PTEN IHC Research Articles

PI3K/AKT pathway biomarkers analysis from the phase III IPATential150 trial of ipatasertib plus abiraterone in metastatic castration-resistant prostate cancer.

13 Background: In IPATential150 (NCT03072238), ipatasertib (ipat) + abiraterone (abi) as first-line treatment for metastatic castration-resistant prostate cancer (mCRPC) significantly reduced the risk for disease worsening or death vs placebo (pbo) + abi in patients (pts) with tumors with PTEN loss by immunohistochemistry (IHC; HR, 0.77 [95% CI: 0.61, 0.98]; P = 0.0335) but not in the intention-to-treat population (de Bono, ESMO 2020). In patients with PTEN loss tumors by IHC, median radiographic progression-free survival (rPFS) was 16.5 mo (95% CI: 13.9, 17.0) with pbo + abi and 18.5 mo (95% CI: 16.3, 22.1) with ipat + abi. Here, we present exploratory analyses evaluating putative biomarker associations with rPFS. Methods: Before randomization, tumor samples ( > 90% archival) were tested for PTEN loss by VENTANA PTEN (SP218) IHC assay (N = 1101). PTEN loss was pre-defined as ≥ 50% of tumor cells with no specific cytoplasmic IHC staining. Exploratory analysis evaluated different IHC staining cutoffs. Tumor genomic alterations were profiled with next-generation sequencing (NGS) using the Foundation Medicine FoundationOne CDx NGS assay (Shi, ASGO-GU 2020; n = 743 evaluable by NGS, of which n = 518 were PTEN evaluable). rPFS was determined by the investigator. Results: Consistent benefit with the combination arm vs pbo + abi was observed when PTEN loss by IHC was defined more stringently (rPFS at ≥ 60% tumor cells with PTEN loss: HR, 0.72 [95% CI, 0.56, 0.92]; ≥ 70%: HR, 0.72 [95% CI, 0.56, 0.93]; ≥ 80%: HR, 0.71 [95% CI, 0.54, 0.92]; ≥ 90%: HR, 0.72 [95% CI, 0.53, 0.97]; 100%: HR, 0.65 [95% CI, 0.39, 1.08]). In contrast, ipat + abi was not associated with improved rPFS in pts with PTEN intact by IHC tumors ( < 50% no staining; stratified HR, 0.91 [95% CI: 0.72, 1.16]); the median rPFS was 19.1 mo (95% CI: 16.4, 21.9) with pbo + abi and 19.7 mo (95% CI: 16.4, 26.3) with ipat + abi. By NGS assessment, pts with tumors with PTEN loss and with genomic alterations in PIK3CA/AKT1/PTEN had a larger magnitude of rPFS benefit with ipat + abi than pts with no detectable alterations (Table). Conclusions: Analyses of more-stringent biomarkers associated with activation of the PI3K/AKT pathway further support ipat + abi as a treatment option for first-line mCRPC with PI3K/AKT pathway alterations, a mCRPC subtype with a worse prognosis. Clinical trial information: NCT03072238. [Table: see text]

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.

Abstract P4-10-20: Protein quantitation assays for AKT and PTEN to better understand sensitivity and resistance of breast cancer patients to treatment with AKT inhibitor capivasertib

Abstract Context: The PI3K/AKT/PTEN pathway is frequently aberrantly activated in breast cancer (BC) and involved in resistance to hormonal therapy. Multiple drugs targeting this pathway are approved or in development, including the pan-AKT inhibitor capivasertib (AZD5363). Early phase studies of capivasertib as monotherapy for solid cancers (e.g. NCT01226316, NCT01625286) have used genetic testing to select patients considered most likely to respond based on AKT1, PIK3CA or PTEN mutations. However, not all selected patients responded. Randomised phase 2 studies on Capivasertib as combination treatment showed the importance of context; a more pronounced response in combination with paclitaxel was seen in TNBC patients whose tumors harboured an alteration on AKT1, PIK3CA or PTEN (Schmid et al. ASCO 2018), whereas the same combination in ER+ HER2- advanced/metastatic BC patients did not show a clinical benefit in the overall population nor in the PIK3CA-mutation subgroup (Turner et al. Ann Oncol 2019). The combination with fulvestrant demonstrated an effect in unselected ER+ HER2- advanced BC patients resistant to aromatase inhibitors irrespective of PIK3CA mutations or PTEN IHC status (Jones et al. ASCO 2019). DNA-based assays also have the limitation that potential responders may be missed, e.g., tumors with epigenetic pathway activation but no detectable mutations. Techniques such as IHC and Western Blot (WB) lack the sensitivity, reproducibility and standardization required to precisely quantify these proteins. We hypothesize that precise mass spectrometry (MS)-based methods may facilitate a better understanding of sensitivity and resistance to inhibitors of the PI3K/AKT pathway in future studies. Methods: Protein extracted from tumor tissue lysate is subjected to proteolytic digestion to generate proteotypic peptides used for protein quantitation. Synthetic stable isotope-labeled peptides are added to the digest as an internal standard, after which the target peptides are immuno-enriched with antibodies coupled to magnetic beads. MS analysis is performed by either eluting peptides for measurement with liquid chromatography multiple reaction monitoring tandem MS (LC-MRM-MS/MS) or by spotting beads onto a plate for matrix-assisted laser desorption/ionization MS (MALDI-MS). Calibration curves enable accurate quantitation. A new method for PTEN was applied to 13 breast cancer PDXs. Results were compared to IHC and WB. In a retrospective study, we are applying these methods to test samples from HR+ metastatic breast or gynecological cancer patients (n=25 with PIK3CA-mutations, capivasertib monotherapy; n=26 with PTEN alterations, capivasertib + fulvestrant; n=9 with no PIK3CA, AKT1 or PTEN mutations, not receiving capivasertib). AKT and PTEN quantitation data will be analyzed for a relationship to treatment response as determined by RECIST guidelines (NCT01226316). Results: We validated MS methods to precisely and reproducibly determine AKT and PTEN concentrations in tumor samples. AKT concentrations varied significantly between mutation-positive tumor samples (AKT1: 0.06-0.45, AKT2: 0.05-0.20 fmol/µg total protein). Significant phosphorylation of AKT1-Ser473 or AKT2-Ser474 was observed in 2 out of 17 tested samples. At least 1 mutation-negative sample had a high AKT1 concentration of 0.42 fmol/ug total protein. PTEN concentrations from PDXs were consistent across biological replicates and agreed with IHC and WB. Furthermore, we were able to detect and quantify PTEN from 3 samples that were negative by IHC and/or WB. Conclusion: AKT and PTEN quantitative proteomics assays have the potential to enable more accurate assessment of PI3K/AKT/PTEN pathway activation in tumor tissues from patients treated with inhibitors of this key pathway. Citation Format: Sahar Ibrahim, Constance A Sobsey, Robert Popp, René P. Zahedi, Gerald Batist, Christoph H. Borchers. Protein quantitation assays for AKT and PTEN to better understand sensitivity and resistance of breast cancer patients to treatment with AKT inhibitor capivasertib [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 P4-10-20.

