P53 Loss Of Heterozygosity Research Articles

Correlation of 1p/16q loss of heterozygosity and 1p gain with clinicopathological characteristics and prognosis in Wilms tumor

Objective: To investigate the relationship between 1p/16q loss of heterozygosity (LOH) and 1p gain in Wilms tumor and their clinicopathologic characteristics and prognosis. Methods: A total of 175 Wilms tumor samples received from the Department of Pathology, Beijing Children's Hospital from September 2019 to August 2022 were retrospectively analyzed. The histopathologic type and presence of lymph node involvement were evaluated by two pathologists. The clinical data including patients'gender, age, tumor location, preoperative chemotherapy, and tumor stage were summarized. Fluorescence in situ hybridization (FISH) was done to detect 1p/16q LOH and 1p gain and their correlation with the clinicopathological features and prognosis were analyzed. Results: Among the 175 samples, 86 cases (49.1%) were male and 89 (50.9%) were female. The mean age was (3.5±2.9) years, and the median age was 2.6 years. There were 26 (14.9%) cases with 1p LOH, 28 (16.0%) cases with 16q LOH, 10 (5.7%) cases of LOH at both 1p and 16q, and 53 (30.3%) cases with 1q gain. 1q gain was significantly associated with 1p LOH (P<0.01) and 16q LOH (P<0.01). There were significant differences (P<0.01) between 1q gain, 1p LOH and 16q LOH among different age groups. The rate of 16q LOH in the high-risk histopathological subtype (50.0%) was significantly higher than that in the intermediate-risk subtype (13.6%, P<0.05). The frequency of 1q gain, 1p LOH, and 16q LOH in children with advanced clinical stages (Ⅲ and Ⅳ) was significantly higher than that in children with early clinical stages (Ⅰ and Ⅱ). 1q gain, 1p LOH, and 16q LOH showed no significant correlation with gender, unilateral or bilateral disease, chemotherapy, or lymph node metastasis. The progression-free survival (PFS) time for patients with 1q gain and 1p LOH was significantly shorter than those without these aberrations (P<0.05). Additionally, the PFS time of patients with 16q LOH was slightly shorter than those with normal 16q, although the difference was not statistically significant. Patients with stage Ⅲ to Ⅳ disease exhibiting 1q gain or 1p LOH had a significantly higher relative risk of recurrence, metastasis, and mortality. Conclusions: 1p/16q LOH and 1q gain are associated with age, high-risk histological type, and clinical stage in Wilms tumor. 1q gain and 1p LOH are significantly correlated with the prognosis of Wilms tumor.

Loss of Heterozygosity of Chromosome 9p and Progression in MPNs

Introduction Copy-neutral loss of heterozygosity (LOH) of chromosome 9p, concomitant with mutated JAK2, can occur in myeloproliferative neoplasms (MPNs), and may be the most common chromosomal aberration in patients with polycythemia vera (PV). The prevalence of LOH of 9p varies amongst the different MPNs and is highest in PV where it has been reported to be between 30-70%. The reported prevalence of LOH of 9p is 20-50% and 10-20% in patients with myelofibrosis (MF) and essential thrombocythemia (ET), respectively. A hallmark feature of MPNs is the tendency to progress to more aggressive disease phenotypes (i.e., PV/ET to MF to accelerated- or blast-phase MPN) through the acquisition or loss of cytogenetic and molecular clonal markers. Although loss of LOH of 9p likely contributes to progression in MPNs, its pathologic role is still incompletely defined. Methods We conducted a retrospective review of patients at a single-center, tertiary-care institution who were diagnosed with an MPN, either chronic, accelerated or blast-phase (MPN-AP/BP), from January 1 st, 2017 until July 1 st, 2023. All patients underwent a microarray-based chromosome analysis using the I Scan System with the Global Diversity Array-8 (GDA) v1.0 Array BeadChip. Mutational profiling was done using the NextGENe software (SoftGenetics LLC) and MiSeq Reporter, and variant annotation and filtration was performed using VarSeq (Golden Helix). Results Fifty-two patients with MF were identified. Twenty-eight patients (53.8%) were found to have LOH of 9p, 15 of whom had secondary MF (7 with preceding ET and 8 with preceding PV). Eight of 9 patients (88.9%) with PV-MF harbored LOH of 9p, vs. 6 of 13 (46.2%) with ET-MF. The median cell fraction affected was 70% (range, 5-95%) and the median JAK2 variant allele frequency (VAF) was 75% (range, 16-97%). Eleven of the 24 patients without LOH of 9p harbored JAK2 mutations, at a median VAF of 40%. Nine patients (32.2%) with LOH of 9p had abnormal cytogenetics by conventional karyotyping, compared to 8 (33.3%) without LOH of 9p. Eleven patients (39.3%) with LOH of 9p were MIPSS-70 v2 high or very high risk, compared to 10 (41.7%) patients without LOH of 9p. Nine patients (32.1%) with LOH of 9p were DIPSS-plus intermediate-2 or high-risk, compared with 9 (37.5%) patients without LOH of 9p. Twenty patients with MPN-AP/BP were identified, of whom 11 had MPN-AP and 9 had MPN-BP. Four patients (20%), all with MPN-BP, had LOH of 9p. Median JAK2 VAF in these patients was 50% (range, 38-80%), vs. 4% (range, 3-48%) in the 6 patients with JAK2 without LOH of 9p. Two of 19 patients (10.5%) with ET were found to have LOH of 9p, with a corresponding JAK2 VAF of 70% and 20%. Twelve of the 17 patients (70.6%) who did not have LOH of 9p had a JAK2 mutation, at a median VAF of 27% (range, 4-44%). One patient with LOH of 9p had a thrombotic event, vs. 4 of 17 patients (23.5%) without LOH of 9p. Three of 9 patients (33%) with PV were found to have a LOH of 9p, with a median corresponding JAK2 VAF of 61% (range, 55-81%). Median JAK2 VAF for the other 6 patients (all with a JAK2 mutation) was 34% (range, 26-46%). One patient with LOH of 9p had a thrombotic event vs. 3 of 6 patients (50%) without LOH of 9p. Discussion The highest prevalence of LOH of 9p in patients with an MPN was seen in those with MF (&amp;gt;50%), followed by PV, MPN-AP/BP and ET. These results are generally in line with those previously reported, though the prevalence in PV was lower than other studies (likely due to small sample size). The prevalence of LOH of 9p in ET-MF was more than four times higher than in ET, suggesting that LOH of 9p may be an acquired cytogenetic aberration involved in ET progression. LOH of 9p was seen in almost 90% of PV-MF vs. 33% of PV patients, further supporting that LOH of 9p may be implicated in MPN progression. Notably, LOH-9p was seen in far fewer patients with accelerated- or blast-phase disease. It is likely that clonal evolution of MPNs to MPN-AP/BP is driven more so by the acquisition of high-risk mutations (e.g., TP53, ASXL1, U2AF1) and less by changes such as LOH of 9p and increased JAK2 VAF. In summary, our data largely confirms previous reports regarding the prevalence of LOH of 9p in MPNs while suggesting a role for the gain or loss of LOH of 9p in the progression of these hematologic cancers.

