Homozygous Deletion Of PTEN Research Articles

Abstract 2963: A novel uterine leiomyosarcoma mouse model

Abstract leiomyosarcoma is an extremely rare but clinically aggressive uterine cancer arising from the smooth muscle of the uterus. Each year, it is estimated to occur in 6 out of every 1,000,000 women in the United States. The average age at diagnosis is 51 and women with uterine leiomyosarcomas have a poor prognosis. Current therapies for uterine leiomyosarcoma are not very effective and the primary treatment of uterine leiomyosarcoma is surgery. Due to the intrinsic rarity of this disease, prospective randomized clinical trials examining the outcome in individuals with uterine leiomyosarcoma is limited. Moreover, a poor understanding of the pathogenesis of this disease is also a major knowledge gap to develop effective treatment strategies. Thus, a preclinical uterine leiomyosarcoma models that recapitulate the human disease is desperately needed to support clinical trial readiness. Since both p53 mutations and Pten homozygous deletion have been observed in 61% and 64% human uterine leiomyosarcomas respectively [3], we are generating genetic mouse models with Amhr2-Cre Ptenfl/fl p53R245Wfl/fl (homozygous p53 mutation), Amhr2-Cre Ptenfl/fl p53R245Wfl/+ (heterozgyous p53 mutation) and Amhr2-Cre Ptenfl/fl (control) genotypes. Our unpublished preliminary data showed that mouse with homozygous p53 mutation and homozygous deletion of Pten developed metastatic uterine leiomyosarcoma at 24 weeks of age with 100% penetrance. However, mouse with heterozygous p53 mutation and homozygous deletion of Pten did not develop uterine leiomyosarcoma at the age of 24 weeks. From the public available human uterine leiomyosarcoma RNAseq data, 80% of those tumors with p53 mutations had homozygous mutations. Previous mouse model with homozygous deletion of p53 gene and/or BRCA1 with Amhr2-Cre only had uterine leiomyosarcoma developed in approximately 50% of mice around 13 months without metastasis. RNAseq and RPPA functional data of the uterine leiomyosarcomas from our novel mouse models are being generated for comparison with known genomic profiles of human uterine leiomyosarcomas. The RNAseq and RPPA functional proteomic data from comparing uterine tissues from homozygous and heterozygous p53 mutated mice provide insight in the understanding the development of uterine leiomyosarcoma and for future clinical trials. Citation Format: Chun Wai Ng, Kwong-Kwok Wong. A novel uterine leiomyosarcoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2963.

PATH-34. GENETIC PROFILING OF AGGRESSIVE MENINGIOMAS REVEAL DIVERSE SPECTRUM OF ACCOMPANYING ALTERATIONS BEYOND NF2 INACTIVATION

Abstract BACKGROUND Meningiomas are the most common primary central nervous system tumor in adults. The majority are clinically indolent and WHO grade I. However, 20-30% are WHO grade II and 1-3% are WHO grade III, which have shorter progression-free (PFS) and overall survival. A subset of grade I meningiomas also follow an aggressive course, requiring serial resection and/or radiotherapy, similar to high-grade meningiomas. The objective of this study was to characterize the genetic alterations in meningiomas with an aggressive clinical course. METHODS Targeted next-generation sequencing (NGS) of 40 aggressive meningiomas was performed using the UCSF500 cancer panel, which assesses ~500 cancer-related genes. Information on patient demographics, tumor histopathology, and treatment history was also collected. RESULTS Meningiomas analyzed included 15 (38%) WHO grade I, 11 (28%) WHO grade II, and 13 (33%) WHO grade III. At the time of genetic profiling, 71% of patients had received prior treatment (68% surgery, 48% fractionated radiotherapy, 23% radiosurgery). The most commonly altered gene was NF2, with 71% of tumors demonstrating biallelic inactivation. Other common alterations included biallelic CDKN2A/B deletion (15%) and TERT amplification or promoter mutation (13%). Other recurrent alterations involved SUFU (8%), and ARID1A, ATM, BAP1, DMD, KDM6A, and PTEN (each 5%). Genes which are commonly altered in indolent WHO grade I meningiomas, such as AKT1, KLF4, PIK3CA, SMO, and TRAF7, were intact in this cohort. CONCLUSIONS The additional genetic alterations beyond NF2 inactivation that drive aggressive meningiomas are diverse and include homozygous deletion of CDKN2A (negative regulator of cyclin-dependent kinases 4/6), inactivating mutations in SUFU (a negative regulator of Hedgehog signaling), homozygous deletion of PTEN (a negative regulator of mTOR signaling), and mutations/deletions in epigenetic regulatory factors (e.g. ARID1A, KDM6A). Clinical trials are needed to assess the efficacy of therapeutics targeting these specific pathways in patients with meningiomas refractory to conventional treatments.

Locally invasive, castrate-resistant prostate cancer in a Pten/Trp53 double knockout mouse model of prostate cancer monitored with non-invasive bioluminescent imaging.

Here we have improved an existing mouse model of prostate cancer based on prostate-specific deletion of Pten and Trp53 by incorporating a Cre-activatable luciferase reporter. By coupling the deletion of those genes to the activation of a luciferase reporter, we were able to monitor tumor burden non-invasively over time. We show that, consistent with previous reports, deletion of both Pten and Trp53 on a C57BL/6 background accelerates tumor growth and results in both the loss of androgen receptor expression and castrate resistant tumors as compared with loss of Pten alone. Loss of Trp53 results in the development of sarcomatoid histology and the expression of markers of epithelial-to-mesenchymal transition Zeb1 and vimentin, with kinetics and penetrance dependent on whether one or both alleles of Trp53 were deleted. Homozygous deletion of Trp53 and Pten resulted in uniformly lethal disease by 25 weeks. While we were able to detect locally invasive disease in the peritoneal cavity in aggressive tumors from the double knockout mice, we were unable to detect lymphatic or hematogenous metastatic disease in lymph nodes or at distant sites.