1168P - Trastuzumab plus docetaxel in patients with advanced HER2-positive salivary duct carcinoma: Exploratory biomarker analyses

BackgroundA phase II trial of trastuzumab plus docetaxel in patients with advanced HER2–positive salivary duct carcinoma (SDC) showed 70% of the overall response rate. However, biomarkers which predict survival in this population remain unknown. MethodsA total of 91 patients with HER2–positive SDC treated with trastuzumab plus docetaxel were included. Age, sex, ECOG performance status (PS), status of visceral metastases, previous treatment, previous docetaxel exposure, previous combined androgen blockade, the best overall response to the treatment, pretreatment serum C-reactive protein (CRP) level, modified Glasgow Prognosis Score (mGPS), HER2 status (immunohistochemistry [IHC] score, HER2/CEP17 ratio, HER2 copy number, overall positivity according to ASCO/CAP Guidelines), mutational status of PIK3CA, HRAS, and TP53, IHC score of AR, Ki-67, CK5/6, p53, HER3, Akt, PI3K, FOXA1, adipophilin, mTOR and PTEN were assessed and correlated with progression-free survival (PFS) and overall survival (OS). ResultsTreatment response (PR or CR vs. SD or PD) and PTEN IHC score (1+ – 3+ vs. 0) were related with favorable PFS (hazard ratio [HR], 0.25 and 0.39, respectively) and OS (HR, 0.29 and 0.36, respectively). CRP (≥ 0.50mg/dL) was related with shorter PFS (HR, 2.28) and OS (HR 4.46). Presence of previous treatment and mTOR IHC (1+ – 3+ vs. 0) had significant predictive value of better PFS (HR 0.47 and 0.22, respectively) but not significantly related with OS. ECOG PS of 1-2 (vs. 0), presence of visceral metastasis, and mGPS of 1-2 (vs. 0) had significant relationship with shorter OS but not with PFS. Neither previous docetaxel exposure nor previous combined androgen blockade did not affect the survival outcome. HER2, PIK3CA, HRAS and TP53 status did not related with survival. ConclusionsAlthough HER2 status did not correlated with survival, its downstream factors PTEN and mTOR can serve as predictive biomarkers in patients with advanced SDC treated with trastuzumab plus docetaxel. Serum CRP level may predict survival of this population. Clinical trial identificationUMIN000009437, Released on 03/12/2012. Legal entity responsible for the studyThe authors. FundingMEXT/JSPS. DisclosureAll authors have declared no conflicts of interest.

PTEN Expression, Not Mutation Status in TSC1, TSC2, or mTOR, Correlates with the Outcome on Everolimus in Patients with Renal Cell Carcinoma Treated on the Randomized RECORD-3 Trial.

Genomic alterations in key components of PI3K/mTOR pathway have been proposed as candidate predictive markers for rapalog therapy in renal cell carcinoma (RCC). We tested this hypothesis in patients from a randomized phase II trial of everolimus versus sunitinib. Archival specimens collected at baseline were analyzed with targeted next-generation sequencing (NGS). Focus of interest were alterations in key PI3K pathway components. PTEN expression was assessed by IHC. Association between molecular findings and treatment outcomes was investigated; same associations were tested for 2 everolimus-treated trial cohorts in gastric and hepatocellular carcinoma (HCC). Among 184 everolimus-treated patients with RCC with NGS data, mutation rates in genes of interest were 6% (TSC1), 4.4% (TSC2), and 8.2% (mTOR); 44% harbored alterations in ≥1 PI3K pathway component. For subjects with presence versus absence of mutations in TSC1, TSC2, or mTOR progression-free survival (PFS) neither differed on univariate analysis (HR, 1.0; P = 0.895) nor on multivariate testing stratified by MSKCC risk group and other established prognostic factors (HR, 1.1; P = 0.806). Everolimus-treated patients with retained (n = 50) versus lost (n = 50) PTEN IHC expression had median PFS of 5.3 months versus 10.5 months (HR, 2.5; P < 0.001). Such differences were not seen with sunitinib (10.9 months vs. 10.3 months; HR, 0.8; P = 0.475). Molecular findings did not correlate with outcomes in gastric and HCC cohorts. Association between mutation status for TSC1/TSC2/mTOR and therapeutic outcome on everolimus was not confirmed. Clinically meaningful differences in PFS were seen based on PTEN expression by IHC, lost in >50% of patients.

