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Background: The primary aim of the NRG Oncology/NSABP B-52 clinical trial was to test if estrogen deprivation (ED) administered concomitantly with neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP), would improve the pCR rate in patients with HER2+/ER+ early breast cancer. A numerical increase in the pCR rate was observed with ED (46.1% v 40.9%), but the difference was not statistically significant. The purposes of this study were to assess the association of sTILs in pretreatment biopsies with pCR in the total population and within the molecular subtypes of breast cancer and to assess changes in sTILs between pre- and on-treatment biopsies. The secondary endpoints of recurrence-free interval (RFI) and overall survival (OS) are currently being analyzed and will be presented along with association of these endpoints with sTILs in pretreatment biopsies in the total cohort and within molecular subtypes. Methods: Scoring of sTILs on routine H&E slides from pre-treatment biopsies with sufficient tumor from 249 of the 315 patients (79%) entered in B-52 were performed by one of two pathologists (SKM, RSM). Both pathologists scored sTILs on a subset of 64 patients to document concordance. Wilcoxon two-sided test, box and whisker plots, and forest plots were used to assess associations with pCR. Molecular subtypes were determined utilizing RNA-seq data and AIMS subtyping method. On-treatment biopsies were available in 46 patients and were scored and compared to paired baseline samples. Results: Good concordance between pathologists was established with an inter-pathologist difference of ˂20% difference between scores in 92% of cases. sTILs in pre-treatment samples were associated with pCR across both arms of the trial (p=0.0074) and in the TCHP+ED arm (p=0.033), but not in the TCHP arm (p=0.093). The distribution of intrinsic subtypes was 34% luminal B, 29% luminal A, 28% HER2E, 5.8% normal, and 2.7% basal, with no significant differences between the arms. Presence of sTILs showed a trend for association with pCR in HER2E pre-treatment samples (p=0.054) but not in non-HER2E (p=0.75). Similarly, sTILs were associated with pCR in non-luminal tumors (p=0.055) but not in luminal tumors (p=0.44). Stratification by treatment arm and menopausal status suggested sTILs are associated with pCR in premenopausal women treated with TCHP (OR: 1.04, 95% CI=1.00-1.09). Interestingly, decreases in the sTIL scores with treatment were associated with pCR in the TCHP+ED arm (p=0.01) but not in the TCHP arm. Analysis of RFI and OS on B-52 is ongoing and will be presented along with associations of sTILs with intrinsic subtypes for RFI and OS. Conclusions: Although a positive correlation between sTILs and pCR was observed, the clinical utility appears limited because of the extensive overlap in the TIL scores between pCR and non-pCR tumors. Significance for a positive association of sTILs with pCR was detected in HER2E but not in luminal tumors. This may be due to the molecular differences of the subtypes, or the make-up of the TILs, or both. The association of a decrease in sTILs with TCHP+ED treatment needs further investigation. The small number of samples is a limitation of the study; however, the B-52 protocol specified that the collection of the B-52 samples was for the purpose of exploratory analysis. Our results highlight the molecular heterogeneity of the HER+/ER+ patient population and suggests that different treatment strategies may be required in future treatment regimens for this patient population. Support: NSABP Foundation; BCRF; 3U10CA180868-03S2, -180822; UG1CA189867; Genentech. Citation Format: Katherine L. Pogue-Geile, Sai K. Maley, Rim S. Kim, Ying Wang, Roberto Salgado, Corey Lipchik, Huichen Feng, Reena S. Cecchini, Samuel A. Jacobs, Ashok Srinivasan, Eleftherios (Terry) Mamounas, Charles E. Geyer Jr, Priya Rastogi, C. Kent Osborne, Soonmyung Paik, Norman Wolmark, Peter C. Lucas, Mothaffar Rimawi. Association of stromal tumor infiltrating lymphocytes (sTILs) in pretreatment biopsies in different molecular subtypes of HER2+/ER+ breast cancer: Assessment of NRG Oncology/NSABP B-52 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-04-10.

Purpose:In metastatic colorectal cancer (mCRC), HER2 (ERBB2) gene amplification is implicated in anti-EGFR therapy resistance. We sought to determine the recommended phase II dose (RP2D) and efficacy of neratinib, a pan-ERBB kinase inhibitor, combined with cetuximab, in patients with progressive disease (PD) on anti-EGFR treatment.Patients and Methods:Twenty-one patients with quadruple-wild-type, refractory mCRC enrolled in this 3+3 phase Ib study. Standard dosage cetuximab was administered with neratinib at 120 mg, 160 mg, 200 mg, and 240 mg/day orally in 28-day cycles. Samples were collected for molecular and pharmacokinetic studies.Results:Sixteen patients were evaluable for dose-limiting toxicity (DLT). 240 mg was determined to be the RP2D wherein a single DLT occurred (1/7 patients). Treatment-related DLTs were not seen at lower doses. Best response was stable disease (SD) in 7 of 16 (44%) patients. HER2 amplification (chromogenic in situ IHC) was detected in 2 of 21 (9.5%) treatment-naïve tumors and 4 of 16 (25%) biopsies upon trial enrollment (post-anti-EGFR treatment and progression). Compared with matched enrollment biopsies, 6 of 8 (75%) blood samples showed concordance for HER2 CNV in circulating cell-free DNA. Five SD patients had HER2 amplification in either treatment-naïve or enrollment biopsies. Examination of gene-expression, total protein, and protein phosphorylation levels showed relative upregulation of ≥2 members of the HER-family receptors or ligands upon enrollment versus matched treatment-naïve samples.Conclusions:The RP2D of neratinib in this combination was 240 mg/day, which was well tolerated with low incidence of G3 AEs. There were no objective responses; SD was seen at all neratinib doses. HER2 amplification, detectable in both tissue and blood, was more frequent post-anti-EGFR therapy.

Supplemental Figure Legends

Figure SF3 Comparisons of the expression of the NRG family of ERBB ligands, at the RNA level, between post-anti-EGFR metastatic biopsies collected upon enrollment into FC-7, and post-neratinib residual tumor tissue samples.

