PTEN Locus Research Articles

Abstract 2820: The effect of Pten haploinsufficiency on genomic and transcriptomic profiles in combined genetically-engineered mouse models for colonic neoplasia based on Apc inactivation

Abstract Background and Aim: We have reported genetically-engineered mouse models for colonic neoplasia by using our novel CDX2P-Cre mice, in which Cre is restrictedly expressed in colonic epithelial cells. Although colonic tumors are generated in CDX2P-Cre;Apcflox/+ (CPC;Apc) mice with highly penetrant rate, greater number and deeper invasion of colonic tumors were observed in CDX2P-Cre;Apcflox/+;Ptenflox/+ (CPC;Apc+Pten), possibly resulting from haploinsufficiency of Pten gene. We have also confirmed shorter lifespan in CPC;Apc+Pten than that in CPC;Apc (AACR Annual Meeting 2018). Here, we test if these phenotypes come from one copy loss of Pten itself or secondary genetic and/or transcriptomic alterations induced by Pten loss by using whole genome sequence (WGS) and RNA sequence from macroscopically-dissected colonic tumors (bulk RNA-seq). Methods: WGS for five tumors and five livers as counterpart controls in each mouse model was performed by DNBSEQ platforms. Bulk RNA-seq was performed for three tumors in each model. Results: In the analysis using B-allele frequency, copy-neutral LOH occurred in chromosome 18 including Apc loci specifically in colonic neoplasia in both CPC;Apc and CPC;Apc+Pten mice. In contrast, LOH was not observed in chromosome 19 including Pten loci in CPC;Apc+Pten mice. We also confirmed remarkably decreased coverage of reads in reference mapping in Apc locus, and there was a slight decrease of coverage in Pten locus. There were no additional driver gene mutations in CPC;Apc or CPC;Apc+Pten in WGS. All these results suggested just one copy loss of Pten resulting in aggressive tumor phenotype in CPC;Apc+Pten mice. In RNA-seq and subsequent GSEA comparing colonic tumors in CPC;Apc+Pten with those in CPC;Apc revealed upregulation of some signal pathways including mTORC1. Supporting this observation, number of colon tumors, especially adenocarcinomas, were decreased by rapamycin administration to CPC;Apc+Pten. Similarly, rapamycin was more effective in tumorids from CPC;Apc+Pten those from CPC;Apc. Conclusion: WGS reveals one copy loss of Pten locus is the only genetic alteration in CPC;Apc+Pten when compared with CPC;Apc. One copy loss of Pten is sufficient to upregulate mTORC1 pathway in a CPC;Apc+Pten mouse model, resulting in promotion of tumor formation and invasion. Citation Format: Haruki Sada, Hiroaki Niitsu, Hikaru Nakahara, Naoya Sakamoto, Hirotaka Tashiro, Hideki Ohdan, Takao Hinoi. The effect of Pten haploinsufficiency on genomic and transcriptomic profiles in combined genetically-engineered mouse models for colonic neoplasia based on Apc inactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2820.

Detection of PSMA expression on circulating tumor cells by blood-based liquid biopsy in prostate cancer.

For patients with mCRPC, PSMA-targeted radioligand treatment has significantly improved the clinical outcome. A blood-based liquid biopsy assay for recognizing PSMA protein expression on circulating tumor cells may be beneficial for better informing therapeutic decision-making and identifying the patients most likely to benefit from PSMA-targeted radioligand therapy. Using high-throughput imaging and digital AI pathology algorithms, a four-color immunofluorescence assay has been developed to find PSMA protein expression on CTCs on a glass slide. Cell line cells (LNCaP/PC3s/22Rv1) spiked into healthy donor blood were used to study the precision, specificity, sensitivity, limit of detection, and overall accuracy of the assay. Clinical validation and low-pass whole-genome sequencing were performed in PSMA-PET-positive patients with high-risk mCRPC (N = 24) utilizing 3 mL of blood. The PSMA CTC IF assay achieved analytical specificity, sensitivity, and overall accuracy above 99% with high precision. In the clinical validation, 76% (16/21) of the cases were PSMA positive with CTC heterogeneity, and 88% (21/24) of the patients contained at least one conventional CTC per milliliter of blood. Thirty-six low-pass-sequenced CTCs from 11 individuals with mCRPC frequently exhibited copy number increases in AR and MYC and losses in RB1, PTEN, TP53, and BRCA2 locus. The analytical validation utilizing Epic Sciences' liquid biopsy CTC platform demonstrated the potential to detect PSMA protein expression in CTCs from patients with mCRPC. This assay is positioned as an effective research tool to evaluate PSMA expression, heterogeneity, and therapeutic response in many ongoing clinical studies to target tumors that express PSMA.

