Loss Of Heterozygosity Research Articles

Molecular taxonomy of myelodysplastic syndromes and its clinical implications

AbstractMyelodysplastic syndromes (MDS) are clonal hematologic disorders characterized by morphologic abnormalities of myeloid cells and peripheral cytopenias. Although genetic abnormalities underlie the pathogenesis of these disorders and their heterogeneity, current classifications of MDS rely predominantly on morphology. We performed genomic profiling of 3233 patients with MDS or related disorders to delineate molecular subtypes and define their clinical implications. Gene mutations, copy-number alterations, and copy-neutral loss of heterozygosity were derived from targeted sequencing of a 152-gene panel, with abnormalities identified in 91%, 43%, and 11% of patients, respectively. We characterized 16 molecular groups, encompassing 86% of patients, using information from 21 genes, 6 cytogenetic events, and loss of heterozygosity at the TP53 and TET2 loci. Two residual groups defined by negative findings (molecularly not otherwise specified, absence of recurrent drivers) comprised 14% of patients. The groups varied in size from 0.5% to 14% of patients and were associated with distinct clinical phenotypes and outcomes. The median bone marrow (BM) blast percentage across groups ranged from 1.5% to 10%, and the median overall survival ranged from 0.9 to 8.2 years. We validated 5 well-characterized entities, added further evidence to support 3 previously reported subsets, and described 8 novel groups. The prognostic influence of BM blasts depended on the genetic subtypes. Within genetic subgroups, therapy-related MDS and myelodysplastic/myeloproliferative neoplasms had comparable clinical and outcome profiles to primary MDS. In conclusion, genetically-derived subgroups of MDS are clinically relevant and might inform future classification schemas and translational therapeutic research.

O-095 Assessment of genome editing outcomes in human preimplantation embryos subjected to CRISPR-Cas9 – most loss of heterozygosity (LOH) events are caused by DNA repair deficiency

Abstract Study question What is the cause of loss of heterozygosity, frequently observed in embryos subjected to CRISPR-Cas9, an interhomolog-homologous recombination repair (IH-HR) or deficiency in DNA repair? Summary answer Vast majority of LOH observed in human zygotes subjected to CRISPR-based editing is explained by deficiency in DNA repair prior to genome activation, not IH-HR. What is known already Genome editing (GE) at the preimplantation stage, intended to correct disease-causing mutations, is controversial due to safety and ethical concerns. It has been suggested that a high frequency of LOH seen in zygotes subjected to GE could be explained by resolution of the double-stranded DNA breaks (DSBs) created using CRISPR-Cas9 by interhomolog-homologous recombination repair (IH-HR), a process with potential to correct deleterious heterozygous mutations. However, such hypotheses are controversial and it is unclear whether embryos at this stage development can utilise IH-HR. CRISPR-Cas9 targeted sites are difficult to interrogate with traditional methods, risking a misinterpretation of editing outcomes. Study design, size, duration We hypothesised that the frequently observed LOH affecting the CRISPR-targeted sites in human zygotes is caused by the deficiency in DNA repair at this developmental stage, resulting in complex genotypes which traditional molecular methods have failed to detect, and not IH-HR which uses the second un-cleaved parental allele as a template for repair. DSBs were induced at three genomic sites in embryos microinjected at the time of fertilisation (n = 27) in this IRB-approved study. Participants/materials, setting, methods Research embryos were cultured for 60 hours, then disaggregated. Individual cells underwent an array of genetic tests, including NGS, allowing determination of the repair pathway utilised and revealing any cytogenetic abnormalities. A novel state-of-the-art analytical workflow was developed to interrogate 25kb around CRISPR cleavage sites. This approach provided a means of detecting complex genomic rearrangements as well as unresolved DNA damage, which would appear as LOH when employing traditional PCR-based approaches for assessment. Main results and the role of chance Induction of DSBs using CRISPR-Cas9 was 100% efficient when applied at the time of fertilisation. LOH was confirmed to be common at targeted sites, affecting 69/216 (32%) loci examined in 108 blastomeres. Remarkably, the great majority of LOH events (>90%) were a direct consequence of inappropriate/failed DNA repair (unresolved DNA breaks or complex genomic rearrangements). Strikingly, not a single instance of IH-HR was observed when CRISPR reagents were introduced at fertilisation. This may be explained by the physical separation of the two parental genomes in distinct pronuclei at that time. Allele-drop out (ADO), a technical problem, accounted for <10% of all LOH events. In total, 46% of DSBs failed to undergo appropriate repair, resulting in segmental aneuploidy (21%), large-scale deletions (10%), inversions/translocations (10%), or loss of the targeted chromosome (5%). 45% of DSBs were resolved using error-prone non-homologous end joining (NHEJ), while only 9% utilised the homology directed repair (HDR) pathway required for correction of mutations. Application of CRISPR-Cas9 to embryos on day-3 of development (after embryonic genome activation - EGA) was less efficient, only producing a DSB in 6/37 embryos (16%). However, the mode of repair differed markedly, 100% employing HDR (p = 0.0001). In 4 cases, this involved IH-HR. Limitations, reasons for caution This data provides strong evidence that LOH recorded in earlier studies was incorrectly attributed to IH-HR and was actually due to abnormal/failed repair. CRISPR appears more effective after EGA, with efficient DNA repair dominated by desirable HDR. However, a larger number of embryos is required to corroborate this finding. Wider implications of the findings The use of CRISPR-Cas9 at the time of fertilisation is frequently associated with problematic, unintended outcomes, rendering the technique clinically inapplicable in the current format. Careful study design which allows determination of all possible editing outcomes is essential and advances our understanding of the underlying mechanisms of DNA repair. Trial registration number not applicable