Background: The NRG Oncology/NSABP B-52 neoadjuvant clinical trial was conducted to test if the addition of estrogen deprivation (ED) would improve the pCR rate in HER2+/ER+ breast cancer patients (pts) treated with docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP). A numerical increase in pCR rate was observed with ED (46.1% v 40.9%), but the difference was not statistically significant. We have previously quantitated T cells (CD8, FOXP3), macrophages (CD68), and immune checkpoint proteins (PD-1, PD-L1) with multiplex immunofluorescence in B-52 and shown that CD68 and FOXP3 cells were associated with pCR but not CD8 cells. Our purpose was to determine the associations of FCGR genotypes and immune cells with pCR. Methods: A single baseline, pre-treatment FFFPE tissue section per case (N=181) was used to perform a 7-plex multiplex immunofluorescence procedure using opal fluorophores for staining. The Vectra Pathology System and inForm analysis software (Akoya Biosciences) was used for imaging and quantitation of CD8, CD68, FOXP3, PD-1, and PD-L1 cells in both the tumoral and stromal regions. Stromal data is reported here. Favorable- and unfavorable- FcGγR genotypes for FCGR2A-131H/R and FCGR3A-158V/F alleles were determined via the Sequenom MassARRAY iPLEX platform. Rates of pCR with pts with 1 or 2 favorable alleles was compared to pts who were homozygous for the unfavorable allele. Within each genotype, Wilcoxon rank sum test was used to test the association of markers with pCR and within each treatment. Results: No significant association of FCGR2A and 3A alleles with pCR was detected in the entire B-52 cohort, however, among pts with favorable FCGR genotypes (FCGR2A-131-HH, or H/R, FCGR3A-158- VV, or VF HR) the median value of the % CD68 cells was significantly higher in tumors that achieved pCR v those that did not (p=0.0004, p=0.0006), respectively. In pts who were homozygous for the FCGR2A or FCGR3A unfavorable alleles, there was no significant difference in the median values of the % of CD68 cells between pCR and no-pCR tumors. Further stratification of tumors by treatment showed that pts with an FCGR2A or FCGR3A favorable genotype and whose tumors achieved pCR had a higher median value of CD68 only in the TCHP + ED arm (p=0.0007, p=0.0003), respectively and not in the TCHP arm (p=0.059; p=0.21). Higher levels of PD-L1 were associated with pCR in pts with FCGR3A- favorable genotypes, but higher levels of FOXP3 were associated with pCR regardless of genotype. In contrast to the other cell types, higher PD-1 or CD8 cells showed no association with genotypes. Conclusions: This is an exploratory study examining the potential role of ADCP in HER2+/ER+ breast cancer and supports the notion that ADCP may be one mechanism that promotes the elimination of tumor cells in a subset of pts in the neoadjuvant setting. Tumors that achieve pCR have higher % of CD68 cells, in pts with favorable FCGR2A and 3A genotypes than pts who do not. However, in pts with unfavorable FCGR3A or FCGR2A genotypes there was no difference in the median CD68 levels in pCR v no-pCR tumors. When tumors were further stratified by CD68 levels, FCGR3A genotypes, and treatment, the association of pCR in tumors with high CD68 and FCGR3A favorable genotypes was seen only in the TCHP+ED arm. This may indicate that ED may improve pCR rates in some tumors with more macrophages and favorable genotypes. Macrophages are known to have estrogen receptors, and estrogen has been shown to promote the alternative activation of macrophages, potentially dampening down the immune response. Thus, one could speculate that ED may block the estrogen-induced alternative activation of macrophages, allowing the classically activated macrophages to phagocytize tumor cells. Support: BCRF, U10CA180868 & Admin Sup, U24CA196067, Genentech, NSABP Foun. Citation Format: Katherine L Pogue-Geile, Ying Wang, Huichen Feng, Corey Lipchick, Patrick Gavin, Rim S Kim, Reena S Cecchini, Samuel A Jacobs, Ashok Srinivasan, Sandra M Swain, Eleftherios Mamounas, Charles E Geyer, Jr, Priya Rastogi, Peter C Lucas, C. Kent Osborne, Soonmyung Paik, Norman Wolmark, Mothaffar F Rimawi. Potential role of the antibody-dependent cellular phagocytosis (ADCP) in tumors achieving pCR in NRG Oncology/NSABP B-52 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-04.

Purpose: In metastatic colorectal cancer (mCRC), HER2 (ERBB2) gene amplification is implicated in anti-EGFR therapy resistance. We sought to determine the recommended phase II dose (RP2D) and efficacy of neratinib, a pan-ERBB kinase inhibitor, combined with cetuximab, in patients with progressive disease (PD) on anti-EGFR treatment. Patients and Methods: Twenty-one patients with quadruple-wild-type, refractory mCRC enrolled in this 3+3 phase Ib study. Standard dosage cetuximab was administered with neratinib at 120 mg, 160 mg, 200 mg, and 240 mg/day orally in 28-day cycles. Samples were collected for molecular and pharmacokinetic studies. Results: Sixteen patients were evaluable for dose-limiting toxicity (DLT). 240 mg was determined to be the RP2D wherein a single DLT occurred (1/7 patients). Treatment-related DLTs were not seen at lower doses. Best response was stable disease (SD) in 7 of 16 (44%) patients. HER2 amplification (chromogenic in situ IHC) was detected in 2 of 21 (9.5%) treatment-naïve tumors and 4 of 16 (25%) biopsies upon trial enrollment (post-anti-EGFR treatment and progression). Compared with matched enrollment biopsies, 6 of 8 (75%) blood samples showed concordance for HER2 CNV in circulating cell-free DNA. Five SD patients had HER2 amplification in either treatment-naïve or enrollment biopsies. Examination of gene-expression, total protein, and protein phosphorylation levels showed relative upregulation of ≥2 members of the HER-family receptors or ligands upon enrollment versus matched treatment-naïve samples. Conclusions: The RP2D of neratinib in this combination was 240 mg/day, which was well tolerated with low incidence of G3 AEs. There were no objective responses; SD was seen at all neratinib doses. HER2 amplification, detectable in both tissue and blood, was more frequent post-anti-EGFR therapy.

PURPOSE: In metastatic colorectal cancer (mCRC), HER2 (ERBB2) gene amplification is implicated in anti-EGFR therapy resistance. We sought to determine the recommended phase II dose (RP2D) and efficacy of neratinib, a pan-ERBB kinase inhibitor, combined with cetuximab, in patients with progressive disease (PD) on anti-EGFR treatment. EXPERIMENTAL DESIGN: Twenty-one patients with quadruple-wild-type, refractory mCRC enrolled in this 3+3 phase-Ib study. Standard dosage cetuximab was administered with neratinib at 120mg, 160mg, 200mg, and 240mg/day orally in 28-day cycles. Samples were collected for molecular and pharmacokinetic studies. RESULTS: Sixteen patients were evaluable for dose-limiting toxicity (DLT). 240mg was determined to be the RP2D wherein a single DLT occurred (1/7 patients). Treatment-related DLTs were not seen at lower doses. Best response was stable disease (SD) in 7/16 (44%). HER2 amplification (CISH) was detected in 2/21 (9.5%) treatment-naïve tumors and 4/16 (25%) biopsies upon trial enrollment (post-anti-EGFR treatment and progression). Compared to matched enrollment biopsies, 6/8 (75%) blood samples showed concordance for HER2 CNV in cfDNA. Five SD patients had HER2 amplification in either treatment-naïve or enrollment biopsies. Examination of gene-expression, total protein, and protein phosphorylation levels showed relative upregulation of ≥2 members of the HER-family receptors or ligands upon enrollment versus matched treatment-naïve samples. CONCLUSIONS: The RP2D of neratinib in this combination was 240mg/day, which was well tolerated with low incidence of G3 AEs. There were no objective responses; SD was seen at all neratinib doses. HER2 amplification, detectable in both tissue and blood, was more frequent post-anti-EGFR therapy.

Background: We previously described a predictive signature for trastuzumab benefit which was validated in the adjuvant setting in an independent cohort within NSABP B-31 (the 8-gene signature) (Pogue-Geile et al JNCI, 2013) and in Alliance/NCCTG N9831 (SABCs 2017). The 8-gene signature subtyped B-31 patients into three trastuzumab benefit groups: high HR=0.27, intermediate HR=0.56 and no benefit HR=1.56 based on disease free survival. The 8 gene signature was also predictive of trastuzumab benefit in N9831. HRs were 0.47, P Methods: RNA-Seq data from FB-7 pretreatment biopsies was used to predict the trastuzumab benefit groups (high, intermediate, and no) for each patient's tumor using the 8 gene signature using methods and cut-offs as previously described (Pogue-Geile et al 2013). The pCR rates (percentages) were tested for treatment interaction with a chi-square test. Results: The pCR rates were 75%, 53%, and 22%, in the high (N=12), intermediate (N=32) and no benefit groups (N=9), respectively, when analyzed without regard to treatment arm. The pCR rates for the no benefit group and the high benefit groups were significantly different (p=0.030) and there was a significant treatment interaction with the 8-gene benefit group (intp=0.0081). The predicted low and intermediate groups were combined to test whether the 8 gene signature could identify a group of patients whose pCR rates might improve by adding N to T, and referred to it as the low benefit group. This was necessary due to the small numbers of patients in each group. The pCR rate in the low benefit group was higher in patients treated with T+N (9/15, 60%) than in the T arm (6/11, 45%) but these differences were not significant. Conclusions: This is the first test of the 8-gene signature in the neoadjuvant setting and interpretations of these data should be interpreted cautiously due to the small numbers. However, if these results were validated in another neoadjuvant trial then the 8 gene signature could provide a rationale for selecting patients who would be appropriate for the addition of neratinib or other TKIs to trastuzumab and chemotherapy. SUPPORT: PUMA Biotechnology, NCI U10CA180868, -180822, UG1-189867, and U24-196067; The Pennsylvania Department of Health. The Department specifically disclaims responsibility for any analysis,interpretations, or conclusions. Citation Format: Pogue-Geile KL, Wang Y, Srinivasan A, Gavin PG, Kim RS, Song N, Feng H, Lipchik C, Costantino JP, Wolmark N, Lucas PC, Paik S, Jacobs SA. The fully validated NSABP/NRG 8-gene signature which predicted the degree of benefit in the adjuvant setting (B-31 and NCCTG N9831) associates with pCR in the neoadjuvant setting in NSABP clinical trial FB-7 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-04.

