PTEN Protein Loss Research Articles

Genetic and epigenetic alterations in precursor lesions of endometrial endometrioid carcinoma.

The hyperplasia-carcinoma sequence is a stepwise tumourigenic programme towards endometrial cancer in which normal endometrial epithelium becomes neoplastic through non-atypical endometrial hyperplasia (NAEH) and atypical endometrial hyperplasia (AEH), under the influence of unopposed oestrogen. NAEH and AEH are known to exhibit polyclonal and monoclonal cell growth, respectively; yet, aside from focal PTEN protein loss, the genetic and epigenetic alterations that occur during the cellular transition remain largely unknown. We sought to explore the potential molecular mechanisms that promote the NAEH-AEH transition and identify molecular markers that could help to differentiate between these two states. We conducted target-panel sequencing on the coding exons of 596 genes, including 96 endometrial cancer driver genes, and DNA methylome microarrays for 48 NAEH and 44 AEH lesions that were separately collected via macro- or micro-dissection from the endometrial tissues of 30 cases. Sequencing analyses revealed acquisition of the PTEN mutation and the clonal expansion of tumour cells in AEH samples. Further, across the transition, alterations to the DNA methylome were characterised by hypermethylation of promoter/enhancer regions and CpG islands, as well as hypo- and hyper-methylation of DNA-binding regions for transcription factors relevant to endometrial cell differentiation and/or tumourigenesis, including FOXA2, SOX17, and HAND2. The identified DNA methylation signature distinguishing NAEH and AEH lesions was reproducible in a validation cohort with modest discriminative capability. These findings not only support the concept that the transition from NAEH to AEH is an essential step within neoplastic cell transformation of endometrial epithelium but also provide deep insight into the molecular mechanism of the tumourigenic programme. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract A049: Immunohistochemical detection of ERG, ETV1, ETV4, and PTEN in prostate tumor specimens reveals the heterogeneity of multi-focal prostate cancer

Abstract Introduction: ETS gene rearrangements involving ERG, ETV1 and ETV4, as well as PTEN deletions are among the most common genomic alterations in prostate cancer. Although these events are found to be mutually exclusive, the multifocality of prostate cancer suggests that they may co-occur in some tumors. The immunohistochemistry (IHC) detection of both ERG overexpression and PTEN protein loss are reliable substitutes for fluorescence in situ hybridization (FISH) assay to probe genomic alterations. The lack of specific antibodies that can discriminate between multiple ETS factors, however, has limited the evaluation for the co-occurrence of these genomic alterations in prostate cancer. Improving our ability to identify the multifocality or inter-focal heterogeneity of tumors can enhance our understanding of biological mechanisms of tumor initiation and progression, and help stratify patients for treatment. Methods: Using monoclonal antibodies (mAbs) that we developed against ETV1 and ETV4, we examined their expression in relation to ERG and PTEN expression by IHC using two TMAs constructed from primary prostate cancer specimens of independent patient cohorts: one was constructed from multiple 1 mm cores representing distinct tumor focus from multifocal tumors from 50 African American (AA) and 50 Caucasian American (CA) men; the other, from a single 2 mm core of individual tumors from a cohort of 152 AA and 304 CA men. Positive expression for each ETS factor and PTEN were evaluated for association with race and clinico-pathologic features that include PSA at diagnosis, Gleason score, pathologic T stage, biochemical recurrence, and metastasis. Univariate analysis of BCR free survival as a function of ERG, ETV1 and ETV4 and PTEN expression was performed for both patient cohorts. Results: ERG(+) expression alone or in combination with PTEN loss were significantly higher in CA men in both patient cohorts. When expression of ETS factors were evaluated together with PTEN status, positive expression of ETS factors and PTEN loss were significantly more prevalent among CA men in both patient cohorts ETV1 and ETV4 expression were detected at 5% or less in both patient cohorts. Cases harboring tumor foci that were positive for more than one marker evaluated frequently showed either intra-tumor or inter-tumor heterogeneity. Univariate analysis of PTEN-ETS protein expression status and BCR free survival showed that patients with loss of PTEN expression trends towards poorer BCR free survival regardless of ETS expression status. Conclusion: We showed that the IHC detection of multiple aberrantly expressed proteins such as ERG, ETV1, ETV4 and PTEN in prostate tumor specimens using highly specific mAbs can reveal the heterogeneity and multi-focality in prostate cancer, which could be used to enhance the stratification of patients for novel treatment strategies. Citation Format: Shyh-Han Tan, Cara Schafer, Denise Young, Jiji Jiang, Yingjie Song, Albert Dobi, Gyorgy Petrovics, Bettina F. Drake, Gregory T. Chesnut, Isabell A. Sesterhenn. Immunohistochemical detection of ERG, ETV1, ETV4, and PTEN in prostate tumor specimens reveals the heterogeneity of multi-focal prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A049.