P-805 Sperm and embryo genome editing by CRISPR-Cas9 in a mouse model

Abstract Study question We wonder whether it is possible to edit a specific gene in individual mammalian spermatozoa as well as embryo by using CRISPR-Cas9? Summary answer We were able to successfully edit the coat pigment gene on individual spermatozoa as well as embryos by injecting CRISPR-Cas9 during Piezo-ICSI. What is known already Earlier investigations into embryo genome editing with CRISPR-Cas9 were conducted during the S-phase or zygote stage, encountering obstacles such as mosaicism and loss of heterozygosity. To overcome these challenges, performing genome editing at the gamete level is considered more favorable. While it may be achievable in oocytes, sperm genome editing can be ambitious due to the tightly supercoiled DNA around the protamine cores. In initial attempts, simple permeabilization of the sperm membrane allowed CRISPR-Cas9 penetration into the cell; however, the attempts at genomic editing proved to be unsuccessful. Study design, size, duration In the past several months, oocytes were divided into two groups: half of them were used to perform embryo genome editing, whilst the other half was used to edit exclusively the sperm genome through oocyte-mediated sperm decondensation (OMSD), where a single spermatozoon was injected into an enucleated oocyte and used the ooplasmic machinery for DNA decondensation and editing. In both models, CRISPR-Cas9 was used to knock out Tyr gene to create an albino phenotype. Participants/materials, setting, methods B6D2F1 mice were used to retrieve oocytes and spermatozoa. A cohort of oocytes used for the OSMD approach were enucleated, whilst the others were maintained with an intact nucleus. All oocytes, enucleated or not, were injected with a single spermatozoon with Tyr-sgRNA and Cas9 protein. Embryos were cultured until the 8-cell stage and processed for T7E1 cleavage analysis. Genome editing efficiency was calculated following the manufacturer protocol. Main results and the role of chance Of the 40 oocytes used for the study, 20 intact oocytes were used to perform embryo genome editing in the standard fashion, while 20 were enucleated for OMSD experiments. After undergoing Piezo-ICSI with CRISPR-Cas9 solution, 90.0% (18/20) of intact oocytes fertilized. After 48 hours in culture, diploid embryos reached the 8-cell stage at 88.9% (16/18). The 584-bp region of the target site of extracted DNA was amplified and gene modification of the edited diploid embryo cohort was confirmed in 87.5% (14/16). Compared to the OMSD cohort, enucleated oocytes fertilized at 85.0% (17/20) while cleavage rate was lower at 64.7% (11/17) and sperm genome modification at 81.8% (9/11). Both diploid embryos and haploid androgenetic embryos evidence an editing efficiency of 36.8%. Therefore, we verified that genome editing on embryos and sperm achieved the same editing yield. Limitations, reasons for caution While the experimental observations are still limited, it is important to note that these results in the embryo model may be underestimated by the T7E1 cleavage assay that only recognize heterozygous modification. The technique needs further refinement to optimize targeting and editing efficiency. Wider implications of the findings Sperm genome decondensation through an OMSD approach allows CRISPR-Cas9 to edit the sperm genome in order to correct eventual pathogenic mutations present in the gamete. Moreover, these would be the only method to absolutely ensure uniform generation of resulting embryos. Trial registration number not applicable

The impact of comparative genomic hybridization/single-nucleotide polymorphism microarray in risk stratification of pediatric acute lymphoblastic leukemia.

The objective of this study is to assess the concordance and added value of combined comparative genomic hybridization plus single-nucleotide polymorphism microarray (CGH/SNP) analyses in pediatric acute lymphoblastic leukemia (ALL) risk stratification compared to conventional cytogenetic methods. This is a retrospective study that included patients aged 1-18years diagnosed with de novo ALL at Sainte-Justine Hospital between 2016 and 2021. Results from conventional cytogenetic and molecular analyses were collected and compared to those of CGH/SNP. A total of 135 ALL patients were included. Sample failures or non-diagnostic analyses occurred in 17.8% cases with G-banding karyotypes versus 1.5% cases with CGH/SNP. The mean turnaround time for results was significantly faster for CGH/SNP than karyotype with 5.8 versus 10.7days, respectively. The comparison of ploidy assessment by CGH/SNP and G-banding karyotype showed strong concordance (r=.82, p<.001, r2=.68). Furthermore, G-banding karyotype did not detect additional clinically relevant aberrations that were missed by the combined analysis of CGH/SNP and fluorescence in situ hybridization. The most common gene alterations detected by CGH/SNP were deletions involving CDKN2A (35.8%), ETV6 (31.3%), CDKN2B (28.4%), PAX5 (20.1%), IKZF1 (12.7%), and copy-neutral loss of heterozygosity (CN-LOH) of 9p (9.0%). Among these, only ETV6 deletion was found to have a significant prognostic impact with superior event-free survival in both univariate and multivariate analyses (adjusted hazard ratio 0.08, 95% confidence interval: 0.01-0.50, p=.02). CGH/SNP provided faster, reliable, and highly concordant results than those obtained by conventional cytogenetics. CGH/SNP identified recurrent gene deletions in pediatric ALL, of which ETV6 deletion conferred a favorable prognosis.