Abstract 4602: PTEN and ERG expression in MRI-ultrasound guided fusion biopsy correlated with radical prostatectomy findings in men with prostate cancer

Abstract Introduction &amp; Objectives We previously compared ERG and PTEN protein expression in systematic biopsies with radical prostatectomy (RP) findings. MRI-Ultrasound (MRI-US) guided biopsies are expected to more accurately reflect the biological changes in the RP specimens. Here, we studied the correlation of PTEN and ERG status in MRI-US fusion biopsies (FBx) and RP specimens, and assessed the predictive value of FBx on disease stage and grade at RP. Material &amp; Methods The study included patients who had undergone RP in Helsinki University Hospital between June 2015 and May 2017, and had preoperative FBx tissue available (n=110). Matched FBx specimens and tissue microarrays constructed from RP were immunostained with ERG and PTEN antibodies, and scored as either negative or positive. The RP TMA contained one to three clinically relevant cancer foci per patient. Results A high correlation of both PTEN and ERG status was observed between FBx and RP materials (Table 1). The correlation of PTEN status was particularly better compared with our previous findings in systematic 12-core biopsies (Lokman et al. 2017), where there was no significant concordance. High grade RP (grade group 4&amp;5) correlated with ERG negativity in FBx (p=0.021), whereas PTEN was lost in high stage RP (pT≥3) to a higher extent than in low stage RP (pT2, p= n.s). Higher FBx grade group was associated with stage increase in RP (p=0.014). Conclusions FBx is feasible method to assess ERG and PTEN status correctly in RP specimens. More validation studies, accompanied with MRI features, are warranted. Table 1A. Concordance of ERG IHC status in fusion biopsies (FBx) and radical prostatectomy (RP) specimens.RP ERG NegativeRP ERG PositiveP-valueFBx ERG Negative54 (90.0)6 (10.0)&amp;lt;0.001aFBx ERG Positive3 (7.1)39 (92.9)Table 1B. Concordance of PTEN IHC (Any PTEN Loss) status in FBx and RP specimens.RP Any PTEN LossRP PTEN IntactP-valueFBx Any PTEN Loss14 (73.7)5 (26.3)0.002aFBx PTEN Intact22 (32.4)46 (67.6)Table 1C. Concordance of PTEN IHC (Complete PTEN Loss) status in FBx and RP specimens.RP Complete PTEN LossRP PTEN IntactP-valueFBx Complete PTEN Loss6 (50.0)3 (50.0)&amp;lt;0.001aFBx PTEN Intact3 (4.0)72 (96.0)aFisher''s exact test. All p-values two-tailed. Citation Format: Andrew Erickson, Utku Lokman, Kanerva Lahdensuo, Sara Tornberg, Harri Visapää, Robin Bergroth, Henrikki Santti, Anssi Petas, Tuomas Mirtti, Antti Rannikko. PTEN and ERG expression in MRI-ultrasound guided fusion biopsy correlated with radical prostatectomy findings in men with prostate 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 4602.

Abstract 2582: A novel PI3K/Akt-pathway activation biomarker using comprehensive genomic profiling (CGP) for clinical trial assay

Abstract Introduction Patients with PI3K/Akt-pathway activation may be sensitive to selective Akt-inhibitors that are currently under development. We have developed a novel next-generation sequencing (NGS)-based composite biomarker assay that identifies patients with PI3K/Akt-pathway activated tumors by identifying activating PIK3CA and AKT1 alterations, and inactivating alterations in PTEN. This assay was analytically validated, and applied to triple-negative breast cancer (TNBC) patients in the LOTUS trial (NCT02162719), a placebo-controlled phase II clinical trial to assess the safety and efficacy of adding ipatasertib to paclitaxel treatment in patients with metastatic TNBC (Kim et al., 2017). Methods DNA extracted from FFPE tumor tissue underwent whole-genome shotgun library construction and hybridization-based capture, followed by sequencing using Illumina HiSeq 4000. Sequence data were processed using a proprietary analysis pipeline designed to detect base substitutions, indels, copy number alterations, genomic rearrangements, microsatellite instability, and tumor mutational burden. The assay further evaluated the PI3K/Akt-pathway activation biomarker status that consists of six features: 1-2) AKT1 and PIK3CA activating mutations, 3) PTEN homozygous deletion, 4) PTEN heterozygous deletion (HE), 5) PTEN dominant negative mutations, and 6) bi-allelically inactivated (BI) PTEN mutations defined as mutation plus loss of heterozygosity (LOH). We evaluated the limit of detection (LoD) and the precision of the biomarker for two novel genomic features: HE and BI, with the other four features previously validated. Results Analytical validation of novel biomarker features: The LoD of detecting PTEN HE and BI was determined to be 30%, the lowest tumor content at which the features can be detected at 90% probability. In the precision study, 100% (81/81) agreement was achieved across different replicates within the same sequencer run and across different sequencer runs for biomarker positive samples, demonstrating high reproducibility in calling PTEN HE and BI. Conclusions We developed and analytically validated an NGS-based assay that identifies complex and novel genomic alterations (heterozygous deletion and bi-allelic inactivation) in PTEN that is part of a composite PI3K/Akt-pathway activation biomarker. This assay identified patients that appeared to derive greater benefit in the Phase II LOTUS study as compared to using PTEN IHC to only identify patients with PTEN protein loss (Kim et al., 2017). This assay could be generalized to identify other biomarkers with similar types of genetic alterations. It also demonstrates that NGS-based CGP can broaden the intent to treat population to be more specifically related to the mechanism of action of a drug, while also being more selective to patients with potential to respond. Citation Format: Yuting He, Matthew Wongchenko, Joel Skoletsky, Christine Burns, Yali Li, Paula Maness, Doris Kim, Doron Lipson, Philip Stephens, Vincent Miller, Jeffrey Ross, Steven Gendreau, James Sun. A novel PI3K/Akt-pathway activation biomarker using comprehensive genomic profiling (CGP) for clinical trial assay [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 2582.