Multiplatform Analysis of Intratumoral PTEN Heterogeneity in Melanoma

Loss of protein expression of the tumor suppressor PTEN is associated with increased cancer aggressiveness, decreased tumor immune infiltration, and resistance to immune and targeted therapies in melanoma. We assessed a unique cohort of eight melanoma samples with focal loss of PTEN protein expression to understand the features and mechanisms of PTEN loss in this disease. We compared the PTEN-negative (PTEN[-]) areas to their adjacent PTEN-positive (PTEN[+]) areas using DNA sequencing, DNA methylation, RNA expression, digital spatial profiling, and immunohistochemical platforms. Variations or homozygous deletions of PTEN were identified in PTEN(-) areas that were not detected in the adjacent PTEN(+) areas in three cases (37.5%), but no clear genomic or DNA methylation basis for loss was identified in the remaining PTEN(-) samples. RNA expression data from two independent platforms identified a consistent increase in chromosome segregation gene expression in PTEN(-) versus adjacent PTEN(+) areas. Proteomic analysis showed a relative paucity of tumor-infiltrating lymphocytes in PTEN(-) versus adjacent PTEN(+) areas. The findings add to our understanding of potential molecular intratumoral heterogeneity in melanoma and the features associated with the loss of PTEN protein in this disease.

BAT26 Only Microsatellite Instability with High Tumor Mutation Burden-A Rare Entity Associated with PTEN Protein Loss and High PD-L1 Expression.

Detecting microsatellite instability (MSI) in advanced cancers is crucial for clinical decision-making, as it helps in identifying patients with differential treatment responses and prognoses. BAT26 is a highly sensitive MSI marker that defines the mismatch repair (MMR) status with high sensitivity and specificity. However, isolated BAT26-only instability is rare and has not been previously reported. Of the 6476 cases tested using pentaplex MSI polymerase chain reaction, we identified two BAT26-only instability cases (0.03%) in this study. The case #1 patient was diagnosed with endometrial adenocarcinoma without MMR germline mutations. The endometrial tumor showed BAT26-only instability, partial loss of MLH1/PMS2 protein expression, and a high programmed cell death ligand 1 (PD-L1) combined positive score (CPS = 8). The tumor exhibited a somatic phosphatase and tensin homolog (PTEN) R303P missense mutation and loss of the PTEN protein. On a comprehensive cancer panel sequencing with ≥500 genes, the tumor showed an MSI score of 11.38% and high tumor mutation burden (TMB) (19.5 mt/mb). The case #2 patient was diagnosed with colorectal carcinoma with proficient MMR and PTEN protein loss without PTEN alteration, as well as a high PD-L1 CPS (CPS = 10). A pathogenic KRAS A146T mutation was detected with an MSI score of 3.36% and high TMB (13 mt/mb). In conclusion, BAT26-only instability is very rare and associated with PTEN protein loss, high TMB, and a high PD-L1 score. Our results suggest that patients with BAT26-only instability may show good responses to immunotherapy.

Abstract 2220: Immunohistochemical detection of prostate cancer heterogeneity by using ETS and PTEN monoclonal antibodies

Abstract The multifocality and highly variable molecular pathology of prostate cancer are underlying features of this clinically heterogeneous disease. Most patients harbor multiple molecularly distinct cancer foci at diagnosis that likely arose as independent clones. There is a critical need for molecular biomarkers that can distinguish the multifocality and inter-focal heterogeneity of tumors and help stratify patients for treatment. PTEN loss and ETS gene rearrangements are among the most prevalent genomic alterations in prostate carcinogenesis. The detection of both ERG overexpression and PTEN protein loss by immunohistochemistry (IHC) have proven to be reliable substitutes for detecting genomic alterations by fluorescence in situ hybridization (FISH) assay. We developed monoclonal antibodies against ETV1 and ETV4 and evaluated their performance in prostate cancer specimens. The expression of ETV1, ETV4, were examined in relation to ERG and loss of PTEN expression in TMAs constructed from primary prostate cancer specimens of two independent patient cohorts. The first TMA was constructed from multiple 1 mm cores representing distinct tumor focus from multifocal tumors from 50 African American (AA) and 50 Caucasian American (CA) men. The second was constructed from a single 2 mm core of individual tumors from an independent cohort of 152 AA and 304 CA men. We present results on the expression of each protein biomarker in the context of patient race and their association with clinico-pathologic features, together with concurrence or mutual exclusiveness for each event. These results support the application of ETS monoclonal antibodies in IHC assays to detect prostate cancer tumor heterogeneity and to identify subsets of prostate cancer. Citation Format: Cara Schafer, Denise Young, Yingjie Song, Jiji Jiang, Albert Dobi, Gyorgy Petrovics, Bettina F. Drake, Gregory T. Chesnut, Isabell A. Sesterhenn, Shyh-Han Tan. Immunohistochemical detection of prostate cancer heterogeneity by using ETS and PTEN monoclonal antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2220.

