Paraffin-embedded Tumor Specimens Research Articles

PIK3CA alterations and benefit with neratinib: analysis from the randomized, double-blind, placebo-controlled, phase III ExteNET trial

BackgroundNeratinib is an irreversible pan-HER tyrosine kinase inhibitor that inhibits PI3K/Akt and MAPK signaling pathways after HER2 receptor activation. The ExteNET study showed that neratinib significantly improved 5-year invasive disease-free survival (iDFS) in women who completed trastuzumab-based adjuvant therapy for early breast cancer (EBC). We assessed the prognostic and predictive significance of PIK3CA alterations in patients in ExteNET.MethodsParticipants were women aged ≥ 18 years (≥ 20 years in Japan) with stage 1–3c (modified to stage 2–3c in February 2010) operable breast cancer, who had completed (neo)adjuvant chemotherapy plus trastuzumab ≤ 2 years before randomization, with no evidence of disease recurrence or metastatic disease at study entry. Patients were randomized to oral neratinib 240 mg/day or placebo for 1 year. Formalin-fixed, paraffin-embedded primary tumor specimens underwent polymerase chain reaction (PCR) PIK3CA testing for two hotspot mutations in exon 9, one hot-spot mutation in exon 20, and fluorescence in situ hybridization (FISH) analysis for PIK3CA amplification. The primary endpoint (iDFS) was tested with log-rank test and hazard ratios (HRs) estimated using Cox proportional-hazards models.ResultsAmong the intent-to-treat population (n = 2840), tumor specimens were available for PCR testing (991 patients) and PIK3CA FISH (702 patients). Overall, 262 samples were PIK3CA altered: 201 were mutated (77%), 52 (20%) were amplified, and 9 (3%) were mutated and amplified. iDFS was non-significantly worse in placebo-treated patients with altered vs wild-type PIK3CA (HR 1.34; 95% CI 0.72–2.50; P = 0.357). Neratinib’s effect over placebo was significant in patients with PIK3CA-altered tumors (HR 0.41; 95% CI 0.17–0.90, P = 0.028) but not PIK3CA wild-type tumors (HR 0.72; 95% CI 0.36–1.41; P = 0.34). The interaction test was non-significant (P = 0.309).ConclusionsAlthough there was a greater absolute risk reduction associated with neratinib treatment of patients with PIK3CA-altered tumors in ExteNET, current data do not support PIK3CA alteration as a predictive biomarker of response to neratinib in HER2-positive EBC.Trial registrationClinicalTrials.gov, NCT00878709. Trial registered April 9, 2009.

Targeted sequencing of a specific gene panel detects a high frequency of ARID1A and PIK3CA mutations in ovarian clear cell carcinoma

BackgroundThe objective of this study was to assess the mutational profile in epithelial ovarian cancer using formalin-fixed, paraffin-embedded (FFPE) tumor specimens from a Taiwanese population by performing targeted sequencing of 9 cancer-associated genes. MethodsTargeted sequencing was performed on 32 formalin-fixed, paraffin embedded (FFPE) tumor specimens, consisting of matched samples from 16 epithelial ovarian cancer patients. Genetic alterations in the 9 cancer-associated genes were detected using a deep sequencing (>1000×) approach. ResultsARID1A and PIK3CA were the most frequently mutated genes. Specifically, ARID1A mutations and PIK3CA mutations were detected in 77.8% and 66.7% of ovarian clear cell carcinoma patients, respectively. Mutations in other genes, including MLH1 (6.3%) and CREBBP (6.3%), were detected in the Taiwanese population. We also identified coexisting ARID1A-PIK3CA mutations (43.8%) and ARID1A-KRAS mutations (12.5%) in tumors. It should also be noted that we identified the presence of three coexisting mutations, the ARID1A-KRAS-PIK3CA mutations and the ARID1A-CREBBP-PIK3CA mutations. ConclusionsIn summary, we identified novel genetic alterations in patients with epithelial ovarian carcinoma (EOC) in a Taiwanese populations. Further studies are needed to elucidate the mechanism of chromatin remodeling to examine the role of the PI3K/AKT pathway, to determine the critical roles of these mechanisms in tumor development and the progression of ovarian malignancy and to investigate new targeted therapies. Overall, our findings were reliable and are worthy of further study.

PTK2 promotes cancer stem cell traits in hepatocellular carcinoma by activating Wnt/β-catenin signaling

Emerging evidence indicates that cancer stem cells (CSCs) are involved in tumorigenesis, tumor recurrence, and therapeutic resistance in hepatocellular carcinoma (HCC). However, the mechanisms underlying HCC CSC regulation remain largely unknown. Here we report our analysis of 97 paraffin-embedded HCC tumor specimens. We found that protein tyrosine kinase 2 (PTK2) expression correlated with liver CSC marker expression, overall survival, and recurrence-free survival in HCC patients. Our results further showed that PTK2 activated Wnt/β-catenin signaling by promoting nuclear accumulation of β-catenin in HCC cells. In this manner, PTK2 activates CSC traits and drives tumorigenicity in HCC cells, leading to HCC recurrence and sorafenib resistance. Moreover, PTK2 expression was negatively correlated with its level of promoter methylation. PTK2 apparently acts as an oncogene by increasing CSC traits and tumorigenicity in HCC. The present data suggest that PTK2 may be a novel prognostic biomarker for HCC recurrence, and a therapeutic target for HCC treatment.

Next generation sequencing of primary prostate cancer tumors to reveal potentially actionable opportunities for clinical management: The UNC experience.