The primary aim of NSABP FB-7 was to determine the pathologic complete response (pCR) rate in locally advanced HER2-positive (HER2+) breast cancer patients treated with neoadjuvant trastuzumab or neratinib or the combination and weekly paclitaxel followed by standard doxorubicin plus cyclophosphamide. The secondary aims include biomarker analyses. pCR was tested for association with treatment, gene expression, and a single nucleotide polymorphism (SNP) in the Fc fragment of the IgG receptor IIIa-158V/F (FCGR3A). Pre-treatment biopsies and residual tumors were also compared to identify molecular changes. The numerical pCR rate in the trastuzumab plus neratinib arm (50% [95%CI 34–66%]) was greater than that for single-targeted therapies with trastuzumab (38% [95%CI 24–54]) or neratinib (33% [95%CI 20–50]) in the overall cohort but was not statistically significant. Hormone receptor-negative (HR−) tumors had a higher pCR rate than HR+ tumors in all three treatment arms, with the highest pCR rate in the combination arm. Diarrhea was the most frequent adverse event and occurred in virtually all patients who received neratinib-based therapy. Grade 3 diarrhea was reported in 31% of patients; there were no grade 4 events. Our 8-gene signature, previously validated for trastuzumab benefit in two different clinical trials in the adjuvant setting, was correlated with pCR across all arms of NSABP FB-7. Specifically, patients predicted to receive no trastuzumab benefit had a significantly lower pCR rate than did patients predicted to receive the most benefit (P = 0.03). FCGR genotyping showed that patients who were homozygous for the Fc low-binding phenylalanine (F) allele for FCGR3A-158V/F were less likely to achieve pCR. Combining trastuzumab plus neratinib with paclitaxel increased the absolute pCR rate in the overall cohort and in HR− patients. The 8-gene signature, which is validated for predicting trastuzumab benefit in the adjuvant setting, was associated with pCR in the neoadjuvant setting, but remains to be validated as a predictive marker in a larger neoadjuvant clinical trial. HR status, and the FCGR3A-158V/F genotype, also warrant further investigation to identify HER2+ patients who may benefit from additional anti-HER2 therapies beyond trastuzumab. All of these markers will require further validation in the neoadjuvant setting. ClinicalTrials.gov, NCT01008150. Retrospectively registered on October 5, 2010.

Increased EGFR (epidermal growth factor receptor) expression has been reported in many types of human cancer and its levels are positively associated with advanced cancers. Recently, upregulation of Id-1 (inhibitor of differentiation or DNA binding) protein was found in over 70% of ovarian cancer samples and correlated with poor survival of ovarian cancer patients. However, the molecular mechanisms responsible for the role of Id-1 in ovarian cancer are not clear. The aim of this study was to investigate the effect of Id-1 on ovarian cancer proliferation and its association with the EGFR pathway. To achieve this, we transfected an Id-1 expression vector into three ovarian cancer cell lines and examined cell proliferation rate by flow cytometry and bromodeoxyuridine staining. We found that ectopic Id-1 expression led to increased cell proliferation demonstrated by increased BrdU incorporation rate and S-phase fraction. The Id-1-induced cell growth was associated with upregulation of EGFR at both transcriptional and protein levels. In contrast, inactivation of Id-1 through transfection of an Id-1 antisense vector resulted in downregulation of EGFR. Our results indicate that increased Id-1 in ovarian cancer cells may promote cancer cell proliferation through upregulation of EGFR. Our findings also implicate that Id-1 may be a potential target for the development of novel strategies in the treatment of ovarian cancer.

The NSABP FB-7 trial enrolled HER2-positive breast cancer patients (pts) with locally advanced disease who were randomly assigned to trastuzumab (T) or neratinib (N) or the combination (T+N) with weekly paclitaxel (P) followed by standard AC. A total of 126 pts were randomized to Arm 1 (T+P→AC), Arm 2 (N+P→AC), or Arm 3 (N+T+P→AC). Eligibility criteria included women >18 years of age, ECOG PS 0-1, stage IIB-IIIC invasive breast cancer, HER2-positivity by IHC 3+, FISH, or CISH as determined by local laboratories, hormone receptor positive or negative, LVEF >50%, and adequate laboratory parameters . The primary endpoint was pathologic complete response (pCR) in breast and nodes and was reported at SABCS 2015 (Jacobs et al, abstract # PD5-04). Dual anti-HER2 therapy yielded greater pCR rates than single targeted therapies in the overall cohort, and the magnitude of pCR was highest in HR-negative patients. Since this initial report, an in depth correlative biomarker analysis with clinical outcome has been performed and will be presented. Methods: In an effort to discover candidate predictive markers of response to study drugs, pre-treatment core biopsy samples (n=59) and post treatment surgical samples (n=17) were obtained from a subset of patients. Samples were collected prospectively from 2011-2014 and stored prior to batch analysis. Retrospective correlation with clinical outcome was performed after datalock of 1st Sept. 2015. Expression and activation of HER family RTKs, including truncated HER2, as well as downstream components of PI3K/AKT and MAPK pathways, were quantified using the multiplexed microarray CEERTMplatform (Collaborative Enzyme-Enhanced Reactive immuno-assay). pCR data were available in n=51 pts from the biomarker cohort. After excluding low tumor content non-evaluable samples, correlative biomarker analysis was performed in n=42 patients. Breast cancer subtypes were assessed with RNA-Seq to assess changes in gene expression and subtypes before and after treatment and association of pathways with pCR. The distribution, in our study population, of MammaPrint and BluePrint scores (Agendia) and their association with pCR will be presented. Results from a custom AmpliSeq (Thermo-Fisher) gene-panel used to assess the variant status of 117 genes potentially relevant to the development of resistance to anti-HER2 therapies, will be presented. Conclusions: Different patterns of baseline expression and activation levels of several key drivers, such as p95HER2, HER2, HER3, and PI3K, were observed between pCR in Arm1 (T+P→AC) and Arm 2 (N+P→AC). In particular, Arm 2 pCR displayed higher baseline levels of truncated HER2 compared to non-pCR. This exploratory marker analysis generates the hypothesis that high p95HER2 levels as measured by CEERTM may predict the likelihood of response to a neratinib containing regimen in the neoadjuvant setting in locally advanced breast cancer. Other correlatives will be discussed. Support: Puma Biotechnology, Inc. Citation Format: Gavin PG, Kim PS, Lipchick C, Langley E, Feng H, Meyer GR, Rim Kim S, Michel JP, Jacobs SA, Srinivasan A, Pogue-Geile KL. An exploratory correlative biomarker analysis of NSABP FB-7, a phase II randomized trial evaluating neoadjuvant therapy with weekly paclitaxel (P) plus neratinib (N) or trastuzumab (T) or neratinib and trastuzumab (N+T) followed by doxorubicin and cyclophosphamide (AC) with postoperative T in women with locally advanced HER2-positive breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-12.