PTEN loss confers sensitivity to rapalogs in clear cell renal cell carcinoma.

Rapalogs (everolimus and temsirolimus) are allosteric mTORC1 inhibitors and approved agents for advanced clear cell renal cell carcinoma (ccRCC), although only a subset of patients derive clinical benefit. Progress in genomic characterization has made it possible to generate comprehensive profiles of genetic alterations in ccRCC; however, the correlations between recurrent somatic mutations and rapalog efficacy remain unclear. Here, we demonstrate by using multiple patient-derived ccRCC cell lines that compared to PTEN-proficient cells, PTEN-deficient cells exhibit hypersensitivity to rapalogs. Rapalogs inhibit cell proliferation by inducing G0/G1 arrest without inducing apoptosis in PTEN-deficient ccRCC cell lines. Using isogenic cell lines generated by CRISPR/Cas9, we validate the correlation between PTEN loss and rapalog hypersensitivity. In contrast, deletion of VHL or chromatin-modifying genes (PBRM1, SETD2, BAP1, or KDM5C) fails to influence the cellular response to rapalogs. Our mechanistic study shows that ectopic expression of an activating mTOR mutant (C1483F) antagonizes PTEN-induced cell growth inhibition, while introduction of a resistant mTOR mutant (A2034V) enables PTEN-deficient ccRCC cells to escape the growth inhibitory effect of rapalogs, suggesting that PTEN loss generates vulnerability to mTOR inhibition. PTEN-deficient ccRCC cells are more sensitive to the inhibitory effects of temsirolimus on cell migration and tumor growth in zebrafish and xenograft mice, respectively. Of note, PTEN protein loss as detected by immunohistochemistry is much more frequent than mutations in the PTEN gene in ccRCC patients. Our study suggests that PTEN loss correlates with rapalog sensitivity and could be used as a marker for ccRCC patient selection for rapalog therapy.

Abstract PO-146: Copy number landscape of primary African-American and European-American prostate tumors

Abstract African-American (AA) men are more likely to be diagnosed with and to die of prostate cancer than their European-American counterparts. Much of this disparity is due to access to care and social determinants of health; however, there is emerging evidence of molecular differences in prostate tumors arising in patients of different genetic ancestry. We investigated the copy number landscape of 290 grade and race-matched primary prostate tumors through interrogation of Infinium MethylationEPIC Array (Illumina) data derived from primary prostate tumors at radical prostatectomy. Our cohort was composed of 145 self-identified AA patients and 145 self-identified EA surgically-treated patients with accompanying genetic ancestry estimation via SNP array (GSAv3). EPIC array-derived copy number data estimated through the Conumee package on R was significantly correlated with genomic losses and gains identified by exome sequencing of a panel with 100 cancer driver genes. We also observed a significant association between genomic losses of the PTEN gene and PTEN protein loss by immunohistochemistry (P<0.001). Similar observations were made for p53 protein nuclear accumulation detected by immunohistochemistry and TP53 gene copy loss (P<0.001). Next, we derived percent genome altered (PGA) from EPIC array data for our 290 patients. PGA from EPIC array was significantly correlated with PGA obtained from targeted sequencing of a subset of AA tumors (n=119, P<0.0001, R²=0.50). No difference in PGA was found between self-identified AA and EA patients (P<0.05), though PGA levels were significantly associated with Gleason Grade groups for both AA (P<0.0001) and EA (P=0.003). We then used the genetic ancestry estimation derived from SNP arrays to identify regions of genomic loss and gain differing by percent African ancestry. We used a comprehensive genome annotation with >80k coding and non-coding regions from FANTOM CAT. Adjusted LIMMA models with age, Gleason Grade Group, and pre-operative PSA levels showed significant copy number differences by percent African ancestry in chromosomes 6p, 10q, 11p, 12p, and 17p. PTEN losses (as expected) and WT1 gains were more frequent in patients with lower percent African ancestry. Gene expression data from TCGA data showed that WT1 expression was significantly increased for self-identified EA patients. Multivariable Cox regression models adjusted for age, Gleason Grade Group, and pre-operative PSA levels revealed that chromosome 8q gains (including MYC, GRHL2, and FZD6) were significantly associated with biochemical recurrence and metastasis. However, when we stratified the models by self-identified race, only AA tumors showed significant associations with 8q gains and poor outcome. Further in silico and mechanistic validation will be conducted to confirm whether chromosome 8 gains may be a potential biomarker for prostate cancer outcome in AA men. Citation Format: Thiago Vidotto, Eddie Imada, Farzana Faizal, Siqun Zheng, Jianfeng Xu, Karen S. Sfanos, Luigi Marchionni, Tamara L. Lotan. Copy number landscape of primary African-American and European-American prostate tumors [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-146.