Abstract B080: Update on the NCI-Molecular Analysis for Therapy Choice (NCI-MATCH/EAY131) precision medicine trial

Abstract Purpose: NCI-Molecular Analysis for Therapy Choice (NCI-MATCH/EAY131), by ECOG-ACRIN (EA) and NCI, is the first national signal-finding trial to incorporate centralized NGS testing to direct patients (pts) to molecularly targeted parallel phase 2 treatment arms. We report status of accrual from opening on 08/12/15 thru 07/16/17 and future plans. Methods: Eligible pts have advanced/refractory solid tumors, lymphoma, or myeloma. Drug treatments and molecular targets require stringent levels of evidence (LOE). Each arm (N-35) has a phase 2 dose, a molecular abnormality believed to predict response, and evidence of clinical activity. Pts must have enrolled by 05/22/17 with intent to submit fresh tissue or recent clinical biopsy. Tumor cores are shipped to the EA Central Biorepository &amp; Pathology Facility at MD Anderson Cancer Center for evaluation of histopathology and % tumor; PTEN, MLH1, and MSH2 IHC; and RNA/DNA extraction. NGS is performed in one of 4 CLIA-accredited laboratories using the NCI-MATCH adapted Oncomine™ panel (Version 1.0; 143 genes with &amp;gt;4000 reported variants including SNVs, indels, amplifications, and gene fusions). When an actionable mutation of interest (aMOI) or relevant IHC result is identified, the pt is assigned to treatment by a custom-designed informatics system (MATCHbox). If &amp;gt;1 aMOI present, the pt is assigned by the variant with the highest LOE. Results: As of 07/16/17, 5963 tumors were screened for 30 treatment arms. The assay success rate is 93%; median turnaround is 15 days (from sample receipt to return of results). 38.2% of pts have common cancers: colorectum (15.4%), breast (12.8%), lung (7.4%), and prostate (2.6%); 61.8% have less common tumors. The current overall match rate to aMOI’s is 18% (95% CI 17%, 19%); aMOI prevalence rates range from 3.47% to zero. The match rate also varies across tumor types: &amp;lt;10% in pancreatic &amp; SCLC; &amp;gt;30% in bladder/urinary tract, uterine, and head/neck cancer. 998 pts have been assigned to Rx; 69% have enrolled. Of 30 arms, 8 have enrolled ≥ 35 pts; some arms with higher prevalence rates have been expanded to N=70 to accommodate pts with matching aMOIs. Arms with less frequent aMOIs will not complete accrual within the 6,000-pt central screening goal, leading to identification of pts for screening based on approved high-volume NGS labs’ assay results, verified centrally. Conclusions: NCI-MATCH screened ~6000 pts at a rate that far exceeded expectations, and with acceptable toxicity; NGS was successful in 93%, well above the industry average of ~80%. The pace of enrollment, along with the trial’s availability at 1100+ sites, reflects the broad interest in the promise of genomics and the ability of such a trial to deliver on that promise. Lower accrual to "rare variant" arms led to use of high-volume NGS laboratories to complete the study. Follow-up will determine whether matching drugs to molecular targets results in meaningful response rates and improved patient outcome. Citation Format: Lyndsay Harris, Alice Chen, Peter O'Dwyer, Keith Flaherty, Stanley Hamilton, Lisa McShane, Robert Gray, Shuli Li, Edith Mitchell, Diane Dragaud, Mickey Williams, Jeffrey Sklar, A. John Iafrate, David Patton, Richard F. Little, James Zweibel, Jeffrey Abrams, James Doroshow, Barbara Conley. Update on the NCI-Molecular Analysis for Therapy Choice (NCI-MATCH/EAY131) precision medicine trial [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B080.

Characterization of PD-L1 expression in Chinese non-small cell lung cancer patients with PTEN expression as a means for tissue quality screening.

The goal of this study is to evaluate PD-L1 prevalence and its association with major clinical characteristics in Chinese non-small cell lung cancer (NSCLC) patients to inform the clinical development of anti-PD1/PD-L1 agents in this population. We used phosphatase and tensin homolog (PTEN) expression through IHC as a surrogate tissue quality marker to screen surgical NSCLC samples in tissue microarray (TMA; 172 cases) or whole-section (268 cases) format. The samples were then analyzed with a clinically validated PD-L1 IHC assay. The results were correlated with baseline characteristics and clinical outcomes. PTEN IHC showed that 108 TMA samples and 105 whole-section samples qualified for PD-L1 IHC. With a clinically relevant cutoff, 41.7% of the TMA samples were PD-L1 positive. PD-L1 level was much lower in EGFR-mutant patients and seemed to be a favorable prognostic factor for both overall survival (OS) and recurrence-free survival (RFS). These findings were confirmed in the whole-section samples except that their survival data were not mature enough for correlation analysis. In summary, PD-L1 expression was detected in approximately 40% of PTEN-qualified Chinese NSCLC samples, negatively correlated with EGFR mutation and seemed to be a favorable prognostic factor for both OS and RFS. Notably, the different results from PTEN-qualified and PTEN-disqualified samples underscore the importance of tissue quality control prior to biomarker testing.