Abstract P1-19-14: Results from plasmaMATCH trial treatment cohort D: A phase II trial of capivasertib in patients with an AKT activation basket mutation identified via ctDNA testing or tumour sequencing (CRUK/15/010)

Abstract Background: Activation of the AKT pathway can result from diverse rare genetic events, including mutation of AKT1, AKT2/3 E17K, and through inactivating mutation or homozygous deletion of PTEN. AKT1 mutations and genetic loss of PTEN are associated with activation of AKT signalling and selective sensitivity to AKT inhibition in pre-clinical models. Capivasertib is a potent catalytic inhibitor of AKT1, AKT2 and AKT3 with activity both in vivo and in the clinic. The plasmaMATCH trial assessed the efficacy of capivasertib in BC patients with rare AKT activating mutations. Methods: The plasmaMATCH trial is an open-label, multi-centre, multi-cohort platform trial, consisting of ctDNA testing in ~1000 patients with advanced BC. Patients with AKT1 mutations in ER negative BC; or AKT2/3 E17K mutations, PIK3R1 or PTEN inactivating mutations orhomozygous deletion of PTEN in both ER positive and ER negative BC were recruited. Mutations were identified in ctDNA testing in plasmaMATCH or in prior tumour sequencing. Patients were treated with capivasertib 480mg BID 4 days on - 3 days off. The primary endpoint for Cohort D is confirmed objective response rate as defined by RECIST v1.1. Using a single stage A’Hern design with a target response rate of 25%, unacceptable response rate of 5%, alpha=5% and power=80%, at least 3 responses out of 16 evaluable patients were required to infer efficacy for capivasertib. Results: In total 19 patients were recruited in Cohort D, 12 following ctDNA testing and 7 on tumour testing. Fifteen (79%) were ER positive, all were HER2 non-amplified, and 14 (74%) had visceral metastases. Mutations were AKT1 E17K (5 patients), AKT1 L52R (1), PTEN inactivating mutation (12), and PTEN homozygous deletion (1). All patients were evaluable with overall confirmed response rate of 10.5% (95%CI 1.3-33.1%, 2/19) (first 16 evaluable patients: 2/16, 12.5% (95%CI 1.6-38.3)). Two further patients had unconfirmed responses. Median progression free survival was 3.4 months (IQR 1.8-5.5 months) and median duration of response 3.9 months (IQR 3.7-4.2 months) with 1 patient continuing treatment. In patients with AKT1 mutations there was a 33% response rate (2/6) with 2 further unconfirmed responses. There were no responses in patients with PTEN mutations (0/13). The most common clinically significant grade 3 or 4 adverse events were rash (26%), hypertension (11%), transaminase increase (11%) and vomiting (11%). Conclusions: Capivasertib monotherapy did not meet pre-specified criteria for efficacy in this group of patients with a range of AKT pathway activating mutations. In exploratory analysis, capivasertib was active in patients with AKT1 mutations. Citation Format: Richard D Baird, Lucy Kilburn, Sarah Kernaghan, Andrew M Wardley, Iain Macpherson, Rebecca Roylance, Peter Stephens, Olga Oikonomidou, Jeremy P Braybrooke, Mark Tuthill, Jacinta Abraham, Matthew C Winter, Belinda Kingston, Katie Wilkinson, Nicholas Turner, Alistair Ring, Judith M Bliss, on behalf of the plasmaMATCH Trial Management Group. Results from plasmaMATCH trial treatment cohort D: A phase II trial of capivasertib in patients with an AKT activation basket mutation identified via ctDNA testing or tumour sequencing (CRUK/15/010) [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 P1-19-14.

Association of ERG/PTEN status with biochemical recurrence after radical prostatectomy for clinically localized prostate cancer.

We have previously demonstrated a significant correlative relationship between PTEN deletion and ERG rearrangement, both in the development of clinically localized prostate cancers and metastases. Herein, we evaluate the cooperative role of ERG and PTEN in oncological outcomes after radical prostatectomy for clinically localized prostate cancer. We evaluated ERG and PTEN status using three previously described cohorts. The first cohort included 235 clinically localized prostate cancer cases represented on tissue microarrays (TMA), evaluated using previously validated FISH assays for ERG and PTEN. The second cohort included 167 cases of clinically localized prostate cancer on TMAs evaluated for PTEN by FISH, and for PTEN and ERG by dual IHC. The third cohort comprised 59 clinically localized prostate cancer cases assessed by array comparative genomic hybridization (aCGH). Kaplan-Meir plots and long rank tests were used to assess the association of ERG and PTEN status with biochemical recurrence after radical prostatectomy for clinically localized prostate cancer. Of the 317 cases eligible for analyses with evaluable ERG and PTEN status, 88 (27.8%) patients developed biochemical recurrence over a median follow-up of 5.7years. Overall, 45% (142/317) of cases demonstrated ERG rearrangement and 20% (62/317) of cases demonstrated PTEN loss. Hemizygous and homozygous deletion of PTEN was seen in 10% (18/175) and 3% (5/175) of ERG-negative cases, respectively. In contrast, hemizygous and homozygous deletion of PTEN was seen in 11% (15/142) and 17% (24/123) of ERG-positive cases, respectively. PTEN loss (heterozygous or homozygous) was significantly associated with shorter time to biochemical recurrence compared to no PTEN loss (p < 0.001). However, ERG rearrangement versus no rearrangement was not associated with time to PSA recurrence (p = 0.15). Patients who exhibited ERG rearrangement and loss of PTEN had no significant difference in time to recurrence compared to patients with wild-type ERG and loss of PTEN (p = 0.30). Our findings confirm a mutual cooperative role of ERG and PTEN in the pathogenesis of prostate cancer, particularly for homozygous PTEN deletion. ERG did not stratify outcome either alone or in combination with PTEN in this cohort.