Multiple unilateral angiofibromas with a hypopigmented patch as a possible presentation of tuberous sclerosis complex (TSC)

Tuberous sclerosis complex (TSC), known as Bourneville’s disease or epiloia, is an autosomal dominant inherited disorder that may affect numerous organ systems. TSC has many forms of clinical presentation. The cutaneous features of TSC include facial angiofibromas, shagreen patches, ash leaf hypopigmented macules, periungual fibromas, forehead fibrous plaques, confetti hypopigmentation, and poliosis. Rarely, the angiofibromas in TSC have a segmental distribution. It has been suggested that the segmental expression of tuberous sclerosis may result from a postzygotic mutation. The aim of this concise communication was to describe a patient with unilateral angiofibromas associated with a hypopigmented patch. No other features of TSC were present. We think this could have been a rare presentation of TSC. Key words: Angiofibroma, Hypopigmented patch, Loss of heterozygosity (LOH), Tuberous sclerosis complex (TSC)

The Variable Genomic Landscape During Osteosarcoma Progression: Insights From a Longitudinal WGS Analysis.

Osteosarcoma is a primary bone tumor that exhibits a complex genomic landscape characterized by gross chromosomal abnormalities. Osteosarcoma patients often develop metastatic disease, resulting in limited therapeutic options and poor survival rates. To gain knowledge on the mechanisms underlying osteosarcoma heterogeneity and metastatic process, it is important to obtain a detailed profile of the genomic alterations that accompany osteosarcoma progression. We performed WGS on multiple tissue samples from six patients with osteosarcoma, including the treatment naïve biopsy of the primary tumor, resection of the primary tumor after neoadjuvant chemotherapy, local recurrence, and distant metastases. A comprehensive analysis of single-nucleotide variants (SNVs), structural variants, copy number alterations (CNAs), and chromothripsis events revealed the genomic heterogeneity during osteosarcoma progression. SNVs and structural variants were found to accumulate over time, contributing to an increased complexity of the genome of osteosarcoma during disease progression. Phylogenetic trees based on SNVs and structural variants reveal distinct evolutionary patterns between patients, including linear, neutral, and branched patterns. The majority of osteosarcomas showed variable copy number profiles or gained whole-genome doubling in later occurrences. Large proportions of the genome were affected by loss of heterozygosity (LOH), although these regions remain stable during progression. Additionally, chromothripsis is not confined to a single early event, as multiple other chromothripsis events may appear in later occurrences. Together, we provide a detailed analysis of the complex genome of osteosarcomas and show that five of six osteosarcoma genomes are highly dynamic and variable during progression.

Somatic Mutations in KEAP1-NRF2 Complex in Breast Cancer.

Breast cancer remains the leading cause of cancer deaths for women. Long-term estrogen exposure is considered carcinogenic due to semiquinone production and to compromised detoxification. Metabolic regulator polymorphisms, such as KEAP1 (rs1048290) and NRF2 (rs35652124, rs6721961, rs6706649), can be valuable in understanding the individual cytoprotection profile. Thus, we aim to genotype these polymorphisms in blood, tumours and surrounding tissue, to identify somatic mutations and correlate it to prognoses. A total of 23 controls and 69 women with histological confirmed breast cancer were recruited, and DNA from blood/surrounding/tumour tissue was genotyped. Genotyping and clinicopathological data were correlated. We verified that rs35652124 presents different genotype distribution between the blood/surrounding tissue (p-value = 0.023) and tumour/surrounding tissues (p-value = 0.041). Apart from rs35652124 and considering the histological grade, the other four polymorphisms have different distributions among different tissues. There is a tendency towards the loss of heterozygosity in the surrounding tissue when compared to blood and tumour tissues, and higher genotype variability in histologic grade 2. These somatic mutations and different distribution patterns may indicate a heterogeneous and active microenvironment, influencing breast cancer outcome. Additionally, it would be pertinent to evaluate the predictive value of the histologic grade 2 considering somatic mutation profiles and distributions.

Chromosome 7 Isodisomy in a Child with Silver-Russell Síndrome

Silver-Rusell syndrome is a rare genetic disease. There is evidence that the genetic causes of the disorder are heterogeneous, with predominant alterations in the imprinted regions of chromosomes 11 and 7, in addition to other genomic alterations, such as chromosomal structural aberrations, single nucleotide polymorphisms, copy number variations, and small insertions and deletions. The most prevalent clinical manifestations include prenatal and postnatal growth retardation, dysmorphic features, and feeding difficulties. We present a case of a 4-year-old boy with phenotypic features consistent with Silver-Russell syndrome. The sample was subjected to conventional karyotyping analysis. The analysis was also conducted using the SALSA MLPA Probemix ME032-A1 UDP7-UDP14 and Applied Biosystems CytoScan 750K Suite. MS-MLPA analysis revealed the presence of hypermethylation in the &lt;em&gt;GRB-10&lt;/em&gt; and &lt;em&gt;MEST&lt;/em&gt; genes on chromosome 7. SNP-array analysis revealed a loss of heterozygosity (LOH) at 7q11.22q31.1 (38.7 Mb). The methylation of the genes involved in this epigenetic event, in conjunction with LOH and the clinical characterization of this child, indicates that the origin of the disease is due to an isodisomy of maternal chromosome 7. This report of a child who exhibits the clinical characteristics of SRS and presents a UPD of chromosome 7, most likely originating from the mother, once again demonstrates the involvement of these genes in SRS despite the incomplete understanding of the underlying mechanism. A multidisciplinary strategy has been proposed for the follow-up and treatment of this disease according to its etiology in the proband.