Abstract C030: Gene expression profiling tumors of patients with clear cell renal carcinoma: May identify race-specific therapeutic targets

Abstract Black/African Americans are second only to Native Americans in having the highest incidence of renal cancer. Despite this statistic, very few studies have examined whether patients’ tumors exhibit racial/ethnic differences in both gene and/or protein expression. Using KIRC (clear cell renal carcinoma) TCGA dataset (n=542), we identified several genes that were significantly overexpressed in ccRCC derived from Blacks. Genes which showed significantly higher expression in tumors from Blacks (n=56) relative to Whites (n=463) included ADCK5 (adj p<0.001) ADRM1 (adj p=0.013), ANKRD13D (adj p=0.003), ARFGAP1 (adj p=.003), LZTS2 (adj p<0.001), FAM160B2 (adj p<0.001), and NCKAP5L (adj p=0.029). In addition, these genes were also significantly overexpressed (p<0.05) in advanced stages, higher grades as well as in metastatic tissues. Multivariate cox regression analysis of gene expression, ethnicity, racial demographics and clinicopathological features relative to overall survival time revealed that ARFGAP1 (HRadj=2.40 [95% CI: 1.37 to 4.22]), ANKRD13D (HRadj=2.56 [95% CI:1.43 to 4.59]), FAM160B2 (HRadj= 1.74 [95%CI: 1.01 to 2.99]), and NCKAP5L (HRadj= 1.88 [95% CI:1.04 to 3.41]) were independent prognostic markers for survival. Higher expression of ADCK5 (HRadj= 5.98[CI:1.26-28.35], p=0.012), ADMR1 (HRadj= 1.74 [95%CI: 1.05 to 11.38], p=0.03), ANKRD13D (HRadj= 9.05 [95%CI: 1.13 to 72.66], p=0.014), ARFGAP1 (HRadj=3.79 [95% CI: 0.097-14.54], p=0.041), FAM160B2 (HRadj=5.09 [95%CI: 1.01-25.75], p=0.033), LZTS2 (HRadj=3.32 [95%CI: 1.01-10.99], p=0.037) was associated with lower ccRCC survival in Blacks. ROC analysis indicated that expression of FAM160B2 and LZTS2 are strong predictors of ccRCC survival among Blacks (AUC= 0.737, p<0.001, AUC= 0.731, p<0.001, respectively), whereas ADCK5 and ADRM1, (AUC=0.692, p<0.001, AUC=0.625, p=0.001, respectively) were moderate predictors of ccRCC survival among Blacks. FAM160B2 (family with sequence similarity 160 member B2) is a protein kinase A anchoring protein (AKAP) and participates in PKA (protein kinase A) phosphorylation of HSP70 (heat shock protein 70). FAM160B2 is also involved in hepatocellular carcinoma and promotes tumorigenesis. LZTS2, which encodes leucine zipper tumor suppressor 2, was identified as a tumor susceptibility gene within the 10q24.3 chromosomal region and is approximately 15Mb from the PTEN locus. This region containing both loci is frequently deleted in a variety of human malignancies, including prostate cancer. Taken collectively our data suggest that the aforementioned genes, FAM160B2 and LZTS2 may be useful both as clinical biomarkers and should be explored as potential therapeutic targets for ccRCC, with possible greater impact among Black patients. Citation Format: Romonia Renee Reams, Simone O. Heyliger, Marilyn Saulsbury, Emanuela Taioli, Diana Wilkie, John Carpten. Gene expression profiling tumors of patients with clear cell renal carcinoma: May identify race-specific therapeutic targets [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C030.

PRC2-Mediated Apoptosis Evasion Is a Therapeutic Target of MDS/AML Harboring Inv(3)/t(3;3) and -7