305 Background: The Strata trial (NCT03061305) is a multi-institutional precision oncology collaboration structured as an observational protocol that aims to match patients to genomically-guided therapies. Methods: Selected University of North Carolina (UNC) metastatic prostate cancer (mPC) patients were enrolled on this IRB-approved study. Formalin fixed paraffin-embedded primary tumor specimens, without matched germline controls, were sent for targeted next generation sequencing (NGS) to detect actionable variants, including: mutations in 87 genes, copy number variations in 31 genes, and gene fusions in 46 gene drivers. mPC-related genes of particular interest included: AR, ATM, BRCA1/2, ERG, MSH2, MSH6, PTEN, RB1, and TP53. Results: Of the 92 cases sequenced, 5 [5%] failed testing. Of the 87 mPC patients (median age 69 years [47-86]) enrolled: 53 [61%] were white, 28 [32%] were black, 1 [1%] was Asian, and 5 [6%] declined to be identified. NGS data revealed 106 variants in 27 genes: 62 patients (71%) had at least one variant, 21 (24%) had 2 variants, 7 (8%) had 3 variants, and 4 (3%) had 4 variants. Among the 62 patients with at least 1 identified variant, TMPRSS2-ERG fusion occurred most frequently (50%), followed by TP53 (40%), and PTEN (16%). 6% of all sequenced patients had variants in DNA damage repair genes including ATM (3%), BRCA2 (2%) and MSH2 (1%). One patient had a SLC45A3-ERG fusion combined with PTEN deep deletion, which has been associated with a more aggressive phenotype. One patient with a microsatellite-instability high tumor was treated with pembrolizumab. Conclusions: The UNC experience shows that a high proportion of primary prostate cancer tumors from mPC patients have genomic variants, and one patient was treated based on these data. Limited actionability may reflect the landscape of currently FDA approved mPC treatments, and available clinical trials. It may also be due to a short follow-up, and these data could inform treatment planning upon progression.

Eukaryotic translation initiation factor 5A2 is highly expressed in prostate cancer and predicts poor prognosis.

Eukaryotic translation initiation factor (EIF) 5A2 exerts important functions that regulate the development and progression of cancers. The present study aimed to investigate the expression of EIF5A2 in prostate cancer (PCa) and its association with biological and prognostic significance. EIF5A2 mRNA and protein levels were analyzed in three paired samples of freshly resected PCa and adjacent non-tumor tissues. Immunohistochemical staining was used to detect the expression of EIF5A2 protein levels in 72 paraffin-embedded PCa tumor specimens. Subsequently, the association between EIF5A2 protein expression and clinicopathological parameters was assessed. Semi-quantitative reverse transcription-polymerase chain reaction and western blot analyses showed both EIF5A2 mRNA and protein levels were elevated in PCa compared with adjacent non-tumor tissues. Elevated EIF5A2 protein levels were observed in 73.6% (53/72) of the clinical PCa tissues using immunohistochemical staining. EIF5A2 expression was significantly associated with tumor stage (P=0.011) and biochemical recurrence status (P=0.032). Additionally, high levels of EIF5A2 predicted worse progression-free survival (P=0.007). Multivariate Cox regression analysis indicated that high expression of EIF5A2 was an independent prognostic factor for poor progression-free survival (hazard ratios, 0.366; 95% confidence interval, 0.349-0.460; P=0.021). The present study demonstrated that EIF5A2 is overexpressed in prostate cancer and may be a potential predictor and therapeutic target in PCa patients.

Comprehensive Analysis of Somatic Mutations in Colorectal Cancer With Peritoneal Metastasis

To analyze for genetic mutations which may presage peritoneal metastasis by using targeted next-generation sequencing (NGS). Formalin-fixed, paraffin-embedded primary tumor specimens were obtained from 10 patients with small obstructing colorectal cancer and peritoneal metastasis (group A) and five with large non-obstructing colorectal cancer and no recurrence (group B). DNA was extracted for the sequencing of 409 cancer genes. The distribution of genetic mutations was compared between the two groups to find genetic mutations related to peritoneal metastasis. When the samples were sorted based on similarity of gene expression by hierarchical clustering analysis, the samples were well divided between the two study groups. Mutations in AT-rich interactive domain-containing protein 1A (ARID1A), polycystic kidney and hepatic disease 1 (PKHD1), ubiquitin-protein ligase E3 component n-recognin 5 (UBR5), paired box 5 (PAX5), tumor protein p53 (TP53), additional sex combs like 1 (ASXL1) and androgen receptor (AR) genes were detected more frequently in group A. A number of somatic mutations presumed to be relevant to colorectal cancer with peritoneal metastasis were identified in our study by NGS.

Expression of CD117, MITF and NAT10 and their prognostic values in sinonasal mucosal melanoma

Objective: To investigate the correlation between the expression of CD117, MITF, NAT10 and clinical parameters in sinonasal mucosal melanoma (SNMM). Methods: Formalin-fixed paraffin-embedded tumor specimens of 80 cases of SNMM at the Eye, Ear, Nose and Throat Hospital, Fudan University, from December 1999 to November 2013 were analyzed for CD117, MITF and NAT10 expression by immunohistochemistry. Results: There were 40 men and 40 women. The median age was 61 years, age 26 to 85 years. There was no correlation of the expression of CD117, MITF and NAT10 with the patients' age, gender, tumor site, stage, therapy method and brain metastases (P>0.05). The expression of MITF and NAT10 was associated with lymph node metastasis and the tumors were more likely to metastasize when MITF and NAT10 were positive. However, expression of CD117 had no correlation with lymph node metastasis. Log-rank test revealed that the expression of CD117 was correlated with both three-year and five survival rate (P=0.012, P=0.023; respectively) and patients with tumor having low expression of CD117 had the worse outcome. COX test revealed that low CD117 expression, advanced age and lymph node metastasis were independent risk factors (P<0.05). No significant association was found between the expression of CD117, MITF and NAT10 with disease free survival (P>0.05). Conclusions: Patients with SNMM expressing low level of CD117 have decreased survival rate. Tumors with high level of MITF and NAT10 expression are more likely to metastasize. The expression level of CD117 can be used as an important indicator for the patient survival, and the expression of MITF and NAT10 can be used as a predictor of tumor metastasis.