Evaluation of somatic copy number variation detection by NGS technologies and bioinformatics tools on a hyper-diploid cancer genome

BackgroundCopy number variation (CNV) is a key genetic characteristic for cancer diagnostics and can be used as a biomarker for the selection of therapeutic treatments. Using data sets established in our previous study, we benchmark the performance of cancer CNV calling by six most recent and commonly used software tools on their detection accuracy, sensitivity, and reproducibility. In comparison to other orthogonal methods, such as microarray and Bionano, we also explore the consistency of CNV calling across different technologies on a challenging genome.ResultsWhile consistent results are observed for copy gain, loss, and loss of heterozygosity (LOH) calls across sequencing centers, CNV callers, and different technologies, variation of CNV calls are mostly affected by the determination of genome ploidy. Using consensus results from six CNV callers and confirmation from three orthogonal methods, we establish a high confident CNV call set for the reference cancer cell line (HCC1395).ConclusionsNGS technologies and current bioinformatics tools can offer reliable results for detection of copy gain, loss, and LOH. However, when working with a hyper-diploid genome, some software tools can call excessive copy gain or loss due to inaccurate assessment of genome ploidy. With performance matrices on various experimental conditions, this study raises awareness within the cancer research community for the selection of sequencing platforms, sample preparation, sequencing coverage, and the choice of CNV detection tools.

Analysis of Genetic Alterations in TP53 Gene in Breast Cancer – A Secondary Publication

Tumor protein p53 (TP53) mediates DNA repair and cell proliferation in growing cells. The TP53 gene is a tumor suppressor that regulates the expression of target genes in response to multiple cellular stress factors. Key target genes are involved in crucial cellular events such as DNA repair, cell cycle regulation, apoptosis, metabolism, and senescence. TP53 genetic variants and the activity of the wild-type p53 protein (WT-p53) have been linked to a wide range of tumorigenesis. Various genetic and epigenetic alterations, including germline and somatic mutations, loss of heterozygosity, and DNA methylation, can alter TP53 activity, potentially resulting in cancer initiation and progression. This study was designed to screen three reported mutations in the DNA-binding domain of the p53 protein in breast cancer, to evaluate the relative susceptibility and risk associated with breast cancer in the local population. Genomic DNA was isolated from 30 breast tumor tissues along with controls. Tetra and Tri ARMS PCR were performed to detect mutations in the TP53 coding region. For SNPs c.637C&gt;T and c.733C&gt;T, all analyzed cases were homozygous for the wild-type allele ‘C,’ while for SNP c.745A&gt;G, all cases were homozygous for the wild-type allele ‘A.’ These results indicate no relevance of these three SNPs to cancer progression in our study cohort. Additionally, the findings from whole exon sequencing will help to predict more precise outcomes and assess the importance of TP53 gene mutations in breast cancer patients.

HGG-36. ASSESSMENT OF CHROMOSOMAL ABERRATIONS IN DIFFUSE ASTROCYTOMAS OF CHILDHOOD USING THE ARRAY COMPARATIVE GENOMIC HYBRIDIZATION (ACGH+SNP) TECHNIQUE – PRELIMINARY STUDY

Abstract BACKGROUND Comprehensive knowledge of the genomic changes underlying cancer is a key basis for diagnosis, prognosis and targeted therapies. With recent advances, the technique of comparative genomic hybridization (aCGH+SNP) facilitates genome-wide screening for genome copy number changes and rapid examination of numerical and structural chromosomal changes, as well as loss of heterozygosity at high resolution down to several tens of kilobases. METHODS The study included 12 patients aged 6 months to 15 years, operated on at the Department of Pediatric Neurosurgery in Krakow due to paediatric-type diffuse gliomas (according to WHO classification 5th Ed). Tumor tissue samples were collected from all included participants as remnants of routine examination. Pangenomic profiling of diffuse astrocytomas was performed with using SurePrint G3 Cancer CGH+SNP Kit 4x180K (Agilent, Santa Clara USA) at the Molecular Genetics Laboratory of the University Children’s Hospital in Krakow. Genomic DNA was extracted automatically from deep-frozen tumor tissue. Descriptive statistics were used to describe the basic characteristics of the data included in the study due to the small number of participants. RESULTS Of the 12 tumors examined by microarray, 5 patients had abnormalities in the form of diffuse high grade gliomas. In 1 patient, only numerical changes in chromosomes were observed, in 1 case only structural chromosomal abnormalities were detected. Combined numerical and structural aberrations were presented in 3 samples. Additionally, we detected 1 case of triploidy and 1 case of loss of heterozygosity. CONCLUSIONS Significant changes were observed in paediatric-type diffuse high-grade gliomas. Interestingly, we detected new undescribed alterations that were not only of a chromosomal nature, but also concerned individual genes. This may constitute the basis for further research aimed at establishing new diagnostic and prognostic markers, as well as effective therapy. A limitation of this study is the small patient sample, and further research is recommended.