Myelodysplastic syndromes and acute myeloid leukemia (MDS/AML) with both inversion/translocation of chromosome 3 (inv(3)/t(3;3)) and monosomy 7 (-7) is an extremely poor prognostic entity. To explore potential therapeutic target of MDS/AML harboring both inv(3)/t(3;3) and -7, we performed drug screen using YCU-AML1, a high-risk MDS/AML cell line harboring t(3;3) and -7 (Kunimoto et al. Hemasphere 2020), as well as OCI-AML20, another AML cell line with inv(3) and -7, and found that both YCU-AML1 and OCI-AML20 showed high response to EZH2 inhibitors valemetostat and tazemetostat. Previous study has shown that EVI1, an oncogenic transcription factor highly expressed in MDS/AML with inv(3)/t(3;3), directly binds to EZH2 and thereby recruits PRC2 complex to PTEN locus, leading to epigenetic silencing of PTEN expression and activation of PI3K/AKT/mTOR pathway in leukemia with 3q rearrangement (Yoshimi et al. Blood 2011). Together with the fact that EZH2 locus is on chromosome 7q, we hypothesized that the survival of MDS/AML cells with inv(3)/t(3;3) and -7 may be highly dependent on residual allelic EZH2-mediated silencing of specific targets which drive cell death. We further validated in colony-forming unit and cell growth assays that YCU-AML1 and OCI-AML20 are both highly sensitive to valemetostat and tazemetostat, whereas FKH-1, Kasumi-3 and SKM-1, MDS/AML cell line with -7, 3q rearrangement, and complex karyotype without chromosome 3 and 7 abnormalities respectively, are resistant to these drugs. Apoptosis analysis revealed that valemetostat and tazemetostat efficiently induced apoptosis in YCU-AML1 and OCI-AML20 but not in FKH-1, Kasumi-3 and SKM-1. To seek molecular basis of EZH2 inhibitor-mediated apoptosis induction, we performed CUT&Tag sequence for H3K27me3 using vehicle or valemetostat-treated cells. Strikingly, promoter region of GADD45 γ was the most robustly and significantly decreased annotated peak locus of H3K27me3 in valemetostat-treated OCI-AML20. H3K27me3 peak in GADD45 γ locus was also significantly decreased in valemetostat-treated YCU-AML1 but not in FKH-1 and Kasumi-3. As expected, valemetostat treatment induced increased expression of GADD45 γ in OCI-AML20. Moreover, transcriptomic analysis also demonstrated GADD45 γ as the most robustly upregulated gene in valemetostat-treated YCU-AML1 compared to vehicle-treated cells. GADD45γ is known to be an upstream regulator of stress-activated protein kinases such as p38 and JNK in which signaling pathways activation induce apoptosis upon various cellular stresses. GADD45 γ promoter region possessed putative EVI1 binding site predicted by rVista2.0 software, indicating that EVI1 may directly bind and recruit PRC2 complex to GADD45 γ locus. We further confirmed phosphorylation of Thr180/Tyr182 (T180/Y182) residues of p38α in valemetostat-treated YCU-AML1 and OCI-AML20 but not in SKM-1 and Kasumi-3. p38 is known to phosphorylate Ser33 (S33) and Ser46 residues of TP53, leading to apoptosis in lung cancer cells (Yogosawa et al. Cancer Sci. 2018). In line with this finding, valemetostat treatment induced phosphorylation of Ser33 residue of TP53 in YCU-AML1 and OCI-AML20. Phosphorylations of p38α (T180/Y182) and TP53 (S33) were also noted in tazemetostat-treated YCU-AML1 and OCI-AML20, suggesting that EZH2 inhibition directly activates GADD45γ-p38α-TP53 axis leading to apoptosis preferentially in MDS/AML cells with inv(3)/t(3;3) and -7. As a proof of concept, p38MAPK inhibitor SB203580 restored valemetostat-induced colony growth inhibition as well as apoptosis induction in YCU-AML1 and OCI-AML20. Importantly, valemetostat treatment significantly reduced leukemic burden and improved overall survival in xenotransplant mouse model of YCU-AML1. Moreover, primary MDS/AML patient bone marrow (BM) sample harboring inv(3) and -7 exhibited preferential sensitivity to valemetostat compared to BM samples derived from healthy control or MDS/AML patients with or without -7 in vitro. Taken together, our study unraveled PRC2-mediated inactivation of GADD45γ-p38α-TP53 axis as a molecular basis for evasion of apoptosis in MDS/AML with inv(3)/t(3;3) and -7, which can be preferentially abrogated by EZH2 inhibition leading to efficient induction of apoptosis in this high-risk MDS/AML.

Distinct mechanisms of PTEN inactivation in dogs and humans highlight convergent molecular events that drive cell division in the pathogenesis of osteosarcoma

A hallmark of osteosarcoma in both human and canine tumors is somatic fragmentation and rearrangement of chromosome structure which leads to recurrent increases and decreases in DNA copy number. The PTEN gene has been implicated as an important tumor suppressor in osteosarcoma via forward genetic screens. Here, we analyzed copy number changes, promoter methylation and transcriptomes to better understand the role of PTEN in canine and human osteosarcoma. Reduction in PTEN copy number was observed in 23 of 95 (25%) of the canine tumors examined leading to corresponding decreases in PTEN transcript levels from RNA-Seq samples. Unexpectedly, canine tumors with an intact PTEN locus had higher levels of PTEN transcripts than human tumors. This variation in transcript abundance was used to evaluate the role of PTEN in osteosarcoma biology. Decreased PTEN copy number and transcript level was observed in - and likely an important driver of - increases in cell cycle transcripts in four independent canine transcriptional datasets. In human osteosarcoma, homozygous copy number loss was not observed, instead increased methylation of the PTEN promoter was associated with increased cell cycle transcripts. Somatic modification of PTEN, either by homozygous deletion in dogs or by promoter methylation in humans, is clinically relevant to osteosarcoma, because the cell cycle related transcripts are associated with patient outcomes. The PTEN gene is part of a syntenic rearrangement unique to the canine genome, making it susceptible to somatic loss of both copies of distal chromosome 26 which also includes the FAS death receptor. Significance StatementPTEN function is abrogated by different mechanisms in canine and human osteosarcoma tumors leading to uncontrolled cell cycling. Somatic loss of this canine specific syntenic region may help explain why the canine genome appears to be uniquely susceptible to osteosarcoma. Syntenic arrangement, in the context of copy number change, may lead to synergistic interactions that in turn modify species specific cancer risk. Comparative models of tumorigenesis may utilize different driver mechanisms.

Generation and Validation of an Anti-Human PANK3 Mouse Monoclonal Antibody.

