Copy-neutral Loss Of Heterozygosity Research Articles

Abstract 5214: Expanding the spectrum of germline-driven cancers by leveraging population-scale targeted tumor and normal sequencing

Abstract Large case-control and familial studies have established clear cancer-specific risk profiles for several key cancer predisposition genes (CPGs). For example, germline pathogenic variants (PVs) in BRCA1/2 (gBRCA) are associated with increased risk for developing breast, ovarian, pancreatic, and prostate cancers. However, the extent to which gBRCA mutations are involved in mediating the tumorigenesis of other cancer types remains challenging to characterize. We hypothesized that integrating orthogonal features such as selection for biallelic inactivation of the PVs and depletion of canonical somatic drivers among the carriers can enrich the signal for identifying novel gene and cancer type associations. We then extend this framework to identify novel CPGs as well as to understand how tumors arise in patients with PVs in oncogenes. To study this, we leveraged the prospective MSK-IMPACT matched tumor-normal sequencing cohort of 49,291 patients across 77 major cancer types. We study 90 well-known CPGs as well as >300 cancer genes not previously associated with cancer predisposition. Overall, 8% (N=3,964) of patients harbored a PV in high or moderate penetrance CPGs. We identified 90 gene and cancer type associations with enrichment for biallelic inactivation (q<0.05), including 19 novel findings among clinically actionable genes. For example, we find enrichment for biallelic inactivation of BRCA1/2 in unexpected lineages such as hepatobiliary, endometrioid, and ampullary cancers. These tumors were also significantly depleted for somatic gain-of-function driver alterations. Hepatobiliary cancers with gBRCA1 mutations were also enriched for somatic loss of NF1. Among carriers of PVs in oncogenes, we observe two possible mechanisms of first somatic hit towards malignant transformation. We find enrichment for copy number gain or copy neutral loss of heterozygosity of the germline PV in thyroid cancers with a PV in RET. We also find that lung cancers with a germline PV in EGFR frequently developed additional somatic point mutations located in cis with the PV. Investigating genes with no prior association with germline predisposition to cancer, we find evidence for KEAP1 and CIC as likely novel CPGs. Lung (n=8) and thyroid (n=4) cancers with deleterious germline variants in KEAP1 were characterized by loss of the wild-type allele, co-occurring somatic STK11 mutations, and depletion of canonical drivers such as EGFR. We also found biallelic loss of CIC in two patients with Neuroblastoma, each carrying a different germline loss-of-function mutation in CIC. Both tumors were also negative for MYCN and ALK defects. Collectively, our findings expand our understanding of cancer predisposition in cancer, shed new insights into how tumors arise in germline carriers, and provide a framework for identifying new CPGs using population scale tumor-normal paired clinical sequencing data. Citation Format: Miika Mehine, Rebecca Caeser, Yelena Kemel, Daniel Muldoon, Sebastià Franch-Expósito, A. Rose Brannon, Aijazuddin Syed, Ozge Ceyhan-Birsoy, Maksym Misyura, Panieh Terraf, David B. Solit, Marc Ladanyi, Kenneth Offit, Zsofia K. Stadler, Diana L. Mandelker, Yonina R. Murciano-Goroff, Charles M. Rudin, Michael F. Berger, Chaitanya Bandlamudi. Expanding the spectrum of germline-driven cancers by leveraging population-scale targeted tumor and normal sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5214.

Genomic landscape of follicular lymphoma across a wide spectrum of clinical behaviors.

While some follicular lymphoma (FL) patients do not require treatment or experience prolonged responses, others relapse early, and little is known about genetic alterations specific to patients with a particular clinical behavior. We selected 56 grade 1-3A FL patients according to their need of treatment or timing of relapse: never treated (n=7), non-relapsed (19), late relapse (14), early relapse or POD24 (11), and primary refractory (5). We analyzed 56 diagnostic and 12 paired relapse lymphoid tissue biopsies and performed copy number alteration (CNA) analysis and next generation sequencing (NGS). We identified six focal driver losses (1p36.32, 6p21.32, 6q14.1, 6q23.3, 9p21.3, 10q23.33) and 1p36.33 copy-neutral loss of heterozygosity (CN-LOH). By integrating CNA and NGS results, the most frequently altered genes/regions were KMT2D (79%), CREBBP (67%), TNFRSF14 (46%) and BCL2 (40%). Although we found that mutations in PIM1, FOXO1 and TMEM30A were associated with an adverse clinical behavior, definitive conclusions cannot be drawn, due to the small sample size. We identified common precursor cells harboring early oncogenic alterations of the KMT2D, CREBBP, TNFRSF14 and EP300 genes and 16p13.3-p13.2 CN-LOH. Finally, we established the functional consequences of mutations by means of protein modeling (CD79B, PLCG2, PIM1, MCL1 and IRF8). These data expand the knowledge on the genomics behind the heterogeneous FL population and, upon replication in larger cohorts, could contribute to risk stratification and the development of targeted therapies.

Large cell calcifying Sertoli cell tumour: molecular and immunohistochemical assessment of a series comprising non-metastasising and metastasising neoplasms.

Large cell calcifying Sertoli cell tumour (LCCSCT) is a type of testicular sex cord-stromal tumour that may occur sporadically or in the context of Carney complex and other genetic syndromes. A subset is clinically malignant, and the molecular mechanisms that drive such aggressive behaviour remain unknown. METHODS AND RESULTS: We analysed 21 samples from 20 patients with LCCSCT (12 non-metastasising and eight metastasising) using PRKAR1A immunohistochemistry (IHC) and next-generation sequencing. Alltumours except two (cases 17 and 20, both metastasising) demonstrated loss of PRKAR1A expression. Among 11 cases with interpretable sequencing results, all harboured pathogenic single nucleotide variants of PRKAR1A. Evidence of loss of heterozygosity (LOH) of PRKAR1A was present in all tumours with interpretable zygosity data, but the mechanisms of LOH were different for non-metastasising and metastasising tumours. Non-metastasising tumours demonstrated only copy-neutral LOH, while metastasising tumours demonstrated a spectrum of mechanisms of LOH, including copy-loss LOH, two concurrent mutations or copy-neutral LOH. Relevant molecular findings in non-metastasising LCCSCT were limited to PRKAR1A variants. In contrast, all metastasising LCCSCTs with interpretable data harboured additional pathogenic variants, including (but not restricted to) BRCA2 mutations with evidence of LOH and bi-allelic CDKN2A/B deletions. Three patients harboured PRKAR1A variants of inferred germline origin, including one with Carney complex and two without known syndromic features. CONCLUSIONS: This study further confirms that PRKAR1A IHC is a useful diagnostic tool for both non-metastasising and metastasising tumours and suggests that molecular analyses can be helpful to identify non-metastasising tumours with malignant potential in selected patients. Importantly, these results highlight that germline assessment could be beneficial for all patients presenting with LCCSCT.

Calorie restriction alters the mechanisms of radiation-induced mouse thymic lymphomagenesis.