LncRNA RP11-295G20.2 regulates hepatocellular carcinoma cell growth and autophagy by targeting PTEN to lysosomal degradation

PTEN is a crucial tumor suppressor and loss of PTEN protein is involved in various cancers. However, the detailed molecular mechanisms of PTEN loss in cancers remain elusive, especially the involvement of lncRNAs. Here, lncRNA RP11-295G20.2 is found to be significantly upregulated in hepatocellular carcinoma (HCC) and promotes the growth of liver cancer cells both in vitro and in vivo. Furthermore, RP11-295G20.2 inhibits autophagy in liver cancer cells. Interestingly, RP11-295G20.2 directly binds to the PTEN protein and leads to its degradation. RP11-295G20.2 expression is inversely correlated with PTEN protein expression in 82 TCGA/TCPA-LIHC samples. Surprisingly, RP11-295G20.2-induced PTEN degradation occurs through the lysosomal pathway instead of the proteasome pathway. RP11-295G20.2 binds to the N terminus of PTEN and facilitates the interaction of p62 with PTEN. Thus, PTEN is translocated into lysosomes and degraded. RP11-295G20.2 also influences AKT phosphorylation and forkhead box O 3a (FOXO3a) translocation into the nucleus, in turn regulating the transcription of autophagy-related genes. Collectively, RP11-295G20.2 directly binds to PTEN and enables its lysosomal degradation. This newly identified RP11-295G20.2/PTEN axis reveals an unexplored molecular mechanism regarding PTEN loss in liver cancer and might provide new therapeutic benefits for liver cancer patients.

SPOP and CHD1 alterations in prostate cancer: Relationship with PTEN loss, tumor grade, perineural infiltration, and PSA recurrence.

In the non-ETS fusion of prostate cancer (PCa) pathway, SPOP mutations emerge as a distinct oncogenic driver subclass. Both SPOP downregulation and mutation can lead to SPOP target stabilization promoting dysregulation of key regulatory pathways. CHD1 gene is commonly deleted in PCa. CHD1 loss significantly co-occurs with SPOP mutations, resulting in a PCa subclass with increased AR transcriptional activity and with a specific epigenetic pattern. In this study, SPOP alterations at mutational and protein levels and CHD1 copy number alterations have been analyzed and correlated with ERG and PTEN protein expression and with the clinical pathological features of the patients. SPOP protein loss has been detected in 42.9% of the cases, and it has been strongly associated with PTEN protein loss (p < .001). CHD1 gene loss has been detected in 24.5% and SPOP mutations in 5.9% of the cases. Loss of CHD1 has been strongly associated with SPOP mutations (p = .003) and has shown a trend to be associated with ERG wt cancers (p = .08). The loss of SPOP protein (p = .01) and the combination of PTEN and SPOP protein loss (p = .002) were both statistically more common in grade group 5 cancers, with a prevalence of 60% and 37.5%, respectively. Furthermore, SPOP loss/PTEN loss and SPOP wt/PTEN loss phenotypes were strongly associated with extraprostatic perineural infiltration (p = .007). Strong CHD1 loss was associated with a shorter time to PSA recurrence in the univariate (p = .04), and showed a trend to be associated with the PSA recurrence risk in the multivariate analysis (p = .058). The results of the present study suggest that the loss of SPOP protein expression, either alone or in combination with loss of PTEN and, on the other hand, a marked loss of the CHD1 gene are very promising prognostic biomarkers in PCa.

530P Phase II study of PI3K inhibitor copanlisib in cancer patients with deleterious PTEN mutations and retained PTEN protein expression: Results from the NCI-MATCH Trial (EAY131) sub-protocol Z1H

The NCI-MATCH trial, the largest national study (1173 sites) for patients with relapsed/refractory solid tumors, lymphomas and myeloma, assigns targeted therapies based on individual tumor molecular alterations detected using clinical targeted next-generation sequencing and immunohistochemistry. It was hypothesized that patients with deleterious PTEN mutations without PTEN protein loss may benefit from treatment with the pan-PI3K inhibitor copanlisib.