59P Characterization of PD-L1 expression in Chinese non-small cell lung cancer patients with PTEN IHC as a means for sample quality screening

59P Characterization of PD-L1 expression in Chinese non-small cell lung cancer patients with PTEN IHC as a means for sample quality screening

59P Characterization of PD-L1 expression in Chinese non-small cell lung cancer patients with PTEN IHC as a means for sample quality screening

59P Characterization of PD-L1 expression in Chinese non-small cell lung cancer patients with PTEN IHC as a means for sample quality screening

Abstract P2-08-13: Integrating multiplex and next generation sequencing (NGS) platforms in routine molecular profiling of metastatic breast cancer (MBC) patients (pts): Trends for enrollment in genotype-directed clinical trials (GDTs)

Abstract Background/aims: Multiplex or NGS platforms increase the number of mutations (mut) detected in tumor samples respect to single-gene sequencing techniques. We aimed to assess the actionable molecular alteration (ActMA) detection rate and the enrollment in GDTs derived from the integration of these platforms in routine molecular profiling of MBC pts, in addition to FISH and IHC techniques already in use. Methods: Consecutive MBC pts screened for gene mut by Sequenom (Seq) or AmpliconSeq (ASeq) were identified. Data on FGFR1 amplification (amp), PTEN IHC, and enrollment in GDTs were collected. ActMA: any mut, PTENnull (IHC score=0), or FGFR1/HER2amp for which a matched targeted drug might be available. Targeted therapy: treatment with PI3K/mTOR, novel anti-HER2, FGFR, or AKT inhibitors (inh) irrespective of having ActMA. GDT: treatment matched to ActMA. Results: From Oct2010-Apr2015, 260 pts screened (Seq 207, ASeq 53). IHC subtype: HR+/HER2- (LUM) 65%, HER2+ 13.5%, TN 19.6%, unk 1.9%. 84 samples from a metastatic site (32.3%). ActMA / n (%)LUMHER2+TNP value (Fisher's exact test)TotalTP53*11 (31.4)1 (50)9 (52.9)0.3421 (38.9)PIK3CA44 (26)10 (28.6)4 (7.8)0.158 (22.7)FGFR1amp21 (17.4)1 (5.3)6 (16.7)0.4728 (15.9)PTENnull12 (9.3)2 (7.7)9 (25)0.0323 (12)AKT110 (5.9)1 (2.9)-0.1511 (4.3)ERBB23 (1.8)---3 (1.2)EGFR1 (0.6)-2 (4.1)-3 (1.2)ESR1*1 (3)---1 (2)KRAS2 (1.2)---2 (0.8)Denominators vary according to platform. *Amplicon only Proportion of PIK3CAmut was similar irrespective of the site of analysis (primary 25.5%, metastasis 21.4%; P=0.63) and platform (Seq 22.2%, ASeq 24.5%, P=0.72). ASeq detected more mutations in actionable genes than Seq (36% vs. 29%, P=0.01). At least 1 ActMA (range 0-3) was found in 53.5% of pts, with non-significant differences in HER2- subtypes (LUM 48.5% vs. TN 39.2%, P=0.32). Subtype* / ActMA n (%)≥10123All139 (53.5)121 (46.5)111 (42.7)25 (9.6)3 (1.2)LUM82 (48.5)87 (51.5)71 (42)9 (5.3)2 (1.2)HER2+35 (100)-22 (62.9)12 (34.3)1 (28)TN20 (39.2)31 (60.8)16 (31.4)4 (7.8)-*5 pts with unk subtype not shown Pts with ≥2 ActMA (excluding HER2amp): 11 LUM (interestingly, 3 pts with PIK3CAmut+FGFR1amp), 1 HER2+, and 4 TN. Overall, 56% of pts received ≥1 targeted therapy (range 0-4). From the 139 pts with ≥1 potential ActMA (including HER2amp if treated with a novel anti-HER2), 61.8% received a targeted therapy and 42.4% were enrolled in a GDT: PI3K/mTOR inhibitor (inh) 54 (64.3%), novel anti-HER2 16 (19.1%), FGFR inh 8 (9.5%), AKT inh 6 (7.1%). Of the 121 pts that did not have potentially ActMA, 50% received a targeted therapy. The OR for receiving targeted therapy if ActMA was present was 1.59 (95%CI 0.94-2.70, P=0.08). Conclusion: Integration of multiplex and NGS platforms in routine molecular profiling of MBC pts yields a detection rate of ActMA &amp;gt;50%, which translates into higher probability of receiving a targeted agent and enrollment in a GDT. This suggests that physicians are pushing towards matched targeted therapies for pts that participate in molecular screening programs and have ActMA. Results on the outcome of these pts will be presented. Citation Format: Oliveira M, Dienstmann R, Bellet M, Pérez-Garcia JM, Gómez P, Muñoz-Couselo E, Vidal M, Ortega V, Zamora E, Soberino J, Meire A, Nuciforo P, Vivancos A, Cortés J, Saura C. Integrating multiplex and next generation sequencing (NGS) platforms in routine molecular profiling of metastatic breast cancer (MBC) patients (pts): Trends for enrollment in genotype-directed clinical trials (GDTs). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-08-13.

Histopathological features of intra-ductal carcinoma of prostatic and high grade prostatic intraepithelialneoplasia and correlation with PTEN and P63.