Additional file 1: Methods. Table S1. Paclitaxel, trastuzumab, and neratinib drug intensity during weekly paclitaxel phase: NSABP FB-7. Table S2. Pathologic complete response (pCR) rates in neoadjuvant HER2+ breast cancer trials: NSABP FB-7. Figure S1. MammaPrint Scores for patients enrolled in NSABP FB-7. Randomized patients were assigned a MammaPrint score based on the propriatory test from Agendia. Figure S2. Intrinsic subtypes differ in residual tumors compared to the pretreatment biopsy: NSABP FB-7.

Background: NRG Oncology/NSABP B-52 neoadjuvant clinical trial was conducted to test if the addition of estrogen deprivation (ED) would improve the pCR rate in HER2+/ER+ breast cancer pts treated with docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP). A numerical increase in the pCR rate was observed with ED (46.1% v 40.9%), but the difference was not statistically significant. The purpose of this study was to determine the utility of using stromal tumor infiltrating lymphocytes (sTILs), mutations, established, and novel signatures, to assess their value for predicting pCR, particularly in the TCHP + ED arm. Methods: Whole transcriptome RNA-Seq and Ampli-Seq libraries were sequenced on the Ion Torrent platform. Mutations were assessed with a custom Ampli-Seq panel of 117 genes, including HER2-activated pathways and/or trastuzumab (T)-resistance markers. The 8-gene T-benefit signature was prospectively tested for association with pCR, and was previously validated in the adjuvant setting in B-31 and NCCTG N9831. Hot-spot mutations, sTILs, and signatures for immune cells, intrinsic subtypes, risk of recurrence proliferation (RORP), and MammaPrint, were also tested for associations with pCR. RNA-Seq data was also used to identify ED-predictive genes. Differential expression was assessed in normalized RNA-Seq data using DESeq2 and each sample was subtyped with the AIMS classifier. Wilcoxon two-sided test, chi-square, or Fisher's exact tests were used to assess associations with pCR. Results: Subtypes were determined with RNA-Seq data from pretreatment biopsies (N=230). The 8-gene T-benefit signature was associated with pCR. The high-, medium-, and low- T-benefit groups had pCR rates of 55%, 44%, and 6.8%, respectively (p=1.3e-13). Intrinsic subtypes were associated with pCR by comparing HER2E to all other subtypes combined (69% v 28%, p=3.9e-08). Hot spot mutations in PIK3CA alone (p=0.014), or combined with hot spot mutations in ERBB2, ERBB3, AKT1, PTEN, and MAP3K1, were associated with no pCR (p=0.0007) and may be useful as resistance markers. sTILs, MammaPrint, RORP, and some immune cell signatures also showed statistically significant associations with pCR but did not identify a subset of pts with an increased pCR rate in the TCHP + ED arm. In contrast, expression of the SH3BP2 gene was associated with pCR only in the TCHP + ED arm (interaction p=0.03). Conclusion: The previously validated 8-gene T-benefit signature identifies a subset of pts with very low pCR rate (6.8%) with TCHP. PIK3CA and other activating mutations were associated with no pCR. These findings may identify pts with resistant disease who may require a different treatment. Exploratory analyses suggest that SH3BP2 expression may identify pts who may benefit from TCHP + ED, but validation is required for clinically utility. Support: BCRF, U10CA180868, -180822; UG1CA189867, Genentech, PUMA Biother Citation Format: Katherine L. Pogue-Geile, Ying Wang, Huichen Feng, Corey Lipchik, Rim S. Kim, Reena S. Cecchini, Samuel A. Jacobs, Ashok Srinivasan, Joseph P. Costantino, Eleftherios P. Mamounas, Charles E. Geyer, Priya Rastogi, Peter C. Lucas, Soonmyung Paik, C. Kent Osborne, Norman Wolmark, Mothaffar F. Rimawi. Association of molecular signatures, mutations, and sTILs, with pCR in breast cancer patients in NRG Oncology/NSABP B-52 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4064.

BACKGROUND: Colorectal cancer (CRC) pts with deficient mismatch repair (dMMR) tumors have greater immune cell infiltration than pts with proficient (pMMR) tumors and is thought to be partly responsible for the robust response of dMMR pts to PD-1/PD-L1 blockade. Not all pts with dMMR tumors respond to PD-1 blockade. Therefore, we conducted exploratory analyses to understand the complexity of the tumor immune microenvironment. We developed a gene expression signature predictive for MMR status and tested it for associations with prognosis. METHODS: The one-sided Wilcoxon signed-rank test was used to select genes that were significantly over expressed in dMMR v pMMR tumors in a randomly selected discovery cohort in NSABP C-08 (N=500). Six chemokine genes were selected to build a predictive signature for MMR status because they were among the top 20 most significantly differentially expressed genes and were part of an immune chemo-attractant pathway. Addition of other genes did not improve prediction accuracy. The signature was tested in an independent cohort of C-08 (N=454) and in three other data sets: NSABP C-07 (N=1603), TCGA colon (N=619), and the NSABP Molecular Profiling Registry (MPR-1) (N=263). MPR-1 tissue samples were from primary tumors in pts who had recurred. Survival analyses were performed in C-07, C-08, and TCGA data, using Cox proportional hazards models. MMR status was determined for MPR-1 tissues with IHC using antibodies for MSH2, MLH1, PMS2, and MSH6. RESULTS: Higher chemokine scores (CKS) were significantly associated with dMMR tumors in all three independent data sets composed of treatment-naïve early-stage CRC pts (P CONCLUSIONS: The CKS score has a strong association with MMR status in three different data sets and is associated with a better prognosis in early-stage CRC. In these pts, dMMR tumors had significantly higher CKS than pMMR tumors, but primary tumors from MPR1 pts who had all become metastatic showed a trend for association in the reverse direction. We conclude that dMMR primary tumors, which are likely to become metastatic, have a very different immune microenvironment than those that do not because the CKS is composed of chemo attractants for a variety of immune cells including NK, T, and B cells. Such a signature could be useful for prospectively identifying dMMR pts with early-stage CRC who are at high risk for relapse. SUPPORT: Genentech; sanofi; U10CA180868, UG1CA189867, U24CA196067, PA DoH; NSABP Foundation. Citation Format: Ying Wang, Rim S. Kim, Corey Lipchik, Ashok Srinivasan, Huichen Feng, Nan Song, Carmen J. Allegra, Patrick G. Gavin, Samuel A. Jacobs, Norman Wolmark, Peter C. Lucas, Katherine L. Pogue-Geile. Development of a chemokine signature identifying dMMR patients (pts) with poor prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3407.