Any Role of PIK3CA and PTEN Biomarkers in the Prognosis in Oral Squamous Cell Carcinoma?

Oral squamous cell carcinoma (OSCC) accounts for 95% of the lesions in the oral cavity. Despite development in OSCC management, the outcome is still unsatisfactory. Identification of new therapies in OSCC is urgently needed. One objective of such treatment may be a signaling pathway of phosphatidylinositol 3-kinase. The study group included 92 patients treated for OSCC at the University Clinical Centre in Gdańsk, Poland. Study was performed on formalin-fixed paraffin-embedded samples from primary OSCC. Phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA) and phosphatase and tensin homolog encoded on chromosome 10 (PTEN) protein expression was assessed by immunohistochemistry (IHC). PIK3CA gene copy number was analyzed using chromogenic and silver in situ hybridization where molecular probes are marked by chromogens and silver ions. PIK3CA IHC H-score ≥ 70 was found in 51.65% patients, and loss of PTEN protein was noticed in 31.46% cases. PIK3CA amplification was detected in 5 tumors. In the case of PTEN protein expression, there was an inverse correlation with the T stage of the primary tumor (r = −0.243) and positive correlation with a 5-year survival (r = 0.235). The number of copies of the PIK3CA gene was associated with the tumor grading (r = 0.208). The present study shows that loss of PTEN protein and the grading (p = 0.040), distant metastases (p = 0.033), smoking (p = 0.016), and alcohol abuse (p = 0.042) were prognostic factors for the survival of patients with OSCC. In contrast, the presence of amplification and OSCC on the floor of the mouth resulted in a nearly six-fold increase in the risk of shortening survival (p = 0.037). Our finding suggests a potential prognostic significance of PTEN loss and PIK3CA amplification in OSCC. Future studies are needed to confirm our results.

Upregulation of DJ-1 expression in melanoma regulates PTEN/AKT pathway for cell survival and migration.

Cutaneous melanoma is known to be one of the most dangerous skin cancers because of its metastatic functions. Today, it is essential to investigate specific biomarkers for the target treatment in many diseases including cancers. DJ-1 protein, also known as Parkinson disease 7, has various functions associated with cancer progression including cell survival and migration. Phosphatase and tensin homolog (PTEN) is a tumor suppressor that regulates the PI3K/AKT signaling pathway and its mutations have been reported to frequently occur in many cancers such as thyroid, breast and skin. Recently, DJ-1 has been identified as a negative regulator of PTEN in many human cancer cells. However, the impacts and relationship of DJ-1 and PTEN have not been studied yet in melanoma. To confirm the expression of DJ-1 and PTEN in melanoma compared to normal skin tissues and find out functions of DJ-1 in melanoma cells, Western blot analysis and immunohistochemical staining were used. Transfection of G361 cells with DJ-1-specific small interfering RNA was performed to figure out the roles of DJ-1 and the relationship between DJ-1 and PTEN in melanoma cells. In our study, the DJ-1 protein was significantly increased with loss of PTEN protein in melanoma compared to that in normal skin. Inhibition of DJ-1 in G361 cells induced apoptosis, and suppressed cell survival and migration. Furthermore, suppression of DJ-1 in G361 cells increased the expression of cleaved caspase-3, cleaved PARP, Bax, p53, and Daxx as well as PTEN, while it decreased expression of survivin, caspase-3, and PARP. Also, downregulated DJ-1 inhibited phosphorylation of AKT in G361 cells. Collectively, DJ-1 overexpression could affect the proliferative and invasive capabilities of melanoma cells via regulating the PTEN/AKT pathway and apoptosis-related proteins. This study suggests that DJ-1 may be a potential target for the treatment of melanoma.