The association of genomic lesions and PD-1/PD-L1 expression in resected triple-negative breast cancers

BackgroundElevated PD-L1 expression on tumor cells, a context associated with an adaptive immune response, has been linked to the total burden of copy number variants (CNVs) in aneuploid tumors, to microsatellite instability (MSI), and to specific genomic driver lesions, including loss of PTEN, MYC amplification, and activating mutations in driver oncogenes such as KRAS and PIK3CA. Triple-negative breast cancers (TNBCs) typically have high levels of CNVs and diverse driver lesions in their genomes. Thus, there is significant interest in exploiting genomic data to develop predictive immunotherapy biomarkers for patients with TNBC.MethodsWhole tissue samples from 55 resected TNBCs were screened by immunohistochemistry (IHC) for PD-1 and PD-L1 by using validated antibodies and established scoring methods for staining of tumor and non-tumor cells. In parallel, we interrogated biopsies from each resection with DNA content flow cytometry and sorted the nuclei of diploid, tetraploid, and aneuploid cell populations. CNVs were mapped with CNV oligonucleotide arrays by using purified (>95%) tumor populations. We generated whole exome data for 12 sorted tumor samples to increase the resolution within loci of interest and to incorporate somatic mutations into our genomic signatures.Results and ConclusionsPD-L1 staining was detected on tumor cells in 29 out of 54 (54%) evaluable cases and was associated with increased overall survival (P = 0.0024). High levels of PD-1 and PD-L1 (IHC ≥4) were present in 11 out of 54 (20%) and 20 out of 54 (37%) cases with staining of PD-L1 primarily on tumor cells for 17 out of 20 (85%) cases. The latter included tumors with both high (>50) and low (<20) numbers of CNVs. Notably, homozygous deletion of PTEN (n = 6) or activating mutation in PIK3CA (n = 1) was not associated with increased expression of either immune checkpoint activator in TNBC. In contrast, two treatment-naïve cases with EGFR driver amplicons had high PD-L1 tumor staining. High mutational load and predicted neoepitopes were observed in MSI+ and high CNV burden TNBCs but were not associated with high PD-L1 expression on tumor cells. Our results challenge current models of genomic-based immunotherapy signatures yet suggest that discrete genomic lesions may complement existing biomarkers to advance immune checkpoint therapies for patients with TNBC.

Abstract 2341: Sleeping Beauty transposon mutagenesis screen identifies cancer genes of uterine leiomyosarcoma driving sarcomagenesis and lung metastasis

Abstract Introduction Uterine leiomyosarcoma (ut-LMS) is one of the most aggressive malignancies with extremely poor prognosis. The 5-year survival rate is 0-20% for patients with tumors beyond uterus and 50% for even early stage patients due to high hematological metastatic potential. Because of its rarity, large-scale genetic profiling could not be done and thus its molecular mechanisms remain largely unknown. To solve this problem, we conducted in vivo sleeping beauty (SB) transposon mutagenesis screen and identified the key genetic drivers of ut-LMS. Methods We crossed Amhr2-Cre knock-in mice, transposon transgenic mice (T2Onc2), SB transposase knock-in mice (Rosa-LSL-SBase), Pten floxed mice, and Kras G12D knock-in mice, generating experimental mice (Amhr2-Cre Tg/+;T2Onc2 Tg/+;SBase KI/+;Pten fl/fl;Kras LSL-G12D/+) and control mice (Amhr2-Cre Tg/+; Pten fl/fl;Kras LSL-G12D/+). The experimental mice have homozygous deletion of Pten, constitutive activation of Kras and SB transposon mobilization in uterine smooth muscle cells. Fifty-nine experimental mice and 14 control mice were aged until moribund. Results All of the experimental mice died of multiple uterine tumors by 2 months of age, while none of the control mice developed uterine tumors. These uterine tumors were histologically diagnosed as ut-LMS by their morphology and immunohistochemical positivity of desmin and alpha-SMA. These results clearly indicated that transposon mutagenesis was required for sarcomagenesis of ut-LMS. To identify genes mutagenized by SB transposon in these tumors, we performed splink HiSeq sequence of gDNA from 80 primary uterine tumors and 24 normal uterus of mice with uterine tumors. Gene-centric common insertion site analysis (gCIS) identified 19 CIS genes in primary uterine tumors but no CIS genes in normal uterus, indicating the selective enrichment of transposon insertions in cancer genes in tumors. By further focusing on the transposon insertions with high sequence read counts, we identified trunk mutations driving sarcomagenesis that include inactivating mutations of NF1 and activating mutations of one of zinc fingers gene family. Its inhibition via siRNA in human ut-LMS cell lines, SK-LMS1, SK-UT1, and SKN, impaired cell proliferation and sphere formation, suggesting the importance of this gene for the survival of ut-LMS. Next, to identify the metastatic drivers of ut-LMS, we induced the lung metastasis by tail vein injection of 9 cell lines established from SB uterine tumors. After the sequential in vivo passage of metastatic SB tumors, we have obtained 50 lung metastatic tumors in immunocompetent mice. Sequencing and gCIS analysis of 50 lung metastases identified 3 potential driver genes of lung metastases. Conclusion SB mutagenesis screen discovers multiple cancer genes of ut-LMS driving sarcomagenesis and lung metastasis. Citation Format: Michiko Kodama, Takahiro Kodama, Justin Y. Newberg, Jean C. Tien, Roberto Rangel, Aya Nakae, Kae Hashimoto, Seiji Mabuchi, Kenjiro Sawada, Tadashi Kimura, Nancy A. Jenkins, Neal G. Copeland. Sleeping Beauty transposon mutagenesis screen identifies cancer genes of uterine leiomyosarcoma driving sarcomagenesis and lung metastasis [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 2341.

