Somatic TP53 Mutations Research Articles

Germline TP53 and MSH6 mutations implicated in sporadic triple-negative breast cancer (TNBC): a preliminary study

BackgroundGermline BRCA1/2 prevalence is relatively low in sporadic triple-negative breast cancer (TNBC). We hypothesized that non-BRCA genes may also have significant germline contribution to Chinese sporadic TNBC, and the somatic mutational landscape of TNBC may vary between ethnic groups. We therefore conducted this study to investigate germline and somatic mutations in 43 cancer susceptibility genes in Chinese sporadic TNBC.Patients and methodsSixty-six Chinese sporadic TNBC patients were enrolled in this study. Germline and tumor DNA of each patient were subjected to capture-based next-generation sequencing using a 43-gene panel. Standard bioinformatic analysis and variant classification were performed to identify deleterious/likely deleterious germline mutations and somatic mutations. Mutational analysis was conducted to identify significantly mutated genes.ResultsDeleterious/likely deleterious germline mutations were identified in 27 (27/66, 40.9%) patients. Among the 27 patients, 9 (9/66, 13.6%) were TP53 carriers, 5 (5/66, 7.6%) were MSH6 carriers, and 5 (5/66, 7.6%) were BRCA1 carriers. Somatic mutations were identified in 64 (64/66, 97.0%) patients. TP53 somatic mutations occurred in most of the patients (45/66, 68.2%) and with highest mean allele frequency (28.1%), while NF1 and POLE were detected to have the highest mutation counts.ConclusionsOur results supported our hypotheses and suggested great potentials of TP53 and MSH6 as novel candidates for TNBC predisposition genes. The high frequency of somatic NF1 and POLE mutations in this study showed possibilities for clinical benefits from androgen-blockade therapies and immunotherapies in Chinese TNBC patients. Our study indicated necessity of multi-gene testing for TNBC prevention and treatment.

TP53 somatic mutations are associated with somatic mitogenome substitutions but not indels in colorectal cancer cells.

p53 is a tumour suppressor protein that is involved in many cancer-related processes. Growing evidence suggests that p53 also plays an important role in mitochondrial (mtDNA) maintenance. Somatic mitogenome mutations are frequently observed in colorectal cancer (CC) cells. Thus, it was important to determine whether somatic mtDNA changes are associated with TP53 mutational status. In the present study, we analysed the TP53 gene in 67 CC patients, for whom mitogenome haplotypes were previously described. In total, 134 TP53 sequences (of cancer and matched normal specimens) were determined using the dideoxy method. Nine hereditary polymorphisms in the TP53 gene were detected in normal colon cells. None of them (neither alleles, nor genotypes) was associated with somatic mitogenome mutations in CC cells. Moreover, 42 somatic TP53 mutations were found in approximately 36% of CC tissues. These somatic changes were significantly more frequent in CC cells with somatic mtDNA mutations (p=0.0069). Furthermore, we show that only mitochondrial somatic substitutions (p=0.0017), but not indels (p>0.05), were associated with somatic TP53 mutations. The results of the present study suggest that changes in TP53 may modify p53 properties, which may result in the accumulation of somatic substitutions in CC mitogenomes.

Prospective study of the efficacy and utility of TP53 mutations in circulating tumor DNA as a non-invasive biomarker of treatment response monitoring in patients with high-grade serous ovarian carcinoma.

ObjectiveSomatic TP53 mutation (TP53mut) is a characteristic finding in high-grade serous ovarian cancer (HGSOC). The aim of this study was to assess the clinical efficacy and utility of TP53mut circulating tumor DNA (ctDNA) monitoring as a biomarker for managing HGSOC.MethodsTP53muts were evaluated in patients who received primary treatment for suspected ovarian cancer at Asan Medical Center. In patients diagnosed with HGSOC and with TP53mut, ctDNA, cancer antigen 125 (CA 125), and computed tomography were followed up according to the treatment course.ResultsDirect sequencing analysis of 103 tumor tissues from 61 HGSOC patients confirmed TP53muts in 41 patients (67.2%). All these patient-specific somatic mutations were detected in plasma cell-free DNA. The mean value of preoperative TP53 mutant allele count (TP53MAC) in stage III patients was 12.2 copies/µL and in stage IV patients was 45.3 copies/µL. TP53MAC was significantly reduced by treatment and there was no significant difference in the rate of decrease compared to CA 125 by the generalized linear mixed model. When patients were divided into a low TP53MAC group (<0.2 copies/µL) and a high TP53MAC group (≥0.2 copies/µL) based on the TP53MAC value at 3 months after the end of chemotherapy, there was a significant difference in time to progression between the two groups (p=0.038).ConclusionTP53mut ctDNA shows potential as a tumor-specific biomarker for treatment response monitoring in HGSOC. TP53mut ctDNA levels at 3 months post treatment has a significant prognostic utility than that of CA 125.

The molecular genetic make-up of male breast cancer.

Male breast cancer (MBC) is extremely rare and accounts for less than 1% of all breast malignancies. Therefore, clinical management of MBC is currently guided by research on the disease in females. In this study, DNA obtained from 45 formalin-fixed paraffin-embedded (FFPE) MBCs with and 90 MBCs (52 FFPE and 38 fresh-frozen) without matched normal tissues was subjected to massively parallel sequencing targeting all exons of 1943 cancer-related genes. The landscape of mutations and copy number alterations was compared to that of publicly available estrogen receptor (ER)-positive female breast cancers (smFBCs) and correlated to prognosis. From the 135 MBCs, 90% showed ductal histology, 96% were ER-positive, 66% were progesterone receptor (PR)-positive, and 2% HER2-positive, resulting in 50, 46 and 4% luminal A-like, luminal B-like and basal-like cases, respectively. Five patients had Klinefelter syndrome (4%) and 11% of patients harbored pathogenic BRCA2 germline mutations. The genomic landscape of MBC to some extent recapitulated that of smFBC, with recurrent PIK3CA (36%) and GATA3 (15%) somatic mutations, and with 40% of the most frequently amplified genes overlapping between both sexes. TP53 (3%) somatic mutations were significantly less frequent in MBC compared to smFBC, whereas somatic mutations in genes regulating chromatin function and homologous recombination deficiency-related signatures were more prevalent. MDM2 amplifications were frequent (13%), correlated with protein overexpression (P = 0.001) and predicted poor outcome (P = 0.007). In conclusion, despite similarities in the genomic landscape between MBC and smFBC, MBC is a molecularly unique and heterogeneous disease requiring its own clinical trials and treatment guidelines.