Coenzyme A (CoA) is an essential co-factor at the intersection of diverse metabolic pathways. Cellular CoA biosynthesis is regulated at the first committed step—phosphorylation of pantothenic acid—catalyzed by pantothenate kinases (PANK1,2,3 in humans, PANK3 being the most highly expressed). Despite the critical importance of CoA in metabolism, the differential roles of PANK isoforms remain poorly understood. Our investigations of PANK proteins as potential precision oncology collateral lethality targets (PANK1 is co-deleted as part of the PTEN locus in some highly aggressive cancers) were severely hindered by a dearth of commercial antibodies that can reliably detect endogenous PANK3 protein. While we successfully validated commercial antibodies for PANK1 and PANK2 using CRISPR knockout cell lines, we found no commercial antibody that could detect endogenous PANK3. We therefore set out to generate a mouse monoclonal antibody against human PANK3 protein. We demonstrate that a clone (Clone MDA-299-62A) can reliably detect endogenous PANK3 protein in cancer cell lines, with band-specificity confirmed by CRISPR PANK3 knockout and knockdown cell lines. Sub-cellular fractionation shows that PANK3 is overwhelmingly cytosolic and expressed broadly across cancer cell lines. PANK3 monoclonal antibody MDA-299-62A should prove a valuable tool for researchers investigating this understudied family of metabolic enzymes in health and disease.

TB-8 Genetic and molecular properties of long-term proliferating tumorsphere -forming glioma derived cells

Long-term proliferating tumorsphere-forming glioma derived cells (LTP-TS-GDCs) and patient derived xenografts (PDXs) are essential tools for translational research for glioma. However, only small subsets of glioma samples are established as LTP-TS and/or PDXs and little is known about the genetics and molecular properties of LTP-TS -forming GDCs and PDX. In this study, we aim to analyze the characteristics of LTP-TS -forming GDCs and PDXs. We tried primary sphere cultures from 56 glioma patient-derived samples and established 11 LTP-TS-GDCs out of 45 glioblastoma samples and no long-term sphere culture was isolated from grade3 and grade 2 gliomas. LTP-TS-GDCs had self-renewal ability and possessed certain multipotency. However, they significantly less expressed SOX1 FOXG1 and TUBB3, whereas they expressed LGALS1 and EN1 significantly higher than normal neural stem/progenitor cells. In addition, we found that LTP-TS-GDCs shared the same genetic profiles with original patients’ tumors. Furthermore, we investigated the genetic differences between the glioma tissues which were successfully established as LTP-TS-GDCs and those which were not. We found that glioma tissues with TERT promotor mutations and triple copy number alteration (CNA) [EGFR, CDKN2A, and PTEN loci] are significantly established as LTP-TS-GDCs. Lastly, we next investigated in vivo characteristics of glioma PDXs. We have injected glioma PDXs lines into immunodeficient mice brains and histopathologically analyzed the characteristics of xenografts. Each xenograft well recapitulated histological features of original patients’ tumors and tumor cells remarkably invade through subventricular zone. In conclusion, each LTP-TS-GDCs and PDXs had various gene expression profiles, reflecting intratumoral and interpatient heterogeneities of glioma. In addition, TERT promotor mutations and triple CNA significantly correlated with success rate of LTP-TS-GDCs. These findings will be of use and advance the preclinical and translational researches of glioma.

TMOD-05. GENETIC AND MOLECULAR PROPERTIES OF LONG-TERM PROLIFERATING TUMORSPHERE -FORMING GLIOMA DERIVED CELLS

Abstract Long-term proliferating tumorsphere (LTP-TS)-forming glioma derived cells (GDCs) and patient derived xenografts (PDXs) are essential tools for translational research for glioma. However, only small subsets of glioma samples are established as LTP-TS and/or PDXs and little is known about the genetics and molecular properties of LTP-TS -forming GDCs and PDX. In this study, we aim to analyze the characteristics of LTP-TS -forming GDCs and PDXs. We tried primary sphere cultures from 56 glioma patient-derived samples and established 14 LTP-TS -forming GDCs out of 48 glioblastoma samples and no long-term sphere culture was isolated from grade3 and grade 2 gliomas. LTP-TS -forming GDCs had self-renewal ability and possessed certain multipotency. However, they significantly less expressed SOX1 FOXG1 and TUBB3, whereas they expressed LGALS1 significantly higher than normal neural stem/progenitor cells. In addition, we found that LTP-TS -forming GDCs shared the same genetic profiles with original patients’ tumors. Furthermore, we investigated the genetic differences between the glioma tissues which were successfully established as LTP-TS -forming GDCs and those which were not. We found that glioma tissues with TERT promotor mutations and triple CNA (EGFR, CDKN2A, and PTEN loci) are significantly established as LTP-TS -forming GDCs. Lastly, we next investigated in vivo characteristics of glioma PDXs. We have injected glioma PDXs lines into immunodeficient mice and histopathologically analyzed the characteristics of xenografts. Each xenograft well recapitulated histological features of original patients’ tumors and tumor cells remarkably invade through subventricular zone. In conclusion, each LTP-TS -forming GDCs and PDXs had various gene expression profiles, reflecting intratumoral and interpatient heterogeneities of glioma. In addition, TERT promotor mutations and triple CNA significantly correlated with success rate of LTP-TS -forming GDCs. These findings will be of use and advance the preclinical and translational researches of glioma.

Redefining the PTEN promoter: identification of novel upstream transcription start regions.