Prognostic impact of lymph node involvement and loss of heterozygosity of 1p or 16q in stage III favorable histology Wilms tumor: A report from Children's Oncology Group Studies AREN03B2 and AREN0532.

The prognostic impact of positive lymph nodes (LN+) and/or singular loss of heterozygosity (LOH) of 1p or 16q were assessed in children with stage III favorable histology Wilms tumor (FHWT) enrolled on AREN0532 or AREN03B2 alone. A total of 635 stage III FHWT vincristine/dactinomycin/doxorubicin (DD4A)-treated patients met inclusion criteria. Event-free survival (EFS) and overall survival are reported overall and by LN sampling, LN status, LOH 1p, LOH 16q, and a combination of LN status and singular LOH. Patients with unknown or positive combined LOH of 1p and 16q status and AREN03B2-only patients with unknown outcomes or treatment other than DD4A were excluded. EFS did not differ by study, supporting pooling. Lack of LN sampling (hazard ratio [HR],2.12; p=.0037), LN positivity (HR,2.78; p=.0002), LOH 1p (HR,2.18; p=.0067), and LOH 16q (HR,1.72; p=.042) were associated with worse EFS. Compared with patients with both LN- and LOH-, those with negative nodes but positive LOH 1p or 16q and those with LN+but LOH- for 1p or 16q had significantly worse EFS (HR,3.05 and 3.57, respectively). Patients positive for both LN and LOH had the worst EFS (HR,6.33; overall group factor, p<.0001). Findings confirm LN+status as an adverse prognostic factor amplified by presence of singular LOH 1p or 16q, supporting study of intensified therapy for patients with LN+ in combination with singular LOH in a prospective clinical trial.

Novel high–risk acute myeloid leukemia subgroup with ERG amplification and Biallelic loss of TP53

ETS-related gene (ERG) amplification, observed in 4–6% of acute myeloid leukemia (AML), is associated with unfavorable prognosis. To determine coincident effects of additional genomic abnormalities in AML with ERG amplification (ERGamp), we examined 11 ERGamp cases of 205 newly diagnosed AML using chromosomal microarray analysis and next generation sequencing. ERGamp cases demonstrated a distinct pattern of high genetic complexity: loss of 5q, chromothripsis and TP53 loss of function variants. Remarkably, allelic TP53 loss or loss of heterozygosity (LOH) co-occurring with TP53 inactivating mutation dramatically effected ERGamp tumor patient outcome. In the presence of homozygous TP53 loss of function, ERGamp patients demonstrated no response to induction chemotherapy with median overall survival (OS) of 3.8 months (N = 9). Two patients with heterozygous loss of TP53 function underwent alloSCT without evidence of relapse at one year. Similarly, a validation TCGA cohort, 6 of the 8 ERGamp cases with TP53 loss of function demonstrated median OS of 2.5 months. This suggests that with TP53 mutant ERGamp AML, successive loss of the second TP53 allele, typically by 17p deletion or LOH identifies a specific high-risk subtype of AML patients who are resistant to standard induction chemotherapy and need novel approaches to avert the very poor prognosis.