Calorie restriction (CR) suppresses not only spontaneous but also chemical- and radiation-induced carcinogenesis. Our previous study revealed that the cancer-preventive effect of CR is tissue dependent and that CR does not effectively prevent the development of thymic lymphoma (TL). We investigated the association between CR and the genomic alterations of resulting TLs to clarify the underlying resistance mechanism. TLs were obtained from previous and new experiments, in which B6C3F1 mice were exposed to radiation at 1 week of age and fed with a CR or standard (non-CR) diet from 7 weeks throughout their lifetimes. All available TLs were used for analysis of genomic DNA. In contrast to the TLs of the non-CR group, those of the CR group displayed suppression of copy-neutral loss of heterozygosity (LOH) involving relevant tumor suppressor genes (Cdkn2a, Ikzf1, Trp53, Pten), an event regarded as cell division-associated. However, CR did not affect interstitial deletions of those genes, which were observed in both groups. In addition, CR affected the mechanism of Ikzf1 inactivation in TLs: the non-CR group exhibited copy-neutral LOH with duplicated inactive alleles, whereas the CR group showed expression of dominant-negative isoforms accompanying a point mutation or an intragenic deletion. These results suggest that, even though CR reduces cell division-related genomic rearrangements by suppressing cell proliferation, tumors arise via diverse carcinogenic pathways including inactivation of tumor suppressors via interstitial deletions and other mutations. These findings provide a molecular basis for improved prevention strategies that overcome the CR resistance of lymphomagenesis.

Next-Generation Sequencing of Newly-Diagnosed Primary CNS Lymphoma Reveals Alterations in BTG1, ETV6, and 6p Are Associated with Chemoresistance and Inferior Progression-Free Survival

Introduction: There is a significant unmet need for the development of reliable genetic biomarkers that identify patients with newly-diagnosed primary CNS lymphoma (PCNSL) who are at high risk of progression, both during standard methotrexate-based induction as well as delayed progression after receipt of dose-intensive consolidation. Currently, little is known about the genetic subtypes of PCNSL and the basis for differences in chemo-immunotherapeutic response. We hypothesized that an integrated clinical-genomic analysis may reveal new determinants of tumor progression and may ultimately guide a precision medicine-based approach to the selection of effective treatment regimens. Methods: We performed targeted next-generation sequencing of 529 pan-cancer genes of pre-treatment, formalin-fixed, paraffin-embedded tumors from 52 patients with CNS diffuse large B-cell lymphoma. We identified structural variants including single nucleotide variants, small insertions and deletions, and copy-number alterations. Clinical records were retrospectively reviewed to determine patients' baseline characteristics, IELSG index, and progression-free survival (PFS). 44 EBV-negative PCNSL patients treated with methotrexate, temozolomide, rituximab (MTR) induction with adequate genomic samples were available for complete analysis. Statistical testing was performed using R. Results: We identified three candidate genomic alterations that were significantly associated with chemotherapy resistance in PCNSL: (1) Alterations at 6p21.3, including both copy neutral loss of heterozygosity (CN LOH) at the HLA Class I (HLA-A, B, C) and Class II loci (HLA-DR, DP, DQ, TAP1) as well as homozygous deletion of HLA Class II; (2) Mutations of tumor suppressor genes BTG-1 and ETV6 (Fisher's exact, p < 0.01). Overall, these genomic alterations were detected in 18 out of 19 progression events in the cohort (Fisher's exact, p < 0.001). 7 of 9 tumors with 6p CN LOH also contained mutations at BTG-1 and/or ETV6. Kaplan-Meier survival analysis shows three subgroups stratified by alteration type with distinct PFS outcomes (Figure 1). The subgroup with either CN LOH or homozygous deletion at 6p had the earliest progression, followed by the subgroup with mutations at either BTG-1/ETV6 and no 6p CN LOH or homozygous deletion, compared to no alterations at these 3 loci (Log-rank, p < 0.01). Patients with hemizygous loss at 6p exhibited a trend towards worse outcome compared to no 6p loss. Furthermore, multivariate Cox proportional hazard analysis demonstrated a statistically significant 2.7-fold increased risk of progression with 6p CN LOH/homozygous deletion or mutations involving BTG-1/ETV6 (Table 1). While etoposide-cytarabine consolidation and maintenance lenalidomide were each significantly associated with a decreased risk of progression, age at diagnosis, Karnofsky performance status, intraocular disease status, and IELSG index were not significantly associated with PFS. Conclusions: Through harmonizing clinical and next-generation sequencing data, in this retrospective analysis we identify pathogenic alterations involving BTG-1 and ETV6, as well as major alterations at 6p21.3, that correlated with accelerated disease progression and an early tumor resistance phenotype in PCNSL. While 6p loss has been identified as a mechanism of tumor immune escape in PCNSL, our results suggest that 6p CN LOH may be a novel mechanism associated with early progression. Of note, in a parallel investigation using patient-derived CNS lymphoma tumor cells engrafted in a murine orthotopic xenograft model, we have demonstrated that lenalidomide significantly induces transcription of 6p21.3 genes involved in antigen presentation and immunomodulation, including HLA-A, TAP-1, and lymphotoxins. Taken together, 6p21.3 copy number loss, in particular CN LOH and homozygous deletion, as well as mutations in BTG-1 and ETV6 may be a promising biomarker set, potentially useful in risk stratification in clinical trials as well as to identify high risk patients more likely to derive benefit from lenalidomide. Prospective validation studies are planned to confirm these results. Supported by NCI. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LNK/SH2B3 As a Novel Driver in Juvenile Myelomonocytic Leukemia

Introduction Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive myelodysplastic/myeloproliferative disorder in toddlers. Ninety-five percent of patients have mutations detected in the Ras/MAPK signaling pathway including CBL, KRAS, NF1, NRAS, RRAS, RRAS2, and PTPN11. Oncogenic fusion proteins leading to hyperactive Ras signaling and other mutations in genes encoding for proteins upstream the Ras pathway have also been described. One of these upstream proteins is LNK which is encoded by SH2B3. Loss of function mutations in SH2B3 result in abnormal proliferation of hematopoietic cells due to cytokine hypersensitivity and activation of JAK/STAT signaling. Mutations in SH2B3 have been detected in a variety of hematologic malignancies, specifically in myeloproliferative neoplasms in adults. We previously identified secondary mutations in SH2B3 in JMML. Here we report a case series of four JMML patients with initiating mutations in SH2B3. We also show that SH2B3 mutant induced pluripotent stem cell (iPSC)-derived JMML like hematopoietic progenitor cells (HPC) are sensitive to JAK inhibitors including ruxolitinib. Patients and Methods Genomic DNA from peripheral blood, bone marrow or buccal swabs was extracted using standard protocols. DNA samples were sequenced using a custom amplicon-based targeted sequencing approach. Ficoll-purified mononuclear cells from bone marrow of JMML patients and control samples were reprogrammed by using Sendai virus expressing doxycycline-regulated OCT4, KLF4, MYC, and SOX2. Control and JMML iPSCs were differentiated to HPCs by culturing in serum-free media with sequential combinations of growth factors to support multipotent hematopoietic progenitor formation. The half-maximal inhibitory concentration (IC50) for each kinase inhibitor was determined by performing Cell Titer Glo assays (Promega). Results Clinical characteristics of patients with SH2B3 mutant JMML described in this study are highlighted in Table 1. iPSC-derived JMML-like HPCs with different mutational backgrounds were analyzed in cell proliferation assays after exposure to multiple different JAK inhibitors (ruxolitinib, momelotinib, tofacitinib). HPCs with alterations in SH2B3 were more sensitive to chemical JAK inhibition compared to HPCs not harboring mutations in SH2B3. This finding was observed for all JAK inhibitors but was most prominent with ruxolitinib (Figure 1). Patient 3 received ruxolitinib monotherapy after being refractory to 6-mercaptopurine. Treatment resulted in resolution of splenomegaly but no reduction in the variant allele frequency of SH2B3. The patient is now alive more than one year after hematopoietic stem cell transplantation (HSCT). Discussion In a previous study of 100 patients with JMML we first identified seven patients with secondary SH2B3 mutations. Here, we report four additional patients with JMML that appear to be driven by initiating mutations in SH2B3 (Table 1). The mutations identified here result in a truncated protein (patient 1, 3 and 4) or affect the SH2 domain (patient 2). Interestingly, copy neutral loss of heterozygosity of SH2B3 in patient 1, also known as uniparental isodisomy, is a mechanism that has been observed in other genes (e.g. CBL and NF1) in JMML. Since in vitro data showed that loss of LNK results in increased JAK/STAT signaling, we hypothesized that this cohort of patients may benefit from JAK inhibitor therapy. Our data from iPSC-derived JMML like HPCs shows, that those cells with an additional SH2B3 mutation are more sensitive to JAK inhibitors, specifically ruxolitinib, a JAK1/2 inhibitor that is already approved or under investigation in multiple clinical trials. In summary, we expand the list of initiating mutations in JMML to include SH2B3 and raise the possibility of targeting the JAK pathway in these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical and Molecular Characterization of Newly Diagnosed IDH/TP53 Co-Mutated AML and Impact of Genomically Sensitive Treatment Strategies

