial analysis further qualified the cross-sectional analysis and allowed for categorization of subclonal events. For instance, CMML-1 cases initially presented with dominant TET2 followed by subtype specific subclonal SRSF2 and progression event IDH2 progressed to sAML with new NPM1 acquisition and expansion of IDH2 c lone. In our serial sample analysis we observed that increasing ASXL1 and RUNX1 clones correspond to clinical progression, ancestral events may remain stable (TET2, SRSF2, SETBP1), while non-permissive subclones can smolder or even decline.In sum, deep NGS allows for identification of specific ancestral events, which may determine the subsequent secondary mutational events in CMML. Classification of CMML based on ancestral events and subclonal events rather than on the global mutational spectrum correlates with clinical features and prognosis and may contribute to further clinical resolution of CMML based on the presence of specific founder mutations ultimately help establish therapeutic interventions. DisclosuresSekeres:Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees.

Abstract LB-175: Deep clonal profiling identifies distinct mechanisms of heterogeneity and evolution in breast cancer

Abstract Background: Breast tumors exhibit intratumor heterogeneity resulting in targeted therapy resistance and other challenges in disease management. To address the sources of heterogeneity, we performed a unique, in-depth analysis of clonal architecture in primary chemoradiation-naïve breast cancers. We combined DNA content-based flow cytometry and ploidy analysis with aCGH and whole exome next-generation sequencing (NGS) in multiple biopsies from the tumors and involved lymph nodes (LNs). Material and methods: We sorted nuclei from distinct populations of diploid, tetraploid, and aneuploid cells in surgical tumor samples from three chemoradiation-naïve patients. Each sorted tumor cell population was interrogated with aCGH and whole exome NGS. In Patient #1, we sorted and interrogated the genomes of tumor populations from 12 fresh frozen sections morphologically mapped from within a HER2+, ER+, PR- primary invasive ductal carcinoma (IDC) of histological grade 3 with LN involvement and 2-3 sections from 2 out of 5 LNs. In Patient #2, 11 morphologically mapped fresh frozen sections were analyzed from a grade 2, ER+, PR+, HER2+, BRCA2 mutant LN- IDC. In parallel, matching samples were processed for IHC assays. In Patient #3 biopsies from a grade 2 ER+, PR+ primary tumor were profiled. Results: The 18 primary and LN biopsies from Patient #1 fell into 6 distinct ploidy groups albeit with aberrant but homogenous aCGH profiles, characterized by SARC amplification and homozygous deletions in ROBO1 and ROBO2. In contrast a dominant ploidy was identified throughout 10 biopsies in Patient #2 but with heterogeneous aCGH profiles. Patient #3 had a clonal homozygous deletion in Numb in each of 4 tumor biopsies. Mutation profiles obtained through exome sequencing further confirmed that ploidy was the main driver in Patient #1 whereas copy number aberrations played the key role in Patient #2 with the BRCA2 mutation (R3129X). A dendrogram based on exome variant calls of the aneuploid populations in Patient #1 strongly correlated with ploidy group and further revealed the specific clonal population characterized by a 5N ploidy and homozygous mutations in TP53 and PIK3CA as the progenitor to the ploidies present in the distant LNs. Strikingly, patients #1 and #2 were HER2 wild type across their clonal populations, contradicting IHC staining in a single core biopsy. Conclusions:Rather than inferring the presence of distinct tumor cell populations, our flow-sorting based approach of first identifying clonal populations and then interrogating their genomes, provides an objective method of exploring the sources and clinical significance of tumor heterogeneity. Our approach of clonal analysis has broad implications in the study of tumor heterogeneity and the identification of drivers in breast and other solid tumors that can advance more effective treatment and clinical management of patients with this disease. Citation Format: MARIACARLA ANDREOZZI, Princy Francis, Elizabeth Lenkiewicz, Mia Champion, Brady Laughlin, Karen Anderson, Heather Cunliffe, Ann E. McCullough, Michael T. Barrett, Barbara Pockaj. Deep clonal profiling identifies distinct mechanisms of heterogeneity and evolution in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-175. doi:10.1158/1538-7445.AM2015-LB-175