Gastric-type Adenocarcinoma of the Cervix: Tumor With Wide Range of Histologic Appearances

Gastric-type endocervical adenocarcinoma (GAS) is a recently described diagnostic entity originally characterized as a tumor with (1) voluminous cytoplasm that is (2) clear or pale eosinophilic, and (3) cells showing distinct cell borders. Since the initial tumor description there has been accumulating experience that the neoplasm, in addition to classic features, may show a wide spectrum of morphologic appearances. This paper describes and illustrates cases of GAS with focal or diffuse findings that include: densely eosinophilic cytoplasm, foamy cytoplasm, goblet cells, glands with elongated, stratified nuclei, glands with small cuboidal cells, glands with flattened cells, papillary growth, single cell infiltration and infiltration with microcystic elongated and fragmented pattern. All these patterns may bring up a differential diagnosis with other cervical malignancies such as usual, intestinal, endometrioid, clear cell, serous, and mesonephric adenocarcinoma. The paper describes the patterns of immunostaining of respective lesions that may aid in the diagnostic process and summarizes the main points of the differential diagnosis. GAS is associated with somatic and germline STK11 mutations and TP53 mutations but is invariably negative for human papilloma virus when tumor only is tested. It shows variation in incidence between countries. Awareness of the spectrum of morphologic appearances in GAS is important for accurate and confident diagnosis. Correct identification of GAS is important due to its propensity for ovarian and other distant metastases, markedly worse prognosis as compared with usual endocervical adenocarcinoma, and its relative resistance to chemotherapy.

Genomic and transcriptomic characterisation of undifferentiated pleomorphic sarcoma of bone.

Undifferentiated pleomorphic sarcoma of bone (UPSb) is a rare primary bone sarcoma that lacks a specific line of differentiation. There is very little information about the genetic alterations leading to tumourigenesis or malignant transformation. Distinguishing between UPSb and other malignant bone sarcomas, including dedifferentiated chondrosarcoma and osteosarcoma, can be challenging due to overlapping features. To explore the genomic and transcriptomic landscape of UPSb tumours, whole-exome sequencing (WES) and RNA sequencing (RNA-Seq) were performed on UPSb tumours. All tumours lacked hotspot mutations in IDH1/2 132 or 172 codons, thereby excluding the diagnosis of dedifferentiated chondrosarcoma. Recurrent somatic mutations in TP53 were identified in four of 14 samples (29%). Moreover, recurrent mutations in histone chromatin remodelling genes, including H3F3A, ATRX and DOT1L, were identified in five of 14 samples (36%), highlighting the potential role of deregulated chromatin remodelling pathways in UPSb tumourigenesis. The majority of recurrent mutations in chromatin remodelling genes identified here are reported in COSMIC, including the H3F3A G34 and K36 hotspot residues. Copy number alteration analysis identified gains and losses in genes that have been previously altered in UPSb or UPS of soft tissue. Eight somatic gene fusions were identified by RNA-Seq, two of which, CLTC-VMP1 and FARP1-STK24, were reported previously in multiple cancers. Five gene fusions were genomically characterised. Hierarchical clustering analysis, using RNA-Seq data, distinctly clustered UPSb tumours from osteosarcoma and other sarcomas, thus molecularly distinguishing UPSb from other sarcomas. RNA-Seq expression profiling analysis and quantitative reverse transcription-polymerase chain reaction showed an elevated expression in FGF23, which can be a potential molecular biomarker for UPSb. To our knowledge, this study represents the first comprehensive WES and RNA-Seq analysis of UPSb tumours revealing novel protein-coding recurrent gene mutations, gene fusions and identifying a potential UPSb molecular biomarker, thereby broadening the understanding of the pathogenic mechanisms and highlighting the possibility of developing novel targeted therapeutics. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Mutational Profile of Pediatric Therapy-Related Myeloid Neoplasms