TP53 is mutated in ~4% of IDH mutated newly diagnosed (ND) acute myeloid leukemia (AML) and the clinical and prognostic impact of IDH/TP53 co-mutated AML is not well defined. We analyzed patients (pts) with IDH1, IDH2, and/or TP53 mutated, ND AML treated at our institution from 2012 to 2022. Multihit TP53 status was defined by 1) multiple TP53 muts 2) TP53 mut(s) and concomitant 17p/TP53 deletion 3) TP53 mut variant allele frequency (VAF)>50% suggesting mutation+ copy neutral loss of heterozygosity. Treatment (trt) regimens were classified as IDH-targeted therapy (IDHi), intensive chemotherapy (IC), or low intensity therapy (LIC). All regimens were employed with or without the addition of venetoclax (Ven) per treatment protocol. Among 628 pts with ND AML, 35 (5%) had IDH/TP53co-mut[23/35 (66%) IDH/TP53co-mut with multihit TP53], 100 (16%) had IDH1 mut, 208 (33%) had IDH2 mut, and 285 (45%) had TP53 mut [among 217/285 pts with evaluable TP53 allelic state, 185 (85%) had multihit TP53] (Table 1). Compared to pts withTP53 mut , pts with IDH/TP53co-mut had higher platelet count and higher bone marrow blasts and diploid karyotype (all p<0.05) more closely resembling features of IDHmut AML. However, pts with IDH/TP53co-mut had higher TP53 mut VAF [55%, interquartile range (IQR), (28%-67%)] compared with TP53 mut AML (33%, IQR, (10%-50%) (p=0.01). The CR/CRi rates in IDH/TP53co-mut, IDH1 mut, IDH2 mut, and TP53 mut AML were 37%,77%,72%, and 48% respectively, regardless of trt regimen. Among pts with IDH/TP53co-mut, CR/CRi rates were significantly higher in non-multihit TP53 mut than pts with multihit TP53 mut (66% vs 9%, P=0.0005). Among pts who received Ven or IDHi based therapy, pts with IDH/TP53co-mut had significantly lower CR/CRi rates compared with pts with IDH mut AML (20% vs 87% ,respectively, P<0.05). In contrast, among pts treated with IC, pts with IDH/TP53co-mut had numerically superior CR/CRi rates compared with pts with TP53 mut (75% vs 47% vs respectively, P>0.05). Among pts treated with LIC, CR/CRi rates were similar between IDH/TP53co-mut , IDH mut ,and TP53 mut AML(31% ,48%,43%,respectively). The 60-day mortality rates in IDH/TP53co-mut, IDH1 mut, IDH2 mut, and TP53 mut were 11%,10%,7%,and 19%,respectively. Hematopoietic stem cell transplant rates in IDH/TP53co-mut, IDH1 mut, IDH2 mut, and TP53 mut AML were 17%, 20%, 25%, and 12%, respectively. The median OS for pts with IDH/TP53co-mut, IDH1 mut, IDH2 mut, and TP53 mut was 8.7m,15.5m,30.3m, and 6.3m, respectively. The median OS was similar between those with IDH1/TP53co-mut and IDH2/TP53co-mut (7.8m vs. 9.5m; p=0.50). Compared to pts with IDH1 mut or IDH2 mut , those with IDH1/TP53co-mut (median OS-15.5m vs 7.8m) and IDH2/TP53co-mut (median OS-30.3 vs 9.5m) had inferior OS, respectively. There was no difference in OS between patients with IDH1/TP53co-mut and TP53 mut (median OS:7.8m vs 6.3m,p=0.3) but OS was superior in those with IDH2/TP53co-mut compared to those with TP53 mut (median OS 9.5m vs. 6.3m; p=0.03). We additionally analyzed the outcomes of IDH/TP53co-mut and TP53 mut by multi-hit TP53status. Multihit TP53status was associated with inferior survival in those with IDH/TP53co-mut (median OS:5.4m vs 41.2m, p=0.0008) (Figure-1A) and TP53 mut (median OS: 5.8 m vs 10.1m, p=0.05) compared with non-multihit status. Among pts with non-multihit TP53 status, those with IDH/TP53co-mut had superior OS compared with those with TP53 mut AML (Median OS:41.2m vs 10.1m, P=0.01)(Figure-1B) Among pts who received IDH-sensitive therapy (i.e., Ven and/or IDHi), IDH/TP53co-mut had inferior survival compared with either IDH1 mut (median OS: 5.4m vs 24.4m, p<0.05) or IDH2 mut (median OS: 5.4m vs 35.5m, p<0.05) alone. Among pts who received Ven-based therapy, OS was similar in pts with IDH/TP53co-mut and TP53 mut (median OS: 5.4m vs 5.3m, p=0.72). Among pts who received LIC, there was no difference in OS between pts with IDH/TP53co-mut and TP53 mut (median OS: 5.0m vs 6.3m, p=0.92). However, among pts who received IC, IDH/TP53co-mut had superior OS compared with TP53 mut AML (median OS: 43.8m vs 5.1m, P=0.01). Notably all pts with IDH/TP53co-mut had non-multihit TP53 status. IDH/TP53co-mut AML is a unique subtype with clinical features resembling IDHmut AML and prognosis driven by TP53 mut and the allelic status of TP53. Genomically sensitive therapies targeting both IDH and TP53 may improve the outcomes in this unique molecular subset of AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pediatric-Specific Patterns of Clonal Evolution Arising from Acquired Aplastic Anemia