Background Monotherapy with ICB has not been effective in MSS mCRC. Preclinical data demonstrate that immunomodulators synergize with radiotherapy (RT) resulting in tumor regression at irradiated sites and rarely at non-irradiated sites. Anecdotal reports in pts have confirmed an abscopal effect of RT and ICB. This phase II, open-label, single-arm study is testing dual ICBs following SBRT in pts with MSS mCRC who have progressed on chemotherapy. We report here efficacy and safety results. Methods Eligible pts have: MSS mCRC, progressed on prior oxaliplatin and irinotecan-based regimens, measurable lesions with at least one amenable to SBRT and another for core biopsy, and performance status Results FC-9 is open at six academic centers. From October 2017 to November 1, 2018, 33 pts enrolled with 20 pts evaluable. Median age was 58 y (range, 37-71). Toxicity was assessed without regard to attribution. One pt had grade 5 unexplained sudden death (3%); two had grade 4 (6%, colitis, dehydration, pneumonitis); and 12 had grade 3 (38%, primarily GI, other than colitis, and nutrition). Partial responses were seen in 2 pts lasting 44 wks and 44+ wks; two pts had stable disease of 12 and 16 wks duration. Correlative studies will be presented including PD-L-1 expression, CD8+ T cell infiltration into tumor tissues, and gene expression of immune pathways. Conclusions The combination of SBRT and dual immunotherapy was safe and well tolerated following immunotherapy standard guidelines. In this refractory group of MSS mCRC pts, we observed 2 partial responses of 44 and 44+ wks among 20 evaluable pts. Correlative analyses will be presented. Support: Astra-Zeneca; NSABP Foundation, Inc. Citation Format: James J. Lee, Greg Yothers, Thomas J. George, Marwan G. Fakih, Atrayee Basu Mallick, Edith P. Mitchell, James L. Wade, John C. Krauss, Omar R. Kayaleh, Dwight E. Heron, Carmen J. Allegra, Corey Lipchik, Huichen Feng, Marion Joy, Ashok Srinivasan, Katherine L. Pogue-Geile, Peter C. Lucas, Sarah E. Warren, Alessandra Cesano, Samuel A. Jacobs. Phase II study of dual immune checkpoint blockade (ICB) with durvalumab (Durva) plus tremelimumab (T) following palliative hypofractionated radiotherapy (SBRT) in patients (pts) with microsatellite-stable (MSS) metastatic colorectal cancer (mCRC) progressing on chemotherapy: NSABP FC-9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2257.

3503 Background: MOSAIC and C-07 showed that oxaliplatin (OX) added to 5-fluorouracil plus leucovorin significantly improved disease free survival (DFS). However, OX is associated with neurotoxicity and the vast majority of patients do not receive OX-benefit; highlighting the importance of an OX-benefit predictor. In C-07, colon tumors with a CRCA (Colorectal Cancer Assigner) stem-like subtype were associated with a poor prognosis and no OX-benefit, but stage III patients with an enterocyte subtype did. We tested the association of CRCA and CMS molecular subtypes with prognosis and OX-benefit in stage III MOSAIC patients. Also, recombination proficiency scores (RPS) were tested for the same associations. RPS scores quantify the efficiency of DNA-damage repair. Low RPS have been associated with inferior overall survival (OS) in non-small cell lung carcinoma patients treated with surgery alone but better OS for patients who received surgery plus chemotherapy. Methods: Gene expression profiles from 590 stage III pts with follow up were successfully profiled by a custom designed nCounter code set. CRCA subtypes were determined by a locked down algorithm based on a re-estimated centroid using 72 genes (Song et al 2016). CMS subtypes were determined by modified single sample predictor (SSP) using 84 genes. RPS scores were determined as previously described by Pitroda et al (2014). Signature predictions were made while blinded to clinical outcome and signature performance was evaluated while blinded to gene expression. Results: The stem-like subtype was associated with a very poor prognosis (Stem-like vs others HR=1.56, p

Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high mortality rate. The majority of MCC (70-80%) harbor clonally integrated Merkel cell polyomavirus (MCV) in the tumor genome and express viral T antigen oncoproteins. The characterization of an early passage MCV-positive MCC cell line MS-1 is described, and its cellular, immunohistochemical, and virological features to MCV-negative (UISO, MCC13, and MCC26) and MCV-positive cell lines (MKL-1 and MKL-2) were compared. The MS-1 cellular genome harbors integrated MCV, which preserves an identical viral sequence from its parental tumor. Neither VP2 gene transcripts nor VP1 protein are detectable in MS-1 or other MCV-positive MCC cell lines tested. Mapping of viral and cellular integration sites in MS-1 and MCC tumor samples demonstrates no consistent viral or cellular gene integration locus. All MCV-positive cell lines show cytokeratin 20 positivity and grow in suspension. When injected subcutaneously into NOD scid gamma (NSG) mice, MS-1 forms a discrete macroscopic tumor. Immunophenotypic analysis of the MS-1 cell line and xenografts in mice show identical profiles to the parental tumor biopsy. Hence, MS-1 is an early passage cell line that provides a useful in vitro model to characterize MCV-positive MCC.

Merkel cell polyomavirus (MCV) is the recently discovered cause of most Merkel cell carcinomas (MCCs), an aggressive form of nonmelanoma skin cancer. Although MCV is known to integrate into the tumor cell genome and to undergo mutation, the molecular mechanisms used by this virus to cause cancer are unknown. Here, we show that MCV small T (sT) antigen is expressed in most MCC tumors, where it is required for tumor cell growth. Unlike the closely related SV40 sT, MCV sT transformed rodent fibroblasts to anchorage- and contact-independent growth and promoted serum-free proliferation of human cells. These effects did not involve protein phosphatase 2A (PP2A) inhibition. MCV sT was found to act downstream in the mammalian target of rapamycin (mTOR) signaling pathway to preserve eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1) hyperphosphorylation, resulting in dysregulated cap-dependent translation. MCV sT–associated 4E-BP1 serine 65 hyperphosphorylation was resistant to mTOR complex (mTORC1) and mTORC2 inhibitors. Steady-state phosphorylation of other downstream Akt-mTOR targets, including S6K and 4E-BP2, was also increased by MCV sT. Expression of a constitutively active 4E-BP1 that could not be phosphorylated antagonized the cell transformation activity of MCV sT. Taken together, these experiments showed that 4E-BP1 inhibition is required for MCV transformation. Thus, MCV sT is an oncoprotein, and its effects on dysregulated cap-dependent translation have clinical implications for the prevention, diagnosis, and treatment of MCV-related cancers.

Merkel cell polyomavirus (MCV) is a virus discovered in our laboratory at the University of Pittsburgh that is monoclonally integrated into the genome of ≈80% of human Merkel cell carcinomas (MCCs). Transcript mapping was performed to show that MCV expresses transcripts in MCCs similar to large T (LT), small T (ST), and 17kT transcripts of SV40. Nine MCC tumor-derived LT genomic sequences have been examined, and all were found to harbor mutations prematurely truncating the MCV LT helicase. In contrast, four presumed episomal viruses from nontumor sources did not possess this T antigen signature mutation. Using coimmunoprecipitation and origin replication assays, we show that tumor-derived virus mutations do not affect retinoblastoma tumor suppressor protein (Rb) binding by LT but do eliminate viral DNA replication capacity. Identification of an MCC cell line (MKL-1) having monoclonal MCV integration and the signature LT mutation allowed us to functionally test both tumor-derived and wild type (WT) T antigens. Only WT LT expression activates replication of integrated MCV DNA in MKL-1 cells. Our findings suggest that MCV-positive MCC tumor cells undergo selection for LT mutations to prevent autoactivation of integrated virus replication that would be detrimental to cell survival. Because these mutations render the virus replication-incompetent, MCV is not a “passenger virus” that secondarily infects MCC tumors.

Merkel cell carcinoma (MCC) is a rare but aggressive human skin cancer that typically affects elderly and immunosuppressed individuals, a feature suggestive of an infectious origin. We studied MCC samples by digital transcriptome subtraction and detected a fusion transcript between a previously undescribed virus T antigen and a human receptor tyrosine phosphatase. Further investigation led to identification and sequence analysis of the 5387–base-pair genome of a previously unknown polyomavirus that we call Merkel cell polyomavirus (MCV or MCPyV). MCV sequences were detected in 8 of 10 (80%) MCC tumors but only 5 of 59 (8%) control tissues from various body sites and 4 of 25 (16%) control skin tissues. In six of eight MCV-positive MCCs, viral DNA was integrated within the tumor genome in a clonal pattern, suggesting that MCV infection and integration preceded clonal expansion of the tumor cells. Thus, MCV may be a contributing factor in the pathogenesis of MCC.