We and others recently showed that the mutational spectrum of de novo pediatric myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is different than those in adults. MDS and AML also occur in children as a consequence of cytotoxic therapies used to treat childhood malignancies and are collectively referred to as therapy-related myeloid neoplasms (tMN). The incidence of pediatric tMN is ~1% in the pediatric cancer population. These secondary malignancies are usually resistant to conventional chemotherapy and managed with hematopoietic cell transplantation (HCT). These patients have a dismal prognosis. TP53 mutations and somatic alterations in chromatin modifiers predominate in adults with tMN, yet whether children with tMN have a similar constellation of genetic alterations remains unclear since comprehensive genomic profiling has not been completed in a large pediatric tMN cohort. We hypothesize that the mutational profile of pediatric tMN will be different than adult tMN given the patients' younger age and the different spectrum of primary tumor types and chemotherapies.Here we describe the somatic mutational profile of pediatric tMN (including tMDS & tAML) using whole exome (WES) and RNA-sequencing. We evaluated 65 diagnostic bone marrow samples from 61 unique patients, obtained from the St. Jude Children's Research Hospital Tissue Bank from patients diagnosed between 1987 & 2018. The cohort contains 26 tMDS and 39 tAML cases; in 4 patients both tMDS and tAML samples were included. Primary tumors included hematological malignancies (n=45), bone and soft tissue solid tumors (n=14), and brain tumors (n=2); acute lymphoblastic leukemia (ALL) was the most common primary tumor (n = 38, 62%). WES was completed for 61 tumor/normal pairs using Nextera Rapid Capture Expanded Exome (Illumina), while WGS was completed on 4 pairs. Normal comparator genomic DNA was obtained from flow-sorted lymphocytes. Median sequencing coverage for the tumor and normal samples were 107x and 95x, respectively. An average of 49 variants/patient (range: 6-217) was observed in the tMN cohort, including coding, non-coding, silent, and splice site variants, which is significantly different than our previously reported 5 variants/patient in pediatric primary MDS (p = 1x10-6). There was not a significant difference in the number of mutations/patient when tMDS was compared to tAML. Mutational signature analysis (https://cancer.sanger.ac.uk/cosmic/signatures) identified 3 major signatures, the most predominant was characterized by a strong bias for C>A mutations (Signature 24), followed by a signature with strong transcriptional strand bias for T>A mutations (Signature 27) and then a smaller subset resembling MDS and AML (Signature 1). Interestingly, patients with Signature 1 had an inferior 2-year overall survival than the other mutational signatures, with a median survival of 0.3 years (p = 0.0005). WES data and conventional karyotyping showed that chromosome 7 deletions (del(7)) were frequent (n=21, 32%), followed by deletions involving chromosome 5 (del(5)) (n=10, 15%). All of the cases with del(5) had complex cytogenetics and 6 of the 10 cases also had del(7). Ras/MAPK pathway mutations were present in 37% of the cases (40 total mutations in 27 cases). Canonical KRAS (n = 14), NF1 (n = 8), and NRAS (n = 7) mutations were the most frequent coding mutations present overall. Only 5 patients had somatic TP53 mutations, all of which had complex karyotypes. RNA sequencing was performed on 55 samples with suitable RNA. KMT2A rearrangements (KMT2Ar) were common (n = 29, 53%), 4 of which were cytogenetically cryptic. KMT2A rearrangements were more common in tAML (n = 25) but were present in tMDS (n = 4). Nearly half of these KMT2Ar cases also harbored an additional Ras/MAPK mutation. Fusions involving NUP98, RUNX1, MECOM, and ETV6 were also detected.In conclusion, we show that the mutational profile of pediatric tMN has fewer TP53 mutations and more KMT2Ar than adults, as well as a unique set of mutational signatures. These differences are likely a reflection of age-specific chemotherapeutic strategies and fewer pre-existing TP53 mutant hematopoietic clones in children. Future studies understanding the clonal evolution of pediatric tMN development will be helpful in describing pediatric tMN further. DisclosuresNo relevant conflicts of interest to declare.

PD-L1 Gene Alterations Identify a Unique Subset of Diffuse Large B Cell Lymphoma That Harbors a T Cell Inflamed Phenotype

Background: Programmed death - ligand 1 (PD-L1) expression is a dominant immune escape mechanism across human cancers. Ubiquitous copy gains of chromosome 9p24.1 - a region containing the PD-L1 and PD-L2 loci - lead to PD-L1 expression on Hodgkin-Reed-Sternberg cells in classical Hodgkin lymphoma. PD-L1 gene alterations also occur in diffuse large B cell lymphoma (DLBCL), albeit less commonly. Hypothesizing that PD-L1 gene alterations identify DLBCLs in which potent anti-lymphoma immune responses have been activated, we aimed to characterize the immune landscape of PD-L1 gene-altered DLBCLs, describe the key clinical features of these patients, and determine the degree to which PD-L1 gene alterations predict for response to PD-1 blockade therapy in DLBCL.Methods: FISH was performed on 105 formalin-fixed, paraffin-embedded (FFPE) DLBCL specimens with DNA probes to PD-L1, a region centromeric to PD-L2, and centromere 9. Lymphoma-infiltrating T cell numbers (68 cases), HLA class I/II expression (74 cases), and PD-L1 protein expression (93 cases) were assessed by IHC. RNA sequencing (seq) was performed on FFPE samples utilizing the Illumina HiSeq 2000 platform. Differentially-expressed genes were identified between PD-L1-altered and not altered DLBCLs using a false discovery rate (FDR) of 0.05 and log2 fold change of >1.5. Whole exome seq (WES) was performed on DNA from DLBCL specimens and 22 matched blood samples using the Illumina HiSeq exome kit in collaboration with Theragen Etex Bio Institute.Results:PD-L1 gene alterations were identified by FISH in 28/105 samples (27% - 16% relative PD-L1 copy gains, 7% PD-L1 amplifications, 2% PD-L1 translocations, 2% chromosome 9 polysomy), were enriched in non-germinal center (GC) DLBCLs (75% non-GC vs 25% GC; p=0.001), and were associated with robust PD-L1 protein expression (Fig 1A-B). PD-L1 gene-altered DLBCLs were more heavily infiltrated by CD8+ T cells compared to PD-L1 not altered DLBCLs (22.6 vs 13.8 CD8+ T cells/hpf; p<0.05), and TCR-β seq revealed a more clonal TCR repertoire among PD-L1-amplified DLBCLs (Fig 1C). Consistent with heightened T cell surveillance, HLA class I expression was absent or decreased more frequently among DLBCLs with vs without PD-L1 gene alterations (42% vs 20%; p<0.05). Furthermore, RNAseq of 12 PD-L1 gene-altered and 12 not altered DLBCLs (controlled for COO) identified differential expression of ~140 genes (Fig 1D). Gene ontology (GO) terms enriched in PD-L1 gene-amplified DLBCLs (p<0.05) included those related to negative regulation of T cell activation, while ingenuity pathway analysis (IPA) identified that a TH1 response and NF-κB activation were significantly enriched in PD-L1 gene-altered vs not altered DLBCLs (z-score p-value <0.05). Pathway-level up-regulation of NF-κB-associated genes in PD-L1 gene-altered DLBCLs was confirmed by GSEA (NES=1.65; p=0.009). WES performed on the same 24 cases revealed a 2-3-fold increase in somatic mutations in genes involved in antigen presentation (HLA, B2M, CIITA) and T cell co-stimulation (CD70, CD83) in PD-L1 gene-altered vs not altered DLBCLs (p=0.02), consistent with the activation of additional genetic mechanisms of immune escape in these lymphomas. Somatic TP53 and TNFAIP3 (A20) mutations, among others, were enriched in DLBCLs with PD-L1 gene alterations (p<0.05) (Fig 1E). Total tumor mutational burden and predicted neo-antigen numbers were similar in PD-L1 gene-altered and not altered DLBCLs (p=ns), arguing that PD-L1 gene-altered DLBCLs are not inherently more antigenic. Patients with PD-L1 gene-altered DLBCLs had higher-risk clinical features and inferior progression-free survival following initial treatment (Fig 1F). Finally, in the relapsed/refractory setting, PD-L1 gene alterations in an exploratory retrospective analysis were associated with response to PD-1 blockade therapy with pembrolizumab among 29 DLBCL patients enrolled in the KEYNOTE-013 study with tissue available (50% objective responses in PD-L1 gene-altered DLBCLs vs 8% in not altered cases), suggesting that PD-L1 gene alterations may be a useful predictive biomarker of response to anti-PD-1 antibody therapy in DLBCL.Conclusions: Our results indicate that PD-L1 gene alterations identify a unique biological subset of DLBCL in which endogenous anti-lymphoma immune responses have been activated, and which is associated with responsiveness to PD-1 blockade therapy. DisclosuresKline:Merck: Honoraria, Research Funding; iTeos: Research Funding. Orlowski:Merck & Co., Inc.: Employment. Smith:Portola: Honoraria; BMS: Consultancy.