HGG-21. DISTRIBUTION AND MUTATIONAL PATTERNS OF TP53 ALTERATIONS ACROSS SUBTYPES OF PEDIATRIC HIGH-GRADE GLIOMAS

Abstract Inactivating alterations of tumor suppressor gene TP53 are a hallmark of tumor progression in various cancer entities. Unfortunately, TP53 inactivation has not yet provided an actionable vulnerability. To investigate distribution, mutational pattern and impact of TP53 alterations in pediatric high-grade gliomas (pedHGG), we investigated TP53 alterations in 407 pedHGG by analyzing genomic sequencing and survival data collected through the INFORM and MNP molecular diagnostic pipelines. Nearly half of pedHGG (47%) showed signs of genomic TP53 inactivation. A second hit suggesting complete loss of function was observed in 84% of tumors, with loss of heterozygosity as the most common mechanism (70%). The frequency of TP53 alterations ranged from 97% in H3 G34-mutated tumors, 70% in IDH-mutated tumors and 61% in H3 K27-mutated tumors down to 40% in H3-/IDH-wildtype subtypes and none in infant-type hemispheric gliomas. Alterations in TP53 were associated with significantly shorter mean overall survival of 11 vs. 15 months in patients diagnosed with H3 K27-mutated tumors. Previously described TP53 hotspot mutations, such as amino acid substitutions at position 17, 248, 273, 282 and 342 were also prominent in the studied cohort (NM_000546). Mutations at position 342 were enriched in H3 G34-mutated tumors, with one-third carrying a stopgain mutation at this position rarely seen in other pedHGG. G34-mutated tumors also showed an abundance of nonsense, splicing or frameshift mutations in contrast to the predominance of missense mutations in other subtypes. The functional impact of these differences is currently under investigation. Lastly, we observed potential evidence for a genotype-phenotype link with Li-Fraumeni syndrome (LFS)-associated (constitutional) TP53 mutations in pedHGG, including an absence of the R248 hotspot mutation in 15 LFS-associated tumors.

ETMR-21. PRIMARY DIFFUSE LEPTOMENINGEAL MELANOMATOSIS (PDLM) ASSOCIATED WITH INTRACRANIAL BLEEDING AND RAPID PROGRESSION IN A CHILD

Abstract BACKGROUND PDLM is a rare and aggressive disease with limited cases reported worldwide and few cases in the pediatric population. Herein we report on clinical, histologic, and molecular/genetic findings of a young child with PDLM and rapid clinical progression. CASE STUDY A 12-year-old right-handed male presented with two months of intermittent fevers, progressive frontal headaches, lethargy, vomiting, and weight loss, and one month of focal seizures with altered awareness, speech arrest, eye deviation, and tonic posturing. Examination revealed bilateral papilledema and signs of increased intracranial pressure. A brain CT demonstrated increased density of the anterosuperior right frontal lobe and punctate focus of increased density along the left frontal lobe suggestive of hemorrhage. MRI revealed diffuse leptomeningeal thickening and enhancement throughout the brain and spinal cord. At biopsy, the frontal lesion was noted to be grossly abnormal and discolored, and on pathology demonstrated a melanocytic neoplasm infiltrating the subarachnoid space and extending into Virchow-Robin spaces. Tumor cells stained strongly for HMB45, vimentin, Melan-A, and PRAME and were BRAFV600E negative. RNA sequencing was negative for structural variants, Whole exome sequencing revealed a Tier I missense variant in NRAS (c.181C&amp;gt;A) among other copy number variations and loss of heterozygosity. Comprehensive ophthalmologic and dermatologic examinations as well as whole body PET/CT did not reveal any other sites of disease. He was due to commence immunotherapy with combination PD-1/CTLA-4 inhibitors but developed status epilepticus and raised intracranial pressure, warranting ventriculoperitoneal shunt insertion. Three days later, he had further deterioration with altered level of consciousness, hypertension, eye deviation, and vomiting. CT brain revealed large bifrontal hematomas with mass effect and care was redirected to palliation. CONCLUSION PDLM can be challenging to diagnose with a rapidly progressive course. Advanced molecular diagnostics may provide new targets for precision therapy, though overall outcomes remain dismal.

METB-14. INTEGRATED GERMLINE AND SOMATIC PROFILING OF PEDIATRIC BRAIN TUMORS REVEALS INCIDENTAL FINDINGS AND NOVEL TUMOR BIOLOGY

Abstract BACKGROUND The contribution of rare pathogenic germline variation to central nervous system (CNS) tumor formation in pediatric patients without a family history of cancer remains unclear. METHODS We characterized the prevalence of pathogenic germline variants in 214 cancer predisposition genes (CPGs) in 837 patients profiled in the Pediatric Brain Tumor Atlas by whole genome or exome sequencing (n=820 and n=17, respectively). Rare SNVs and small INDELs were annotated as pathogenic (P) or likely pathogenic (LP) consistent with American College of Medical Genetics criteria using AutoGVP, our open-source automated pathogenicity assessment tool. We classified pathogenicity of germline structural variants (SVs) using ClassifyCNV and AnnotSV. Somatic alterations and mutational signatures from matched tumor sequencing were integrated to identify functional consequences associated with germline pathogenic variation. RESULTS We observed 206 germline P/LP SNVs/small INDELs and 18 SVs (16 deletions, 2 duplications) within 78 unique CPGs in 195 patients (23.3%). We detected syndrome-associated P/LP variants in 45/58 patients with a clinically-reported cancer predisposition syndrome and incidentally in 41 patients. Ninety-five (42%) germline P/LP variants co-occurred with at least one somatic alteration in the same gene in matched tumors: n=6 oncogenic SNVs, n=28 CNVs, n=54 loss of heterozygosity (LOH), n=26 differential gene or protein expression, and n=18 alternative splicing. NF1, TSC1, and TSC2 germline P/LP carriers exhibited significantly higher tumor LOH scores and lower gene expression in tumors relative to non-P/LP carriers. Patients with germline splice region P/LP variants exhibited significantly increased skipping of most the proximal exon in matched tumor relative to patients with non-splice region P/LP variants (W=401, p=1.1E-04). CONCLUSION We have identified rare germline P/LP variants associated with cancer predisposition syndromes and diverse functional consequences in pediatric CNS tumor patients. Efforts are underway to extend this work to include 1,801 additional probands and 1,105 parents to further characterize the prevalence and heritability of germline pathogenic variation in children diagnosed with CNS tumors.