Germline mutation of PTEN is causally observed in Cowden syndrome (CS) and is one of the most common, penetrant risk genes for autism spectrum disorder (ASD). However, the majority of individuals who present with CS-like clinical features are PTEN-mutation negative. Reassessment of PTEN promoter regulation may help explain abnormal PTEN dosage, as only the minimal promoter and coding regions are currently included in diagnostic PTEN mutation analysis. Therefore, we reanalyzed the architecture of the PTEN promoter using next-generation sequencing datasets. Specifically, run-on sequencing assays identified two additional transcription start regions (TSRs) at -2053 and -1906 basepairs from the canonical start of PTEN, thus extending the PTEN 5'UTR and redefining the PTEN promoter. We show that these novel upstream TSRs are active in cancer cell lines, human cancer and normal tissue. Furthermore, these TSRs can produce novel PTEN transcripts due to the introduction of new splice donors at -2041, -1826 and -1355, which may allow for splicing out of the PTEN 5'UTR or the first and second exon in upstream-initiated transcripts. Combining ENCODE ChIP-seq and pertinent literature, we also compile and analyze all transcription factors (TFs) binding at the redefined PTEN locus. Enrichment analyses suggest that TFs bind specifically to the upstream TSRs may be implicated in inflammatory processes. Altogether, these data redefine the architecture of the PTEN promoter, an important step toward a comprehensive model of PTEN transcription regulation, a basis for future investigations into the new promoters' role in disease pathogenesis.

Clinicopathologic Characteristics of Thyroid Nodules Positive for PTEN Mutations on Preoperative Molecular Testing

Somatic and germline mutations of PTEN tumor suppressor gene are associated with follicular-pattern thyroid tumors and PTEN Hamartoma Tumor Syndrome (PHTS). The incidence of cancer in thyroid nodules positive for PTEN mutations on fine-needle aspiration (FNA) is not well defined. The aim of this study was to characterize diagnostic and phenotypic features of thyroid nodules with preoperatively detected PTEN mutations and their impact on management. Thyroid nodules with PTEN mutations on ThyroSeq v3 GC testing of FNA and core needle biopsy specimens from November 2017 to July 2020 were identified from the ThyroSeq Molecular Database. Demographic and clinicopathologic data were obtained through retrospective chart review. We identified 49 PTEN mutation-positive nodules from 48 patients. Patients were 57 years old on average (range 14-88) and 80% female. Cytology was predominantly indeterminate (73% atypia of undermined significance, 18% follicular neoplasm). There were 18 (29%) frameshift, 6 (10%) splice site, and 39 (62%) single nucleotide variant PTEN mutations. Fourteen (29%) nodules had two PTEN mutations, 5 (10%) had copy number alterations, and single cases had concurrent BRAF K601N, EZH1, and NRAS mutations. Surveillance was pursued for 27 (56%) and surgery for 21 (44%) patients (16 lobectomies, 5 total thyroidectomies). There were 14 follicular adenomas (FA), 4 oncocytic FA’s, 1 oncocytic hyperplastic nodule, and 1 encapsulated follicular variant papillary thyroid carcinoma (EFVPTC). The EFVPTC had two low-frequency PTEN mutations, PTEN locus loss, an NRAS mutation, and was a low-risk tumor with capsular but no angiolymphatic invasion. Four (8.3%) patients had confirmed or suspected PHTS, all with multiple nodules. Two had surgery finding no malignancies (2 FA). One PHTS patient had a prior thyroidectomy for a MET mutation-positive nodule that was follicular carcinoma. On US, the mean nodule size of patients who had surgery was larger than the surveillance group (3.2 cm vs. 2.3 cm, p=0.02) but there was no difference in TI-RADS level (p=0.54). There was no difference in mean nodule size (3.5 cm vs. 2.6 cm, p=0.35) or TI-RADS level (p=0.81) between PHTS and non-PHTS patients. Among surveillance patients, follow-up US was done at 1 year in 13/19 (68%) and 2 years in 3/6 (50%) of eligible cases. Only 1/19 (5%) underwent repeat FNA for increased nodule size. No thyroid malignancy was found with a mean of 1.75 years of follow-up (range 1.00-2.78). The EFVPTC patient had no recurrence after 1.05 years of follow-up. In summary, thyroid nodules with isolated somatic PTEN mutations are primarily benign and can be safely followed with serial imaging. Nodules with multiple PTEN mutations were only associated with malignancy when accompanied by an additional NRAS mutation. About 8% of patients with PTEN mutations may be PHTS patients who may be at greater risk for malignancy.

Clinical and Molecular-Based Approach in the Evaluation of Hepatocellular Carcinoma Recurrence after Radical Liver Resection.