TP53 Allelic State in Myelodysplastic Syndromes with 5q Deletion

Introduction: Around 10% of de novo MDS harbor mutations in tumor protein p53 (TP53) gene. Recently, it has been shown that two-thirds of patients with MDS and mutated TP53 gene exhibit multi-hit mutations of TP53, this being associated with poor clinical features and outcome. Taking into consideration that MDS with isolated deletion of chromosome 5 (MDS-del5q) are particularly enriched in TP53 mutations (around 20% exhibit TP53 aberrations), we decided to analyze the TP53 allelic state in patients with MDS-del(5q). Methods: Patients from seven centers belonging to the Spanish Group of MDS (GESMD) and diagnosed with MDS-del5q according to the WHO 2017 classification were included. Therapy related MDS-del5q, as well as patients on disease modifying therapy prior the genetic analysis were excluded. Clinical characteristics and laboratory data were collected at the moment of diagnosis. Genetic profiling included conventional G-banding analyses, FISH for TP53 deletion, next-generation sequencing (NGS) analysis of a panel of 27 genes recurrently mutated in MDS (ASXL1, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, WT1, ZRSR2), and Sanger sequencing. Variant filtering and categorization were performed according to the Spanish Guidelines (Palomo et al., 2019). Based on TP53 mutations and allelic imbalances in chromosome 17p, patients were classified as follows (Bernard et al, 2020): 1) TP53 wild-type (TP53-wt) when no TP53 mutations were found; 2) TP53 monoallelic (TP53-monoallelic) when only 1 mutation was present; and 3) TP53 multi-hit (TP53-multihit) when multiple mutations or mutation(s) and concomitant deletion or copy number neutral loss of heterozygosity (LOH) were identified. Statistical analysis was performed by R (4.2.0). Results: We included 140 MDS-del(5q). Patient's characteristics are detailed in Table 1. Median overall survival (OS) was 96.7 months. Conventional cytogenetic analysis and/or FISH were performed in all cases. NGS was done in 76% (n=106), whereas in the remaining 24% (n=34) analysis of TP53, TET2, CBL and IDH1/2 was performed by Sanger sequencing. Eighty-six per cent (n=120) of patients were TP53-wt, and 14% (n=20) presented TP53 abnormalities: 12% (n=17) TP53-monoallelic and, interestingly, only 2% (n=3) TP53-multihit (Figure 1). In most TP53-monoallelic patients (n=10/17), TP53 mutation was detected as a single genetic event, while the most frequent concomitant mutation among the remaining MDS was DNMT3A (n=3). All TP53-multihit cases harbored two TP53 mutations, but no deletions or LOH in 17p were detected. In 2 out of these 3 patients, no other concomitant mutations were detected. As per the general mutational landscape of the MDS-del(5q) TP53-wt patients, the most frequently affected genes were SF3B1 (15%), DNMT3A (14%), TET2 (13%), ASXL1 (8%), JAK2 (4%), SETBP1 (2%) and CBL (2%). Finally, median OS was 96.7 months for MDS-del(5q) TP53-wt, 62.8 months for MDS-del(5q) TP53-monoallelic, and 56.9 months for MDS-del(5q) TP53-multihit; p= 0.3, with a trend for a worse OS of TP53-monoallelic compared with TP53-wt (HR, 0.5; p=0.106). Conclusions: This study shows that, as opposite to other MDS subgroups, the presence of multi-hit TP53 status is uncommon in patients with MDS-del5q. Larger series are currently collected to further ascertain the prognostic impact of TP53 aberrations in patients with MDS-del(5q). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Reduced sister chromatid cohesion acts as a tumor penetrance modifier.

Sister chromatid cohesion (SCC) is an important process in chromosome segregation. ESCO2 is essential for establishment of SCC and is often deleted/altered in human cancers. We demonstrate that esco2 haploinsufficiency results in reduced SCC and accelerates the timing of tumor onset in both zebrafish and mouse p53 heterozygous null models, but not in p53 homozygous mutant or wild-type animals. These data indicate that esco2 haploinsufficiency accelerates tumor onset in a loss of heterozygosity (LOH) sensitive background. Analysis of The Cancer Genome Atlas (TCGA) confirmed ESCO2 deficient tumors have elevated number of LOH events throughout the genome. Further, we demonstrated heterozygous loss of sgo1, important in maintaining SCC, also results in reduced SCC and accelerated tumor formation in a p53 heterozygous background. Surprisingly, while we did observe elevated levels of chromosome missegregation and micronuclei formation in esco2 heterozygous mutant animals, this chromosomal instability did not contribute to the accelerated tumor onset in a p53 heterozygous background. Interestingly, SCC also plays a role in homologous recombination, and we did observe elevated levels of mitotic recombination derived p53 LOH in tumors from esco2 haploinsufficient animals; as well as elevated levels of mitotic recombination throughout the genome of human ESCO2 deficient tumors. Together these data suggest that reduced SCC contributes to accelerated tumor penetrance through elevated mitotic recombination.