Prevalence of APC and PTEN Alterations in Urachal Cancer

Urachal carcinoma (UrC) is a rare tumor with remarkable histological and molecular similarities to colorectal cancer (CRC). Adenomatous polyposis coli (APC) is the most frequently affected gene in CRC, but the prevalence and significance of its alterations in UrC is poorly understood. In addition, loss of phosphatase and tensin homologue (PTEN) was shown to be associated with therapy resistance in CRC. Our primary aim was to assess specific genetic alterations including APC and PTEN in a large series of UrC samples in order to identify clinically significant genomic alterations. We analyzed a total of 40 UrC cases. Targeted 5-gene (APC, PTEN, DICER1, PRKAR1A, TSHR, WRN) panel sequencing was performed on the Illumina MiSeq platform (n = 34). In addition, ß-catenin (n = 38) and PTEN (n = 30) expressions were assessed by immunohistochemistry. APC and PTEN genes were affected in 15% (5/34) and 6% (2/34) of cases. Two of five APC alterations (p.Y1075*, p.K1199*) were truncating pathogenic mutations. One of the two PTEN variants was a pathogenic frameshift insertion (p.C211fs). In 29% (11/38) of samples, at least some weak nuclear ß-catenin immunostaining was detected and PTEN loss was observed in 20% (6/30) of samples. The low prevalence of APC mutations in UrC represents a characteristic difference to CRC. Based on APC and ß-catenin results, the Wnt pathway seems to be rarely affected in UrC. Considering the formerly described involvement of PTEN protein loss in anti-EGFR therapy-resistance its immunohistochemical testing may have therapeutic relevance.

PTEN Protein Loss and Loss-of-Function Mutations in Gastric Cancers: The Relationship with Microsatellite Instability, EBV, HER2, and PD-L1 Expression.

Inactivation of phosphatase and tensin homolog (PTEN) is caused by multiple mechanisms, and loss of PTEN activity is related to the progression of various cancers. In gastric cancer (GC), the relationship between the loss of PTEN protein expression and various genetic alterations remains unclear. The effects of microsatellite instability (MSI), Epstein–Barr virus (EBV), HER2 overexpression, and PD-L1 expression on PTEN mutation have not been fully explored. We performed comprehensive cancer panel tests with a cohort of 322 tumor samples from patients with advanced GC. Immunohistochemistry for PTEN protein was performed in all cases, and the loss of protein expression was defined as a complete absence of nuclear staining. In total, 34 cases (10.6%) had pathogenic PTEN mutations, of which 19 (55.9%) showed PTEN protein loss. The most common PTEN variants associated with protein loss were p.R130 (n = 4) followed by p.R335, p.L265fs, and deletions (n = 2). All the ten nonsense mutations identified in the samples resulted in PTEN inactivation. In the remaining 288 GC cases with wild-type PTEN, protein loss was found in 35 cases (12.2%). Thus, PTEN mutations were significantly associated with PTEN protein loss (p = 5.232 × 10−10), high MSI (p = 3.936 × 10−8), and EBV-positivity (p = 0.0071). In conclusion, our results demonstrate that loss-of-function mutations in PTEN are a frequent genetic mechanism of PTEN inactivation in GC.

Association between BRCA2 status and histologic variants (intraductal [IDC] and cribriform [CRIB] histology) in prostate cancer (PC).

5579 Background: IDC histology in PC has been suggested to associate with germline BRCA2 mutations (gBRCA2) in small series, leading to the potential recommendation of genetic testing for all PC patients with IDC in the primary tumor. Methods: We conducted a case-control study in which primary PC from 58 germline BRCA2 mutation carriers ( gBRCA2) and 116 from non-carriers (NC) were matched 1:2 by Gleason score and specimen type (core biopsy/prostatectomy). Samples were independently reviewed by two expert pathologists blinded to genetic status who established the presence of IDC and/or CRIB morphology with supportive immunohistochemical stains in a subset of cases. Next-generation sequencing, aCGH and/or FISH were used to assess for somatic mono-/bi-allelic BRCA2 alterations. PTEN protein loss was determined by IHC, and TMPRSS2-ERG was detected by FISH/qRT-PCR. Chi-square tests were used to compare the frequency of IDC and cribriform histology in gBRCA2 vs NC, as well as to assess the associations with other variables. Logistic regression models were built to identify independent factors associated with IDC and CRIB histology. Results: gBRCA2 cases were younger at diagnosis (median 61.3 vs 64, p &lt; 0.01) and had T3-4 disease more often than NC cases (31% vs 10.5%, p &lt; 0.01), but the two groups did not differ in any other clinical-pathologic characteristics. After independent histopathological review, 79 cases demonstrated IDC histology and 81 had CRIB histology. No differences in the prevalence of IDC (50% NC vs 36% gBRCA2, p = 0.09) or CRIB (43% NC vs 53% gBRCA2, p = 0.20) patterns were observed. The probability of IDC was higher in PC with bi-allelic BRCA2 alterations (OR 5.1, 95%CI 2.1-12.6), PTEN loss (OR 5.1, 95%CI 1.9-13.5) or both (OR 23.0, 95%CI 4.9-107.2) compared to those without these alterations. Bi-allelic BRCA2 alteration was also associated with higher probability of CRIB histology (OR 7.2, 95%CI 3.1-16.4). TMPRSS2-ERG fusions were not associated with IDC or CRIB histology. MVA confirmed the independent association of bi-allelic BRCA2 alteration (p &lt; 0.01) and PTEN loss (p &lt; 0.01) with IDC histology. Bi-allelic BRCA2 alteration (p &lt; 0.01) and Gleason &gt;8 (p &lt; 0.01) were independent risk factors for CRIB histology. Conclusions: Primary PC with bi-allelic BRCA2 alterations was significantly associated with IDC and CRIB histology, independent of other clinical-pathologic factors (while gBRCA2 status alone was not). PTEN loss in primary PC was also independently associated with IDC, but not CRIB, histology.