Simple SummaryHepatocellular carcinoma (HCC) recurrence is still a major issue after liver resection. While several clinical factors were found to be associated with tumor recurrence, HCC pathogenesis is a complex process of accumulation of somatic genomic alterations, which has not been completely understood, especially with respect to tumor recurrence. The aim of this study is to complement potentially predictive clinical factors with next-generation sequencing genomic profiling and loss of heterozygosity analysis. We confirmed that serum bilirubin level, number of HCC nodules and size of the larger nodule are linked to a higher risk of tumor recurrence. Loss of heterozygosity in the PTEN loci was found to be associated with a lower risk of HCC recurrence.Background: Hepatic resection remains the treatment of choice for patients with early-stage HCC with preserved liver function. Unfortunately, however, the majority of patients develop tumor recurrence. While several clinical factors were found to be associated with tumor recurrence, HCC pathogenesis is a complex process of accumulation of somatic genomic alterations, which leads to a huge molecular heterogeneity that has not been completely understood. The aim of this study is to complement potentially predictive clinical and pathological factors with next-generation sequencing genomic profiling and loss of heterozygosity analysis. Methods: 124 HCC patients, who underwent a primary hepatic resection from January 2016 to December 2019, were recruited for this study. Next-generation sequencing (NGS) analysis and allelic imbalance assessment in a case-control subgroup analysis were performed. A time-to-recurrence analysis was performed as well by means of Kaplan–Meier estimators. Results: Cumulative number of HCC recurrences were 26 (21%) and 32 (26%), respectively, one and two years after surgery. Kaplan–Meier estimates for the probability of recurrence amounted to 37% (95% C.I.: 24–47) and to 51% (95% C.I.: 35–62), after one and two years, respectively. Multivariable analysis identified as independent predictors of HCC recurrence: hepatitis C virus (HCV) infection (HR: 1.96, 95%C.I.: 0.91–4.24, p = 0.085), serum bilirubin levels (HR: 5.32, 95%C.I.: 2.07–13.69, p = 0.001), number of nodules (HR: 1.63, 95%C.I.: 1.12–2.38, p = 0.011) and size of the larger nodule (HR: 1.11, 95%C.I.: 1.03–1.18, p = 0.004). Time-to-recurrence analysis showed that loss of heterozygosity in the PTEN loci (involved in the PI3K/AKT/mTOR signaling pathway) was significantly associated with a lower risk of HCC recurrence (HR: 0.35, 95%C.I.: 0.13–0.93, p = 0.036). Conclusions: multiple alterations of cancer genes are associated with HCC progression. In particular, the evidence of a specific AI mutation presented in 20 patients seemed to have a protective effect on the risk of HCC recurrence.

Significance of P16INK4A Expression and PTEN Loss of Heterozygosity in Human Papilloma Virus-related Oral Squamous Cell Carcinoma.

p16 and PTEN are tumor suppressor genes. Loss of these molecules in oral squamous cell carcinoma (OSCC) has been studied worldwide. In this study, we explored whether p16 cooperates with inactive PTEN during the pathogenesis of OSCC, especially in regard to tumor aggressiveness and proliferation. Immunocytochemistry and western blot analysis were used to examine the levels of p16 and PTEN. Sequencing analysis was performed to identify mutations in the PTEN gene and HPV infection. Fluorescence in situ hybridization was used to examine the presence of the PTEN locus. PTEN analysis showed high positivity in T4 samples. HPV-positive tumors correlated with tabagism, tumor size 3 and 4, disease stages 3 and 4, presence of lymph node metastasis (N1) and poor differentiation. Immunoexpression of p16 was strongly correlated with the presence of HPV. PTEN demonstrated a higher reactivity in advanced disease stages and p16 was strongly associated with HPV. Viral presence decreases tumor aggressiveness. Patients with advanced stage lesions demonstrated lower survival rate.

Deficiency of PTEN and CDKN2A Tumor-Suppressor Genes in Conventional and Chondroid Chordomas: Molecular Characteristics and Clinical Relevance.

IntroductionChordoma is a malignant tumor predominantly involving the skull base and vertebral column. This study aimed to investigate the molecular characteristics of PTEN and CDKN2A in conventional and chondroid chordomas and their correlation with clinical prognosis.Materials and MethodsA total of 42 patients were enrolled, including 26 patients with conventional chordoma and 16 patients with chondroid chordoma. Clinicopathological profiles and tissue specimens were collected. Gene sequencing and fluorescence in situ hybridization were performed to identify genetic alterations in the PTEN and CDKN2A genes. Immunohistochemical staining was used for semiquantitative evaluation of PTEN and CDKN2A expression.ResultsGene sequencing revealed an intron SNP (c.80–96A>G) and a missense mutation (c.10G>A; p.Gly4Arg) in the PTEN gene and a missense mutation (c.442G>A; p.Ala148Thr) in the CDKN2A gene. Loss of the PTEN locus was identified in 25 (59.5%) cases, and loss of the CDKN2A locus was found in 28 (66.7%) cases. There was no significant correlation between progression-free survival (PFS)/overall survival (OS) and loss of PTEN or CDKN2A. The patients with lower PTEN expression showed significantly shorter PFS and OS than those with higher expression, while there was no significant difference in PFS or OS between patients with lower CDKN2A expression and those with higher CDKN2A expression.ConclusionOur findings delineated the genetic landscape and expression of PTEN and CDKN2A in chordomas. PTEN expression may serve as a prognostic and predictive biomarker for chordomas.

Oncogenic ERG Represses PI3K Signaling through Downregulation of IRS2.