A novel heptasomy 21 associated with complete loss of heterozygosity and loss of function RUNX1 mutation in acute myeloid leukemia

Chromosomal aberrations are among the most important prognostic parameters in AML, and conventional cytogenetic analysis remains essential for risk stratification. In this report, we describe an adult male patient with a high percentage of circulating blasts, pathologically confirmed as AML with maturation. Cytogenetic analysis of a bone marrow sample revealed heptasomy 21 and trisomy 13 within a complex karyotype of 52,XY,der(2)t(2;13)(q33.3;q32.1),+13,+21,+21,+21,+21,+21 in all 20 cells examined, which was confirmed by metaphase FISH. Chromosomal microarray analysis (CMA) revealed complete loss of heterozygosity (LOH) of chromosome 21, supporting a common origin. In addition, LOH of chromosome 1p, trisomy 13, and partial tetrasomy of 13q and partial monosomy of 2q as a result of an unbalanced translocation between chromosomes 2 and 13 were observed. Molecular analysis identified two pathogenic missense variants: RUNX1 p.D198Y and SRSF2 p.P95R. The clonal allele ratio of RUNX1 p.D198Y was consistent with all copies of chromosome 21 in the leukemic clone carrying the mutation. Within the medical literature, there are no reports of heptasomy 21 for comparison; however, there are reports of AML with either polysomy 21 or trisomy 13. Our results suggest that even relatively ‘common’ AML aneuploidies may be associated with much more complex genomic changes, including loss of heterozygosity, which impact prognosis.

Abstract 1504: Mouse model and human patient data suggest critical roles for Pten and p53 in suppressing POLE mutant tumor development

Abstract Mutations in the exonuclease domain of POLE are associated with tumors harboring very high mutation burdens. The mechanisms linking this significant mutation accumulation and tumor development remain poorly understood. Pole+/P286R;Trp53+/- mice showed accelerated cancer mortality compared to Pole+/P286R;Trp53+/+ mice. Cells from Pole+/P286R mice showed increased p53 activation, and subsequent loss of p53 permitted rapid growth, implicating canonical p53 loss of heterozygosity in POLE mutant tumor growth. Somewhat surprisingly, however, p53 status had no effect on tumor mutation burden or single base substitution signatures in POLE mutant tumors from mice or humans. Pten has important roles in maintaining genome stability. We find that PTEN mutations are highly enriched in human POLE mutant tumors, including many in POLE signature contexts. One such signature mutation, PTEN-F341V, was previously shown in a mouse model to specifically decrease nuclear Pten and lead to increased DNA damage. We found tumors in Pole+/P286R mice that spontaneously acquired PtenF341V mutations and were associated with significantly reduced nuclear Pten and elevated DNA damage. Further analysis in cells suggests the presence of the mutant polymerase triggers DNA damage in specific contexts. Taken together, our analysis supports the idea that POLE-mediated hypermutagenesis is necessary, but not entirely sufficient, for tumorigenesis. Disabling surveillance of nuclear DNA damage is a likely sufficient factor. Citation Format: Meijuan J. Sun, Vivian S. Park, Wesley D. Frey, Leonard G. Williams, Karl P. Hodel, Juliet D. Strauss, Sydney J. Wellens, James G. Jackson, Zachary F. Pursell. Mouse model and human patient data suggest critical roles for Pten and p53 in suppressing POLE mutant tumor development [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 1504.

Genetic changes associated with relapse in favorable histology Wilms tumor: A Children’s Oncology Group AREN03B2 study

SummaryOver the last decade, sequencing of primary tumors has clarified the genetic underpinnings of Wilms tumor but has not affected therapy, outcome, or toxicity. We now sharpen our focus on relapse samples from the umbrella AREN03B2 study. We show that over 40% of relapse samples contain mutations in SIX1 or genes of the MYCN network, drivers of progenitor proliferation. Not previously seen in large studies of primary Wilms tumors, DIS3 and TERT are now identified as recurrently mutated. The analysis of primary-relapse tumor pairs suggests that 11p15 loss of heterozygosity (and other copy number changes) and mutations in WT1 and MLLT1 typically occur early, but mutations in SIX1, MYCN, and WTX are late developments in some individuals. Most strikingly, 75% of relapse samples had gain of 1q, providing strong conceptual support for studying circulating tumor DNA in clinical trials to better detect 1q gain earlier and monitor response.

MYCN amplification plus 1p36 loss of heterozygosity predicts ultra high risk in bone marrow metastatic neuroblastoma.