High Resolution Melt Analysis for Rapid and Cost-Effective Screening of TP53 Mutations in Patients with Myeloid Malignancies

BackgroundRecent reports have highlighted an adverse impact of TP53 mutations on the prognosis of patients with myeloid malignancies. TP53 mutational analysis is useful in classifying these patients with respect to treatment strategies. High Resolution Melt (HRM) analysis is based on differences in the patterns of melting curves obtained when the targeted double strand DNA (dsDNA) dissociates during heating. HRM analysis is more cost- and time-effective than next-generation sequencing (NGS). In this study, we used HRM analysis to evaluate the impact of TP53 mutations on the clinical features and prognosis of patients at our institute with myeloid malignancies.MethodsIn this retrospective case study, HRM analysis was performed on 23 patients with MDS and 131 with AML, from October 2011 to January 2018. We targeted ten genes recurrently mutated in myeloid malignancies (TP53, NPM1, FLT3-ITD, IDH1, IDH2, NRAS, KRAS, WT1, DNMT3A, CEBPA) using HRM analysis for screening. For TP53 mutation detection, the entire coding region of TP53 gene from exons 1 to 11 was analyzed using the HRM method. Positive samples were validated using Sanger sequencing. We excluded frequently reported single nucleotide polymorphisms (P47S and R72P), silent mutations, and mutations which were not reported in the Catalogue of Somatic Mutations in Cancer database. NGS of TP53 mutations was done in 18 cases to validate mutations found by HRM analysis. Other non-TP53 gene mutations were similarly analyzed. The primary clinical endpoint of this study was overall survival (OS), measured from the time of diagnosis till time of death due to any cause, or till the last follow-up. The secondary clinical endpoint was relapse-free survival (RFS), measured from time of complete remission to relapse, or the last follow-up. The OS and RFS were compared using the log-rank test.ResultsWe evaluated gene mutations in 131 AML and 23 cases of MDS using HRM analysis. Costs for one HRM analysis were $25 (USD, excluding labor) and required approximately 7 hours processing time per sample. If the screen was positive, add-on cost was an additional $20 with 2 hours of processing time. In total, we identified 28 TP53 somatic mutations in 27 cases from this cohort using HRM analysis (17 newly-diagnosed AML, 4 refractory/relapsed AML, and 6 MDS). Out of the total 154 cases, 18 were also analyzed by NGS; TP53 mutations were detected in 4 cases (median variant allele frequencies = 0.42) and no TP53 mutations were detected in the remaining 14 cases, consistent with the results of HRM analysis. Among the 28 somatic TP53 mutations, 26 were missense, 1 frameshift, and 1 affecting splice sites. Most missense mutations detected (22/26) were localized to the DNA-binding domain of the gene. Cases with TP53 mutations frequently had a complex karyotype (P=0.001), but rarely had NPM1 mutations (P=0.039). In comparison with patients with myeloid malignancies but no TP53 mutations, patients with TP53 mutations had lower counts of white blood cells (3,000 ·μL-1 vs 12,700 ·μL-1 ; P=0.006), lower hemoglobin (7.9 g·dL-1 vs 9.3 g·dL-1 ; P=0.048), and lower platelet counts (2.7×104 ·μL-1 vs 5.7×104 ·μL-1 ; P=0.024). TP53 mutations were associated with shorter OS (median OS, 260 days vs 687 days; P=0.02). In newly-diagnosed AML patients, TP53 mutations were associated with lower counts of both white blood cells (3,200 ·μL-1 vs 26,550 ·μL-1; P=0.006) and lower platelet counts (2.0×104 ·μL-1 vs 6.2×104 ·μL-1; P=0.008) in the peripheral blood. Analysis of bone marrow aspirate revealed that more cases with TP53 mutations had a low myeloid:erythroid ratio than those with TP53 wildtype (53.8% vs 12.7%; P=0.02), suggesting that cases with TP53 mutations have a higher proportion of erythroid precursors, such as in acute erythroid leukemia or erythroid/myeloid leukemia (M6) , as per the FrenchAmerican-British (FAB) classification. Among the patients treated with intensive therapy (either conventional chemotherapy or allogeneic stem cell transplantation), cases with TP53 mutations had shorter overall survival (median OS, 265 days vs not reached; P= 0.04) and shorter relapse-free survival (median RFS, 188 days vs not reached; P=0.02).ConclusionPatients with TP53 mutations have a much poorer prognosis than those without, and exhibit refractoriness to conventional therapies. HRM analysis is a cost- and time-effective method to detect TP53 mutations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High frequency of POLE mutations in synchronous endometrial and ovarian carcinoma