KRAS mutation and DNA repair and synthesis genes in non-small-cell lung cancer.

The aim of the present study was to assess the expression of select DNA repair and synthesis genes in non-small-cell lung cancer (NSCLC) according to KRAS mutation status. ERCC1, TS, RRM1, and BRCA1 mRNA expression levels were assessed from either primary or metastatic tumor specimens of patients diagnosed with epidermal growth factor receptor (EGFR) wild-type (WT) advanced NSCLC. Total RNA was isolated from paraffin-embedded tumor specimens using the RNeasy FFPE kit and automatically purified using a QiaCube instrument. Quantification levels were analyzed by real-time one-step RT-PCR using QuantiFast technology, and the results were compared considering β-actin as the internal reference gene. One hundred and eighty-four patients with advanced NSCLC were evaluated for the analysis, of which 92 were KRAS-mutants. Nearly all patients had adenocarcinoma histology (96.7%). Among KRAS-mutants, the majority had a KRAS codon 12 mutation (88%), the most common being G12C (44.4% of cases). Mean ERCC1 levels were indicated to be significantly higher in KRAS-mutants when compared with KRAS WT patients (3,234±6.63 vs. 184±1.24; P=0.05). However, mean TS levels were significantly lower in the KRAS-mutant subgroup compared with the KRAS WT subgroup (4,481±3.756 vs. 5,941±6.4; P=0.039). KRAS-mutant NSCLCs are more likely to express high ERCC1 and low TS levels. This finding may suggest different sensitivity to cytotoxic chemotherapy according to KRAS mutation status.

Abstract A20: Analysis of tumor infiltrating lymphocytes and expression of PD1 and PD-L1 in breast tumors prior to and after neoadjuvant chemotherapy

Abstract Introduction: The effect of chemotherapy on the presence of tumor-infiltrating lymphocytes (TILs) and expression of PD1 and PD-L1 is unclear. We sought to describe the differences in the percentage (%) of TILs, cytotoxic T lymphocytes (CTL), and expression of PD1/PD-L1 in tumors of patients (pts) with operable breast cancer (BC) treated with neoadjuvant chemotherapy (NAC) who were enrolled in a biomarker study at our institution (IRB protocol# 2010C0036). Methods: A multicolor immune-histochemical multiplex assay simultaneously detecting PD1, PD-L1, and CD8 expressing cells was performed on formalin-fixed, paraffin-embedded diagnostic pretreatment biopsy or resected tumor specimen following NAC in 18 of 26 pts participating in the study. A cut-off of ≥1% was considered positive for PD1 and PD-L1 expression. We evaluated stromal and intratumoral CTLs by estimating % of stroma and tumor that contained CD8+ cells. In addition, stromal TILs (sTILs) were identified on full-face hematoxylin and eosin stained sections and defined as the % of tumor stroma containing infiltrating lymphocytes. Pathologic complete or near-complete response (pCR) was analyzed based on residual cancer burden (RCB) score and defined as RCB class 0 or I. Analysis of all slides was performed by an expert breast pathologist. Since the number of pts was limited, we only provide descriptive statistics (mean, range). In addition, because most pts had only a biopsy or only residual tumor available for the analysis, we divided pts into 2 separate cohorts based on what tissue was available. Results: Of 18 pts, biopsy was analyzed in 7 (Bx cohort) and residual tumor was analyzed in 11 pts (RT cohort). The median age of study pts was 48 (range 32-70); 11 (61%), 6 (33%), and 1 (6%) of pts were Caucasian, African American, and Hispanic, respectively. Ten pts (5 in Bx and RT cohorts each) had triple-negative BC (TNBC), 4 had HER2+ BC (1 and 3 in Bx and RT cohorts, respectively), and 4 had hormone receptor-positive, HER2- BC (1 and 3 in Bx and RT cohorts, respectively). Eight pts (44%) had pCR. In the bx cohort, 85% of pts had pCR while 18% of pts in the RT cohort had minimal residual disease (RCB class I). In the Bx cohort, average % of sTILs was 30% (range 2-70%), including 1 pt (14%) with lymphocyte predominant tumor (≥50% of sTILs). The % of sTILs was similar in the 11 residual tumors (mean 22, range 2-60) with 2 pts having lymphocyte predominant tumors. Average % of CTLs was 19 (range 1-50) in the Bx cohort and 14 (range 1-50) in the RT cohort. An average % of intratumoral CTLs was 8 (range 1-30) and 7.5 (range 0-40) while the average % of stromal CTLs was 25 (range 1-60) and 19 (range 1-60) in the Bx and RT cohorts, respectively. Similar % and trends were seen in 10 TNBC pts. PD-L1 expression was seen in 86% and 36% of tumors in the Bx and RT cohorts, respectively, with majority of expression present in the stroma. All cases of intratumoral PD-L1 expression were also positive for stromal PD-L1 expression. This difference was also seen in the TNBC pts (80% vs. 40% of tumors were PD-L1+ in Bx and RT cohorts, respectively). An average PD-L1 intensity was approximately 3% in both cohorts (range 1-20%). Expression of PD1 was very low (1% intensity in 3 pts in Bx cohort and in 1 pt in RT cohort) and it was seen on CD8+ CTLs. Conclusion: Our study preliminarily shows that percent of sTILs and stromal and intratumoral CTLs does not differ between pretreatment biopsy and residual tumors following NAC. Lower proportion of residual tumors were PD-L1+ compared to pretreatment biopsy specimen. The study limitations include small number of subjects and lack of comparison in the same pts. Future studies are needed to confirm these findings. Citation Format: Robert Wesolowski, Zaibo Li, Christopher McQuinn, Maryam Lustberg, Bhuvaneswari Ramaswamy, Anne Noonan, Raquel Reinbolt, Sagar Sardesai, Jeffrey B. VanDeusen, Nicole Williams, William E. Carson, III. Analysis of tumor infiltrating lymphocytes and expression of PD1 and PD-L1 in breast tumors prior to and after neoadjuvant chemotherapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A20.