Introduction Acquired aplastic anemia (aAA) occurs in both children and adults, though pathophysiology and clinical outcomes differ significantly by age. While clinical testing for paroxysmal nocturnal hemoglobinuria (PNH) clones is routine, targeted next generation sequencing (NGS) for myelodysplastic syndrome (MDS) associated variants has only recently been implemented. Furthermore, single nucleotide polymorphism array (SNP-A) assessing for copy neutral loss of heterozygosity in the human leukocyte antigen (HLA) region on chromosome 6p (6p CN-LOH) and HLA NGS assessing for allele specific loss of function (LoF) mutations, which together are seen in up to 25% of aAA cases, are only now being considered for clinical use. Recent studies have reported on the frequency and overlap of these 3 types of clonal evolution (PNH, MDS-associated, HLA-targeting) in adult aAA cohorts, but none focus specifically on pediatric patients, whose MDS-associated clonal evolution patterns are distinct. Delineating patterns of clonal hematopoiesis in pediatric aAA to determine whether evolution driven by immune escape (PNH and HLA loss) affects likelihood of MDS development is critical for personalizing therapeutic options. Thus, we aimed to describe the frequency, clinical significance, and overlap of clonal evolution including PNH, HLA loss, and MDS-associated gene mutations in a pediatric cohort of aAA patients. Methods This study identified 85 pediatric aAA patients treated at Children's Hospital of Philadelphia (CHOP) and enrolled on the IRB-approved CHOP/Penn BMF Registry and Repository in whom clinical testing for all patterns of clonal hematopoiesis was available or could be performed with biorepository samples. PNH flow cytometry was performed clinically in all cases; subjects with PNH > 0.05% in the granulocyte fraction were considered as having a PNH clone. Peripheral blood and bone marrow samples were used for SNP-A, HLA-NGS, and an institutional somatic mutation NGS panel for hematological malignancies encompassing 118 genes. Descriptive and contingency statistical analyses along with Fisher's Exact Test were performed using SAS (Cary, NC). Results Demographic composition of the 85 pediatric subjects with aAA included a median age of 10.7 years (1.7-20.8), male/female ratio of 59%/41%, and a representative race/ethnicity distribution (Table 1). In total, 66% displayed at least one clonal event. As expected, PNH clones were most common, present in 45% (38/85) of the cohort. An additional 16 (19%) exhibited acquired HLA loss by 6p CN-LOH (n=6), allele specific LoF mutations (n=6), or both (n=4). Fifteen (18%) had clonal hematopoiesis associated with MDS, of whom 3 had cytogenetic lesions that progressed to clinical MDS (3.5%), and 12 developed no morphologic features of MDS but demonstrated a total of 14 mutations in MDS-associated genes (BCOR/BCORL1 n=6, KMT2C/KMT2D n=3, RUNX1 n=1, ASXL1 n=1, SRSF2 n=1, PRPF8 n=1, CALR n=1). Critically, pediatric subjects with aAA who developed HLA loss did not develop MDS-associated mutations, with no patient demonstrating both subtypes of clonal events (Figure 1). In contrast, PNH and MDS-associated clonal events overlapped in 11% of the overall cohort. In total, 60% of patients with MDS-associated clonal hematopoiesis also possessed PNH clones, and 23% of patients with detectable PNH clones demonstrated at least one MDS-associated clonal event. Only 3.53% (n=3) of the cohort demonstrated both PNH and HLA loss, and PNH clone size was < 1% in all 3 patients. Furthermore, patients with HLA loss were significantly less likely to have PNH clones than those without HLA somatic alterations (19% vs 51%, p = 0.026). Correlations between patterns of clonal hematopoiesis and clinical outcomes will be presented. Concalusions Pediatric aAA patients treated with IST are at risk for developing MDS and hemolytic PNH. Presence and evolution of clonal hematopoiesis influenced decisions to pursue IST versus alternative donor transplant. Importantly, our study demonstrates that occurrence of clonal hematopoiesis by HLA loss does not increase and may even decrease the risk of developing MDS-associated or PNH clones. This finding strongly suggests that treatment decisions for aAA patients whose only clonal event is HLA loss need not be considered distinctly from patients with no clonal changes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic Impact of Monoallelic Versus Biallelic TP53 Alterations in Intensively-Treated Adults AML Patients: A Retrospective Study from the ALFA Group

Introduction:TP53 is a key tumor suppressor gene involved in apoptotic pathways, metabolism functions, preservation of genomic balance and DNA repairs. TP53 locus is subject to mutations, deletions and copy-neural loss of heterozygosity. TP53 mutations are associated with a reduced overall survival and are assigned to the ELN 2017 adverse-risk group. Recent studies have suggested a worse prognostic impact of biallelic TP53 alterations compared to monoallelic TP53 mutations in myelodysplastic syndromes (Bernard et al, Nat. Medicine, 2020). Whether TP53 allelic state impairs the prognostic in intensively-treated acute myeloid leukemia (AML) patients and may refine the treatment strategy has not been addressed. Here, we analysed TP53 alterations in newly diagnosed AML patients (pts) from 3 prospective Acute Leukemia French Association (ALFA) trials. Methods: A total of 983 adult AML pts enrolled in ALFA-0701 (NCT00927498), ALFA-0702 (NCT00932412) and ALFA-1200 (NCT01966497) trials were retrospectively screened for TP53 alterations by high throughput sequencing and conventional cytogenetics. Pts displaying at least two TP53 mutations or one TP53 mutation associated with either TP53 locus deletion or TP53 locus copy-neutral loss of heterozygosity (CN-LOH) were assigned as biallelic TP53 alteration patients. TP53 locus CN-LOH were approximated based on variant allele frequencies and copy number analysis. Survival was analysed with multivariate Cox models stratified on the trial. Results: Eighty-three patients had TP53 mutations (8%; ALFA-0701: n=21, ALFA-0702: n=28; ALFA-1200: n=34), including 14 pts with monoallelic and 69 pts with biallelic TP53 alterations respectively. Median age of TP53 mutated patients was 62 years (IQR: 57-68). Leukocytes counts and bone marrow blast counts at diagnosis were lower in TP53 mutated patients (3.0 G/L vs. 8.7 G/L, p<0.0001; 40% vs. 58%, p=0.002, respectively). Median variant allele frequency of TP53 mutations was 43% (IQR: 21-69). As compared to TP53 wildtype AML, TP53 mutated pts more often harbored complex karyotype (n=69 (86%) vs n=51 (6%), p<0.0001) and monosomal karyotype (n=65 (81%) vs n=32 (4%), p<0.0001). The median number of additional mutations was 2 (IQR: 0-3). TP53 mutations were associated with mutations in signalling genes (NF1, NRAS, PTPN11, KRAS, FLT3, KIT and JAK2) in 33 pts (40%). Overall, 43 TP53 mutated AML patients (52%) achieved complete remission (CR)/complete remission without platelet recovery (CRp) after one or two courses of induction whereas 684 TP53-wt patients (76%) were in CR/CRp at that time (p<0.0001). TP53 mutations were associated with a significantly reduced OS and DFS in multivariate analysis including age and WBC count (2-year OS, 11% vs 56%, HR: 3.48, 95% CI: 2.71-4.47, p<0.0001; 2-year DFS, 12% vs 50%, HR: 2.97, 95% CI: 2.12-4.16, p<0.0001, respectively). Among TP53 mutated AML, biallelic TP53 alterations were associated with worse OS compared to monoallelic TP53 alterations (2-year OS, 4% vs 43%, HR: 4.13, 95% CI: 1.87-9.13, p=0.0004 in multivariate analysis accounting for age and WBC count). Interestingly, outcome of the 2017 ELN risk stratification intermediate group was not different to that of monoallelic TP53 pts (2-year OS, 43% for monoallelic TP53 pts vs 53%, HR: 1.07, 95% CI: 0.55-2.07, p=0.84). In pts with biallelic TP53 alterations, presence of signalling mutations was associated with a worse survival in a multivariate analysis accounting for age and WBC (HR: 1.82, 95% CI: 1.06-3.11, p=0.03). Conclusion: Allelic status refines the prognostic value of TP53 mutations in AML. While monoallelic TP53 alterations display similar outcome to that of the ELN 2017 intermediate risk group, biallelic TP53 alterations dramatically impair patient outcome. Patients with biallelic TP53 alterations may be good candidates for new first-line treatment strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Detecting the Unusual without Compromising Diagnostic Accuracy - a Prospective WGS/Wts Pilot Study in Acute Leukemias Provided Additional Information for Diagnosis, Prognosis and Treatment