Placental trophoblast cells are unique endocrine cells that play vital roles during the processes of embryonic implantation and placentation. However, research into the function of human trophoblast has been largely restrained mainly due to a lack of adequate cell models. A normal placenta-origin cytotrophoblast cell line (NPC) was previously established by our group, but these cells showed replicating senescence after 50 population doublings (PDs). In this study, the human telomerase catalytic subunit gene (htert) was transferred into B6 strain of NPC cells, and strains with reconstituted telomerase activity (B6Tert) were established. It was shown that B6Tert-1 cells produce various biomarkers of normal extravillous cytotrophoblasts during the early weeks of gestation. Meanwhile, the cell invasiveness was inhibited by transforming growth factor beta (TGFbeta). However, their ability to form syncytium was relatively low when stimulated with fetal calf serum (FCS). The cells maintained normal cell growth properties and failed to elicit tumours in nude mice. They proliferated continuously with no signs of senescence until the final count at 210 PDs. The growth rate of B6Tert-1 cells was increased when compared with the parental cells, which results, at least partly, from facilitating release of the G1/S checkpoint during the cell-cycle regulation. This is the first report of immortalizing human normal cytotrophoblast (CTB) cells by activation of telomerase activity. The cells will provide an ideal in vitro model for the study of human extravillous trophoblast (EVT) functions and consequently the mechanisms of embryonic implantation and placentation.

Background: All-trans retinoic acid (ATRA) is a natural vitamin A derivative that has a profound effect on the regulation of cell growth, differentiation and death. Aim: To investigate the tissue dynamic and cellular invasion effects of ATRA in choriocarcinoma (CCA), an aggressive trophoblastic tumour, by using a three-dimensional organotypic culture model system and cell invasion assay, respectively. Methods: An organotypic culture model of two CCA cell lines, JAR and JEG, was established. The effects of 1 μM ATRA on proliferation, differentiation and apoptosis on this CCA model were assessed by morphological assessment of the mitotic and apoptotic figures as well as by Ki-67 and caspase-related M30 cytoDeath antibody immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay. The effect of ATRA on p53 and its regulated protein product, WAF1/Cip1, was also evaluated with DO7 and p21 WAF1 antibodies, respectively. Moreover, the effect of ATRA on cellular (CCA) invasion was also investigated with Cell Invasion Kit on the JEG cell line. Results: ATRA was found to induce marked apoptosis in organotypic cultures of both cell lines, as evidenced by increased M30-positive cells (p, published_or_final_version

Epstein-Barr virus (EBV) infection is closely associated with the development of nasopharyngeal carcinoma (NPC). The EBV-encoded RNAs (EBERs) are the most abundant EBV transcripts (about 10(7) copies per cell) in EBV infected cells. However, the cellular function of EBER expression, particularly in nasopharyngeal epithelial cells, remains poorly understood. EBERs acquire secondary structures analogous to double-stranded RNA (dsRNA) and may bind to the double-stranded RNA-dependent protein kinase (PKR) and interfere with its function. Activation of PKR involves autophosphorylation resulting in protein synthesis inhibition and cellular apoptosis. Induction of cellular apoptosis by activation of PKR may be an antiviral response adopted by virally infected cells. We have examined the functional properties of EBER expression in an immortalized nasopharyngeal epithelial cell line (NP69). Expression of EBERs was achieved by transfecting the NP69 cells with an EBER-expressing plasmid, pESK10. The EBER-expressing NP69 cells attained a higher growth rate compared to cells transfected with control plasmid (pcDNA3). However, the EBER-expressing NP69 cells did not form colonies in soft agar and were non-tumorigenic in nude mice. To investigate if EBERs may protect the nasopharyngeal epithelial cells from apoptotic insults, we treated the EBER-expressing NP69 cells with a dsRNA analogue, poly(I).poly(C) (pIC), to activate PKR in cells and examined for their responses. Lower level of PKR phosphorylation and elevation of Bcl-2 were observed in EBER-expressing NP69 cells. In addition, other apoptotic markers including the cleaved forms of caspase-3 and poly(ADP)ribose polymerase (PARP) were found to be lower in EBER-expressing NP69 cells after treatment with pIC. Lower phosphorylation levels of p38 MAPK (mitogen-activated protein kinase) and c-jun were also observed in EBER-expressing NP cells. Our results suggest that EBER expression may confer an apoptotic-resistant phenotype in immortalized nasopharyngeal epithelial cells.

Increased EGFR (epidermal growth factor receptor) expression has been reported in many types of human cancer and its levels are positively associated with advanced cancers. Recently, upregulation of Id-1 (inhibitor of differentiation or DNA binding) protein was found in over 70% of ovarian cancer samples and correlated with poor survival of ovarian cancer patients. However, the molecular mechanisms responsible for the role of Id-1 in ovarian cancer are not clear. The aim of this study was to investigate the effect of Id-1 on ovarian cancer proliferation and its association with the EGFR pathway. To achieve this, we transfected an Id-1 expression vector into three ovarian cancer cell lines and examined cell proliferation rate by flow cytometry and bromodeoxyuridine staining. We found that ectopic Id-1 expression led to increased cell proliferation demonstrated by increased BrdU incorporation rate and S-phase fraction. The Id-1-induced cell growth was associated with upregulation of EGFR at both transcriptional and protein levels. In contrast, inactivation of Id-1 through transfection of an Id-1 antisense vector resulted in downregulation of EGFR. Our results indicate that increased Id-1 in ovarian cancer cells may promote cancer cell proliferation through upregulation of EGFR. Our findings also implicate that Id-1 may be a potential target for the development of novel strategies in the treatment of ovarian cancer.

The methylation status of genes in hydatidiform mole and choriocarcinoma and its significance is relatively unexplored. We investigated the methylation status of the promoter regions of six genes, p16, HIC-1, TIMP3, GSTP1, death-associated protein kinase (DAPK), and E-cadherin in 54 hydatidiform moles, five choriocarcinomas, and 10 first trimester placenta by methylation-specific polymerase chain reaction (PCR). Immunohistochemical expression of p16, TIMP3, and E-cadherin, and quantitative real-time RT-PCR of p16 was also performed. Among the six genes examined, the promoter region of four genes (E-cadherin, HIC-1, p16, TIMP3) in choriocarcinoma and three genes (E-cadherin, HIC-1, p16) in hydatidiform mole exhibited aberrant methylation whereas none was hypermethylated in normal placenta. There was a significant correlation between methylation and reduced expression of p16, E-cadherin, and TIMP3 (P < 0.001). Fifteen of the 54 patients with hydatidiform mole developed gestational trophoblastic neoplasia requiring chemotherapy. Promoter hypermethylation of p16 alone, or combined with E-cadherin, was significantly correlated to such development (P = 0.001, 0.0005, respectively). Hypermethylation of multiple genes, especially p16, might be related to the subsequent development of gestational trophoblastic neoplasia.