Overexpression of S100A13 protein is associated with tumor angiogenesis and poor survival in patients with early-stage non-small cell lung cancer.

BackgroundS100A13 plays a key role in tumor growth and metastasis. The purpose of this study was to investigate the prognostic significance of S100A13 expression, microvessel density (MVD), and survival in early stage non‐small cell lung cancer (NSCLC).MethodsIn silico analysis was performed to determine the associations between S100A13 and NSCLC. The data of 82 patients with early‐stage NSCLC who underwent radical resection were evaluated. Paraffin‐embedded tumor specimens were stained with S100A13 and CD31 (a specific endothelial marker) using immunohistochemical methods. Prognostic significance was assessed by univariate and multivariate analyses.ResultsS100A13 messenger RNA was overexpressed in NSCLC, especially in advanced stage. Of the 82 NSCLC specimens examined, 37 (45.1%) cases exhibited S100A13 overexpression and 31 (37.8%) showed high MVD. Univariate analysis indicated that gender, age, smoking status, histology type, tumor differentiation, and T stage were not significantly associated with prognosis. However, the overall and disease‐free survival rates of patients with S100A13 overexpression and high MVD were significantly lower than in the remaining cases. Multivariate analysis demonstrated that only S100A13 overexpression was an independent factor for poor prognosis in early‐stage NSCLC. Statistical analysis demonstrated that the MVD was significantly higher in tumors with high (67.6%, 25/37) compared to low S100A13 expression (13.3%, 6/45) (P < 0.01).ConclusionsHigh S100A13 expression is closely associated with high intratumoral angiogenesis and poor prognosis in patients with stage I NSCLC. Immunohistochemical evaluation of S100A13 expression, along with an examination of the perioperative extent of angiolymphatic invasion, has value for predicting prognosis.

Next-Generation Sequencing Using S1 Nuclease for Poor-Quality Formalin-Fixed, Paraffin-Embedded Tumor Specimens

Next-generation sequencing (NGS) testing of formalin-fixed, paraffin-embedded (FFPE) tissues is widely used in clinical diagnosis. However, the failure of high-quality DNA library construction, a critical step for NGS, often limits NGS application for FFPE tissues, particularly for old FFPE tissues. The aim was to develop a high-quality DNA library construction method optimized for NGS of FFPE specimens. DNA library construction was developed for FFPE specimens by using S1 nuclease and compared with the Covaris method-the widely accepted DNA library construction protocol. The Covaris method includes a DNA shearing step with sonication to generate shortened DNA fragments. DNA shearing was found to be unnecessary, and S1 nuclease treatment only during genomic DNA extraction significantly improved the success rate of library construction from FFPE tissues. Moreover, poor-quality FFPE tissues that failed the Covaris method were rescued with the S1 method. Higher complexity was observed in DNA libraries constructed by the S1 method. Among 223 FFPE specimens, DNA libraries were successfully constructed for 134 cases with the Covaris method (60.0% success rate), whereas the success rate was 86.5% (193 of 223) with the S1 method (P=5.255e-10). Furthermore, we were able to rescue 59 samples, including 25 primary lung cancers, from the 89 failed Covaris method cases. In conclusion, the S1 method is optimal for NGS testing of poor-quality FFPE specimens.

Abstract 1024: Tumor intrinsic properties associate with differential effects on CD8+ tumor-infiltrating lymphocyte density and immune gene expression in non-small cell lung cancer (NSCLC) samples