Background: Following WHO and ELN guidelines, standard diagnostics and risk stratification for acute leukemias (AML and ALL) are based on genetic information gathered by a combination of cytogenetics and targeted molecular profiling. Today, comprehensive techniques, i.e WGS and WTS (whole genome and transcriptome sequencing) and the accompanying clinical bioinformatics workflows may even refine and individualize diagnostics. Aim: To evaluate the clinical utility and added value of WGTS in a real-world setting in comparison to standard of care genetic diagnostics. Patients and Methods: The study enrolled 113 patients, 97 with AML and 16 with ALL, diagnosed according to WHO criteria based on cytomorphology, immunophenotyping, cyto- and molecular genetics. WGS (100x) and WTS (50 Mio reads) were performed on NovaSeq instruments. For selected AML cases CD3+ MAC sorted cells were used as normal controls (30x). Variants were called with Strelka2, Manta and GATK using a tumor w/o normal pipeline, fusions with Arriba, STAR-Fusion and Manta. Results: All patients were analyzed for structural variants (SV), copy number variations (CNV) and copy-neutral loss of heterozygosity (cnLOH) based on WGS data and the presence of fusion transcripts and expression patterns based on WTS data. Samples without a normal control (n = 74) were analyzed for 121 genes recurrently mutated in myeloid/lymphoid neoplasms, whereas for 39 AML cases with CD3+ normal controls the whole exome was assessed. Overall, 308/333 (92%) gene mutations were consistently identified by targeted sequencing and WGS. Due to the higher read depth, 25/333 (8%) sub-clonal mutations were only detected by targeted sequencing, with 23/25 showing a variant allele fraction <10%. However, 40 gene mutations were identified exclusively by WGS due to the unrestricted analytical scope (e.g. PTPN11, DDX54). Every patient carried on average 3.3 gene mutations within the 121 genes of interest. The number increased to 63 variants (mean; range: 46-116) when analyzing the exome. 52/63 variants are most likely germline variants based on occurrence in CD3+ control sample. Hence, the CD3+ control samples reduced the number of somatic variants in the exome to 11 (mean; range: 1-23) mostly patient-specific mutations that can be explored for personalized treatment options. Addressing cytogenetics with relevance to risk stratification and assignment to AML/ALL genetic subgroups, WTS identified all entity defining fusions and WGS and chromosome banding analysis (CBA) showed a very high concordance. In 10 patients WGS missed subclonal aberrations due to small clone size (median clone size detected by IP-FISH analysis: 6%). However, WGS provided additional information in 40 patients, mostly SV, CNV and cnLOH of unknown significance, but also alterations of biological interest and prognostic and/or therapeutic importance: in one AML case WGTS revealed a KMT2A::MLLT10 rearrangement that was cytogenetically cryptic and not detectable by FISH (KMT2A break apart probe), assigning the patient to the adverse risk group. WGS also identified a cytogenetically cryptic SV leading to an ETV6::ABL1 rearrangement in an ALL patient with complex karyotype. Retrospective analysis showed that the aberration had already been present when the patient presented with an MPN and a normal karyotype (CBA) two years earlier. Hence, WGS led to the diagnosis of an ETV6::ABL1-positive neoplasm in blast phase (poor prognosis), which might be treated with tyrosine kinase inhibitors. Of note, in 3 AML patients a 7q deletion or complex SV leading to a 7q deletion resulted in a CDK6::NOM1 fusion transcript and high MNX1 expression. In addition, WGS revealed in one patient with ETP-ALL a cytogenetically cryptic translocation t(7;14)(q36;q32) affecting the IGH locus and the NOM1 gene, accompanied by high MNX1 expression. Elevated MNX1 expression as a result of t(7;12)(q36;p13) is a very rare event in pediatric AML, but our findings might suggest a more important role of MNX1 expression in adult acute leukemia than previously known. Conclusions: This prospective study in a real-world setting 1) demonstrated the diagnostic power of WGTS with an accuracy comparable to gold standard techniques, 2) added important prognostic information, 3) identified potential new therapeutic targets and 4) gave new insights into disease biology. WGTS significantly will improve and refine diagnostics in the future.

Role of TNFRSF17 and GPRC5D Structural and Point Mutations in Resistance to Targeted Immunotherapies in Multiple Myeloma (MM)

Adaptive T cell therapy using chimeric antigen receptor (CAR) T cells and T cell engagers (TCE) targeting BCMA and GPRC5D have demonstrated very encouraging responses in heavily pre-treated multiple myeloma (MM). Recent studies interrogating T cells repertoires and fitness correlated resistance to these immune based therapies with exhausted T cell states. While in lymphoma, antigen escape with CD19 loss is observed in up to 30% of patients with acquired resistance to adaptive therapies, in MM TNFRSF17 or GPRC5D structural mutations are thought to be less prevalent in recipients of BCMA or GPRC5D-targeting CARs or TCEs . Bi-allelic loss (n=2) and a missense truncating mutation (n=1) of TNFRSF17 with CAR T cells, as well as one case of biallelic loss in TNFRSF17 with TCE are reported to date with no reported cases of bi-allelic loss of GPRC5D. Further studies of the role TNFRSF17 or GPRC5D structural or point mutations to the immune escape to these adaptive therapies are needed. Serial BM aspirates were collected from patients treated with BCMA or GPRC5D-targeting TCE or CARs prior to therapy and at relapse. MM cells were profiled with single cell mRNA (scRNAseq), single cell copy number (scCNVseq) using the 10x Genomics GemCode system and whole genome sequencing (WGS). Sequenced reads were aligned to hg38. Single cell reads were processed with CellRanger and downstream analyses with Seurat R package. In order to define the prevalence of TNFRSF17 or GRPC5D structural variants and mutations in MM, we performed WGS (n=19, 14 pre-CAR/TCE, 5 post-CAR/TCE) as well as scCNVseq (n=33, 16 RRMM, 7 pre-CAR/TCE, 8 post-CAR/TCE) on sorted CD138+ cells from relapsed MM patients. WGS (100x coverage) identified at baseline a subclonal deletion at the TNFRSF17 gene locus in only 1 patient; this small subclone subsequently drove the progression post BCMA CAR T cell therapy with an acquired biallelic loss of TNFRSF17. Mono-allelic large and focal deletions of GPRC5D (chr.12p) were detected by WGS in 7 cases (5 deletions, 2 copy neutral LOH). scCNVseq studies identified more frequent subclonal monoallelic copy number losses of TNFRSF17 in 11.2% of the cells (27884 MM cells) prior to anti-BCMA therapy exposure with the deletions ranging from 200 kb to 2.3 Mb. A homozygous deletion of TNFRSF17 was noted in 2 patients at the time of disease progression, one post CAR T and the other post BCMAxCD3 TCE. Structural mutations at the GPRC5D locus were also identified by scCNVseq with monoallelic and biallelic deletions in 11.9% and 2.7% of the cells, respectively. Clonal biallelic loss of GPRC5D post GPRC5DxCD3 TCE was detected in one patient who harbored a monoallelic loss in 79.2% of the cells pre-TCE. With regard to point mutations, in one patient progressing from a durable CR on BCMA TCE, we observed an acquired mono-allelic loss of TNFRSF17 (scCNV) coupled with de novo mutation in TNFRSF17 extracellular domain c.81G>C [p.(R27P)]. This point R27P mutation is predicted to be damaging to BCMA (Polyphen) with disruption of several H-bonds between R27 and C24, Q25, S29, S30 and T32 of BCMA extracellular aa residues. Importantly, molecular dynamic simulations of WT vs. mutant R27P using publicly deposited crystal structural of BCMA bound to anti-BCMA Ab (J22.9-xi) (PBD ID 4ZFO) demonstrated that this mutation disrupts all H-bonds between BCMA and the light chain of chimeric mouse/human anti-BCMA Ab (J22.9-xi). Functionally, primary CD138+ cells with mutant BCMA [p.(R27P)] as well as K562 cells transduced with mutant BCMA [p.(R27P)] were completely resistant to BCMAxCD3 cytotoxicity with teclistamab and elranatamab compared to cells with WT BCMA. While structural alterations of TNFRSF17 and GPRC5D remain relatively rare, we have identified biallelic loss of TNFRSF17 post BCMA CAR T and BCMAxCD3 TCE therapy and herein reported the first case of biallelic loss of GPRC5D post GPRC5DxCD3 TCE treatment. Importantly, we also identified a monoallelic loss of TNFRSF17 (del16p) coupled with BCMA extracellular domain point mutation [p.(R27P)] that confers resistance to two BCMAxCD3 TCEs in MM. Therefore, in addition to structural alterations (biallelic loss of TNFRSF17 or GPRC5D), antigen escape resulting from non-truncating missense point mutations in BCMA extracellular domain also mediate resistance to targeted-immunotherapies.