Abstract P2-07-05: Genomic lesions and PD-1/PD-L1 expression in resected triple negative breast cancers

Abstract Introduction: Striking clinical responses to immunotherapies in subsets of patients with a variety of solid tumors have prompted the search for predictive biomarkers. Recent studies have proposed that gene specific and genome wide mutation and copy number signatures in tumor cells may be predictive of responses to immune checkpoint blockade. Notably high levels of PD-L1 on tumor cells, a context associated with an adaptive immune response, has been linked to specific oncogenic driver lesions including loss of PTEN, activating KRAS mutation, and MYC amplification, and to the total burden of copy number variants (CNVs) in aneuploid tumors. Triple negative breast cancers (TNBCs) typically have multiple driver mutations and high levels of CNVs in their genomes. Thus there is significant interest in exploiting genomic data for the development of prognostic immunotherapy biomarkers for patients with TNBC. Study Design: We interrogated 62 well annotated surgical resections from patients with TNBC and assessed the associations of genomic lesions with expression of PD-1 and PDL-1 in tumor and non-tumor cells in each sample. We applied a systematic approach to rigorously interrogate the genomes of each TNBC sample. Tumor ploidy was initially measured with DNA content flow cytometry followed by sorting the nuclei of distinct diploid, tetraploid, and aneuploid cell populations from each TNBC. The next level of analysis measured genome wide copy number variants (CNV) with oligonucleotide arrays designed for CNV detection using purified (&amp;gt;95%) tumor populations. This enabled the discrimination and mapping of CNVs including single copy losses and gains, focal amplifications, and homozygous deletions across each TNBC genome. Finally we generated whole exome data in a subset of samples to increase the resolution within loci of interest and to incorporate mutations of individual genes into our genomic signatures. This combined approach provides high resolution measures of TNBC genomes from ploidy, whole chromosome and chromosome arm level CNVs, focal amplicons, breakpoints and homozygous deletions, to the level of gene specific indels and mutations. In parallel whole tissue samples were screened by IHC for PD-1 and PD-L1 using validated antibodies and established scoring methods for staining of tumor and non-tumor cells. Results and Conclusions: High levels of PD-1 and PD-L1 were detected in 5/60 (8.3%) and 16/60 (26.7%) evaluable cases with staining of PD-1 exclusively on non tumor cells, while PD-L1 was primarily on tumor cells; 15/16 (93.8%) cases. These data were then used to test the associations of individual recurring genomic lesions, and the extent and nature of chromosome aberrations on the expression patterns of PD-1 and PD-L1. Homozygous deletion of PTEN or activating mutation in PIK3CA did not correlate with increased expression of either immune checkpoint regulator in TNBC cells. Furthermore tumors with highly aberrant aneuploid genomes and distinct oncogenic drivers frequently express relatively low levels of both proteins suggesting an intrinsic escape from immunosurveillance. Citation Format: Barrett MT, Lenkiewicz E, Malasi S, Yearley JH, Annamalai L, McCullough AE, Anderson KS, Pockaj BA. Genomic lesions and PD-1/PD-L1 expression in resected triple negative breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-05.

Abstract 1230: Talazoparib, a second generation PARP inhibitor, is a novel therapy for PTEN mutants in prostate cancer

Abstract Prostate cancer is the most common cancer among men, other than skin cancer, in the United States. When the cancer enters into the metastatic castration resistant stage (mCRPC), few treatment options are currently available and the disease is considered incurable. The purpose of this study is to investigate a new, targeted therapy against prostate cancers, including mCRPC. Talazoparib is a novel PARP inhibitor with up to 100-fold higher potency and PARP affinity when compared to PARP inhibitors such as olaparib, veliparib, and rucaparib. Current literature suggests that PTEN mutants have high sensitivity to talazoparib. PTEN is a tumor suppressor gene that is commonly lost in prostate cancers. A murine prostate cancer cell line, Myc-CaP, which has been modified to overexpress the oncogene Myc, was edited by the CRISPR-Cas9 system to knock out the PTEN gene. Myc-CaP and Myc-CaP PTEN KO cells were treated with olaparib, an FDA approved, first generation PARP inhibitor, and talazoparib. Talazoparib decreased cell viability significantly more in PTEN KO cells versus PTEN wild-type (WT) cells. No such trend was observed when these cells were treated with olaparib. In addition, IC50 values in prostate cancer cell lines with heterozygous or homozygous deletion of PTEN were up to 20 times lower with the talazoparib treatment when compared to olaparib. Not only does this indicate that talazoparib is much more potent than olaparib, but also preferentially impairs cell growth in PTEN mutants. PTEN’s signal transduction prevents the phosphorylation of AKT, which is necessary for cell proliferation. When cells were treated with an AKT inhibitor, MK2206, alone or in combination with talazoparib, pAKT protein levels decreased, but the greatest effect was seen when MK2206 was used in combination with a low dose of talazoparib in the Myc-Cap cell line with wt PTEN. Western blot analysis showed that PTEN was upregulated following talazoparib treatment. These results indicate synergy of talazoparib and MK2206 in reducing the phosphorylation of AKT in a dose-dependent manner. Talazoparib is by far the most potent PARP inhibitor developed to date. With talazoparib potentially acting on prostate cancer with PTEN mutations, talazoparib treatment may expand current PARP inhibitor treatment in advanced prostate cancers and thereby elicit a response in as many as 80% of patients. Thus, talazoparib has vast therapeutic potential in prostate cancer. Citation Format: Zachary Z. Reinstein, Rajita Vatapalli, Jonathan Anker, Sahithi Pamarthy, Benedito Carneiro, Sarki Abdulkadir. Talazoparib, a second generation PARP inhibitor, is a novel therapy for PTEN mutants in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1230. doi:10.1158/1538-7445.AM2017-1230