RB1 Genetic Alterations in Estrogen Receptor–Positive Breast Carcinomas: Correlation With Neuroendocrine Differentiation

Genetic alterations in the retinoblastoma susceptibility gene (RB1) are present in up to 40% of triple-negative breast cancers (BCs) and frequent in tumors with neuroendocrine differentiation, including small cell neuroendocrine carcinoma. Data on RB1 genetic alterations in estrogen receptor (ER)–positive BCs are scarce. In this study, we sought to define the morphologic, immunohistochemical, and genetic features of ER-positive BCs harboring somatic alterations in RB1, with emphasis on neuroendocrine differentiation. ER-positive BCs with pathogenic RB1 genetic alterations were identified in <1% of cases (N = 55) from a cohort of 6026 BCs previously subjected to targeted next-generation sequencing, including 23 primary BCs (pBCs) and 32 recurrent/metastatic BCs (mBCs). In cases where loss of heterozygosity of the wild-type RB1 allele could be assessed (93%, 51/55), most pBCs (82%, 18/22) and mBCs (90%, 26/29) exhibited biallelic RB1 inactivation, primarily through loss-of-function mutation and loss of heterozygosity (98%, 43/44). Upon histologic review, a subset of RB1-altered tumors exhibited neuroendocrine morphology (13%, 7/55), which correlated with expression of neuroendocrine markers (39%, 9/23) in both pBCs (27%, 3/11) and mBCs (50%, 6/12). Loss of Rb protein expression was observed in BCs with biallelic RB1 loss only, with similar frequency in pBCs (82%, 9/11) and mBCs (75%, 9/12). All cases with neuroendocrine marker expression (n = 9) and/or neuroendocrine morphology (n = 7) harbored biallelic genetic inactivation of RB1 and exhibited Rb loss of expression. TP53 (53%, 29/55) and PIK3CA (45%, 25/55) were the most frequently comutated genes across the cohort. Overall, these findings suggest that ER-positive BCs with biallelic RB1 genetic alterations frequently exhibit Rb protein loss, which correlates with neuroendocrine differentiation in select BCs. This study provides insights into the molecular and phenotypic heterogeneity of BCs with RB1 genetic inactivation, underscoring the need for further research into the potential clinical implications associated with these tumors.

Neoadjuvant talazoparib in patients with germline BRCA1/2 mutation-positive, early-stage triple-negative breast cancer: exploration of tumor BRCA mutational status

BackgroundTalazoparib monotherapy in patients with germline BRCA-mutated, early-stage triple-negative breast cancer (TNBC) showed activity in the neoadjuvant setting in the phase II NEOTALA study (NCT03499353). These biomarker analyses further assessed the mutational landscape of the patients enrolled in the NEOTALA study.MethodsBaseline tumor tissue from the NEOTALA study was tested retrospectively using FoundationOne®CDx. To further hypothesis-driven correlative analyses, agnostic heat-map visualizations of the FoundationOne®CDx tumor dataset were used to assess overall mutational landscape and identify additional candidate predictive biomarkers of response.ResultsAll patients enrolled (N = 61) had TNBC. In the biomarker analysis population, 75.0% (39/52) and 25.0% (13/52) of patients exhibited BRCA1 and BRCA2 mutations, respectively. Strong concordance (97.8%) was observed between tumor BRCA and germline BRCA mutations, and 90.5% (38/42) of patients with tumor BRCA mutations evaluable for somatic-germline-zygosity were predicted to exhibit BRCA loss of heterozygosity (LOH). No patients had non-BRCA germline DNA damage response (DDR) gene variants with known/likely pathogenicity, based on a panel of 14 non-BRCA DDR genes. Ninety-eight percent of patients had TP53 mutations. Genomic LOH, assessed continuously or categorically, was not associated with response.ConclusionThe results from this exploratory biomarker analysis support the central role of BRCA and TP53 mutations in tumor pathobiology. Furthermore, these data support assessing germline BRCA mutational status for molecular eligibility for talazoparib in patients with TNBC.

Loss of heterozygosity (LOH) in KEAP1/STK11-mutated lung adenocarcinoma to characterize a new subgroup of hard-to-treat patients with unmet need in the real-world setting.