The main morphologic differential diagnosis of intra-ductal carcinoma of prostate (IDC-P) is high grade prostatic intraepithelialneoplasia (HGPIN). Since IDC-P, unlike PIN, was strongly correlated with aggressive prostate cancer, differentiation of these is too necessary. So we evaluated immunohistopathological patterns and the prognostic factors of IDC-P and HGPIN, in radical prostatectomy samples. We evaluated 250 radical prostatectomy and detected 210 cases of prostatic adenocarcinoma without IDC-P foci, 40 cases with adenocarcinoma concomitant IDC-P, and 40 cases HGPIN; therefore, we evaluated immunohistopathological criteria in these groups. Data were analyzed using SPSS and P-value <0.05 was considered as the statistical significant level. PSA level was significantly higher in IDC-P compared with non-IDC-P patients (15.7 ± 3.1 vs. 10.2 ± 4.3, P = 0.041). All pathological and morphologic features, also invasions factors were higher in IDC-P compared to non-IDC-P groups (P < 0.001). P63 was positive expressed in all IDC-P and HGPIN specimen. PTEN protein was diffusely expressed in the cytoplasm of all HGPIN but in 4 (11.1%) of IDC-P. PTEN and P63 were negative in adenocarcinoma foci. We found that IDC-P had a unique histoclinical feature and was strongly associated with poor prognostic factors. Diagnosis and report of IDC-P should be considered in all prostate specimens. Also, we recommend PTEN IHC application for differentiated IDC-P from HGPIN in biopsies.

MP1-19 A NOVEL LIVE CELL MICROFLUIDIC DIAGNOSTIC USING PHENOTYPIC BIOMARKERS WITH OBJECTIVE ALGORITHMIC ANALYSIS FOR PROSTATE CANCER RISK STRATIFICATION.

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.

Abstract 3864: A transition zone showing highly discontinuous or rapidly oscillating levels of stem cell and proliferation markers characterizes the development of colorectal cancer

Abstract Background: Stepwise acquisition of genetic changes characterize the development of colorectal cancer (CRC). These mutational events are correlated with discrete morphologic transitions from hyperplastic to adenomatous areas followed by in situ transformation and finally invasive carcinoma. The genetic drivers for each stage of this process, however, have not been fully established and can be confounded by tumor heterogeneity. The goal of this study was to identify easily assessed biomarkers of key morphogenetic transitions in CRC. Methods and Findings: We analyzed the pattern of proliferation and expression of growth regulatory and stem cell markers across distinct morphologic transition zones in 726 cases of CRC. In cases with preserved adenoma-carcinoma morphologic transitions, we identified a characteristic zone of adenomatous epithelium, often located immediately adjacent to and extending into the invasive component, that showed rapidly oscillating intraglandular stretches of Ki-67+ and Ki-67- cells. This pattern correlated with oscillating expression of other cell cycle mediators and the growth regulators PTEN and SMAD4. These multifocal stretches of adenomatous epithelium showed alternating abrupt positive/negative expression boundaries. These zones also demonstrated similar abrupt intragland oscillations in the levels and/or subcellular localization of multiple cancer stem cell (CSC) markers including beta-catenin/CTNNB1, MGMT and CD44. In contrast, the expression levels of most of these markers were largely homogenous in the proximal adenomatous and deeper invasive carcinoma surrounding the transitional region. This CSC-like transitional zone, as detected by PTEN IHC, was prominent in 50/726 of all CRC (6.9%) but at least focally present in 97/201 (48.2%) cases with intact adenoma-carcinoma junctions. Genomic microarray (OncoScan HD) and mutation analysis (Ampliseq) on CRC with prominent CSC-like expansions demonstrated complex genomic changes in 12/18 (66.7%) with a similar frequency of KRAS, BRAF and CTNNB1 mutations as expected in unselected CRC cases. The transition zones in these cases also frequently demonstrated unstable genomes from cell-to-cell (as assessed by FISH) indicating high genetic instability in these areas. Conclusions: We show that multiple immunohistochemical markers, including PTEN, SMAD4, CD44 and CTNNB1, highlight a localized CSC-like transition zone of rapidly alternating quiescent/proliferative adenomatous epithelium in primary CRC tumors. This transition zone often begins in pre-invasive adenomatous epithelium adjacent to invasive tumor areas that have more uniform proliferation and more homogeneous genetic and expression profiles. This easily assessed phenomenon appears to represent a commonly occurring genetically unstable forerunner or transitional stage in CRC evolution. Citation Format: Kevin J. Arvai, Ya-Hsuan Hsu, Lobin A. Lee, Dan Jones. A transition zone showing highly discontinuous or rapidly oscillating levels of stem cell and proliferation markers characterizes the development of colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3864. doi:10.1158/1538-7445.AM2014-3864

Abstract 4819: Evaluation of PTEN status in circulating tumor cells (CTCs) and matched tumor tissue from castrate-resistant prostate cancer (CRPC) patients