BackgroundThis study aimed to better understand the prognostic effect of multiple genetic markers and identify more subpopulations at ultra high risk of poor outcome in bone marrow (BM) metastatic neuroblastoma (NB).MethodsWe screened the MYCN, 1p36 and 11q23 loss of heterozygosity (LOH) statuses of 154 patients by interphase fluorescence in situ hybridization of BM cells. The clinical characteristics of patients with the three markers and their associations with prognosis were analysed.ResultsMYCN amplification and LOH at 1p36 and 11q23 were identified in 16.2%, 33.1% and 30.5% of patients, respectively. There were strong associations between MYCN amplification and 1p36 LOH as well as 11q23 LOH. Both MYCN amplification and 1p36 LOH were strongly associated with high levels of lactate dehydrogenase (LDH) and neuron‐specific enolase, more than 3 metastatic organs, and more events. 11q23 LOH occurred mainly in patients older than 18 months, and those who had high LDH levels. In univariate analysis, patients with MYCN amplification had poorer prognosis than those without. Patients with 1p36 LOH had a 3‐year event‐free survival (EFS) and overall survival lower than those without. 11q23 LOH was associated with poorer EFS only for patients without MYCN amplification. In a multivariate model, MYCN amplification was independently associated with decreased EFS in all cohorts. 11q23 LOH was an independent prognostic factor for patients without MYCN amplification, whereas 1p36 LOH was not an independent marker regardless of MYCN amplification. Compared with all cohorts, patients with both MYCN amplification and 1p36 LOH had the worst outcome and clinical features.ConclusionsPatients with both MYCN amplification and 1p36LOH had the worst survival rate, indicating an ultra high‐risk group. Our results may be applied in clinical practice for accurate risk stratification in future studies.

The expression of PHOX2B in bone marrow and peripheral blood predicts adverse clinical outcome in non-high-risk neuroblastoma

Paired-like homeobox 2B (PHOX2B) is a highly sensitive and specific biomarker for diagnosing neuroblastoma, as well as detecting minimal residual disease in neuroblastoma. The clinical significance of PHOX2B expression in bone marrow (BM) and peripheral blood (PB) samples of newly diagnosed patients with very low-, low- and intermediate-risk neuroblastoma remains unknown, to the best of our knowledge. The expression level of PHOX2B in paired BM and PB samples of patients with newly diagnosed neuroblastoma was validated using reverse transcription-quantitative polymerase chain reaction (RTqPCR). Among the 132 patients, 26 exhibited a positive PHOX2B expression BM (19.7%) and 11 in PB (8.3%) samples. PHOX2B was highly expressed in BM and PB samples from patients aged <18 months, with International Neuroblastoma Risk Group Staging System stages M and MS, 1p loss of heterozygosity, and high levels of lactate dehydrogenase, serum ferritin and neuron-specific enolase (p < 0.05). In all eligible patients, the 2-year event-free survival (EFS) and overall survival (OS) rates were 94.7 ± 2.0% and 97.7 ± 1.3%, respectively. However, the 2-year EFS rates were significantly decreased to 76.9 ± 8.3% and 63.6 ± 14.5% in patients with a positive PHOX2B expression in BM and PB samples, respectively (p < 0.05). Similarly, the 2-year OS rates were also decreased to 88.5 ± 6.3% and 81.8 ± 11.6% in patients with a positive PHOX2B expression in BM and PB samples, respectively (p < 0.05). In conclusion, a positive PHOX2B expression in BM and PB samples at diagnosis had a strong adverse prognostic effect on patients with non-high-risk neuroblastoma.

Clinical and Genetic Features of Brainstem Glioma in Adults: A Report of 50 Cases in a Single Center.

Background and PurposeBrainstem gliomas (BSGs) in adults are rare brain tumors with dismal outcomes. The aim of this study was to determine the clinical and genetic features in a series of BSGs and their association with the prognosis.MethodsFifty patients who underwent a stereotactic biopsy between January 2016 and April 2018 at a single institution were collected. Data on clinicopathological characteristics were analyzed and factors associated with patient survival were identified using a Cox regression model.ResultsThe median age at diagnosis was 55.5 years, and 62% of the patients were male. Glioblastoma (44%) accounted for the largest proportion of BSGs, and oligodendroglioma (2 of 50) was rarely encountered. The IDH mutation (6 of 44) occurred infrequently in astrocytomas, and IDH-mutant tumors harbored both ATRX loss and MGMT promoter methylation at a relatively low level. Wild-type IDH astrocytomas were identified as having high rates of 1p/19q codeletion (5 of 38) and loss of heterozygosity 1p (8 of 38) or 19q (8 of 38) only. In diffuse midline glioma H3K27M mutant, MGMT promoter methylation occurred in three of four cases. Patients were offered radiotherapy and/or concurrent/adjuvant temozolomide chemotherapy, and their median survival time was 13 months. Multivariate analysis revealed that a low tumor grade, absence of tumor enhancement, duration of symptoms ≥3 months, Karnofsky performance status ≥70, and ATRX loss conferred a survival advantage.ConclusionsAdult BSGs showed different molecular genetic characteristics, but also resembled supratentorial gliomas in their clinical features associated with oncological outcomes.

Survival of Lung Cancer Patients Dependent on the LOH Status for DMP1, ARF, and p53.