Synchronous endometrial and ovarian carcinomas) represent 5% to 10% of endometrial or ovarian carcinomas. We assessed genetic alterations (in PTEN, CTNNB1, POLE, etc) and evaluated correlations with patient outcomes to determine the utility of clonality analyses for differentiating between metastases and concurrent primary tumors and for determining whether genetic alterations in synchronous tumors are predictive of biological behavior. Genomic DNA was isolated from formalin-fixed, paraffin-embedded tissues and frozen tissues from patients with synchronous endometrial and ovarian carcinomas. Samples were obtained from the Department of Obstetrics and Gynecology at the Shimane University School of Medicine between 2003 and 2017. Sanger sequencing was used to analyze the mutational status of the coding exons in TP53, PTEN, POLE, PIK3CA, KRAS, and CTNNB1 using previously published primers. All patients lived, and 3 had disease recurrence. The frequencies of somatic mutations in TP53, PTEN, CTNNB1, KRAS, and POLE were 3 (37.5%), 2 (25.0%), 3 (37.5%), 0 (0.0%), and 5 (62.5%) of 8 cases in ovarian tumors and 3 (37.5%), 2 (25.0%), 3 (37.5%), 1 (12.5%), and 5 (62.5%) of 8 cases in endometrial tumors, respectively. The frequencies of POLE and CTNNB1 mutations were higher than those in previous reports. A clonal relationship was determined by genomic analyses in 3 of 6 cases that were initially diagnosed as primary independent tumors. We confirmed that these 3 cases were indicated metastatic tumors because the lesion of mutation was the same. This information, provided by the sequencing-based strategy, could be useful for hypothesizing a patient's prognosis and deciding on treatment.

Low PR in ER(+)/HER2(-) breast cancer: high rates of TP53 mutation and high SUV.

On the basis of TP53 mutations and standardized uptake values (SUVs) from 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET), we sought to enhance our knowledge of the biology underlying low progesterone receptor (PR) expression in estrogen receptor (ER)-positive/human epidermal growth factor receptor-2 (HER2)-negative tumors. This study included 272 patients surgically treated for ER-positive, HER2-negative breast cancer and who had undergone TP53 gene sequencing. Of these, 229 patients also underwent 18F-FDG PET or PET/CT. Mutational analysis of exons 5–9 of the TP53 gene was conducted using PCR amplification and direct sequencing. The SUVs were measured using 18F-FDG-PET scan images. Twenty-eight (10.3%) tumors had a somatic TP53 mutation. The TP53 mutation rate was significantly higher in low-PR tumors than in high-PR tumors (17.1% vs 7.9%, P = 0.039). Low-PR tumors had significantly higher median SUVs than high-PR tumors (P = 0.046). The multivariable analysis revealed that SUV and age remained independent variables associated with low PR expression. An adverse impact of low PR expression on recurrence-free survival was observed in the multivariable Cox regression hazard model. We provide clinical evidence that genetic alteration of the TP53 gene and dysregulated glucose metabolism partly involve low PR expression in ER-positive and HER2-negative breast cancer.

Somatic TP53 Mutations Are Detectable in Circulating Tumor DNA from Children with Anaplastic Wilms Tumors

BACKGROUND: Diffuse anaplastic Wilms tumor (DAWT) is a rare, high-risk subtype that is often missed on diagnostic needle biopsy. Somatic mutations in TP53 are associated with the development of anaplasia and with poorer survival, particularly in advanced-stage disease. Early identification of DAWT harboring TP53 abnormalities could improve risk stratification of initial therapy and monitoring for recurrence. METHODS: Droplet digital polymerase chain reaction (ddPCR) was used to evaluate 21 samples from 4 patients with DAWT. For each patient, we assessed TP53 status in frozen tumor, matched germline DNA, and circulating tumor DNA (ctDNA) from plasma, serum, and urine collected throughout treatment. RESULTS: Mutant TP53 was detectable in ctDNA from plasma and serum in all patients. We did not detect variant TP53 in the same volume (200 μl) of urine. One patient displayed heterogeneity of TP53 in the tumor despite both histological sections displaying anaplasia. Concentration of ctDNA from plasma/serum taken prenephrectomy varied significantly between patients, ranging from 0.44 (0.05-0.90) to 125.25 (109.75-140.25) copies/μl. We observed variation in ctDNA throughout treatment, and in all but one patient, ctDNA levels fell significantly following nephrectomy. CONCLUSION: We demonstrate for the first time that ddPCR is an effective method for detection of mutant TP53 in ctDNA from children with DAWT even when there is intratumoral somatic heterogeneity. This should be further explored in a larger cohort of patients, as early detection of circulating variant TP53 may have significant clinical impact on future risk stratification and surveillance.