Objective . To assess the differential gene expression in neoplastic and normal trophoblastic cells and evaluate the effect of methylation on tissue inhibitor of metalloproteinase 3 (TIMP3) expression in choriocarcinoma (CCA) cells. Methods . The Atlas™ Human Cancer 1.2 Array (Clontech) was used to compare differential gene expression in a trophoblastic cell line (B6) established from first term placenta with two choriocarcinoma cell lines (JAR and JEG-3). The differentially expressed candidate genes in the placental and malignant trophoblastic cells in these cell lines were confirmed by real-time polymerase chain reaction (PCR), Western blotting and immunohistochemistry. Differential expression of a specific gene, TIMP3, was confirmed by immunohistochemistry using clinical specimens of choriocarcinoma and placenta. The involvement of promoter methylation in suppression of TIMP3 expression in choriocarcinoma was examined using methylation-specific PCR (MSP) and demethylation treatment. Results . Differential expression of 23 genes was observed in choriocarcinoma cell lines compared to the placental trophoblastic cells using the cDNA array analysis (Atlas™ Human Cancer Array, Clontech). Among these differentially expressed genes, down-regulation of TIMP3, PLAB, IGFBP3 and up-regulation of CCNB1 were confirmed by real-time PCR determination. Reduced expression of TIMP3 was further confirmed in clinical samples of choriocarcinoma by immunohistochemical staining. Methylation of TIMP3 promoter was detected in choriocarcinoma cell lines and clinical samples of choriocarcinoma. Treatment with a demethylation drug, 5-aza-2′-deoxycytidine, in choriocarcinoma cell lines restored TIMP3 expression. Conclusion . Down-regulation of TIMP3, PLAB, IGFBP3 and up-regulation of CCNB1 were observed in choriocarcinoma cells compared to placental trophoblasts. Down-regulation of TIMP3 expression was confirmed in clinical specimens of choriocarcinoma and may play a role in its pathogenesis. Promoter methylation of the TIMP3 is involved in suppression of TIMP3 expression. Differentiation expression of TIMP3 in choriocarcinoma may have potential application in clinical diagnosis and patient treatment.

Nasopharyngeal carcinoma is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded LMP1 has cell transformation property. It suppresses cellular senescence and enhances cell survival in various cell types. Many of the downstream events of LMP1 expression are mediated through its ability to activate NF-kappaB. In this study, we report a novel function of LMP1 to induce Id1 expression in nasopharyngeal epithelial cells (NP69) and human embryonal kidney cells (HEK293). The Id1 is a basic helix-loop-helix (bHLH) protein and a negative transcriptional regulator of p16(INK4a). Expression of Id1 facilitates cellular immortalization and stimulates cell proliferation. With the combination of both specific chemical inhibitors and genetic inhibitors of cell signaling, we showed that induction of Id1 by LMP1 was dependent on its NF-kappaB activation domain at the carboxy-terminal region, CTAR1 and CTAR2. Induction of Id1 by LMP1 may facilitate clonal expansion of premalignant nasopharyngeal epithelial cells infected with EBV and may promote their malignant transformation.

Background: The NSABP FB-7 trial, reported at SABCS 2015 (Jacobs et al., abstract # PD5-04), enrolled HER2-positive breast cancer patients (pts) with locally advanced disease who were randomly assigned to trastuzumab (T) or neratinib (N) or the combination (T+N) with weekly paclitaxel (P) followed by standard AC. Dual anti-HER2 therapy yielded greater pathological complete response (pCR) rates than single targeted therapies in the overall cohort. The purpose of this study was to evaluate the technical qualities of a new custom, targeted AmpliSeq panel and to use it to assess mutations in FB-7 pretreatment biopsies and surgical samples. Methods: To identify mutations that could act as resistance markers to anti-HER2 therapy, we designed a custom panel, termed the NSABP Anti-HER2 Resistance AmpliSeq (NAR) panel, interrogating 117 genes involved in HER2 signaling pathways and/or previously implicated as resistance markers to trastuzumab. The NAR panel, including 3,847 amplicons in 2 pools, was used to sequence HDx™ reference standards with the Ion Torrent sequencing platform. First, the panel and our mutation-calling pipeline were tested for reproducibility and accuracy using the reference samples. Subsequently, DNA from pretreatment biopsies and matched surgical samples (when available) from FB-7 pts were assayed for mutations using the NAR panel. Results: Across the NAR panel, the average target coverage was 94.25%. For the ERBB2 gene, the coverage was 96.0%. HDx™ reference standards, with known allele frequencies as evaluated by ddPCR, were used to assess the accuracy of the NAR panel, using a custom pipeline to filter SNVs. HDx™ reference standards were sequenced with the NAR panel, and the resulting allele frequencies compared with the known allele frequencies. Based on examination of the following mutations, BRAF-V600E, KRAS-G13D, KRAS-G12D, NRAS-Q61K, PIK3CA-H1047R, MET-Del-FS, PDGFRA-G426D, EGF-G719S, we observed a Pearson correlation coefficient of 0.9822. Next, we used our NAR Ampliseq panel to sequence a subset of colon samples collected as part of the NSABP Genome Assessment Guided Medicine (N-GAMe) cohort. Comparison of our NAR AmpliSeq results and those from a CLIA certified Illumina sequencing platform over all ERBB2 polymorphism and mutation sites demonstrated a correlation coefficient of 0.978. We also assessed the association of copy number determined with the Ion Torrent sequencing software with pCR. We confirmed that the pCR rates were higher in patient samples with greater amplification of ERBB2 (P < 0.05). Mutant allele frequencies were compared in the before-treatment biopsies to surgical samples. The mutant allele frequencies of PIK3CA-E545K, MAP2K4-S184L, MTOR-R491Q, and MAP2K4-D150N were increased after treatment, suggesting that these mutations may have provided a selective advantage during treatment, implicating a potential resistance marker. We predicted that N or T+N-treated patients with an ERBB2-V777L mutation would achieve pCR, but such a patient did not achieve pCR. Other mutations in this patient may have been responsible for the failure to achieve pCR. One patient with an ERBB2-S310F mutation and treated with N, did achieve pCR, as predicted. Conclusion: Preliminary analysis of FB-7 suggests that sequence analysis with Ion torrent sequencing using the NAR panel may be clinically useful. Support: Puma Biotechnology and PA state grant, PA DOH disclaims responsibility for analysis, interpretations, or conclusions Citation Format: Huichen Feng, Ying Wang, Peter Lucas, Seong-Rim Kim, Corey Lipchik, Patrick Gavin, Samuel Jacobs, Ashok Srinivasan, Katherine L. Pogue-Geile. Assessment of mutations with a custom NSABP anti-HER2 resistance AmpliSeq (NAR) panel in NSABP clinical trial FB-7 [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 LB-A22.

Inhibitor of differentiation or DNA binding (Id-1), a helix-loop-helix transcription factor, has recently been shown to inactivate the retinoblastoma (RB)/p16INK4a pathway through down-regulation of p16INK4a and increasing phosphorylation of RB in certain cell types. Nasopharyngeal carcinoma (NPC) is a common cancer in Hong Kong, and inactivation of the tumor suppressor RB at transcription level is a rare event in NPC. The objective of this study was to investigate the role of Id-1 in NPC cell proliferation and its expression in NPC samples. An NPC cell line, CNE1, was transfected with a retroviral vector containing a full-length Id-1 cDNA, and six stable transfectant clones were isolated with differential Id-1 expression levels. The effect of ectopic Id-1 expression on serum-independent cell growth, cell-cycle distribution, and expression of proteins associated with RB pathway was studied. The Id-1 expression in five NPC samples was also investigated using immunohistochemistry. Ectopic Id-1 expression in CNE1 cells resulted in an increase in serum-independent cell growth, percentage of cells in S phase, and phosphorylation of RB and cyclin-dependent kinase 2 proteins. In addition, immunohistochemical studies on NPC samples showed that expression of Id-1 was present in NPC cells but absent in normal tissues. This study demonstrates that Id-1 plays an important role in cell proliferation in NPC cells, and our results provide evidence for the first time of the significance of Id-1 expression in NPC cells and suggest a possible role of Id-1 expression in the inactivation of RB and development of NPC. © 2002 Wiley-Liss, Inc.