Abstract P1-09-08: Development and characterization of PTEN IHC assay for testing breast cancer patients specimens

Abstract Background: Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene that is a major negative regulator of the Phosphatidylinositol 3-kinase (PI3K) pathway. Loss of PTEN protein expression has been mechanistically linked to tumor progression. Blocking the PI3K pathway might inhibit the growth and proliferation of cells that have deletions in PTEN. Thus characterization of PTEN expression in patient tumor samples may assist prediction of potential response to PI3K inhibitor therapies. Methods: We developed and validated an immunohistochemical assay on Ventana BenchMark XT to detect PTEN in formalin fixed and paraffin-embedded (FFPE) tissue by utilizing a rabbit monoclonal antibody (clone 138G6) from Cell Signaling Technologies that recognizes the carboxy-terminal domain of PTEN. PTEN immunohistochemical staining was performed on 1577 breast tumor specimens to determine PTEN protein loss. A subset of these cases (n=663) was also assessed for PTEN mutation. Results: Cellular localization of PTEN expression was observed in both the cytoplasm and the nucleus. Both cellular compartments were scored and used in the staining intensity determination. We observed that PTEN staining is sensitive to variation in tissue handling, fixation and antigen retrieval. PTEN staining was affected significantly by antigen retrieval method. Optimal staining conditions were determined to be 1:60 antibody dilution using Citrate pH 6.0 as antigen retrieval buffer. In a cohort of 1577 hormone receptor positive HER2-negative locally advanced or metastatic breast cancer patients, loss of PTEN protein was observed in 8.6% (135/1577) of patients. There was a positive correlation between PTEN mutation rate (17 out of 66 PTEN IHC positive cases vs 14 out of 587 negative cases) and loss of PTEN protein expression. PTEN loss was observed to correlate with better clinical response to PI3K inhibitor. Conclusions: Pre-analytical handling of samples is important for PTEN IHC staining. PTEN mutations and insertions/deletions contribute to PTEN protein loss. This study validates a simple method to interrogate PTEN status in clinical specimens and supports the utility of this test in selecting patients who are likely to respond to PI3K inhibitor treatment. Citation Format: Hua Gong, Emmanuel Pacia, Sharmila Manjeshwar, Beiru Chen, Xun Li, Bashar Dabbas, Jelveh Lameh, Naveen Babbar, Shabnam Tangri. Development and characterization of PTEN IHC assay for testing breast cancer patients specimens [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-09-08.

PTEN in Lung Cancer: Dealing with the Problem, Building on New Knowledge and Turning the Game Around.

Lung cancer is the most common malignancy and cause of cancer deaths worldwide, owing to the dismal prognosis for most affected patients. Phosphatase and tensin homolog deleted in chromosome 10 (PTEN) acts as a powerful tumor suppressor gene and even partial reduction of its levels increases cancer susceptibility. While the most validated anti-oncogenic duty of PTEN is the negative regulation of the PI3K/mTOR/Akt oncogenic signaling pathway, further tumor suppressor functions, such as chromosomal integrity and DNA repair have been reported. PTEN protein loss is a frequent event in lung cancer, but genetic alterations are not equally detected. It has been demonstrated that its expression is regulated at multiple genetic and epigenetic levels and deeper delineation of these mechanisms might provide fertile ground for upgrading lung cancer therapeutics. Today, PTEN expression is usually determined by immunohistochemistry and low protein levels have been associated with decreased survival in lung cancer. Moreover, available data involve PTEN mutations and loss of activity with resistance to targeted treatments and immunotherapy. This review discusses the current knowledge about PTEN status in lung cancer, highlighting the prevalence of its alterations in the disease, the regulatory mechanisms and the implications of PTEN on available treatment options.

Bridging genomics and phenomics of gastric carcinoma.