Abstract B41: Studying the effect of germline polymorphisms on somatic hotspot mutations in TP53 for the treatment of high-grade serous ovarian carcinoma

Abstract Introduction: High-grade serous ovarian carcinomas (HGSOCs) are the most aggressive and histologically important subtype of ovarian cancer. Although over 70% of patients show a complete response to first line of therapy, disease occurrence is observed within 3-5 years and often the subsequent recurrent disease is resistant to platinum-based chemotherapy. Hence it is of importance to identify novel targets and pathways to overcome drug resistance and improve the quality of life of the patient. The gene TP53 encodes the protein p53, a transcription factor that is induced in response to cellular stress and regulates cell cycle arrest, DNA damage repair, and apoptosis, thus inducing an antiproliferative effect on the cell and earning it the title of guardian of the genome. TP53 is found to be mutated in 96.7 % of HGSOC cases, which makes it especially relevant to our study. Most of the somatic mutations found in TP53 are missense mutations that result in amino acid changes at different positions called the hotspot regions and the majority of the studies that understand the relationship of p53 mutations and cancer have focused on hotspot somatic mutations. In addition to somatic mutations, nonsynonymous germline polymorphisms (single-nucleotide polymorphisms) (SNPs) P/R at codon 72 in exon 4 of TP53 play a role in altered p53 function and could pose a risk to cancer susceptibility. Thus, studying the combinatorial role of germline polymorphisms and somatic mutations on cancer progression and recurrence is extremely important to understand HGSOCs and design novel diagnostic and therapeutic tools with a precision medicine approach to overcome the impediments associated with treating HGSOCs. Experimental Procedures and Aim: To characterize the effect of the P72R germline SNP on sensitivity to cancer therapeutics in cells that have hotspot mutations in TP53: We hypothesize that the P72R SNP regulates sensitivity to common chemotherapeutics like paclitaxel and carboplatin and experimental therapeutics. Moreover, we expect the R72 SNP to be more sensitive to cancer therapeutics in comparison to its P72 counterpart in the cell line having the same hotspot mutation. We will use an isogenic p53 null cell line (SKOV3) with exogenous transduction of mutant TP53 in the context of the P72 or R72 SNP and determine the germline effect by using the same genetic background by: A) testing the effect of common cancer therapeutics mentioned above by using cell viability assays like the SRB assay and Cell Titer Glo assay; B) validating cell death observed by using Annexin V staining, caspase activity assays, and immunoblotting for cleaved PARP and caspase 8; and C) studying the differences in cell cycle arrest induced by chemotherapeutic agents by using cell cycle analysis and immunoblotting for cyclins. Summary: In summary, the proposed study indicates that the P72R SNP alters the phenotypic behavior of TP53 somatic mutations with the 72R having a greater proliferation rate, sensitivity to therapy, and cell death with the P72 undergoing a negative selection in the population. Conclusions: 1. Germline polymorphisms at codon 72 in the p53 protein play a significant role in regulating the gain-of-function effect of TP53 somatic hotspot mutations. 2. This regulation encompasses differences in cellular morphology, protein stability, drug resistance, growth potential, cell death, and cell cycle arrest. 3. We aim to further our understanding in elucidating the mechanisms responsible for the relationship between somatic mutations and germline polymorphisms that will ultimately alter the treatment strategies adopted for the patient. Citation Format: Cristabelle Madona N. de Souza, Jeremy Chien, Jill Madden. Studying the effect of germline polymorphisms on somatic hotspot mutations in TP53 for the treatment of high-grade serous ovarian carcinoma. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B41.

Abstract 1080: The effect of germline polymorphisms on somatic hotspot mutations in TP53 for the treatment of high-grade serous ovarian carcinoma

Abstract Introduction: Due to frequent recurrences and acquired resistance to platinum-based chemotherapy in high-grade serous ovarian cancer (HGS OvCa), it is important to identify novel targets and pathways to overcome drug resistance and improve the outcome of patients with ovarian cancer. TP53, which encodes the transcription factor p53, is mutated in 96% of HGS OvCa . Majority of the studies that establish the relationship between mutations in TP53 and cancer have focused on hotspot somatic mutations. In addition to somatic mutations, nonsynonymous germline single nucleotide polymorphisms (SNPs) at codon 72 play a role in altering the function of p53 and a cancer susceptibility. However, the effect of codon 72 (P72R) polymorphism on somatic mutations in TP53 is not well characterized with regard to the Epithelial to Mesenchymal phenotype and cancer progression. Objective: -Characterize the effect of somatic mutations in TP53 in the presence of the P72R SNP on cellular morphology and the Epithelial to Mesenchymal transition (EMT) transcriptional network. -Determine the effect of somatic mutations in TP53 in the presence of the P72R SNP on proliferation and the cell cycle checkpoint transcriptional network Experimental Procedures: We generated 8 pairs of hotspot mutations in TP53 in the context of the P72 or R72 SNP into p53-null SKOV3 cells. Cell morphology was recorded by fluorescence microscopy. Using CHIP-seq, RNA-seq and immunoblotting, we studied the effect of the polymorphism + mutation on EMT markers like BMP1, CALD1, CAMK2N1, CDH2 that are up-regulated during EMT and genes like CAV2, CDH1, DSP, FGFBP1, IL1RN, KRT19, MITF that are down-regulated during EMT. We used techniques like chromatin immunoprecipitation ChIP-seq, gene expression profiling, and computational methods to construct blueprints of the transcription factors like Cyclin D1, p21 that play a role in cell cycle checkpoint regulation which help understand compromised pathways due to the presence of somatic mutations in combination with germline polymorphisms. Summary: - Hotspot mutants with the R72 SNP cause changes in cell morphology resembling mesenchymal cells whereas mutants with the P72 SNP resemble epithelial cells. This effect can be accounted for by changes in the EMT network. - The hotspot missense mutants with R72 have a greater growth potential in comparison to their P72 counterpart which could explain the Gain of Function (GOF) effect of the former. Conclusions: Germline polymorphisms at codon 72 in the p53 protein plays a significant role in regulating the Gain-of-Function (GOF) effect of TP53 somatic hotspot mutations. This is significant because it will be useful in designing a treatment plan for patients with specific somatic mutations and germline polymorphisms. Citation Format: Cristabelle Madona N. De Souza. The effect of germline polymorphisms on somatic hotspot mutations in TP53 for the treatment of high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1080.