Abstract Background: PTEN loss occurs frequently in prostate cancer and may trigger progression to CRPC through activation of the PI3K/AKT pathway. A blood-based assay that determines PTEN status in CRPC patients could enable informed treatment decisions such as the use of a PI3K-targeted therapy. Here we examined the relationship between PTEN status in CTCs and matched archival and fresh tumor biopsies in 43 CRPC patients. Methods: Nucleated cells from CRPC patient blood were plated onto glass slides and subjected to IF staining and CTC identification by high-speed fluorescent scanners at Epic Sciences. CTCs were identified as CK+/CD45- cells with intact DAPI nuclei, and samples with ≥4 CTCs per 2 slides (74%) were then tested for PTEN by FISH. Heterozygous loss was defined as a decrease in PTEN copies (PTEN &amp;lt; CEP10 and &amp;lt; 2 copies) and homozygous loss as zero PTEN copies. PTEN IHC in tissue was stained using CST clone 138G6 and H-scores ≤ 200 counted as loss. Results: Heterozygous or homozygous loss of PTEN by FISH was observed in 16 of 43 patients (37%) by CTC analysis. In addition to loss of PTEN, changes in ploidy were frequently observed and broad heterogeneity seen both within and between patients. The PTEN status in CTCs correlated strongly with the PTEN status in metastatic tissue: All 10 patients that exhibited homozygous PTEN loss in CTCs showed concordant homozygous PTEN loss in fresh biopsies. This correlation extended to three patients that showed correlated mixed homozygous and hemizygous PTEN loss populations in CTCs and tissue. “Drift or change” in PTEN status from archival to fresh biopsies occurred in 15 patients, In 12 of these patients, PTEN status in CTCs was reflective of the status in fresh tissue. Together with PTEN evaluation, the analysis of androgen receptor expression by immunofluorescence and ERG rearrangements by FISH proved feasibility of multiplex biomarker assessment in CTCs. This analysis demonstrated heterogeneity of AR phenotypes and a positive association of ERG rearrangements and PTEN loss in CTCs, consistent with literature reports. Conclusion: Our results illustrate the potential for using CTCs as a non-invasive, real-time biopsy to determine a patient's current PTEN status. PTEN status will be determined using these assays in an ongoing AKT inhibitor Phase II trial. Citation Format: Elizabeth Punnoose, Eric Tucker, Edith Szafer-Glusman, Jin Zhu, Dena Marrinucci, Jessica Louw, Florence Lee, Mikel Kitchen, Natalee Bales, Lukas Amler, Hartmut Koeppen, Premal Patel, Yibing Yan, Ruth Riisnaes, Gerhardt Attard, Johann de Bono. Evaluation of PTEN status in circulating tumor cells (CTCs) and matched tumor tissue from castrate-resistant prostate cancer (CRPC) patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4819. doi:10.1158/1538-7445.AM2014-4819

Abstract PD1-2: PIK3CA mutations and/or low PTEN predict resistance to combined anti-HER2 therapy with lapatinib and trastuzumab and without chemotherapy in TBCRC006, a neoadjuvant trial of HER2-positive breast cancer patients

Abstract We have recently reported that in patients with HER2-positive breast cancer, neoadjuvant targeted therapy with lapatinib and trastuzumab to more completely block the HER receptor layer, combined with endocrine therapy (in ER-positive tumors) and without chemotherapy led to a substantial 27% pathologic complete response (pCR) rate in the breast. Activation of downstream signaling pathways may lead to resistance to therapies targeting the HER pathway receptors. Aberrant activation of the PI3K pathway via decreased levels of PTEN and/or the presence of activating PIK3CA mutations has been implicated in resistance to targeted anti-HER2 therapy, but results of clinical trials are all confounded by the co-administration of chemotherapy and are inconsistent. We sought to clarify the role of these variables in predicting pCR, a surrogate for long-term outcome, in patients treated with potent targeted therapy alone in a prospective Phase II neoadjuvant trial in patients with HER2-positive breast cancer. Patients with large tumors (median 6 cm) were given 12 weeks of lapatinib plus trastuzumab followed by surgery (Rimawi et al. JCO, 2013). Serial tissue biopsies were obtained from study participants. For this study, we focused on baseline pre-treatment characteristics. PTEN protein levels were measured by IHC and scored using the H-score. PIK3CA mutations were identified on extracted DNA using multiplex PCR with targeted next generation sequencing (the Ion Torrent 50-gene cancer mutation panel). Of 64 evaluable patients, tissue was available on 59 for PTEN IHC, and sufficient DNA was available on 33 for the mutation panel. PTEN median H-score was 100 (range 0-300). PTEN status when dichotomized by the median was correlated with pCR (32% in high PTEN vs. 9% in low PTEN, p = 0.04). Activating PIK3CA mutations were identified in 12 out of 33 tumors (36%; 3 mutations in the helical and 9 in the catalytic domain) and were independent of ER status. None of the patients whose tumors harbored a PIK3CA mutation achieved pCR (p = 0.06). There was no association between PTEN status and PIK3CA mutation suggesting they are independent variables (p = 0.44). When PIK3CA mutations were considered together with PTEN status, there were 31 cases with data on both. The overall pCR rate in this cohort was 16% (lower than pCR rate observed in the overall trial). However, 0/17 cases (0%) with a mutation and/or PTEN low expression (&amp;lt;100 H score) had a pCR compared to 5/14 cases (36%) with PI3KCA wild type and high PTEN levels (p = 0.01). We conclude that PI3K pathway activation downstream of HER2 as a result of either low PTEN or activating PIK3CA mutation results in resistance to the combination of lapatinib and trastuzumab. This is the first report on patient tissue samples from a neoadjuvant trial using the combination of lapatinib and trastuzumab without chemotherapy. If validated in a larger cohort, our findings suggest that patients with HER2 positive tumors and who also harbor aberrant downstream PI3K pathway activation may benefit from the addition of PI3K/Akt/mTOR inhibitors to potent HER2 blockade. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD1-2.

Abstract A45: Biomarker assay performance: The impact of the fixation method.