Outcomes of Patients with TP53-Mutated Myeloid Neoplasms (TP53-MN) and the Role of Allogenic Blood or Marrow Transplantation (alloBMT)

Background: TP53 mutations are present in up to 20% of patients with myeloid malignancies and are frequently associated with a complex karyotype (CK) and poor responses to therapy. Even though overall response rates have improved significantly with less intensive induction modalities such as hypomethylating (HMA) agents alone or in combination with venetoclax, most patients succumb to their disease. AlloBMT remains the only curative option and even though the outcomes after alloBMT are disappointing, some patients achieve long-term survival. In the current study, we aimed to determine the clinical outcome of patients with TP53-MN and the impact of therapies including alloHCT on outcomes. Methods: We performed a single-center retrospective review of adult patients with TP53-MN evaluated at Johns Hopkins between November 2015 and February 2020. The analysis of somatic mutations was performed using a 59-gene targeted next-generation sequencing (NGS) assay. Cytogenetic analysis was performed on bone marrow or peripheral blood using standard methods including G-banding by trypsin with Giemsa staining. Results: We identified a total of 93 patients with TP53-MN. Disease subtypes included de novo AML (n = 22), AML arising from MDS/MPN (n = 15), MDS (n = 23), therapy-related myeloid neoplasm (n = 29), MDS/MPN overlap syndrome (n = 2) and MPN (n = 2). The median (Q1, Q3) age was 67 years (59 - 73 years). Cytogenetics was available for 89/93 (95.7%) patients. Of those, 73 (82.0%) patients had complex karyotype (CK). The median maximum TP53 variant allele frequency (VAF) per patient was 42.5%. Sixty-two (66.6%) patients had TP53 null phenotype: 14 (15.1%) patients had multiple TP53 mutations, 37 (41.6%) had deletion 17p and 11 (12.0%) had copy-neutral loss-of-heterozygosity (CnLOH). Overall, 79/93 (85%) patients received therapy: 20 (25.3%) were treated with HMA alone, 30 (38.0%) with HMA+venetoclax, 10 (12.7%) with HMA+others, 15 (19.0%) with intensive chemotherapy, 2 (2.5%) with an immunomodulatory drug, 1 (1.3%) with ivosidenib and 1 (1.3%) with a JAK2 inhibitor. Of those, 29 (36.7%) patients received non-myeloablative alloBMT with post-transplant cyclophosphamide (10 (34.5%) bone marrow and 19 (65.5%) mobilized peripheral blood grafts (PBSCT)). Median overall survival (OS) in patients receiving alloBMT, any therapy, or supportive care was 18.9, 4.1, and 2.5 months respectively (p < 0.001, Figure 1). Among patients in the alloBMT group, there was a significant OS difference between TP53-MN CK vs. non-CK (median OS 13.7 months vs not reached, p = 0.01, Figure 2). Also, patients with TP53-MN CK demonstrated better OS when treated with alloBMT compared to any therapy or supportive care. However, most TP53-MN CK patients ultimately succumbed to their disease regardless of the therapeutic modality. Therapeutic modalities prior to alloBMT did not appear to affect post-alloBMT outcomes but among patients who received PBSCT, OS was significantly better in patients receiving 400cGy vs 200 cGy total body irradiation (median OS 8.9 vs. 21 months, p=0.01). Conclusions: TP53-MN appears to be a heterogeneous disease with a variable prognosis. While OS of patients with TP53 mutations and non-CK who underwent alloBMT resembled that of intermediate-risk AML, patients with TP53 null phenotype associated with CK had a worse outcome. Even though OS appears to be significantly improved with alloBMT, particularly patients receiving PBSCT+400cGy TBI, most patients eventually relapse. Novel therapeutic strategies are urgently needed to improve the outcomes of TP53 CK patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genome-wide identification of copy neutral loss of heterozygosity reveals its possible association with spatial positioning of chromosomes.

Loss of heterozygosity (LOH) is a genetic alteration that results from the loss of one allele at a heterozygous locus. In particular, copy neutral LOH (CN-LOH) events are generated, for example, by mitotic homologous recombination after monoallelic defection or gene conversion, resulting in novel homozygous locus having two copies of the normal counterpart allele. This phenomenon can serve as a source of genome diversity and is associated with various diseases. To clarify the nature of the CN-LOH such as the frequency, genomic distribution and inheritance pattern, we made use of whole-genome sequencing data of the three-generation CEPH/Utah family cohort, with the pedigree consisting of grandparents, parents and offspring. We identified an average of 40.7 CN-LOH events per individual taking advantage of 285 healthy individuals from 33 families in the cohort. On average 65% of them were classified as gonosomal-mosaicism-associated CN-LOH, which exists in both germline and somatic cells. We also confirmed that the incidence of the CN-LOH has little to do with the parents' age and sex. Furthermore, through the analysis of the genomic region including the CN-LOH, we found that the chance of the occurrence of the CN-LOH tends to increase at the GC-rich locus and/or on the chromosome having a relatively close inter-homolog distance. We expect that these results provide significant insights into the association between genetic alteration and spatial position of chromosomes as well as the intrinsic genetic property of the CN-LOH.

Molecular classification of endometrial cancers using an integrative DNA sequencing panel (271)