Abstract 2982: Somatic TP53 mutations alter the immune microenvironmentafter chemotherapy in breast cancer

Abstract Background: Neoadjuvant chemotherapy (NAC), followed by surgery, is the standard of care for triple-negative breast cancer (TNBC) patients. Unfortunately, only 30% achieve a pathologic complete response (pathCR, no evidence of invasive disease in the breast after NAC). In patients who do not achieve a pathCR, tumor-infiltrating lymphocytes (TILS) correlate with improved survival. This suggests there is an immune response to NAC in a subset of patients, which may be augmented by immune checkpoint inhibitors (ICI). Clinical trials are under way to examine the efficacy of ICI in breast cancer; therefore it is vital to identify biomarkers of response to better identify patients who may benefit from these modalities. Because many TNBC patients harbor somatic TP53 mutations and TP53 has been shown to be involved in immunity, we investigated the role of TP53 mutations on the immune microenvironment of breast cancer patients with residual disease. Methods: We examined 34 matched pre- and post-NAC primary breast cancer. TILs were scored by a pathologist. RNA and DNA were extracted and analyzed using nanoString Pan-Cancer Immune panel (&amp;gt;700 genes) and ImmunoSeq T-cell receptor (TCR) sequencing, respectively. CRISPR technology was used in mouse tumor cell lines to generate Trp53 mutations commonly found in TNBC. Cells were treated with chemotherapy in vitro to determine tumor-specific changes in cytokines and PD-L1 expression. To determine alterations to the immune microenvironment, isogenic cell lines were injected into syngeneic mice and expression of immune recruiting cytokines and immune checkpoint molecules by the tumor as well as immune phenotyping of TILs after NAC was performed. Results: TILs in the residual disease of TNBC patients correlated with improved survival after surgery, as expected. A third of patients had increased immune gene signatures after NAC, which correlated with increased TCR clonality, increased overall survival, and increased T cell-recruiting chemokine expression (CXCL9/10, etc.). In isogenic murine breast cancer cells, Trp53 mutant cells had higher levels of immune-recruiting chemokines after chemotherapy treatment in vitro, but demonstrated loss of Cdkn1a, a canonical p53 target gene. Trp53 mutant cells also exhibited increased PD-L1 expression after NAC compared to Trp53 wild-type cells. Mouse studies examining the immune infiltrate are ongoing. Conclusions: Our work suggests that NAC induces immune gene signatures in a subset of TNBC that is correlated with a better prognosis. TP53 mutations in the tumor may contribute to an increased immune infiltrate after chemotherapy through upregulation of chemokine expression and promote T cell exhaustion through upregulation of the immune checkpoint PD-L1. Ongoing in vivo studies with chemotherapy and ICI will indicate if p53-mutant tumors have a better response to ICI after NAC. Citation Format: Mellissa J. Nixon, V. Monica Estrada, Susan R. Opalenki, Donna Hicks, Michael Korrer, Melinda E. Sanders, Roberto Salgado, Young Kim, Rebecca Cook, Carlos L. Arteaga, Justin M. Balko. Somatic TP53 mutations alter the immune microenvironmentafter chemotherapy in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2982.

Abstract 201: Multiregion joint somatic genotyping in ovarian cancer proves optimal tumor sampling for clinical sequencing

Abstract Introduction. Genetic heterogeneity of ovarian tumors is caused by the accumulation of novel mutations through time. A result of this process is a spatially heterogeneous tumor with a fractal-like architecture, comprising of a mixture of spatially separated clones, sub-clones, and single cells. Spatial heterogeneity renders clinical sequencing a challenging task because clinically relevant mutations can go undetected. Here, we compare the genetic composition of single tumors with multiple DNA samples extracted from different regions of the tumor in diverse combinations to examine the effects of spatial separation on tumor heterogeneity. Methods. We derived tumor samples from five patients with ovarian cancer. Three tumor tissue samples were collected from each patient, in each of these, DNA was extracted from a total of eight segments of different sizes (5x3x8 total segments). Exome sequencing was performed and we examined DNA mutations identified from a biopsy sample, from three merged tissue segments with immediate vicinity to the biopsy, and from segments from spatially distant tissue samples merged into one. We compared identified mutations and copy-number variations called from the exome-seq data. Results. Three patients had heterozygous germline alterations affecting the BRCA pathway, paired with somatic TP53 mutations, one patient had multiple somatic mutations affecting the PI3K pathway, the last patient had the most common activating mutations in the PIK3CA and KRAS genes. When comparing the different sequencing runs, increasing the size of the tumor sample did not affect the overall quantity of mutations identified. Clonal mutations were identifiable in all samples, while sub-clonal mutations shifted. In case of a hypermutating phenotype, increasing the sequenced sample size strongly decreased the number of identified somatic mutations, most probably due to dilution of sub-clones. These observations are confirmed by an in silico model, in which we shuffled regions from publicly available multi-region sequencing data into known compositions. Conclusion. In hypermutating tumors the number of detected mutations increases as the size of the sequenced sample decreases. Generally, increasing the tumor sample size did not affect the number of identifiable mutations. Our findings suggest that clinical sequencing using small biopsy samples can generate adequate mutation calling results. Citation Format: Lorinc Pongor, Gyongyi Munkacsy, Ildiko Vereczkey, Imre Pete, Balazs Gyorffy. Multiregion joint somatic genotyping in ovarian cancer proves optimal tumor sampling for clinical sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 201.