Lung cancer is the leading cause of cancer deaths in the world, and accounts for more solid tumor deaths than any other carcinomas. The prognostic values of DMP1, ARF, and p53-loss are unknown in lung cancer. We have conducted survival analyses of non-small cell lung cancer (NSCLC) patients from the University of Minnesota VA hospital and those from the Wake Forest University Hospital. Loss of Heterozygosity (LOH) for hDMP1 was found in 26 of 70 cases (37.1%), that of the ARF/INK4a locus was found in 33 of 70 (47.1%), and that of the p53 locus in 43 cases (61.4%) in the University of Minnesota samples. LOH for hDMP1 was associated with favorable prognosis while that of p53 predicted worse prognosis. The survival was much shorter for ARF-loss than INK4a-loss, emphasizing the importance of ARF in human NSCLC. The adverse effect of p53 LOH on NSCLC patients’ survival was neutralized by simultaneous loss of the hDMP1 locus in NSCLC and breast cancer, suggesting the possible therapy of epithelial cancers with metastatic ability.

COVID-19: crystallising the importance of patient safety.

Background: Mutation of the p53 gene has been implicated in the development of carcinoma in situ (CIS) to invasive solid urothelial carcinomas (UC) whereas loss of heterozygosity (LOH) at chromosome 9 has been suggested to plag part in the development of papillary UCs. Patients and Methods: The p53 mutation and LOH at chromosomes 17p13.1 and 9 were analysed in 120 UCs. Tumor and matched normal DNA were used for microsatellite allelotyping of chromosome 17p and the entire chromosome 9. Results: LOH at 17p13.1 was found in each grade and stage of the UCs, but mutation of the p53 occurred only in the highly malignant G3 tumors including papillary pT1G3 UCs. LOH were found at one or more of the seven tumor suppressor gene loci along chromosome 9 in all but two of the UCs with p53 mutation. Conclusion: Mutation of the p53 gene is not a pathway correlated genetic change, but is associated with the increased cell proliferation of G3 UCs.

Abstract 5118: Proteogenomics characterization of HPV-negative head and neck squamous cell carcinomas

Abstract Patients with head and neck squamous cell carcinomas (HNSCCs) are treated with surgery, radiation, chemotherapy, and limited targeted therapies. Compared to human papillomavirus (HPV)-positive HNSCCs, HPV-negative cases have worse treatment response and prognosis and represent an unmet clinical need. We performed comprehensive proteogenomic characterization of tumor specimens, matched normal adjacent tissues (NATs), and blood samples from 109 HPV-negative HNSCC patients. This cohort is dominated by tumors from oral cavity (45, 41%) and larynx (49, 45%). Somatic mutation and somatic copy number analyses validated previously reported genomic aberrations in HPV-negative HNSCC. Proteomics analysis linked p53 loss of heterozygosity to increased expression of EPCAM, a stemness marker. Additionally, FAT1 truncation mutations were associated with increased expression of proteins involved in keratinization, a key feature of SCC differentiation. Deletions of 3p and 9p led to the loss of genes encoding p16, chemokine receptors, and interferon/JAK/STAT signaling pathway proteins, whereas amplifications of 3q and 11q led to overexpression of proteins involved in cell proliferation and anti-apoptosis pathways. Comparative analysis of tumor and NAT proteomes and phosphoproteomes identified putative diagnostic biomarkers and druggable targets, and proteogenomic integration further identified putative neoantigens. Tumor site-specific characterization associated epigenetic silencing of neurofilaments with laryngeal but not oral cavity SCC. Protein targets of FDA approved or investigational drugs for HNSCC treatment showed high inter-tumor heterogeneity in their protein abundances. DNA copy number and RNA expression level were good surrogates of protein abundance for some targets, such as EGFR and PD-L1, but they failed to reflect protein levels or kinase activities for other targets, such as MMP9 and MTOR. Thus, there is a critical need for protein biomarker-driven treatment stratification. Deconvolution of bulk tumor gene expression data revealed an immune-hot subgroup and an immune-cold subgroup. Immune-hot tumors broadly overexpressed multiple immune checkpoints including PD-L1, IDO1, and CTLA4, underscoring the necessity of combination immune checkpoint inhibition to improve treatment efficacy. Immune-cold tumors were characterized by smoking, chromosomal instability, and activation of the CDK4/6-pRb axis, suggesting they could be targeted by CDK4/6 inhibitors. We also noted that EGFR-amplified tumors frequently harbor copy number aberrations of downstream signaling components of the EGFR pathway. This may explain the low response rate of EGFR-amplified tumors to EGFR inhibitors, and targeting multiple pathway components, including EGFR, PIK3CA and STAT3, may be required for these tumors. In summary, our integrative proteogenomic characterization revealed multiple novel insights into the pathogenesis and treatment of HPV-negative HNSCCs. Citation Format: Chen Huang, Lijun Chen, Yize Li, Sara Savage, Michael Schnaubelt, Felipe V. Leprevost, Marcin Cieslik, Yongchao Dou, Bo Wen, Jonathan T. Lei, Kai Li, Eric Jaehnig, Zhiao Shi, Meenakshi Anurag, Jianbo Pan, Yingwei Hu, Rodrigo V. Eguez, David J. Clark, Matthew Wyczalkowski, Saravana M. Dhanasekaran, Chandan Kumar, Antonio Colaprico, Karsten Krug, Michael Gillette, D. R. Mani, Seungyeul Yoo, Jiayi Ji, Xiaoyu Song, Weiping Ma, Xi Steven Chen, Alex Pico, Nathan J. Edwards, Scott D. Jewell, Mathangi Thiagarajan, Emily S. Boja, Henry Rodriguez, Andrew Sikora, Pei Wang, Matthew Ellis, Gilbert S. Omenn, Li Ding, Alexey I. Nesvizhskii, Adel K. EI-Naggar, Daniel W. Chan, Hui Zhang, Bing Zhang, Clinical Proteomic Tumor Analysis Consortium. Proteogenomics characterization of HPV-negative head and neck squamous cell carcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5118.