<b>Objectives:</b> The Cancer Genome Atlas (TCGA) has identified four molecular subgroups of endometrial cancers (EC) with distinct differences in prognosis and response to adjuvant treatment. Stratification of patients according to molecular profiles is critical for individualized therapy at diagnosis, but adoption of molecular classification into clinical practice remains challenging due to the complexity and costs associated with sequencing. We aimed to develop a simple and affordable molecular technique to classify EC into four TCGA subgroups using our high-depth targeted sequencing panel. <b>Methods:</b> Women with newly diagnosed EC were prospectively recruited from three cancer centers in Ontario, Canada, between 2015 and 2018. Tumors were reflexively assessed for mismatch repair (MMR) protein expression by immunohistochemistry (IHC). Using our custom-designed panel, 181 formalin-fixed paraffin-embedded (FFPE) EC samples were sequenced. Our panel was able to identify somatic mutations, copy number variants, insertions and deletions, copy neutral loss of heterozygosity, structural rearrangements, and promoter methylation from a single aliquot of DNA. Variants were analyzed for pathogenicity, and clinical-pathologic information was collected through medical records. <b>Results:</b> Of 181, 86 (48%) were classified into microsatellite instability-high/hypermutated cohort (MSI-h or MMRd), of which 62 (72%) harbored <i>MLH1</i> promoter methylation, and 24 (27%) had pathogenic variants in MMR genes. Concordance with clinical IHC and methylation testing results was high at 99%. Of cases with a single classifier, three (1.6%; <i>n</i>=181) had pathogenic <i>POLE</i> exonuclease domain mutation (<i>POLE</i>mut), 15 (8%) had a pathogenic p53 variant (p53mut), and 61 (34%) had no specific molecular profile subtype (NSMP). Sixteen (9%; <i>n</i>=181) had more than one molecular classifying feature, with eight (4%) having MMRd-p53mut, six (3%) with MMRd-<i>POLE</i>mut, one (0.5%) with MMRd-<i>POLE</i>mut-p53mut, and one (0.5%) with <i>POLE</i>mut-p53mut. Overall, there were 11 (6%) with <i>POLE</i> mutations (three single and nine multiple classifiers). Survival outcomes of MMRd, <i>POLEmut,</i> p53mut, and NSMP groups stratified similarly to TCGA data, but when the MMRd group was further subclassified according to the mechanism of MMR loss, the MLH1 promoter methylated group did worse than those with somatic MMR pathogenic variants (5-year OS: 0.72 [0.57-0.92] vs 1.00 [1.00-1.00]; p=0.013 and RFS: 0.70 [0.58-0.85] vs 0.92 [0.82-1.00]; p=0.032). In contrast, those with somatic MMR pathogenic variants had similar outcomes to the <i>POLE</i>mut subtype (p=0.396) (Figure 1). <b>Conclusions:</b> Our NGS panel can classify EC into four TCGA subgroups through a simplified process and can be implemented reflexively in clinical practice. The higher rate of multiple classifiers (9%) in this study warrants further investigation. The difference in survival between MLH1 methylated group within MMRd cohort suggests that further subclassification may be required for accurate prognostication.

Primary mediastinal large B-cell lymphoma is characterized by large-scale copy-neutral loss of heterozygosity.

Development of primary mediastinal B-cell lymphoma (PMBL) is driven by cumulative genomic aberrations. We discovered a unique copy-neutral loss of heterozygosity (CN-LOH) landscape of PMBL which distinguishes this tumor from other B-cell malignancies, including the biologically related diffuse large B-cell lymphoma. Using single nucleotide polymorphism array analysis we identified large-scale CN-LOH lesions in 91% (30/33) of diagnostic PMBLs and both investigated PMBL-derived cell lines. Altogether, the cohort showed 157 extra-large (25.3-248.4Mb) CN-LOH lesions affecting up to 14 chromosomes per case (mean of 4.4) and resulting in a reduction of heterozygosity an average of 9.9% (range 1.3-51%) of the genome. Predominant involvement of terminal chromosomal segments suggests the implication of B-cell specific crossover events in the pathogenesis of PMBL. Notably, CN-LOH stretches non-randomly clustered on 6p (60%), 15 (37.2%), and 17q (40%), and frequently co-occurred with homozygous mutations in the MHC I (6p21), B2M (15q15), and GNA13 (17q23) genes, respectively, as shown by preliminary whole-exome/genome sequencing data. Altogether, our findings implicate CN-LOH as a novel and distinct mutational process contributing to the molecular pathogenesis of PMBL. The aberration acting as "second hit" in the Knudson hypothesis, ranks as the major mechanism converting to homozygosity the PMBL-related driver genes. Screening of the cohort of 199 B cell leukemia/lymphoma whole-genomes revealed significant differences in the CN-LOH landscape of PMBL and other B-cell malignancies, including the biologically related diffuse large B-cell lymphoma.

Abstract 5725: HOXA9 demonstrates allelic imbalance in KMT2A-rearranged infant acute lymphoblastic leukemia

Abstract Background: KMT2A-rearrangement is a characteristic finding in approximately 70% of infant acute lymphoblastic leukemia (ALL) cases. We previously utilized a computational screen to find that KMT2A-r infant ALL demonstrated somatic allele specific expression for 431 genes when compared to non-KMT2A-r samples (Abstract Number 3649, AACR 2019). The gene that exhibited the most differential allele specific expression (ASE) pattern was HOXA9. Here, we follow up that analysis with manual examination of additional infant ALL samples. Methods: We performed whole genome sequencing (WGS) and RNA sequencing (RNAseq) on peripheral blood or bone marrow samples from 48 infants with ALL (33 KMT2A-r cases and 15 non-KMT2A-r cases) treated on Children’s Oncology Group protocol AALL0631, at diagnosis (DX), at remission (MD), and at relapse (RL), if applicable. WGS data at MD was phased using a 1000 Genomes reference panel. HOXA9 expression was quantified in transcripts per million (TPM). Biallelic expression was manually calculated on the phased genome for samples with at least 1 germline single nucleotide polymorphism (SNP) within HOXA9 and at least 5 aligned RNAseq reads over the HOXA9 transcript. ASE was defined as minor allele fraction (MAF; allele fraction for the less prevalent allele) of ≤ 0.35. Whole genome bisulfite sequencing data and sequencing-derived copy number data were also analyzed to investigate possible explanations for HOXA9 ASE. Results: We identified diagnostic samples from 21 patients, 9 KMT2A-AFF1, 9 KMT2A-MLLT1, and 3 non-KMT2A-r, that passed manual filtering. Data included 5 patients with paired DX and RL samples. Of the 15 KMT2A-r samples unable to be analyzed for ASE, 13 lacked a SNP for phasing and 2 had &amp;lt; 5 reads. For the 12 non-KMT2A-r samples unanalyzable for ASE, 6 lacked a SNP and 6 had &amp;lt; 5 reads. Average HOXA9 expression was 15.17 TPM (range 0.01 to 44.34) in KMT2A-r samples and 2.91 TPM (range 0 to 29.29) in non-KMT2A-r samples. ASE of HOXA9 (MAF ≤ 0.35) was observed in 14 samples, 72% of KMT2A-r (13 of 18 samples) and 33% of non-KMT2A-r (1 of 3 samples). RL samples were found to have HOXA9 ASE in all cases with ASE of HOXA9 in the DX sample. Of the 18 KMT2A-r samples, 6 of 9 with KMT2A-AFF1 and 7 of 9 with KMT2A-MLLT1 demonstrated HOXA9 ASE. Frequency of HOXA9 ASE at diagnosis was similar between patients known to later relapse versus those known to remain in remission. Allele specific methylation, mono-allelic deletion, or copy-neutral loss of heterozygosity (cn-LOH) for HOXA9 were not observed in KMT2A-r nor non-KMT2A-r samples. Conclusion: Our expanded cohort supports the ASE of HOXA9 in KMT2A-r infant ALL. This phenomenon did not appear to segregate by KMT2A-r partner gene, nor could it be explained by allele specific methylation or copy number variation (e.g., deletion of one HOXA9 allele or cn-LOH). Overall, further investigation into a causal mechanism is needed. Citation Format: Sarah E. Mc Dermott, Byunggil Yoo, Warren A. Cheung, Emily Farrow, Bing Ge, Margaret Gibson, Patrick A. Brown, Erin Guest, Tomi Pastinen, Midhat S. Farooqi. HOXA9 demonstrates allelic imbalance in KMT2A-rearranged infant acute lymphoblastic leukemia [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 5725.

Allelic dosage of RB1 drives CDK4/6 inhibitor treatment resistance in metastatic breast cancer.