Abstract 551: Discovering the co-drivers of mutant TP53 that promote breast cancer progression

Abstract Breast cancer is a heterogeneous disease and has different subtypes. Genetically 30% of all breast tumors and 80% of basal-like breast tumors harbor mutations in TP53 and numerous somatic mutations in other genes. This molecular heterogeneity has posed a challenge in developing effective therapeutic regimens and targeted therapies. Different TP53 missense mutations co-exist with other mutations in tumors and we hypothesize that tumor heterogeneity results from combinatorial effects of neo-morphic functions of specific TP53 driver mutations and a distinct subset of functionally important co-mutations (or ‘co-drivers'). We ectopically expressed the ten most prevalent missense TP53 mutants found in breast cancer tumors in non-transformed mammary epithelial cells and examined their phenotypes associated with the hallmarks of cancer. The results showed wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell invasion and cell polarity. As a proof of concept for the ‘co-driver', we knocked-out PTEN in a non-invasive TP53 Y234C mutant using the CRISPR system and observed increased cell invasion. A subsequent genome-wide CRISPR library-based screen on non-invasive TP53 Y234C expressing cells identified co-driver candidates that were highly enriched in the invasive population of Y234C cells. The top candidates included several known mutated genes in breast cancer patients with TP53 mutations, and were associated with signaling and cytoskeletal pathways that play crucial role in regulating cell invasion. These results demonstrate that this approach can pinpoint the key loss-of-function mutations in clinically important TP53 mutant backgrounds that can promote cancer-like behavior, and identify the combinations of ‘TP53 mutant and co-driver pathways' that can be targeted by specific inhibitors. Citation Format: Anasuya Pal, Jin Park, Joshua LaBaer. Discovering the co-drivers of mutant TP53 that promote breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 551.

PO-489 TP53 mutations and focal genomic amplifications for ctDNA detection in high-grade serous ovarian carcinoma

IntroductionHigh-grade serous ovarian carcinoma (HGSOC) is characterised by ubiquitous TP53 mutations and significant copy-number alterations. Current ctDNA assays for HGSOC have focused on detection of TP53 mutations. In patients with low-stage disease or during therapy, the amount of ctDNA may be below the threshold of reliable detection. We tested whether sensitivity for ctDNA detection could be increased by combining TP53 mutations with focal genomic amplifications.Material and methodsTargeted TP53 sequencing was performed on 264 plasma samples of 137 patients from the BriTROC-1 study with relapsed HGSOC tissue samples. Four patients had no somatic TP53 mutation detected and were excluded from analysis. Shallow WGS (0.1x) was performed on 256 plasma samples and matched tumour samples. The length-adjusted Z-score was calculated for each genomic segment and was used to distinguish significant focal amplifications (FA).Results and discussionsTP53 mutations were detected in 180 plasma samples with median mutant allele fraction (MAF) of 5% (IQR 1.4%–16%). Twenty-five patients had at least one plasma sample with MAF >20%, which could be used for genomic profiling.Focal amplifications were observed in 151 plasma samples with a median of 4 amplifications per sample. The FA with the maximum observed Z-score per sample was significantly correlated with TP53 MAF.Both approaches detected ctDNA in about half of the samples. However, in some plasma samples ctDNA could be detected only by one of the used methods.ConclusionCombining targeted TP53 sequencing with shallow WGS may be a more sensitive approach for ctDNA detection.

Analysis of Mutation and Loss of Heterozygosity by Whole-Exome Sequencing Yields Insights into Pseudomyxoma Peritonei

Pseudomyxoma peritonei (PMP) is a clinical syndrome characterized by gross mucinous ascites originating from a disseminated intraperitoneal neoplasm. Although typically confined to the abdomen, mortality is high if untreated. Biomarkers, including genetic mutation profiles, may aid treatment selection and decision making. We applied whole-exome sequencing to five patients diagnosed with low-grade appendiceal mucinous neoplasms, using paired tumor and germline samples identify biomarkers. Multiple bioinformatic approaches were applied to these data to assess both somatic mutation profiles and loss of heterozygosity events. Mutation profiles of the tumors were consistent with deamination of methylcytosine being the prevailing mechanism. Pathogenic mutations were identified in both KRAS and GNAS in all samples, and further mutations in genes implicated in PMP, namely FGFR2, APC, SMAD2, and FAT4. No TP53 somatic mutations were identified, matching expectations for low-grade tumors. Four of five samples exhibited clonal loss of heterozygosity; these regions were further examined and found to contain genes harboring pathogenic somatic mutations in some samples. RNF43 was hereby implicated in the pathogenesis of PMP of appendiceal origin, having previously been found to increase sensitivity to Wnt signaling and to have involvement in similar mucinous tumors. In conclusion, we have investigated the mutation profile of PMP of appendiceal origin and provided the first report of RNF43 involvement in its progression.

Breast cancer in women with neurofibromatosis type 1 (NF1): a comprehensive case series with molecular insights into its aggressive phenotype.

The purpose of the study was to improve the understanding of NF1-associated breast cancer, given the increased risk of breast cancer in this tumour predisposition syndrome and the limited data. We identified 18 women with NF1 and breast cancer at our institution. Clinical and pathologic characteristics of NF1-associated breast cancers were compared with 7132 breast cancers in patients without NF1 from our institutional database. Next generation sequencing was performed on DNA from blood and breast cancer specimens available. Blood specimens negative for NF1 mutation were subjected to multiplex ligation-dependent probe amplification (MLPA) to identify complete/partial deletions or duplications. Expression of neurofibromin in the NF1-associated breast cancers was evaluated using immunohistochemistry. There was a higher frequency of grade 3 (83.3% vs 45.4%, p = 0.005), oestrogen receptor (ER) negative (66.7% vs 26.3%, p < 0.001) and human epidermal growth factor receptor 2 (HER2)-positive (66.7% vs 23.4%, p < 0.001) tumours among NF1 patients compared to non-NF1 breast cancers. Overall survival was inferior in NF1 patients in multivariable analysis (hazard ratio 2.25, 95% CI 1.11-4.60; p = 0.025). Apart from germline NF1 mutations (11/16; 69%), somatic mutations in TP53 (8/10; 80%), second-hit NF1 (2/10; 20%), KMT2C (4/10; 40%), KMT2D (2/10; 20%), and PIK3CA (2/10; 20%) were observed. Immunohistochemical expression of neurofibromin was seen in the nuclei and/or cytoplasm of all specimens, but without any consistent pattern in the intensity or extent. This comprehensive series of NF1-associated breast cancers suggests that their aggressive features are related to germline NF1 mutations in cooperation with somatic mutations in TP53, KMT2C and other genes.