Abstract 2727: Clinical significance of PTEN in UTUC

Abstract Upper tract urothelial carcinoma (UTUC) is a rare malignancy accounts for about 5% of all urothelial tumours. The cancer cells start in the layer of tissue urothelium, and found in the renal pelvis, renal calyces or ureters. When compare the UTUC to the bladder urothelial carcinoma, the pathological and clinical findings are often look and act alike. Previous studies indicated that deletion of phosphatase and tensin homologue (PTEN) gene is common to be found in bladder cancer. As known tumor suppressor gene PTEN is located on chromosome ten and considered as an important negative regulator for the PIP3/Akt signaling pathway to promote cell proliferation and inhibit apoptosis. Thus, this study we aimed to determine clinical significance of PTEN in UTUC. We collected sixty-eight formalin-fixed paraffin-embedded (FFPE) UTUC samples, the tumor stage was according to American Joint Committee on Cancer (AJCC) with Ta (n=15), T1 (n=11), T2 (n=10), T3 (n=18), T4 (n=7) and seven patients with unknown; in which male no.32, female no.36; the median age is 68 y/o (range 40-85 y/o). Fluorescence in situ hybridization (FISH) was performed to exam the PTEN gene copy number or gene structure alterations of UTUC; besides, immunohistochemistry (IHC) analysis were used to observe the PTEN protein expression in the tumor. Our results showed PTEN mutations were observed in twenty-two tumors with heterozygous PTEN deletion; one tumor with homozygous PTEN deletion, and three tumors with PTEN monosomy. Further, heterozygous PTEN deletion was shown significantly associated with stage of pTa, pT1 and pT2 (P=0.048) and history of smoking (P=0.030). The IHC results showed lower expression level was common to be observed in the tumor area. Our findings suggest loss of PTEN is associated with the low-risk group of UTUC but not the high-risk group, the unknown mechanisms between these two subtypes might be existed. Besides, smoking could also as a factor highly emerge from genetic alterations in UTUC. In conclusion, PTEN genomic loss might act a predictive indicator of classification of UTUC, however, the clinical treatment could be different due to the underlying diversity of the molecular activities. Citation Format: Liang-Chen Li, Cheng-Keng Chuang, Hung-Ying Chiang, Ying-Tzu Chen, Po-Hung Lin, Kai-Jie Yu, See-Tong Pang, Wen Hui Weng*. Clinical significance of PTEN in UTUC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2727. doi:10.1158/1538-7445.AM2017-2727

SPINK1 Overexpression in Localized Prostate Cancer: a Rare Event Inversely Associated with ERG Expression and Exclusive of Homozygous PTEN Deletion.

SPINK1 is proposed as potential prognostic marker in prostate cancer (PCA). However, its relation to PTEN and ERG in localized PCA remains unclear. The study population consisted of two independent cohorts of men treated by radical prostatectomy for localized PCA (discovery n=218 and validation n=129). Patterns of association between SPINK1 and each of ERG and PTEN were evaluated by immunohistochemistry and fluorescence in situ hybridization. Associations between SPINK1 expression and various pathologic parameters and clinical outcome were also investigated. SPINK1 was expressed in 15.3% and 10.9% of cases in the discovery and validation cohort, respectively. SPINK expression was observed in 5.56% of high-grade prostatic intraepithelial neoplasia and 1.1% of adjacent morphologically benign prostatic glands. SPINK1 and ERG expression were almost exclusive, with only 1.0% of the cases co-expressing both in the same core sample. SPINK1 interfocal and within-core heterogeneity was noted in 29.2% and 64.6% of cases, respectively. SPINK1 expression was not significantly associated with PTEN deletion in the two cohorts (p=0.871 for discovery cohort and p=0.293 for validation cohort). While SPINK1 expression did occur with hemizygous PTEN deletion, there was a complete absence of SPINK1 expression in PCA showing homozygous PTEN deletion, which was confirmed in the validation cohort (p=0.02). Despite SPINK1's association with higher Gleason score (>7) (p=0.02), it was not associated with other pathological parameters or biochemical recurrence post-radical prostatectomy. We documented absolute exclusivity between SPINK1 overexpression and homozygous PTEN deletion in localized PCA. SPINK1 and ERG expressions are exclusive events in PCA. SPINK1 is not of added prognostic value in localized PCA.

Abstract 652: Establishment of a novel mouse model of intrahepatic cholangiocarcinoma by liver-specific Kras activation and Pten deletion

Abstract The mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K) pathway are intracellular signaling pathways that are involved in carcinogenesis. Various studies suggest that these pathways are important for the development of liver tumors. To investigate the effect of coactivation of MAPK and PI3K pathways in liver tumorigenesis, we generated three types of mouse models by crossbreeding conditional KrasG12D knockin mice and/or conditional Pten knockout mice with Alb-Cre mice (Alb-Cre KrasG12D/+ Pten flox/flox mice, Alb-Cre KrasG12D/+ Pten flox/+ mice, and Alb-Cre KrasG12D/+ mice). Among the three models, mice carrying liver-specific oncogenic Kras knockin and homozygous Pten deletion (Alb-Cre KrasG12D/+ Pten flox/flox) frequently demonstrated weight loss, abdominal distension, and jaundice with median survival of eight weeks. Macroscopic analysis detected diffuse and firm tumorous lesions mainly in the hepatic hilum. Histological examination of the Alb-Cre KrasG12D/+ Pten flox/flox mice disclosed bile duct hyperplasia at four weeks of age, precancerous lesions of intrahepatic cholangiocarcinoma (ICC) at five weeks of age, and advanced ICC lesions that resemble human ICC at seven weeks of age. On the other hand, the Alb-Cre KrasG12D/+ Pten flox/+ mice, the second model, developed ICCs as well as hepatocellular carcinomas (HCCs), and their average survival was eight months. The third mice model, the Alb-Cre KrasG12D/+ mice, developed HCCs but not ICCs, and survived more than one year. Immunohistochemical analysis of the ICCs in the Alb-Cre KrasG12D/+ Pten flox/flox mice corroborated the activation of the Mapk and the Pi3k pathways in the tumor cells. These data suggest that activity of Pi3k pathway might dictate the fate determination of hepatotumorigenesis into biliary or hepatocyte cell lineage. In addition, oncogenic Kras expression and homozygous Pten deletion cooperate to induce exclusively ICCs. Since a limited number of mouse ICC models have been reported, the Alb-Cre KrasG12D/+ Pten flox/flox mouse model should be a useful tool for the analysis of molecular mechanism(s) underlying ICC. Citation Format: Yumi Terakado, Tsuneo Ikenoue, Kiyoshi Yamaguchi, Yoichi Furukawa. Establishment of a novel mouse model of intrahepatic cholangiocarcinoma by liver-specific Kras activation and Pten deletion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 652.