1010 Background: We recently reported inferior outcomes to CDK4/6 inhibitors and endocrine therapy (CDK4/6i-ET) associated with germline BRCA2 (g BRCA2) in a cohort of estrogen receptor (ER) positive breast cancers. Co-occurrence of gBRCA2 with loss of heterozygosity (LOH) of neighboring RB1 was found to portend particularly poor outcomes. Here, we sought to define the effects of pre-treatment RB1 allelic copy number status on outcomes of CDK4/6i-ET and the likelihood of developing RB1 loss-of-function (LOF) mutations on CDK4/6i through the analysis of an expanded cohort of metastatic ER+ breast cancer patients. Methods: Patients who underwent sequencing on MSK-IMPACT from April 2014 to May 2021 were included. For every sample preceding CDK4/6i-ET, we performed FACETS to infer RB1 allele specific copy number, ploidy, tumor purity and fraction genome altered (FGA). Patients were categorized based on RB1 allelic status: HetLoss (total of one allelic copy), copy neutral LOH (CNLOH), other allelic imbalance including all other aneuploidy states, and diploid. Progression free survival (PFS) was assessed using univariate and multivariate Cox proportional hazard models adjusted for ET partner and FGA. Firth penalized logistic regression was used to study association of pre-treatment RB1 status with acquired RB1 LOF variants in paired post-CDK4/6i samples. Results: Of 2,630 potentially eligible patients, 279 patients had genomic sequencing performed prior to 1st line CDK4/6i-ET. Of these, 75 (26.8%) exhibited RB1 HetLoss, 39 (14.0%) had CNLOH of RB1, 111 (39.7%) exhibited diploid RB1 state, while 54 (19.4%) had other patterns of RB1 allelic imbalance. All non-diploid RB1 states were associated with significantly shortened PFS relative to diploid (univariate HetLoss HR: 2.05, 95% CI: 1.42, 2.97; CNLOH HR: 2.08, 95% CI: 1.32, 3.25; other imbalance HR: 1.70, 95% CI: 1.11, 2.58). Only HetLoss remained significant when adjusted for FGA (HR 1.61, 95% CI: 1.09, 2.38, p = 0.017). RB1 LOF was rare in pre-CDK4/6i tumors (&lt; 1%); excluding these cases did not change our results. Of the 176 patients with paired pre- and post-CDK4/6i samples, only RB1 HetLoss in pre-CDK4/6i sample was significantly associated with development of RB1 LOF mutations in post-CDK4/6i sample (18.4%) as compared to diploid (4.2%, OR 4.25, 95% CI 1.02, 17.7, p = 0.047). These results indicate that tumors with one functional copy of RB1 are more likely to acquire RB1 LOF on CDK4/6i to achieve biallelic RB1 loss as a mechanism of CDK4/6i resistance. Conclusions: We demonstrate that LOH and allelic imbalance of RB1 are associated with shorter PFS on CDK4/6-ET. We postulate this may occur partly as a result of more frequent acquired RB1 LOF mutations under selective pressure of CDK4/6i. These data supports the implementation of more refined allele-specific copy number methods and identifies a high-risk population for escalated monitoring and treatment approaches.

Heterogeneous genetic landscape of congenital neutropenia in Korean patients revealed by whole exome sequencing: genetic, phenotypic and histologic correlations

Congenital neutropenia (CN) is a hematological disease heterogeneous in its genetic, phenotypic and histologic aspects. We aimed to identify the genetic etiology of Korean CN patients in the context of bone marrow (BM) histology and clinical phenotype. Whole-exome sequencing (WES) or targeted sequencing was performed on the BM or peripheral blood specimens of 16 patients diagnosed with CN based on BM exam from 2009 to 2018. Absolute count of myeloperoxidase (MPO)-positive cells was calculated using ImageJ software. Semi-quantitation of MPO-positive cells in BM sections was performed by MPO grading (grades 0–3). Comprehensive retrospective review on real-world data of 345 pediatric patients with neutropenia including 16 patients in this study during the same period was performed. Seven disease-causing variants were identified in ELANE, G6PC3 and CXCR4 in 7 patients. A novel homozygous G6PC3 variant (K72fs) of which the mechanism was copy-neutral loss of heterozygosity was detected in two brothers. A low myeloid-to-erythroid ratio (0.5–1.5) was consistently observed in patients with ELANE mutations, while MPO-positive cells (40%–50%) with MPO grade 1 or 2 were detected in myelokathexis caused by G6PC3 and CXCR4 mutations. Meanwhile, disease-causing variants were detected in ELANE, TAZ and SLC37A4 in 5 patients by retrospective review of medical records. Our results suggest that following the immunological study and BM exam, WES or an expanded next generation sequencing panel that covers genes related to immunodeficiency and other inherited bone marrow failures as well as CN is recommended for neutropenia patient diagnosis.

Tumor and Constitutional Sequencing for Neurofibromatosis Type 1.

PURPOSENF1 variants in tumors are important to recognize, as multiple mechanisms may give rise to biallelic variants. Both deletions and copy-neutral loss of heterozygosity (LOH) are potential mechanisms of NF1 loss, distinct from point mutations, and additional genes altered may drive different tumor types. This study investigates whether tumors from individuals with neurofibromatosis type 1 (NF1) demonstrate additional gene variants and detects NF1 second hits using paired germline and somatic sequencing. In addition, rare tumor types in NF1 may also be characterized by tumor sequencing.MATERIALS AND METHODSSequences of 529 cancer driver genes were analyzed in 6,381 tumors, yielding 391 NF1-mutated tumors in which NF1 LOH analysis was performed. Driver genes were evaluated by tumor type including malignant peripheral nerve sheath tumors and gliomas.RESULTSNF1 LOH was seen in 133 of 391 tumor samples in the cohort. Individuals with NF1 had more prevalent copy-neutral LOH (P < .0001), suggesting somatic intrachromosomal recombination. Osteosarcoma in NF1 also had NF1 LOH and additional p53 alteration. NF1 second hit data from tumors were informative for inferring deleteriousness of missense variants that were conflicting in ClinVar, potentially helping to add to NF1 annotation. Although criteria for evaluating germline and somatic variants are different, deleterious effects on NF1 function may be shared.CONCLUSIONSequencing of NF1-associated tumors demonstrated a spectrum of second hits in NF1 and the prevalence of copy-neutral LOH. Future work may be aimed at further understanding of LOH mechanisms and strategies to mitigate tumor risk.

TP53 copy number and protein expression inform mutation status across risk categories in acute myeloid leukemia

AbstractMutant TP53 is an adverse risk factor in acute myeloid leukemia (AML), but large-scale integrated genomic-proteomic analyses of TP53 alterations in patients with AML remain limited. We analyzed TP53 mutational status, copy number (CN), and protein expression data in AML (N = 528) and provide a compilation of mutation sites and types across disease subgroups among treated and untreated patients. Our analysis shows differential hotspots in subsets of AML and uncovers novel pathogenic variants involving TP53 splice sites. In addition, we identified TP53 CN loss in 70.2% of TP53-mutated AML cases, which have more deleterious TP53 mutations, as well as copy neutral loss of heterozygosity in 5/32 (15.6%) AML patients who had intact TP53 CN. Importantly, we demonstrate that mutant p53 protein expression patterns by immunohistochemistry evaluated using digital image-assisted analysis provide a robust readout that integrates TP53 mutation and allelic states in patients with AML. Expression of p53 by immunohistochemistry informed mutation status irrespective of TP53 CN status. Genomic analysis of comutations in TP53-mutant AML shows a muted landscape encompassing primarily mutations in genes involved in epigenetic regulation (DNMT3A and TET2), RAS/MAPK signaling (NF1, KRAS/NRAS, PTPN11), and RNA splicing (SRSF2). In summary, our data provide a rationale to refine risk stratification of patients with AML on the basis of integrated molecular and protein-level TP53 analyses.