Genomic rearrangements leading to the aberrant expression of ERG are the most common early events in prostate cancer and are significantly enriched for the concomitant loss of PTEN. Genetically engineered mouse models reveal that ERG overexpression alone is not sufficient to induce tumorigenesis, but combined loss of PTEN results in an aggressive invasive phenotype. Here, we show that oncogenic ERG repressed PI3K signaling through direct transcriptional suppression of IRS2, leading to reduced RTK levels and activity. In accordance with this finding, ERG-positive human prostate cancers had a repressed AKT gene signature and transcriptional downregulation of IRS2. Although overexpression of IRS2 activated PI3K signaling, promoting cell migration in a PI3K-dependent manner, this did not fully recapitulate the phenotype seen with loss of PTEN as PI3K signaling is not as robust as observed in the setting of loss of PTEN. Importantly, deletions of the PTEN locus, which promotes active PI3K signaling, were among the most significant copy-number alterations that co-occurred with ERG genomic rearrangements. This work provides insight on how initiating oncogenic events may directly influence the selection of secondary concomitant alterations to promote oncogenic signaling during tumor evolution. SIGNIFICANCE: This work provides insight on how initiating oncogenic events may directly influence the selection of secondary concomitant alterations to promote tumorigenesis.

MPC-15 FEASIBILITY OF GLIOMA SPECIFIC ONCOPANEL IN THE DIAGNOSIS OF GLIOMA

AIMMolecular classification of glioma is a mandatory in the diagnosis of glioma according to the WHO 2016 classification of tumors of the central nervous system. However, WHO does not indicate the molecular methodology to be integrated, and the versatility and cost-effectiveness of molecular diagnosis is a concern. In this study, we evaluate the feasibility of a glioma specific tailored NGS panel where driver gene mutation and 1p/19q codeletion can be analyzed in a single platform.MATERIALS AND METHODSWe developed a glioma specific tailored NGS panel consisting of 48 key driver genes including IDH1/2, TP53, PTEN, EGFR, PDGFR, NF1, RB1, CDKN2A, and the TERT promoter. DNA was extracted from FFPE tumor tissues histologically identified by a pathologist, and from patient derived blood to serve as a control. In this system, gene mutations and copy number alterations can be precisely characterized, thus 1p/19q co-deletion can also be evaluated. We have analyzed 106 glioma patients (Grade II: 19 cases, Grade III: 23 cases, Grade IV: 64 cases) using this system.RESULTSFrom these 106 cases, IDH1 and TERT promoter mutations were detected in 33 cases (28%) and 55 cases (52%), respectively. 1p/19q co-deletion was detected in 19 cases (18%), with IDH1 mutations in all cases. In 57 Grade IV cases, TP53, PTEN, RB1, NF1, PDGFRA mutations were detected in 25 cases (43%), 24 cases (41%), 10 cases (17%), 8 cases (14%) and 6 cases (10%). Although EGFR mutation frequency was low (3%), amplification was detected in 14 cases (24%). As for deletion, PTEN and CDKN2A loci were deleted in 36 cases (62%) and 30 cases (52%), respectively. To note, MET alterations were detected in 2 cases. The cases in which histopathological diagnosis is difficult to make have a tendency to show atypical genetic alterations.CONCLUSIONDiagnosis of glioma patients with this glioma-specific tailored NGS panel is feasible.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Generation of PTEN‑knockout (‑/‑) murine prostate cancer cells using the CRISPR/Cas9 system and comprehensive gene expression profiling

Phosphatase and tensin homolog (PTEN) deficiency is associated with development, progression, and metastasis of various cancers. However, changes in gene expression associated with PTEN deficiency have not been fully characterized. To explore genes with altered expression in PTEN‑deficient cells, the present study generated a PTEN‑knockout cell line (ΔPTEN) from a mouse prostate cancer‑derived cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‑associated protein9 (CRISPR/Cas9) gene editing system. Following transfection of the CRISPR/Cas9 construct, DNA sequencing was performed to identify deletion of the Pten locus and PTEN inactivation was verified by western blotting. The ΔPTEN cell line exhibited enhanced RAC‑alpha serine/threonine‑protein kinase phosphorylation and cyclinD1 expression. In addition, an increase in cell proliferation and colony formation was observed in the ΔPTEN cell line. Gene expression profiling experiments were analyzed with microarray and microRNA (miRNA) arrays. In the microarray analysis, 111genes exhibited ≥10‑fold increased expression compared with the parent strain and mock cell line and 23genes were downregulated. The only miRNA with increased expression of 10‑fold or more was mmu‑miR‑210‑3p. Genes with enhanced expression included genes involved in the development, progression, and metastasis of cancer such as Tet methylcytosine dioxygenase1, twist family BHLH transcription factor2, C‑fos‑induced growth factor and Wingless‑Type MMTV Integration Site Family, Member3, and genes involved in immunosuppression such as Arginase1. The results of the present study suggest that PTEN deficiency mobilizes a variety of genes critical for cancer cell survival and host immune evasion.