A novel mouse model of intrahepatic cholangiocarcinoma induced by liver-specific Kras activation and Pten deletion.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis and its incidence is increasing worldwide. Recently, several types of cells have been considered as the origin of ICC, namely cholangiocytes, liver progenitor cells, and hepatocytes. Here, we have established a novel mouse model of ICC by liver-specific Kras activation and Pten deletion. An activating mutation of Kras in combination with deletion of Pten was introduced in embryonic hepatic bipotential progenitor cells (so-called hepatoblasts) and mature hepatocytes using the Cre-loxP system. As a result, liver-specific Kras activation and homozygous Pten deletion cooperated to induce ICCs exclusively. In contrast, Kras activation in combination with heterozygous Pten deletion induced both ICCs and HCCs, whereas Kras activation alone resulted in HCCs but not ICCs. Furthermore, a cell-lineage visualization system using tamoxifen-inducible Cre-loxP demonstrated that the ICCs did not originate from hepatocytes but from cholangiocytes. Our data suggest that mice carrying liver-specific Kras activation in combination with homozygous Pten deletion should be useful for the investigation of therapeutic strategies for human ICC.

High Sensitivity of an Ha-RAS Transgenic Model of Superficial Bladder Cancer to Metformin Is Associated with ∼240-Fold Higher Drug Concentration in Urine than Serum.

While pharmacoepidemiologic and laboratory studies have supported the hypothesis that the antidiabetic drug metformin may be useful in treating or preventing cancer, there is limited evidence to suggest which specific cancer sites may be particularly sensitive. Sensitivity likely is determined both by features of tumor pathophysiology and by pharmacokinetic factors. We used UPII-mutant Ha-ras transgenic mice that develop hyperplasia and low-grade, papillary urothelial cell carcinoma to determine whether metformin has activity in a model of superficial bladder cancer. Metformin significantly improved survival, reduced urinary tract obstruction, reduced bladder weight (a surrogate for tumor volume), and led to clear activation of AMP α kinase and inhibition of mTOR signaling in neoplastic tissue. We investigated the basis of the unusual sensitivity of this model to metformin, and observed that following oral dosing, urothelium is exposed to drug concentrations via the urine that are approximately 240-fold higher than those in the circulation. In addition, we observed that bladder cancer cell lines (RT4, UMUC-3, and J82) with homozygous deletion of either TSC1 or PTEN are more sensitive to metformin than those (TEU2, TCCSUP, and HT1376) with wild-type TSC1 and PTEN genes. Our findings provide a strong rationale for clinical trials of oral metformin in treatment of superficial bladder cancer.

In prostate cancer needle biopsies, detections of PTEN loss by fluorescence in situ hybridization (FISH) and by immunohistochemistry (IHC) are concordant and show consistent association with upgrading.

The prognostic value of phosphatase and tensin homolog (PTEN) loss in prostate cancer has primarily been evaluated by either fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC). Previously, we found that PTEN loss by IHC was associated with increased risk of upgrading from biopsy (Gleason 3 + 3) to prostatectomy (Gleason 7+). Now, using an evaluable subset of 111 patients with adjacent biopsy sections, we analyzed the association between PTEN deletion in cancer and the odds of upgrading by a highly sensitive and specific four-color FISH assay. We also compared the concordance of PTEN loss by IHC and PTEN deletion by FISH. PTEN deletion was found in 27% (12/45) of upgraded cases compared with 11% (7/66) of controls (P = 0.03). Cancers with PTEN deletions were more likely to be upgraded than those without deletions (adjusting for age odds ratio = 3.40, 95% confidence interval 1.14-10.11). With respect to concordance, of 93 biopsies with PTEN protein detected by IHC, 89 (96%) had no PTEN deletion by FISH, and of 18 biopsies without PTEN protein by IHC, 15 had homozygous or hemizygous PTEN deletion by FISH. Only 4 biopsies of the 93 (4%) with PTEN protein intact had PTEN deletion by FISH. When the regions of uncertainty in these biopsies were systematically studied by FISH, intra-tumoral variation of PTEN deletion was found, which could account for variation in immunoreactivity. Thus, FISH provides a different approach to determining PTEN loss when IHC is uncertain. Both FISH and IHC are concordant, showing consistent positive associations between PTEN loss and upgrading.

Abstract 2650: PI 3-Kinase inhibitors enhance the synthetic lethality of Parp inhibitors