Abstract Introduction: Anti-tumor immune response is controlled by a complex interaction between the immune system, tumor cells, and associated stroma. Expression of major histocompatibility complex class I (MHC I) and MHC class II (MHC II) antigens may be dysregulated in cancer, leading to alterations in the tumor antigen presentation profile. Stage I–III resected NSCLC tumor samples were profiled to study the relationship between MHC I, MHC II, and programmed death ligand 1 (PD-L1) tumor cell (TC) expression with tumor-associated inflammation. Here we report the potential impact of respective profiles on tumor immune response. Methods: 53 adenocarcinoma (NSCLC-AD) and 51 squamous cell carcinoma (NSCLC-SQ) stage I–III resected formalin-fixed, paraffin-embedded tumor specimens from commercial sources were stained by immunohistochemistry for MHC I (HLA-A,B,C), MHC II (HLA-DP,DQ,DR), and PD-L1 and TC expression was assessed by manual pathologist review. CD8+ cell density was quantified using Definiens image analysis algorithms for the intraepithelial tumor region. RNA extracted from the samples was analyzed by RNAseq, with data available for 48 NSCLC-AD and 46 NSCLC-SQ specimens. A 25-gene IFN-gamma gene signature (IFNG score) was used to study the association of T-cell inflammation with other biomarkers. Results: Of all NSCLC-AD and NSCLC-SQ specimens, 85% showed either complete (&amp;lt;20% TC) or partial (20–80% TC) loss of TC MHC I, while TC MHC II was increased (&amp;gt;1%; MHCIIhi) in 60% of NSCLC-AD and 18% of NSCLC-SQ. Quantitative analysis of CD8 in the tumor microenvironment (TME) revealed a significantly reduced intraepithelial density of CD8+ tumor-infiltrating lymphocytes (TILs) in NSCLC-AD (P=0.009) and NSCLC-SQ (P=0.01) with complete MHC I loss. In contrast, intraepithelial CD8+ TILs were significantly increased in MHCIIhi tumors for both NSCLC-AD (P=0.004) and NSCLC-SQ (P=0.006). Tumors displaying both MHCIIhi and retained MHC I demonstrated the highest TIL density. Gene expression associated with IFN-gamma response was increased in tumors with retained MHC I (P=0.0036) and MHCIIhi samples (P&amp;lt;0.001). In NSCLC-AD and NSCLC-SQ, 38% and 59% had PD-L1 expression ≥1%, respectively. PD-L1 expression correlated with CD8 density and IFNG score in NSCLC-AD, but not in NSCLC-SQ. Conclusions: Understanding the role of the antigen presentation machinery in immune activation and evasion in the TME is important to predict responses to immunotherapy. These data suggest that the expression of MHC I, MHC II, and PD-L1 by tumor cells may play a role in guiding the localization of CD8+ T cells in the TME with differential effects dependent on histologic subtype. The lack of correlation between inflammation and PD-L1 expression in stage I–III NSCLC-SQ suggests an alternate PD-L1 induction mechanism in a subset of those tumors. Citation Format: Cyrus Hedvat, Keyur Desai, Dimple Pandya, Peter Szabo, Johannes Zimmermann, Jan Lesniak, Scott Ely, Sujaya Srinivasan, Xi-Tao Wang, Michele French, Robin Edwards. Tumor intrinsic properties associate with differential effects on CD8+ tumor-infiltrating lymphocyte density and immune gene expression in non-small cell lung cancer (NSCLC) samples [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 1024.

Abstract 4208: HOXA9 methylation detection by droplet digital PCR and blood vessel invasion using FFPE tissues for prognostic stratification of stage I lung adenocarcinoma patients

Abstract Surgical resection is the only recommended curative treatment for stage I non-small cell lung cancer (NSCLC) patients, as the benefit of adjuvant chemotherapy remains controversial. After complete resection patients with disease at the same stage experience different outcomes, and within 5 years a third of patients relapse. With the implementation of low-dose computed tomography scan for lung cancer screening, the number of lung cancer patients diagnosed at stage I is projected to rise. Therefore, prognostic biomarkers are urgently needed to more accurately predict recurrence following surgery and potentially guide the decision to administer adjuvant chemotherapy for high-risk patients. In the present study, we evaluated the prognostic significance of two biomarkers, namely HOXA9 promoter methylation and blood vessel invasion (BVI), for risk stratification of stage I lung adenocarcinoma patients. The type of biospecimens and the choice of assay platform are key issues to foster translation of biomarkers to the clinic. Here, we demonstrated the application of a droplet digital PCR (ddPCR)-based assay to analyze HOXA9 promoter methylation in formalin-fixed, paraffin-embedded (FFPE) tumor specimens, generated during routine pathologic assessment of resected patients. In recent years, ddPCR has become increasingly used clinically due to its ability to reliably detect and quantitate rare alleles, as well as its technical simplicity, rapidity and cost effectiveness. We replicated previous observations that HOXA9 promoter is methylated de novo in stage I tumors (P &amp;lt;0.0001). Using 177 FFPE tumor samples, we showed that high methylation was associated with worse cancer-specific survival (Hazard Ratio [HR], 3.37; P=0.0002) and identified high-risk stage IA and IB patients. Importantly, addition of this molecular marker improved a risk model comprising clinical and pathologic parameters (Nested likelihood ratio test; P=0.003). HOXA9 promoter methylation was associated with a transcriptome signature enriched in genes marked by Polycomb in Embryonic Stem Cells, a signature previously associated with poor differentiation and worse overall patient survival. Moreover, BVI was independently associated with poor outcome (HR, 2.62; P=0.054). A score that combined BVI with HOXA9 promoter methylation further stratified high-risk patients (Trend P=0.0001 comparing 0, 1 or 2 positive markers). Collectively, our results support the use of ddPCR to quantify HOXA9 promoter methylation and BVI determination from routine pathology FFPE specimens, to identify patients at high risk of recurrence. If validated in a larger independent study, our findings could help inform patient management in prospective clinical trials that evaluate the benefit of adjuvant chemotherapy in early-stage invasive lung adenocarcinoma to prevent recurrence. Citation Format: Delphine Lissa, Teruhide Ishigame, Rintaro Noro, Marguerite J. Tucker, Valery Bliskovsky, Steven Shema, Elise D. Bowman, Curtis C. Harris, Ana I. Robles. HOXA9 methylation detection by droplet digital PCR and blood vessel invasion using FFPE tissues for prognostic stratification of stage I lung adenocarcinoma patients [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 4208.