Abstract Recent advances in genomic technologies dramatically illustrated the complexity of cancer genomes. These discoveries provide new opportunities for drug development and ultimately better patient care by informing treatment choice decisions in addition to the insights into the mechanisms of response or relapse to therapy. Formalin fixed paraffin embedded (FFPE) tumor tissue specimens routinely collected in Oncology diagnostic practice present a number of challenges for performing molecular biomarker assays. These problems include chemical cross-linking RNA/DNA and protein, nucleic acid fragmentation, and reduced amount of extractable material. We evaluated a new fixative agent (PAXgene, Qiagen) to assess whether an alternative fixation method could alleviate problems presented by the FFPE specimens. PAXgene preservation method was compared to FFPE and frozen tissue utilizing tumor material from xenograft and primary tumor mouse models. We compared the performance of the following techniques across three types of specimens: allele-discrimination PCR, gene expression profiling by QPCR, CNV (copy number variation) analysis, and IHC. DNA and RNA based assay performance including allele-specific PCR, gene expression profiling and CNV demonstrated similar results between frozen and PAXgene tumor specimens. Comparison of transcriptional profiling data between all there type of specimens demonstrated artifacts likely related to the formalin fixation process. Additionally, DNA extracted from PAXgene specimens had higher amount of amplifiable templates compared to DNA extracted from the FFPE specimens. Tissue sections from PAXgene fixed specimens exhibited preserved tissue morphology of acceptable quality. We carried out pAKT and PTEN IHC assays in PAXgene and FFPE tissue sections. The results demonstrated superior performance for pAKT in the PAXgene format. In the case of PTEN IHC assay, the standard FFPE based IHC assay protocol required substantial modifications. Collecting tumor biopsies in the PAXgene format provides an opportunity of conducting both molecular and tissue based biomarker assays from a single tumor specimen. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A45. Citation Format: Sunita Badola. Biomarker assay performance: The impact of the fixation method. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A45.

Prognostic value of PTEN loss in men with conservatively managed localised prostate cancer

Background:The natural history of prostate cancer is highly variable and difficult to predict. We report on the prognostic value of phosphatase and tensin homologue (PTEN) loss in a cohort of 675 men with conservatively managed prostate cancer diagnosed by transurethral resection of the prostate.Methods:The PTEN status was assayed by immunohistochemistry (PTEN IHC) and fluorescent in situ hybridisation (PTEN FISH). The primary end point was death from prostate cancer.Results:The PTEN IHC loss was observed in 18% cases. This was significantly associated with prostate cancer death in univariate analysis (hazard ratio (HR)=3.51; 95% CI 2.60–4.73; P=3.1 × 10−14). It was highly predictive of prostate cancer death in the 50% of patients with a low risk score based on Gleason score, PSA, Ki-67 and extent of disease (HR=7.4; 95% CI 2.2–24.6; P=0.012) ), but had no prognostic value in the higher risk patients. The PTEN FISH loss was only weakly associated with PTEN IHC loss (κ=0.5). Both PTEN FISH loss and amplification were univariately predictive of death from prostate cancer, but this was not maintained in the multivariate analyses.Conclusion:In low-risk patients, PTEN IHC loss adds prognostic value to Gleason score, PSA, Ki-67 and extent of disease.

Phase II, two-stage, two-arm, PIK3CA mutation stratified trial of MK-2206 in recurrent endometrial cancer (EC).

5524 Background: EC has high rates of PI3K pathway alteration including PTEN mutation (50%) or IHC loss (&gt;50%), PIK3CA mutation (25-40%) and PIK3R1 (20%) mutation. MK-2206 is an allosteric inhibitor of AKT, an effector kinase of PI3K signals. We hypothesized that pts whose tumors harbored PIK3CA mutations would be more likely to benefit from MK-2206 than those without PIK3CA mutation. Methods: Pts had recurrent or advanced EC; all histologies except MMMT were eligible. Up to 2 prior chemo lines were permitted; excluding prior treatment with PI3K/MTOR inhibitors. The first 19 pts were treated with MK-2206 200mg QW; due to initial skin toxicity rates, the starting dose was amended to 135mg QW. Co-primary endpoints were objective response and 6 mo PFS. The first 37 pts were stratified retrospectively. PIK3CA MT included R88Q, K111N, E110K, E418K, C420R, E453K, E542K/V,E545K, Q546R, H701P, M1043V, H1047R/L/Y changes. Independent Simon 2-stage tests were planned within PIK3CA MT and WT stratum: for MT, n1=15 and n2=10 pts would allow discrimination of RR&lt;5% and 6moPFS&lt;10% versus RR&gt;25% or 6moPFS&gt;35%; for WT, n1=31 and n2=24 pts would discriminate RR&lt;5% and 6moPFS&lt;10% versus RR&gt;20% or 6moPFS&gt;25%. Results: 37 pts were enrolled (1 ineligible) before accrual was stopped as timely CLIA-compliant prospective mutation analysis was not feasible. By PIK3CA mutation analysis, 9 pts were MT: 1 pt had both PR and 6moPFS. 27 pts were WT: 1 pt had PR and 3 pts had 6moPFS. 2 pts with 6moPFS were treated at 200mg; 2 pts with 6moPFS were treated at 135mg. Each group had 1 PR. The most common toxicity was grade 3 rash (19%). Grade 3 and 4 toxicities occurred in 50% and 8% of pts. Exploratory analysis of histology found all pts with 6moPFS were classified as serous (4 of 8) as compared to all other histologies (0 of 28, p=.001). Targeted exome sequencing and copy number analysis of the PI3K pathway and PTEN IHC are underway. Associative studies will be reported. Conclusions: There is limited single agent activity of MK-2206 in both PIK3CA MT and PIK3CA WT EC populations. Activity was detected in pts with serous histology tumors and warrants further study. Clinical trial information: NCT01307631. [Table: see text]