Abstract Introduction: Clinical trials have shown promising responses of BRCA-linked breast and ovarian cancers to PARP inhibitor therapy, but remissions are often short-lived and incomplete. Frequent loss of INPP4B and PTEN leads to activation in the PI3K pathway in TNBC, making the PI3K pathway an attractive treatment target. Here we present mechanistic data that explain how PI3K- and PARP-inhibition enhance each other. Methods: We examined the effects of PI3K-inhibitors BKM120 or BYL719 and PARP-inhibitor Olaparib on the metabolism of HCC1937 cells (BRCA1 5382C mutation and homozygous deletion of PTEN and p53) measuring lactic acid production, via seahorse analysis, carbon metabolism using targeted Mass Spectrometry (LC-MS/MS) and DNA synthesis. PI3K- but not Akt- or SGK-inhibition induced a DNA damage response in this BRCA1 mutant cell line as well as in a mouse model of BRCA1-related breast cancer (K14-Cre BRCA1f/f p53f/f) mouse model. Summary: Metabolic profiling showed that PI3K-inhibition decreased flux through glycolysis and specifically through the non-oxidative pentose-phosphate pathway, the main source of ribose-5-phosphate required for the de novo synthesis of nucleotides in BRCA1-mutant breast cancer cells. Nucleotide shortage led to replication stress with the appearance of γH2AX, increased poly-ADP-ribosylation and an acute drop in DNA synthesis in cell cultures as well as in vivo. PI3K inhibition led to a reduced and error-prone S-phase due to a decrease in nucleotide biosynthesis which could be rescued upon exogenous supply of nucleosides. In a mouse model (K14-Cre BRCA1f/fp53f/f induced breast cancers) we could confirm that both BKM120 and BYL719 enhanced the efficacy of Parp-inhibitor Olaparib (median PFS for Olaparib 50 days, for BKM120 4 days, for BYL719 6 days and for both combinations, BKM120+Olaparib and BYL719+Olaparib &amp;gt; 120 days). Conclusion: Preceding cell cycle arrest, PI3K-inhibition leads to a decrease in DNA synthesis due to decreased Ribose-5-phosphate production required for nucleotide synthesis. PI3K-inhibitors lower nucleotide pools, lead to impaired DNA damage repair and S-phase progression and further augment the synthetic lethality of Parp-inhibitors in BRCA1-related breast cancer. Citation Format: Ashish P. Juvekar, Sina Yadegarynia, Hai Hu, Costas A. Lyssiotis, Hui Liu, John M. Asara, Ralph Scully, Lewis C. Cantley, Gerburg M. Wulf. PI 3-Kinase inhibitors enhance the synthetic lethality of Parp inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2650. doi:10.1158/1538-7445.AM2015-2650

A multicenter study shows PTEN deletion is strongly associated with seminal vesicle involvement and extracapsular extension in localized prostate cancer.

BACKGROUNDLoss of the phosphatase and tensin homolog (PTEN) tumor suppressor gene is a promising marker of aggressive prostate cancer. Active surveillance and watchful waiting are increasingly recommended to patients with small tumors felt to be low risk, highlighting the difficulties of Gleason scoring in this setting. There is an urgent need for predictive biomarkers that can be rapidly deployed to aid in clinical decision-making. Our objectives were to assess the incidence and ability of PTEN alterations to predict aggressive disease in a multicenter study.METHODSWe used recently developed probes optimized for sensitivity and specificity in a four-color FISH deletion assay to study the Canary Retrospective multicenter Prostate Cancer Tissue Microarray (TMA). This TMA was constructed specifically for biomarker validation from radical prostatectomy specimens, and is accompanied by detailed clinical information with long-term follow-up.RESULTSIn 612 prostate cancers, the overall rate of PTEN deletion was 112 (18.3%). Hemizygous PTEN losses were present in 55/612 (9.0%) of cancers, whereas homozygous PTEN deletion was observed in 57/612 (9.3%) of tumors. Significant associations were found between PTEN status and pathologic stage (P < 0.0001), seminal vesicle invasion (P = 0.0008), extracapsular extension (P < 0.0001), and Gleason score (P = 0.0002). In logistic regression analysis of clinical and pathological variables, PTEN deletion was significantly associated with extracapsular extension, seminal vesicle involvement, and higher Gleason score. In the 406 patients in which clinical information was available, PTEN homozygous (P = 0.009) deletion was associated with worse post-operative recurrence-free survival (number of events = 189), pre-operative prostate specific antigen (PSA) (P < 0.001), and pathologic stage (P = 0.03).CONCLUSIONPTEN status assessed by FISH is an independent predictor for recurrence-free survival in multivariate models, as were seminal vesicle invasion, extracapsular extension, and Gleason score, and preoperative PSA. Furthermore, these data demonstrate that the assay can be readily introduced at first diagnosis in a cost effective manner analogous to the use of FISH for analysis of HER2/neu status in breast cancer. Combined with published research beginning 17 years ago, both the data and tools now exist to implement a PTEN assay in the clinic. Prostate 75: 1206–1215, 2015. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.