Genetic alterations are the starting point leading to numerous changes in clinical and pathologic features (phenotypes) of individual cancers; however, their inter-relationships in gastric cancers (GC) are unclear. We performed massive parallel sequencing of 381 cancer-related genes and compared the results with clinical and pathologic findings in 330 GC. High tumor mutation burden (TMB) accounted for 11% of GC (n = 37) and all 19 MSI-H GCs were high TMB. High TMB was significantly more frequent in intestinal-type by Lauren, tumor with higher host cellular immune response, earlier AJCC stage and favorable prognosis. The most significantly mutated genes were TP53 (54%), ARID1A (23%), CDH1 (22%), PIK3CA (12%), RNF43 (10%) and KRAS (9%). For receptor tyrosine kinases, amplifications detected by immunohistochemistry were higher than sequencing (HER2, 9.1% vs. 5.8%; EGFR, 11.2% vs. 6.1%; FGFR2, 4.6% vs. 3.9%, c-MET, 3.4% vs. 0.9%). PTEN protein loss (22%) correlated well with underlying PTEN alterations while ATM loss (27%) was not significantly correlated with genetic alterations of ATM. p53 protein expression predicted alterations of TP53 with high sensitivity (97.8%) and low (15.9%) specificity. The poorly cohesive histology/CDH1-mutant GC subgroup showed the worst survival (p < 0.001). PD-L1 expression was significantly associated with MSI-H, MLH1 loss, ATM loss, MET positivity, higher host immune response, and genetic alterations of ARID1A, BRD3, PIK3CA, KRAS, MAP3K13, CDH2, PTEN and ESR1. The merged clinical, pathology and genomics of GC provide a better understanding of GC and new insights into the treatment of GC.

PTEN loss in Gleason grade 7 prostate tumors exhibits intratumoral heterogeneity and is associated with unfavorable pathological features

BackgroundPTEN loss is observed in 20–30% of prostate cancers and is associated with a poor outcome, but clinical details of the impact of this biomarker are unclear for intermediate grade tumors.MethodsWe investigated 43 radical prostatectomy-derived grade 7 prostate tumors from the Clinics Hospital of Ribeirão Preto. Tissue microarray (TMA) blocks were constructed and PTEN copy number status was determined for all patients through fluorescence in situ hybridization (FISH). To determine the presence of PTEN protein loss in our study cohort, we performed immunohistochemistry (IHC) in TMA sections. We then developed an automated algorithm in HALO™ to identify regions of PTEN protein loss in whole prostate scanned sections from ten patients with known PTEN deletion status by FISH. Clinical analyses were conducted to determine the associations between PTEN loss and patient outcome. All statistical analyses were conducted in R v3.4.3 with P-values below 0.05 being considered statistically significant.ResultsIn this study of 43 grade 7 tumors, we found PTEN deletions by FISH in 18.9% of tumors, and PTEN protein loss by IHC in 16.3% of tumors. Both techniques were highly concordant and complementary. Clinical analysis demonstrated that PTEN deletion by FISH was significantly associated with positive margin invasion (P = 0.04) and Gleason score upgrade (P = 0.001). Digital image analysis of ten representative tumors demonstrated distinct intratumoral heterogeneity for PTEN protein loss in four tumors.ConclusionsThis study shows that PTEN loss in Gleason grade 7 tumors can be heterogeneous and that a systematic analysis of this biomarker using a combination of FISH, IHC, and digital imaging may identify patients with a greater risk of poor outcome.

Effects of the tumor suppressor PTEN on biological behaviors of activated pancreatic stellate cells in pancreatic fibrosis

Pancreatic stellate cells (PSCs), when activated, are characterized by proliferation and collagen synthesis, and contribute to extracellular matrix deposition in pancreatic fibrosis. Concomitantly, fibrosis is linked with the loss of PTEN (phosphatase and tensin homolog) protein in several organs. This study investigated the association between PTEN protein levels and the activated or apoptotic status of PSCs in a rat model of chronic pancreatitis. In addition, the activation status and biological behaviors of culture-activated PSCs were analyzed after lentiviral transfection with wildtype or mutant (G129E) PTEN for upregulation, or PTEN short hairpin RNA for downregulation, of PTEN. In vivo, PTEN levels gradually decreased during pancreatic fibrosis, which positively correlated with apoptosis of activated PSCs, but negatively with PSC activation. In vitro, activated PSCs with wildtype PTEN showed less proliferation, migration, and collagen synthesis compared with control PSCs, and greater numbers were apoptotic; activated PSCs with mutant PTEN showed similar, but weaker, effects. Furthermore, AKT and FAK/ERK signaling was involved in this process. In summary, activated PSCs during pancreatic fibrosis in vivo have lower levels of PTEN. In vitro, PTEN appears to prevent PSCs from further activation and promotes apoptosis through regulation of the AKT and FAK/ERK pathways.