Abstract 4546: The tumor inflammation signature is predictive of anti-PD1 treatment benefit in the CERTIM pan-cancer cohort

Abstract Purpose of the study : The 18-gene Tumor Inflammation Signature (TIS) (Ayers et al, J Clin Invest 2017; 127:2930) is a clinical research assay that enriches for clinical response to immune checkpoint blockade. In this work, the performance of the TIS was evaluated on standard tumor specimens for its ability to predict clinical benefit of immunotherapy in a cohort of cancer patients treated with PD1 checkpoint inhibitors in routine clinical care. Experimental procedures : Consecutive metastatic cancer patients treated with anti-PD1 in the outpatient monocentric CERTIM cohort with available formalin fixed paraffin embedded tumor specimen were selected for analysis. RNA were extracted from tissue samples with High Pure FFPET RNA Isolation Kit (Roche) and 25-100 ng RNA was hybridized to a pilot version of the NanoString® PanCancer Immuno-oncology 360 Panel. The association between individual gene expression (GE), TIS and other GE signatures with outcome (response to therapy and overall survival) was assessed with logistic resgression model, LogRank tests and Cox model. Results : A total of 69 patients with non-small cell lung carcinoma (NSCLC, n=42), small cell lung carcinoma (n=2), melanoma (n=9), renal cell carcinoma (n=10), urothelial carcinoma (n=5) or colorectal carcinoma (n=1), who had received nivolumab (n=59) or pembrolizumab (n=10) were analyzed. All samples but one SCLC could be successfully analyzed, demonstrating the feasibility of the testing even in small biopsy samples with little available RNA (&amp;gt;30 ng). In the whole cohort, clinical response (PR or CR versus SD or progression) was significantly associated with TIS score [OR=2.002, 95%CI=(1.123,3.977), p =0.029] as well as with the individual expression of PDL1, CTLA4, IDO1 or GE scores characterizing Th1, cytotoxic, NK cells infiltration, immuno-proteasome or antigen processing machinery. Receiver operating characteristics for response status demonstrated the high discriminatory ability of the TIS signature in this unselected series of patients (area under the ROC curve 0.71). Patients with a high TIS score (&amp;gt; 9.39) showed a longer OS (median OS 20.2 months) compared to those with lower scores (median OS 5.6 months, p value = 0.0017). A high TIS score was also significantly associated with clinical response when the analysis was limited to the NSCLC (n=42, [OR=3.156, 95%CI=(1.168,11.455), p-value=0.042] - or showed a strong trend in lung adenocarcinoma (n=29 [OR=11.251, 95%CI=(0.962,131.534), p-value=0.054] patients. Conclusion : Due to costs and toxicity, it is mandatory to identify the patients who benefit of immune checkpoint inhibitors. Here we demonstrate that the Tumor Inflammation Signature, that reflects key processes involved in anti-tumor immune response, may be used in routine samples to predict clinical benefit and prolonged survival of cancer patients. Citation Format: diane damotte, jennifer arrondeau, pascaline boudou-rouquette, audrey lupo, jerome biton, hanane ouakrim, Marco Alifano, Gervais Claire, Audrey Bellesoeur, Nora Kramkimel, Angeline Duche, Franck Letourneur, Xing Ren, Pascale Morel, Alessandra Cesano, Sarah Warren, François Goldwasser, Karen Leroy. The tumor inflammation signature is predictive of anti-PD1 treatment benefit in the CERTIM pan-cancer cohort [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 4546.

The immune-related microRNA miR-146b is upregulated in glioblastoma recurrence.

BackgroundGlioblastoma (GBM) has a high rate of local recurrence despite chemoradiotherapy (CRT). Genome-wide expression profiling was performed on patient tumors before and after chemoradiotherapy to identify genes and gene pathways associated with recurrence.ResultsMedian time to recurrence was 8.9 months with median time to second surgery of 9.6 months. The microRNA (miRNA) analysis identified 9 oncologic and immune-related miRNAs to be differentially expressed, including the hypoxia-related miR-210 and the immune-modulatory miR-146b. More than 1200 differentially-expressed genes were identified with RNA-sequencing (RNA-seq). Gene set enrichment analysis (GSEA) identified p53 signaling, Notch, Wnt, VEGF, and MEK gene sets enriched in recurrent GBM. Consistent with the miRNA profiling data, the miR-146b target gene set from GSEA analysis was also associated with recurrence.MethodsFourteen patients with GBM recurrence after CRT who had available tumor tissue from the initial diagnosis as well as recurrence were selected. Total RNA was isolated from formalin-fixed paraffin-embedded (FFPE) tumor specimens. Genome-wide expression profiling using RT-PCR for miRNA analysis and RNA-seq for messenger RNA (mRNA) analysis were conducted to identify differentially-expressed genes. GSEA was performed on the differential expression data.ConclusionsGenome-wide expression profiling identifies multiple oncologic and immune-related gene sets associated with GBM recurrence. In particular, immune-related miR-146b is upregulated in recurrence and deserves further investigation.

Prognostic and Predictive Value of HER2 Amplification in Patients With Metastatic Colorectal Cancer

PurposeTo evaluate a prognostic and predictive value of HER2 amplification in patients with metastatic colorectal cancer (mCRC). Patients and MethodsPatients with mCRC who underwent surgical resection of the primary tumor and who received best supportive care with or without palliative chemotherapy between 2005 and 2015 were included. HER2 immunohistochemistry was performed using formalin-fixed, paraffin-embedded primary tumor specimens. HER2 amplification was confirmed by fluorescence in-situ hybridization. The RAS and BRAFV600E mutations were centrally assessed using a PCR-based method. Patients were divided into 4 subgroups: R (RAS mutant), B (BRAFV600E mutant), H (wild-type RAS/BRAF with HER2 amplification), and W (wild-type RAS/BRAF without HER2 amplification). Overall survival (OS) and progression-free survival of anti–epidermal growth factor receptor (EGFR) therapy were assessed. ResultsAmong 370 eligible patients, data of 359 were successfully analyzed. Fifteen tumors harbored HER2 amplifications, including 4 tumors with concomitant RAS mutation (group R). The number of patients in groups R, B, H, and W was 204, 13, 11, and 131, respectively. The median OS was 27.4 months, and the median follow-up time was 63.2 months. The median OS for groups R, B, H, and W was 24.0, 14.2, 19.9, and 39.1 months, respectively. The number of patients who received anti-EGFR therapy in groups R, B, H, and W was 17, 4, 5, and 49, respectively. Progression-free survival of anti-EGFR therapy was significantly shorter in groups R, B, and H than in group W. ConclusionHER2 amplification was predictive of anti-EGFR therapy response and appeared to be prognostic in mCRC patients.