Abstract 4484: Synthetic essentiality of chromatin remodeling factor CHD1 in PTEN-deficient cancer

Abstract Background: Prostate cancer (PCa) is the second leading cause of cancer death for men in the United States. Up to 70% of primary prostate tumors show loss of heterozygosity (LOH) at the PTEN locus, and loss of PTEN is a key initiation event in PCa development. Synthetic and collateral lethality have provided conceptual frameworks to identify cancer-specific vulnerabilities. Here, we explored an approach to identify potential synthetic lethal interactions through screening mutually exclusive deletion patterns in cancer genomes. Methods: We sought to identify ‘synthetic essential' genes, which might be occasionally deleted in some cancers but almost always retained in the context of a specific tumor suppressor deficiency, and posited that such synthetic essential genes would be therapeutic targets in cancers harboring specific tumor suppressor deficiencies. Results: In addition to known synthetic lethal interactions, this approach uncovered the chromatin helicase DNA-binding factor CHD1 as a putative synthetic essential gene in PTEN-deleted cancers. In PTEN-deleted prostate and breast cancers, the functional analysis showed that CHD1 depletion profoundly and specifically suppressed cell proliferation, survival, and tumorigenic potential. Mechanistically, functional PTEN stimulates GSK3β-mediated phosphorylation of CHD1 degron domains, which promotes CHD1 degradation via β-TrCP-mediated ubiquitination-proteasome pathway. Conversely, PTEN deficiency results in CHD1 protein stabilization, which in turn engages the H3K4me3 mark to activate transcription of the pro-tumorigenic TNFα/NF-κB gene network. In addition, we found CHD1 depletion significantly inhibits the progression of Pten-deficient prostate cancer genetic engineered mouse model. Conclusions: Together, this study identifies CHD1 as a novel downstream effector in PTEN pathway, and verifies CHD1 as a novel therapeutic target in PTEN deficient prostate cancer and breast cancer. Additionally, this study provides a framework for the discovery of trackable targets in cancers harboring specific tumor suppressor deficiencies. Citation Format: Di Zhao. Synthetic essentiality of chromatin remodeling factor CHD1 in PTEN-deficient 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 4484.

Assessment of a custom designed next generation DNA sequencing gene panel to profile endometrial cancers.

5593 Background: Using whole genome sequencing (WGS), the Cancer Genome Atlas (TCGA) identified four genomic subtypes of endometrial cancer (EC) with survival differences. Limited clinical data is available in the TCGA dataset restricting correlation of WGS with clinical endpoints. We sought to determine if high depth target sequencing allows for genomic classification of EC and prediction of the tumor biology when clinical variables are known. Methods: Using a custom designed sequencing panel targeting the coding regions of 156 EC genes we analyzed 47 EC samples and matching normal controls. Variants were annotated for pathogenicity. Charts were reviewed for clinicopathologic data. Routine statistical analyses were applied. Results: Endometrioid (EEC) tumors had a significantly higher frequency of total and pathogenic SNVs than serous (USC). Pathogenic PTEN and PIK3CA SNVs were more common in EEC while pathogenic TP53 SNVs were more frequent in USC. The presence of pathogenic TP53 SNVs was significantly associated with disease recurrence/progression. A pathogenic SNV at PTEN locus chr10:89692904 was associated with grade 1 EEC. Six hypermutated specimens were identified with 99-272 SNVs (mean 185.8 v 8.7 in non-hypermutated samples). All 6 were grade 3 EEC, of which 4 patients are alive without clinical evidence of disease (mean follow up 35 months). NTRK3, LRP1B, SLIT2, APC, RAD50, PIK3R1, and JAK2 SNVs were more common in grade 3 EEC than the other histologies. Conclusions: Pathogenic variants in EC associated with PTEN, PIK3CA, and TP53 and the number of somatic variants, as identified by a targeted sequencing, can predict histologic features and clinical behavior. Targeted sequencing panels may be a useful tool to predict the clinical phenotype and guide individualized therapeutic intervention. [Table: see text]

LZTS2 and PTEN collaboratively regulate ß-catenin in prostatic tumorigenesis.

The leucine zipper tumor suppressor 2 (LZTS2) was identified as a tumor susceptibility gene within the 10q24.3 chromosomal region, and is approximately 15Mb from the PTEN locus. This region containing the both loci is frequently deleted in a variety of human malignancies, including prostate cancer. LZTS2 is a ß-catenin-binding protein and a negative regulator of Wnt signaling. Overexpression of PTEN in prostate cancer cell lines reduces ß-catenin-mediated transcriptional activity. In this study, we examined the collaborative effect of PTEN and LZTS2 using multiple in vitro and in vivo approaches. Co-expression of PTEN and LZTS2 in prostate cancer cells shows stronger repressive effect on ß-catenin mediated transcription. Using a newly generated mouse model, we further assessed the effect of simultaneous deletion of Pten and Lzts2 in the murine prostate. We observed that mice with both Lzts2 and Pten deletion have an earlier onset of prostate carcinomas as well as an accelerated tumor progression compared to mice with Pten or Lzts2 deletion alone. Immunohistochemical analyses show that atypical and tumor cells from compound mice with both Pten and Lzts2 deletion are mainly composed of prostate luminal epithelial cells and possess higher levels of cytoplasmic and nuclear β-catenin. These cells also exhibit a higher proliferative capacity than cells isolated from single deletion mice. These data demonstrate the significance of simultaneous Pten and Lzts2 deletion in oncogenic transformation in prostate cells and implicates a new mechanism for the dysregulation of Wnt/β-catenin signaling in prostate tumorigenesis.