8641 Background: Recent research suggests that loss of heterozygosity (LOH) in KEAP1/STK11 mutated lung adenocarcinoma (LUAD) may present a new immunological and biological aggressive subtype unresponsive to current standard of care treatment options. Currently, there is limited data on the clinical impact of LOH in KEAP1/STK11 co-alterations in real-world settings. Methods: From November 1st, 2021, to October 1st, 2023, patients with LUAD and mutations detected by FoundationOne CDx in any of the genes KEAP1, STK11, KRAS were enrolled in the study. Clinical data, including overall survival (OS), duration of response to different treatment lines (DOR), and real-world progression-free survival (rwPFS) according to the treatment lines, were retrospectively assessed in patients who had given informed general consent. Multivariate Cox regression models included patient characteristics, selected two-way interactions, OS, LOH, PD-L1 status and rwPFS for patients with STK11, KEAP1, KRAS mutations. Results: A total of 104 patients were included, 63 (60.6%) males with a median age of 64.4 years; [range: 38-90]). 23 (22.1%) patients with STK11/KEAP1/KRASmutations, 15 (14.4%) patients with KEAP1/LOH mutations, 10 (10.4%) patients with STK11/KRAS mutations and six (5.8%) patients with STK11/KEAP1. In the KEAP1/LOH mutated subgroup, median OS (mOS) was 14 months (ms), whereas in the subgroup with KEAP1/KRASmutations mOS was 13 ms. Across all groups mOS was 14 ms. Compared to Keynote 189 with a PFS of 8.8 ms, rwPFS for the first treatment line (rwPFS1) was shorter in patients with KEAP1/LOH status and KRAS/STK11 mutations at 3.5 ms and 3 ms respectively. Additionally, DOR and rwPFS for further treatment lines were analyzed according to PD-L1 and TMB status, as well as the treatment lines patients received. Conclusions: Patients with mutations on STK11/KEAP1/KRAS or with LOH presented an mOS inferior to what is currently expected from historic data for metastatic LUAD, with a worse prognosis and shorter rwPFS1 for the subgroup of K EAP1/LOH, KEAP1/KRAS and KRAS/STK11. Surprisingly, the percentage of patients carrying concurrent mutations of interest was much higher in the analyzed population than previously described, emphasizing the importance of broad NGS panel testing, including STK11 and KEAP1 mutations. Our findings based on real-world data indicate that in addition to the already known STK11/KEAP1/KRASconcomitant mutations, KEAP1/LOH mutations define a new subset of hard-to-treat genetic alterations. These patients represent a subgroup associated with primary therapy resistance, highlighting a new unmet need for tailored therapeutic strategies. Furthermore, our data emphasizes the benefit of upfront panel sequencing, including STK11/KEAP1 mutations and LOH, to identify at-need patients.

Exploring the correlation between high HRD status and immunotherapy response in melanoma.

e21541 Background: Homologous recombination deficiency (HRD) status demonstrates DNA instability in tumor cells and has been implicated as a response marker for immune checkpoint inhibitors (ICI). Here, we explore the correlation between high HRD status and ICI response biomarkers, homologous recombination repair (HRR) mutations, and the prognostic advantage of high HRD status following ICI in cutaneous melanoma patients. Methods: Melanoma samples (N = 2158) were profiled for genomic loss of heterozygosity (LOH) by NGS at Caris Life Sciences (Phoenix, AZ). As a surrogate for HRD, LOH-high was defined as LOH at ≥ 16% of the segments analyzed (up to 552). PD-L1 expression was assessed using IHC (SP142; positive (+): ≥ 1%). Mismatch repair deficiency/microsatellite instability-high (dMMR/MSI-H) was tested by IHC/NGS.Tumor mutational burden (TMB)-high was defined as ≥ 10 mutations/Mb. interferon gamma (IFN-g) and T-cell inflamed scores. Real-world overall survival (rw-OS) information was obtained from insurance claims data, with Kaplan-Meier estimates calculated from the start of immunotherapy until the last date of contact. Mann-Whitney U, Chi-square, and Fisher exact tests were applied where appropriate, with p-values adjusted for multiple comparisons. Results: Melanoma patient samples include - cutaneous: 1853; acral: 130; mucosal: 175, with LOH-high observed in 8.3% of cutaneous specimens, 13.1% of acral, and 30.9% of mucosal. Firstly, most biomarkers associated with response to ICI (PD-L1, TMB-high, total neoantigen load, IFN-g score, T-cell inflamed level, and dMMR/MSI-H) were not significantly different between LOH-high and LOH-low cohorts in all melanoma subtypes. However, TMB-high was significantly more prevalent in LOH-low cutaneous melanoma patients (54.9% vs 63.3%, p = 0.039). Prevalence of HRR gene mutations was not significantly different between high and low LOH groups in all cohorts, except for the ATRX gene which exhibited a significantly higher prevalence in the high LOH cutaneous (8.6% vs 1.3%, p &lt; 0.001) and mucosal (28.8% vs 2.5%, p &lt; 0.00001) melanoma specimens. Analysis of rw-OS showed no improvement in the treatment of LOH-high cutaneous melanoma patients with ICI compared to LOH-low (HR: 1.397 [95% CI: 0.759- 2.569], p = 0.281). Conclusions: This study demonstrates that HRDness reflected by the high-LOH status in melanoma did not correlate with mutations in HRR genes. In addition, melanoma specimens with high HRD status did not exhibit an overall higher prevalence of biomarkers associated with ICI response. Our study did not demonstrate improved survival outcomes in patients with high HRD status following anti-PD1 therapies. Future investigation is needed to assess other therapeutic approaches for these patients such as PARP inhibitors.

EVEREST-1: A seamless phase 1/2 study of A2B530, a carcinoembryonic antigen (CEA) logic-gated Tmod CAR T-cell therapy, in patients with solid tumors associated with CEA expression also exhibiting human leukocyte antigen (HLA)-A*02 loss of heterozygosity (LOH).