Landscape of Tumor Mutation Load, Mismatch Repair Deficiency, and PD-L1 Expression in a Large Patient Cohort of Gastrointestinal Cancers.

The efficacy of immunotherapy varies widely among different gastrointestinal cancers. Response to immune checkpoint inhibitors is shown to correlate with tumor mutation load (TML), mismatch repair deficiency (dMMR) status, and programmed cell death-ligand 1 (PD-L1) expression. Herein, we quantify TML, dMMR, and PD-L1 expression and determine their interrelationship in gastrointestinal cancers. Here, a total of 4,125 tumors from 14 different gastrointestinal cancer sites were studied using validated assays. Next-generation sequencing was performed on genomic DNA isolated from formalin-fixed paraffin-embedded tumor specimens using the NextSeq platform. TML was calculated using only somatic nonsynonymous missense mutations sequenced with a 592-gene panel. Microsatellite instability (MSI) was assessed using direct analysis of altered known MSI loci in the target regions of the sequenced genes. PD-L1 expression was analyzed by IHC. Interestingly, right-sided colon and small-bowel adenocarcinomas had the highest prevalence of TML-high tumors (14.6% and 10.2%, respectively). Pancreatic neuroendocrine tumors and gastrointestinal stromal tumors had the lowest rates of TML-high (1.3% and 0%, respectively). TML-high was strongly associated with MSI-H (P < 0.0001). However, all TML-high anal cancers (8.3%) were microsatellite stable (MSS). Higher PD-L1 expression was more likely to be seen in MSI compared with MSS tumors (20.6% vs. 7.8%, P < 0.0001).Implications: TML-high rate varied widely among gastrointestinal cancers. Although MSI is conceivably the main driver for TML-high, other factors may be involved. Future clinical trials are needed to evaluate whether the integration of TML, MSI, and PD-L1 could better identify potential responders to immunotherapy. Mol Cancer Res; 16(5); 805-12. ©2018 AACR.

PD-1/PD-L1 expression in a series of intracranial germinoma and its association with Foxp3+ and CD8+ infiltrating lymphocytes.

One histopathological characteristic of intracranial germinoma is abundant tumor-infiltrating lymphocytes (TILs) showing a two-cell pattern with large undifferentiated tumor cells. The programmed cell death 1 (PD-1)/programmed cell death 1 ligand (PD-L) axis has recently been recognized as an anti-tumor immune system. To evaluate intratumor immune status in intracranial germinoma, we examined expressions of PD-1 and PD-L1 (clone 28–8) and subtypes of TILs. Expressions of PD-1 and PD-L1 were detected immunohistochemically in 25 formalin-fixed, paraffin-embedded tumor specimens from 24 patients with intracranial germinoma consisting of 22 primary and 3 recurrent tumors. To evaluate subtypes of TILs, quantification of lymphocytes with CD3, CD8, CD4, and Foxp3 was performed. Statistical analyses were performed among PD-1, PD-L1 and subtypes of TILs. In 25 tumor tissue, expressions of PD-1 in TILs and PD-L1 in tumor cells were identified in 96% (24/25) and 92% (23/25), respectively. Expression of PD-1 was associated with CD3+ TIL density. Expression of PD-1 correlated with Foxp3+ TIL density and CD8+ TIL density, but not with CD4+ TIL density. Furthermore, expression of PD-1 correlated strongly with Foxp3+/CD4+ ratio. Taken together, increase of PD-1+ expression is associated with accumulation of Foxp3+ and CD8+ TILs. These findings intimate that PD-1/PD-L1 axis might shape the immune infiltration suggesting a modulation of the immune response and subsequent tumor growth in intracranial germinoma. Anti-PD-1 and anti-PD-L1 are potential immune therapeutic strategies in intracranial germinoma.

Abstract PD3-12: PIK3CA alterations and benefit with neratinib after trastuzumab-based adjuvant therapy in early-stage HER2+ breast cancer: Correlative analyses of the phase III ExteNET trial