Abstract B30: ARID1A is a haploinsufficient tumor suppressor for N-Myc transformation of neural crest cells

Abstract Large segmental chromosomal alterations are common to cancer and a feature of high-risk neuroblastoma (NBL). Though they are early or initiating events in cancer, including often being found in precancerous lesions, their contribution to tumorigenesis is poorly understood. An unproven model is that these changes promote cancer through the cumulative effect of multiple dosage-sensitive genes. Loss of heterozygosity (LOH) at 1p36 is a frequent structural rearrangement in a broad range of human cancers including NBL. Approximately 70% of MYCN amplified NBL have 1p36 LOH with both mutations independently contributing to tumor aggressiveness in NBL. Two tumor suppressor regions have been proposed to exist in 1p36, a distal and proximal region, which have MYCN-independent and dependent roles. Through CRISPR/Cas9 genome editing of primary mouse neural crest cells (NCCs), a source for NBL, we found that loss of the chromatin remodeling factor Chd5 conferred most of the tumor-suppressor effects of 1p36 LOH in in vitro cell transformation assays in cells with endogenous levels of N-Myc. In contrast, when N-Myc was overexpressed, tumor evolution of NCCs genome edited to have randomly sized 1p36 deletions showed a reduction in tumor latency that significantly correlated with deletion of Arid1a. The Arid1a deletions that were selected for ranged from small indels up to large 1p36 deletions, indicating that large deletions are tolerated to achieve loss of a single critical tumor suppressor. Further, using lentiviral Cre-induced deletion of floxed Arid1a in isolated NCCs, we found that Arid1a is a haploinsufficient tumor suppressor in MYCN-driven transformation of NCCs. As Arid1a is a subunit of the chromatin remodeling complex SWI/SNF, which is mutated in 20% of cancers, Arid1a synthetic lethal therapies are being developed for adult malignancies. We are currently verifying those proposed therapies in NBL and are using small-molecule screening of cells lines derived from Arid1a wild-type, het, or null tumors to identify additional synthetic lethalities. Our findings indicate that context, such as the status of MYCN as an oncogene, dictates which 1p36 gene is the critical tumor suppressor, establishing 1p36 LOH as multifaceted not cumulative, as was previously believed. Citation Format: Kirby Wallace, Jesus Garcia-Lopez, Joel Otero, Rachelle Olsen, Chelsea DeVaux, Ashton King, Andrew Davidoff, Kevin Freeman. ARID1A is a haploinsufficient tumor suppressor for N-Myc transformation of neural crest cells [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B30.

SOAT1 promotes mevalonate pathway dependency in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. Altered metabolism is a cancer vulnerability, and several metabolic pathways have been shown to promote PDAC. However, the changes in cholesterol metabolism and their role during PDAC progression remain largely unknown. Here we used organoid and mouse models to determine the drivers of altered cholesterol metabolism in PDAC and the consequences of its disruption on tumor progression. We identified sterol O-acyltransferase 1 (SOAT1) as a key player in sustaining the mevalonate pathway by converting cholesterol to inert cholesterol esters, thereby preventing the negative feedback elicited by unesterified cholesterol. Genetic targeting of Soat1 impairs cell proliferation in vitro and tumor progression in vivo and reveals a mevalonate pathway dependency in p53 mutant PDAC cells that have undergone p53 loss of heterozygosity (LOH). In contrast, pancreatic organoids lacking p53 mutation and p53 LOH are insensitive to SOAT1 loss, indicating a potential therapeutic window for inhibiting SOAT1 in PDAC.

Aneuploidy in targeted endoscopic biopsies outperforms other tissue biomarkers in the prediction of histologic progression of Barrett's oesophagus: A multi-centre prospective cohort study.