TPS2698 Background: Chimeric antigen receptor (CAR) T-cell therapies for treating solid tumors are challenging due to a lack of tumor-specific targets that discriminate cancer from normal cells. Previous studies using CEA T-cell receptors and T-cell engagers have resulted in dose-limiting, on-target, off-tumor toxicities (1,2). A2B530 is an autologous logic-gated, CEA-targeted Tmod CAR T-cell therapy that addresses the challenges of on-target, off-tumor toxicity by combining a CAR-activating receptor with a blocking receptor to discriminate tumor from normal cells (3). The activator recognizes CEA on the surface of both tumor and normal cells while specificity for tumor cells is provided by a blocker that binds HLA-A*02. In patients with both germline HLA-A*02 and tumor-associated HLA-A*02 LOH, the blocker prevents on-target, off-tumor toxicity on normal cells owing to retained HLA-A*02 expression (4). HLA-A*02 LOH can be detected using next-generation sequencing (Tempus AI, Inc.). With this definitive discriminator target, A2B530 can potentially provide a therapeutic window to treat patients with CEA-expressing solid tumors exhibiting HLA LOH. Methods: EVEREST-1 (NCT05736731) is a seamless, phase 1/2, open-label, nonrandomized study to evaluate the safety and efficacy of A2B530 in adult patients. Patients are enrolled through BASECAMP-1 (NCT04981119), a master prescreening study that identifies patients with HLA LOH at any time in the course of their disease. BASECAMP-1–eligible patients undergo leukapheresis and, when clinically appropriate, their banked T cells are manufactured for the EVEREST-1 study. The key inclusion criteria include histologically confirmed recurrent unresectable, locally advanced, or metastatic cancers associated with CEA expression: non-small cell lung, colorectal, or pancreatic cancers. Patients should have received ≥1 line of prior therapy such as checkpoint inhibitor, molecular targeted, or chemotherapy. The primary objective of phase 1 is to evaluate the safety and tolerability of A2B530 and determine the recommended phase 2 dose (RP2D). The dose-escalation portion is based on the Bayesian optimal interval design. The dose-expansion phase will confirm RP2D and collect biomarker data to further characterize A2B530. Phase 2 will assess overall response rate per RECIST v1.1. As of January 29, 2024, 8 patients have been enrolled on EVEREST-1. A2B530 was successfully manufactured for all patients, and all patients have received A2B530 infusion, with the first patient dosed in May 2023. Dose escalation is ongoing. 1. Parkhurst, et al. Mol Ther. 2022. 2. Tabernero, et al. J Clin Oncol. 2017. 3. Hamburger, et al. Mol Immunol. 2020. 4. Sandberg, et al. Sci Transl Med. 2022. Clinical trial information: NCT05736731 .

Correlation of allele-specific HLA loss of heterozygosity in patients with cancer with driver mutations.

e14658 Background: While immunotherapies have revolutionized cancer treatment, the limited predictability of response and resistance mechanisms present challenges to the development of novel therapeutics for solid tumors. Many of these use a sensitive and specific T cell receptor (TCR)-HLA:peptide interaction as a mechanism of action and can be impacted by HLA loss of heterozygosity (LOH). Allele-agnostic HLA LOH analyses assessing loss of any MHC class I allele are relevant for HLA-agnostic approaches such as immune checkpoint inhibitors; however, context-specific studies remain limited. For example, adoptive TCR T cell therapies and bi-specific TCR T cell engagers target neoantigen peptides derived from oncogenic driver mutations, such as those of KRAS and TP53, presented by a specific HLA allele. Thus, allele-agnostic analyses may overestimate the frequency and relevance of HLA LOH for allele-specific modalities. Methods: A real-world comprehensive genomic profiling dataset consisting of 78,418 cases (1), germline heterozygous for one or more HLA class I locus, was analyzed for HLA LOH in the context of frequent driver mutations (KRAS G12C, G12D, G12V and TP53 R175H), the corresponding HLA alleles (A*02:01, A*03:01, and A*11:01), and within tumor subtypes. Results: Allele-agnostic HLA LOH was observed in 16.6% of all samples and varied between indications. Notably, allele-specific loss of HLAs A*02:01, A*03:01, and A*11:01 across all samples was much lower than allele-agnostic loss (6.3%, 6.6%, and 6.3% cases, respectively). Although, a trend for increased allele-specific HLA LOH was observed when analyzed by driver mutation, the frequency remained low. Specifically, across all cases, A*02:01, A*03:01, and A*11:01 allele-specific LOH ranged between 5.9% - 9.7% in relation to KRAS G12C, G12D, and G12V compared to 5.9% - 6.2% for wild-type (WT). Moreover, it did not achieve statistical significance for any HLA-KRAS mutation pairs in colorectal (CRC), non-small cell lung (NSCLC), and pancreatic (PDAC) cancers, except for A*11:01 and KRAS G12C in NSCLC, which was lower in mutated cases (5.3% for G12C vs 10.1% for WT KRAS, p=0.045). Both allele-agnostic and A*02:01-specific HLA LOH in TP53 WT samples were lower than average. The TP53 R175H mutation was associated with limited increase in HLA-A*02:01 LOH (from 4.4% for WT to 8.1% for TP53 R175H) and varied between indications, e.g. no significant increase was observed in R175H-positive breast cancer and NSCLC cases contrary to CRC and PDAC. Conclusions: Based on this real-world dataset, over 90% of cases retain the TCR-targeted allele and, therefore, preserve the molecular complex necessary for HLA/driver mutation-based therapies. Notably, the database is enriched for advanced cancer patients. Further studies should test correlations with treatments and whether tumors in earlier lines of treatment have an even lower rate of HLA LOH. 1. Montesion et al., PMID: 33127846.