Abstract Background: Neratinib is a pan-HER tyrosine kinase inhibitor that blocks the PI3K/Akt and MAPK signaling pathways downstream from HER2. The international, randomized, placebo-controlled phase III ExteNET trial showed that a 1-year course of neratinib after trastuzumab-based adjuvant therapy significantly improved 2-year invasive disease-free survival (iDFS) in early-stage HER2+ breast cancer (HR 0.67; 95% CI 0.50–0.91; p=0.0091) [Chan et al. Lancet Oncol 2016]. Furthermore, the effects of neratinib on iDFS were shown to be durable at 5 years' follow-up (HR 0.73; 95% CI 0.57–0.92; p=0.008) [Martin et al. ESMO 2017]. PIK3CA alterations are common in HER2+ breast cancers, and in general are associated with a worse prognosis. We sought to assess the prognostic and predictive significance of PIK3CA alterations in an exploratory substudy of the ExteNET trial. Methods: ExteNET is an international, multi-center, randomized, double-blind, placebo-controlled phase III trial (Clinicaltrials.gov: NCT00878709). Patients received oral neratinib 240 mg/day or placebo for 1 year. Of the intent-to-treat (ITT) population (n=2840), primary formalin-fixed paraffin-embedded (FFPE) tumor specimens were available from 991 patients for PIK3CA mutation testing by RT-PCR for two hot-spot mutations in exon 9 (E542K, E545K/D) and one hot-spot mutation in exon 20 (H1047R). 702 FFPE tumor slides underwent FISH analysis for PIK3CA amplification with a ratio of ≥2.2 considered as amplified. Primary endpoint: iDFS. iDFS events were tested by 2-sided log-rank tests, and HR (95% CI) were estimated using Cox proportional-hazards models. Data cut-off: March 2017. Results: Baseline demographics and disease characteristics between treatment arms of the correlative cohort (n=1201) were balanced. Overall, 21.2% (n=210) of primary tumors harbored one of the specified PIK3CA mutations, and 8.7% (n=61) were PIK3CA FISH-amplified. Patients with PIK3CA-altered tumors (i.e. PIK3CA mutations or FISH-amplified) had fewer iDFS events with neratinib compared with placebo (HR 0.41; 95% CI 0.17-0.90, p=0.028). The interaction test was not significant (p=0.1842). Results of the various correlative analyses within treatment arms are shown in the table. NeratinibPlacebo iDFS iDFS 2-sidedPopulationnevents, nnevents, nHR (95% CI)P valueaITT142011614201630.73 (0.57–0.92)b0.008bCorrelative cohort59345608700.67 (0.45–0.96)0.0317PIK3CA-mutation positive1047106170.43 (0.17–1.01)0.056PIK3CA-mutation negative38527396420.66 (0.40-1.06)0.089PIK3CA-amplified3312840.20 (0.01-1.33)0.106PIK3CA-non-amplified31629325360.85 (0.52-1.39)0.521PIK3CA-altered1308132200.41 (0.17-0.90)0.028a. Log-rank test; b. Stratified analysis Conclusions: One year of neratinib treatment after trastuzumab-based adjuvant therapy significantly improves iDFS after 5 years in patients with early-stage HER2+ breast cancer. From this modest-sized exploratory cohort, it appears that PIK3CA may be a biomarker for differential sensitivity to neratinib after 1 year of trastuzumab in the adjuvant setting.These exploratory results should be validated in a larger subset. Citation Format: Chia SKL, Martin M, Holmes FA, Ejlertsen B, Delaloge S, Moy B, Iwata H, von Minckwitz G, Mansi J, Barrios CH, Gnant M, Tomašević Z, Denduluri N, Šeparović R, Kim S-B, Hugger Jakobsen E, Harvey V, Robert N, Smith II J, Harker G, Lalani AS, Zhang B, Eli LD, Buyse M, Chan A. PIK3CA alterations and benefit with neratinib after trastuzumab-based adjuvant therapy in early-stage HER2+ breast cancer: Correlative analyses of the phase III ExteNET trial [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 PD3-12.

Abstract P4-15-08: Association of OncotypeDX® DCIS ScoreTM results with local recurrence in patients with DCIS treated on accelerated partial breast radiotherapy (APBI) protocols

Abstract Background: Ductal carcinoma in situ (DCIS) is a proliferation of malignant epithelial cells of the ducts and terminal lobular units of the breast that do not invade the basement membrane. The incidence of DCIS has increased markedly since the early 1980s, chiefly due to screening mammography. Whole breast radiotherapy has largely been used to treat breast DCIS after lumpectomy. More recently, APBI has increasingly been utilized for breast DCIS. Currently updated American Society of Radiation Oncology (ASTRO) APBI guidelines have included "low risk" DCIS (as defined by RTOG 9804 criteria). The following results further explore clinico-pathologic factors, in addition to the DCIS Score, in order to better define an appropriate DCIS population for APBI. Methods: An exploratory analysis aimed to retrospectively measure the association between clinico-pathologic factors and the DCIS Score result, an optimized 12-gene expression algorithm, and risk of any local failure (in situ or IBC recurrence) in a cohort of women treated with local excision and APBI on prospective phase II (NCT01185145) and phase III (NCT01185132) clinical trials. Multifocal tumors were described only by local pathology and not determined or defined centrally. The DCIS Score assay was performed by quantitative RT-PCR on formalin-fixed paraffin-embedded DCIS tumor specimens by Genomic Health (Redwood City, CA). Descriptive statistics of the cohort and assay results overall and by clinical trial were derived. Univariable Cox proportional hazards regression was used to determine whether there was an association between local failure and categorized DCIS Score group (≥39 vs &amp;lt;39) or other clinico-pathologic factors on the pooled cohort of clinical trial patients. Results: This analysis included 104 evaluable patients (N=18 from NCT01185145 and N=86 from NCT01185132). The median age was 60 (range: 41-80), 79% of patients were postmenopausal, and the median span of DCIS was 6 mm (range 2-25 mm). Over two-thirds of the cohort presented with necrosis (71%). The distribution of DCIS Score results ranged from 0 to 82, with 69% of patients having a DCIS Score result &amp;lt;39. The median follow-up time was longer at 8.2 years in NCT01185145 versus 3.0 years in NCT01185132. There was a total of 6 local recurrences. DCIS Score result was significantly associated with local recurrence in univariable modeling (hazard ratio=10.3 for ≥39 vs &amp;lt;39; p=0.0104). None of the other clinico-pathologic characteristics resulted in any significant correlation with locoregional recurrence. All results were highly variable due to the small number of events. Conclusion: The DCIS Score assay demonstrated risk stratification in this cohort of patients treated with local excision and APBI pooled from two clinical trials. These results are consistent with those recently published by Rakovitch et al (J Natl Cancer Inst 2017). The cohort in this study was dominated by those in the phase III trial. Due to the small number of local recurrence events and limited follow-up time in the phase III trial, caution should be taken when interpreting the results. Further investigations are needed to confirm findings. Citation Format: Leonard CE, Fryman SP, Turner MP, Bennett JP, Carter DL, Sing AP. Association of OncotypeDX® DCIS ScoreTM results with local recurrence in patients with DCIS treated on accelerated partial breast radiotherapy (APBI) protocols [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 P4-15-08.