MP1-17 PTEN STATUS DETERMINATION IN PROSTATE CANCER: COMPARISON OF IHC AND FISH IN A LARGE MULTI-CENTER COHORT

You have accessJournal of UrologyProstate Cancer: Markers I1 Apr 2015MP1-17 PTEN STATUS DETERMINATION IN PROSTATE CANCER: COMPARISON OF IHC AND FISH IN A LARGE MULTI-CENTER COHORT Tamara L. Lotan, Carlos L. Morais, Wei Wei, Tamara Jamaspishvili, Ziding Feng, Jesse McKenney, Jeff Simko, Dean Troyer, Lawrence True, James D. Brooks, Jeremy Squire, and CAnary Pathology Team Investigating Tissue Biomarkers (CAPIT) Tamara L. LotanTamara L. Lotan More articles by this author , Carlos L. MoraisCarlos L. Morais More articles by this author , Wei WeiWei Wei More articles by this author , Tamara JamaspishviliTamara Jamaspishvili More articles by this author , Ziding FengZiding Feng More articles by this author , Jesse McKenneyJesse McKenney More articles by this author , Jeff SimkoJeff Simko More articles by this author , Dean TroyerDean Troyer More articles by this author , Lawrence TrueLawrence True More articles by this author , James D. BrooksJames D. Brooks More articles by this author , Jeremy SquireJeremy Squire More articles by this author , and CAnary Pathology Team Investigating Tissue Biomarkers (CAPIT)CAnary Pathology Team Investigating Tissue Biomarkers (CAPIT) More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.180AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES PTEN loss is a promising prognostic and potentially predictive biomarker in prostate cancer. Because PTEN loss in prostate cancer occurs most commonly via gene deletion, we developed and validated an inexpensive immunohistochemical (IHC) assay to detect PTEN protein loss. Here, we studied the sensitivity and specificity of this assay relative to fluorescence in situ hybridization (FISH) for detection of PTEN gene deletion in prostate cancer. METHODS 1275 primary prostate tumors from the Canary Retrospective Prostate Cancer Tissue Microarray (TMA) cohort were analyzed for PTEN loss by IHC. PTEN IHC was performed by autostaining on the Ventana Discovery Ultra platform using a rabbit monoclonal antibody (Cell Signaling, clone D4.3) and scored by two independent reviewers with very good agreement (96.4% agreement over 2783 spots; κ = 0.905; 95% CI=0.887-0.923). FISH was previously performed in the same cohort using a four color probe set (CymogenDx) yielding measurable results for 612 tumors. RESULTS By IHC, 8% (108/1275) of tumors had homogeneous PTEN loss in all sampled tumor glands, 12% (150/1275) had heterogeneous PTEN loss in a subset of sampled tumor glands, 66% (837/1275) had intact PTEN and 14% (180/1275) had ambiguous or missing immunostaining results. 551 tumors had both IHC and FISH data available for comparison. Intact PTEN immunostaining was 91% specific for absence of PTEN gene deletion by FISH, with 413/454 tumors with 2 copies of PTEN showing intact PTEN IHC. PTEN IHC loss was 98% sensitive for homozygous PTEN deletion by FISH, with homogeneous or heterogeneous PTEN protein loss in 48/49 homozygous tumors. PTEN IHC was 62% sensitive for detection of hemizygous PTEN deletion by FISH, with homogeneous or heterogeneous PTEN protein loss in 30/48 tumors with hemizygous deletion. Other inactivating mutations of PTEN (not detectable by FISH) may lead to PTEN protein loss in tumors with normal or hemizygous PTEN copy number. CONCLUSIONS Our validated and automated PTEN immunostaining protocol is a simple and relatively inexpensive test that is 91% specific and 98% sensitive for detection of homozygous PTEN gene deletions in a large multi-center study of prostate cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e7-e8 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tamara L. Lotan More articles by this author Carlos L. Morais More articles by this author Wei Wei More articles by this author Tamara Jamaspishvili More articles by this author Ziding Feng More articles by this author Jesse McKenney More articles by this author Jeff Simko More articles by this author Dean Troyer More articles by this author Lawrence True More articles by this author James D. Brooks More articles by this author Jeremy Squire More articles by this author CAnary Pathology Team Investigating Tissue Biomarkers (CAPIT) More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PTEN mutations and activation of the PI3K/Akt/mTOR signaling pathway in papillary tumors of the pineal region.

Papillary tumors of the pineal region (PTPR) are recognized as a distinct entity in the World Health Organization classification of CNS tumors. Papillary tumors of the pineal region frequently show loss of chromosome 10, but no studies have investigated possible target genes on this chromosome. Chromosome 10 harbors the PTEN (phosphatase and tensin homolog) gene, the inactivation of which, by mutation or epigenetic silencing, has been observed in different brain tumors, including high-grade gliomas. In this study, we investigated copy number changes by molecular inversion probe (MIP) analysis and the mutational status of PTEN in 13 PTPR by direct sequencing. MIP analysis of 5 PTPR showed chromosome 10 loss in all cases. In addition, there were losses of chromosomes 3, 14, 22, and X, and gains of whole chromosomes 8, 9, and 12 in more than 1 case. One case had a homozygous PTEN deletion; and 2 point mutations in exon 7 of PTEN (G251D and Q261stop) were found. Immunohistochemistry revealed decrease or loss of the PTEN protein and increased expression of p-Akt and p-S6. These results indicated that PTEN mutations and activation of the PI3K/Akt/mTOR signaling pathway may play a role in the biology of PTPR. This evidence may lead to the possible use of PI3K/Akt/mTOR inhibitors in therapy for patients with PTPR.

Association of PTEN protein loss with upgrading of prostate cancer from biopsy to radical prostatectomy.

127 Background: Active surveillance is increasingly recommended for men with low-risk Gleason Score 3+3=6 (GS 6) prostate cancer. Yet, approximately one-third of patients with GS 6 cancer on biopsy are upgraded to higher GS at radical prostatectomy (RP). Previous studies have shown that clinical-pathologic parameters (age, prostate-specific antigen [PSA], prostate volume, extent of disease) are weak predictors of GS6 tumor upgrade. Our goal was to investigate the utility of PTEN as a molecular marker to predict upgrading in GS 6 biopsies. Methods: In a retrospective case-control study, 71 patients with GS 6 tumors on needle biopsy that were upgraded to GS 7 or higher cancer at RP (cases) were compared to 103 patients whose GS 6 tumors on needle biopsy were not upgraded at RP (controls). The most extensively involved needle core biopsy from each case was immunostained and scored for PTEN protein loss using a previously validated immunohistochemical (IHC) assay and binary scoring system. Confirmatory fluorescence in situ hybridization (FISH) was used to assess for PTEN gene deletion in biopsies with PTEN protein loss. The correlation of upgrading with PTEN loss and with clinical-pathologic variables was assessed by logistic regression. Results: Patients with upgraded cancers were older than controls (61.8 vs. 59.3 years), had higher mean pre-operative PSA levels (6.53 vs. 5.26 ng/mL), and a higher fraction of biopsy cores involved by tumor (0.42 vs, 0.36). However, of all pathologic variables, PTEN protein loss by IHC was most predictive of upgrading. Overall, PTEN protein loss was found in 18.3% (13 out of 71) of upgraded cases compared to 6.8% (7 out of 103) of controls (p=0.02). In the cases with PTEN protein loss, FISH confirmed homozygous PTEN deletion in 90% (9 out of 10) of upgraded tumors compared to 67% (4 out of 6) of interpretable not upgraded controls. On multivariate analyses, even after adjusting for age, preoperative PSA, clinical stage and race, GS 6 tumors with PTEN protein loss on biopsy were significantly more likely to be upgraded at RP compared to those without PTEN loss with odds ratio (OR) = 3.04 (1.08-8.55; p=0.035). Conclusions: In prostate needle biopsies, PTEN IHC may help distinguish men with low risk cancer from men with intermediate or higher risk cancers.