BackgroundThe cancer risk in Barrett's oesophagus (BO) is difficult to estimate. Histologic dysplasia has strong predictive power, but can be missed by random biopsies. Other clinical parameters have limited utility for risk stratification. We aimed to assess whether a molecular biomarker panel on targeted biopsies can predict neoplastic progression of BO.Methods203 patients with BO were tested at index endoscopy for 9 biomarkers (p53 and cyclin A expression; aneuploidy and tetraploidy; CDKN2A (p16), RUNX3 and HPP1 hypermethylation; 9p and 17p loss of heterozygosity) on autofluorescence-targeted biopsies and followed-up prospectively. Data comparing progressors to non-progressors were evaluated by univariate and multivariate analyses using survival curves, Cox-proportional hazards and logistic regression models.Findings127 patients without high-grade dysplasia (HGD) or oesophageal adenocarcinoma (OAC) at index endoscopy were included, of which 42 had evidence of any histologic progression over time. Aneuploidy was the only predictor of progression from non-dysplastic BO (NDBO) to any grade of neoplasia (p = 0.013) and HGD/OAC (p = 0.002). Aberrant p53 expression correlated with risk of short-term progression within 12 months, with an odds ratio of 6.0 (95% CI: 3.1–11.2). A panel comprising aneuploidy and p53 had an area under the receiving operator characteristics curve of 0.68 (95% CI: 0.59–0.77) for prediction of any progression.InterpretationAneuploidy is the only biomarker that predicts neoplastic progression of NDBO. Aberrant p53 expression suggests prevalent dysplasia, which might have been missed by random biopsies, and warrants early follow up.

Aneuploidy in Targeted Endoscopic Biopsies Outperforms Other Tissue Biomarkers in the Prediction of Histologic Progression of Barrett's Oesophagus: A Multi-Centre Prospective Cohort Study

Introduction: The cancer risk in Barrett’s oesophagus (BO) is difficult to estimate. Confirmed histologic dysplasia has strong predictive power, but can be missed by random biopsies, while other clinical parameters have limited utility for risk stratification. We aimed to assess the utility of a large panel of molecular biomarkers on targeted biopsies to predict neoplastic progression of BO. Methods: In a prospective study in three tertiary centres, 203 patients with BO were tested for 9 biomarkers (p53 and cyclin A expression; aneuploidy and tetraploidy; CDKN2A (encoding for p16), RUNX3 and HPP1 hypermethylation; 9p and 17p loss of heterozygosity) on autofluorescence-targeted biopsies taken at index endoscopy and followed-up as per clinical standard. Data comparing progressors to non-progressors were evaluated by univariate and multivariate analyses using survival curves, Cox-proportional hazards and logistic regression models. Findings: 127 patients with no high-grade dysplasia (HGD) or early oesophageal adenocarcinoma (OAC) were included, of which 42 had evidence of any histologic progression over time. Aneuploidy was the only predictor of progression from non-dysplastic BO (NDBO) to any grade of neoplasia (p=0.013) and HGD/OAC (p=0.002). Aberrant p53 expression correlated with the risk of short-term progression within 12 months, with an odds ratio for histologically missed dysplasia of 6.0 (95% CI: 3.1-11.2). A biomarker panel comprising aneuploidy and p53 had an area under the ROC curve of 0.68 (95% CI: 0.59-0.77) for prediction of any histologic progression. Interpretation: In our study, aneuploidy is the only biomarker that predicts neoplastic progression of NDBO. Aberrant p53 expression suggests prevalent dysplasia, which might have been missed by random biopsies, and warrants early follow up. Funding Statement: This study was funded by a Medical Research Council Program Grant to RCF and by a Lister Institute for Preventive Medicine prize to RCF, with additional clinical research infrastructure support from the Experimental Cancer Medicine Centre and the Cambridge Biomedical Research Centre. Declaration of Interests: KR reports grants, personal fees and non-financial support from Olympus, outside the submitted work. JJGHMB reports grants from Olympus Endoscopy, outside the submitted work. Other authors declare no conflicts of interest. Ethics Approval Statement: This was a prospective study approved by the Cambridgeshire 2 Research Ethics Committee (09/H0308/118) and the Amsterdam University Medical Centre (AUMC) Medical Ethics Committee (MEC 09/073). This was a National Institute of Health research (NIHR) portfolio study (UKCRN ID 7561).

Alterations of chromosome 3p in 24 cases of gastrinomas and their correlations with clinicopathological and prognostic features

Abstract Purpose: The pathogenesis of gastrinomas is largely unknown, and there is a lack of reliable genetic determinants that are useful to distinguish malignant and benign forms of this tumor or predict the prognosis of patients with this disease. Loss of heterozygosity (LOH) on chromosome 3p is reported to occur in pancreatic neuroendocrine tumors (PNETs) as well as in non-PNETs and its presence is reported to correlate with tumor prognosis in non-endocrine tumors. However, little data are available from prospective studies on gastrinomas. Experimental design: We assessed occurrence of 3p LOH in 24 gastrinomas and correlated its presence with tumor biological behavior and other clinicopathological features of gastrinomas. Results: Either 3p LOH or microsatellite instability involving 3p occurred in 11 of 24 tumors (46%). Seven (29%) gastrinomas had 3p LOH. Of the 7 gastrinomas with 3p LOH, 5 (71%) had 3p12 LOH with the marker D3S2406, which was the shortest region of highest overlap (SRO). Chromosome 3p LOH was not associated with aggressive biological behavior of gastrinomas or with poor prognosis of patients with gastrinoma. Similarly, 3p12 LOH (SRO) was not correlated with aggressive growth of tumors and/or liver metastases. Conclusion: Gastrinomas have a relative high frequency of 3p12 LOH suggesting this area may harbor putative tumor suppressor gene(s), which may play a role in the tumorigenesis, but not aggressiveness, of a subset of these tumors.