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, especially in southern China. One of the most striking features of this disease is its close relationship with Epstein–Barr Virus (EBV). However, to date there is no direct study on the mechanisms involved in the role of EBV in the tumorigenesis of NPC, largely due to lack of an experimental model. Available hypotheses on the association between EBV and NPC are generated from non-nasopharyngeal epithelial cell systems such as human keratinocytes or mouse epithelial cells, which may not truly represent the biological properties of nasopharyngeal epithelial (NP) cells. In this study, we report the establishment of two immortalized NP cell lines, NP69SV40T and NP39E6/E7, using SV40T and HPV16E6/E7 oncogenes. We found that NP60SV40T and NP39E6/E7 cell lines not only maintained many characteristics of normal NP cells (i.e. keratin profile and responsive to TGFβ inhibition) but also highly responsive to one of the EBV encoded genes, LMP1. Comparative genome hybridization (CGH) analysis showed that these two cell lines contained multiple genetic alterations, some of which have been described in NPC. The immortalized NP cell lines are non-tumorigenic and exhibit anchorage-dependent growth. These cell lines may provide a possible cell model system for studying the mechanisms involved in the tumorigenesis of NPC.

Human polyomavirus 7 (HPyV7) is one of 11 HPyVs recently discovered through genomic sequencing technologies. Two lung transplant recipients receiving immunosuppressive therapy developed pruritic, brown plaques on the trunk and extremities showing a distinctive epidermal hyperplasia with virus-laden keratinocytes containing densely packed 36-45-nm icosahedral capsids. Rolling circle amplification and gradient centrifugation testing were positive for encapsidated HPyV7 DNA in skin and peripheral blood specimens from both patients, and HPyV7 early and capsid proteins were abundantly expressed in affected tissues. We describe for the first time that HPyV7 is associated with novel pathogenicity in some immunosuppressed individuals.

MicroRNAs (miRNAs) are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA FISH. As a proof of concept, we used this method to differentiate two skin tumors, basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified the tumor-specific miRNAs miR-205 and miR-375 in BCC and MCC, respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and ribosomal RNA (rRNA) retention using model compounds and high-pressure liquid chromatography (HPLC) analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using predefined cutoff values, and all were correctly identified in blinded analysis. Our study establishes a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues.

Merkel cell polyomavirus (MCV) causes ~80% of primary and metastatic Merkel cell carcinomas (MCCs). By comparing digital transcriptome subtraction deep-sequencing profiles, we found that transcripts of the cellular survivin oncoprotein [BIRC5a (baculoviral inhibitor of apoptosis repeat-containing 5)] were up-regulated sevenfold in virus-positive compared to virus-negative MCC tumors. Knockdown of MCV large T antigen in MCV-positive MCC cell lines decreased survivin mRNA and protein expression. Exogenously expressed MCV large T antigen increased survivin protein expression in non-MCC primary cells. This required an intact retinoblastoma protein-targeting domain that activated survivin gene transcription as well as expression of other G(1)-S-phase proteins including E2F1 and cyclin E. Survivin expression is critical to the survival of MCV-positive MCC cells. A small-molecule survivin inhibitor, YM155, potently and selectively initiates irreversible, nonapoptotic, programmed MCV-positive MCC cell death. Of 1360 other chemotherapeutic and pharmacologically active compounds screened in vitro, only bortezomib (Velcade) was found to be similarly potent, but was not selective in killing MCV-positive MCC cells. YM155 halted the growth of MCV-positive MCC xenograft tumors and was nontoxic in mice, whereas bortezomib was not active in vivo and mice displayed serious morbidity. Xenograft tumors resumed growth once YM155 treatment was stopped, suggesting that YM155 may be cytostatic rather than cytotoxic in vivo. Identifying the cellular pathways, such as those involving survivin, that are targeted by tumor viruses can lead to rapid and rational identification of drug candidates for treating virus-induced cancers.

To investigate the correlation between BRI gene expression and the metastatic potential in human non small cell lung cancer cell lines.BRI gene differential expression was detected between human lung adenocarcinoma cell lines AGZY83-a and Anip973 by RT-PCR and Northern blot. Anip973 was isolated from AGZY83-a with a higher metastatic potential than its parent line. Other 6 human non small cell lung cancer cell lines, A549, TKB-18, SPC-A-1, GLC-82, 95D and PAa, were also detected for the relationship between BRI expression and metastatic potential.There was a significant difference in BRI gene expression between AGZY83-a and Anip973 cell lines. BRI was overexpressed in Anip973 cells comparing to AGZY83-a cells. Up regulation of BRI gene was also observed in other 6 lung cancer cell lines, and partly correlated with their metastatic potential. Furthermore, there were two mRNA transcripts in the lung cancer cells, in which the 1.6 kb transcript was the major one.The up-regulated expression of BRI 1.6 kb mRNA transcript may indicate the formation of metastatic potential of NSCLC. BRI is possibly a metastasis related gene.

Merkel cell polyomavirus (MCV) is a newly-discovered human tumor virus found in approximately 80% of Merkel cell carcinoma (MCC). The rate of MCV infection among persons without MCC is unknown. We developed a MCV virus-like particle (VLP) enzyme-linked immunoassay (EIA) that does not cross-react with human BK or murine polyomaviruses. Peptide mapping of the MCV VP1 gene and immunoblotting with denatured MCV VLP are less sensitive than the MCV EIA in detecting MCV antibodies suggesting antibody reactivity in this assay primarily targets conformational but not linear epitopes. Among MCC patients, all 21 (100%) patients tested with MCV-positive tumors had high serum MCV IgG but not high MCV IgM levels. Only 3 of 6 (50%) MCC patients with MCV-negative tumors were positive for MCV antibodies. Sera from most adults, including 107 of 166 (64%) blood donors, 63 of 100 (63%) commercial donors and 37 of 50 (74%) systemic lupus erythematosus patients, show evidence for prior MCV exposure. Age-specific MCV prevalence was determined by examining a cross-sectional distribution of 150 Langerhans cell histiocytosis (an unrelated neoplasm) patient sera. MCV prevalence increases from 50% among children age 15 years or younger to 80% among persons older than 50 years. We did not find evidence for vertical transmission among infants. Although past exposure to MCV is common among all adult groups, MCC patients have a markedly elevated MCV IgG response compared with control patients. Our study demonstrates that MCV is a widespread but previously unrecognized human infection.

Merkel cell polyomavirus (MCV) is a recently discovered human virus closely related to African green monkey lymphotropic polyomavirus. MCV DNA is integrated in approximately 80% of Merkel cell carcinomas (MCC), a neuroendocrine skin cancer linked to lymphoid malignancies such as chronic lymphocytic leukemia (CLL). To assess MCV infection and its association with human diseases, we developed a monoclonal antibody that specifically recognizes endogenous and transfected MCV large T (LT) antigen. We show expression of MCV LT protein localized to nuclei of tumor cells from MCC having PCR quantified MCV genome at an average of 5.2 (range 0.8-14.3) T antigen DNA copies per cell. Expression of this putative viral oncoprotein in tumor cells provides the mechanistic underpinning supporting the notion that MCV causes a subset of MCC. In contrast, although 2.2% of 325 hematolymphoid malignancies surveyed also showed evidence for MCV infection by DNA PCR, none were positive at high viral copy numbers, and none of 173 lymphoid malignancies examined on tissue microarrays expressed MCV LT protein in tumor cells. As with some of the other human polyomaviruses, lymphocytes may serve as a tissue reservoir for MCV infection, but hematolymphoid malignancies associated with MCC are unlikely to be caused by MCV.

Background and Aim: Cyclooxygenase-2 (COX-2) plays an important role in the carcinogenesis of esophageal squamous cell carcinoma (ESCC). However, it is not clear whether COX-2 is involved in the early or late stage of the development of ESCC. The aim of this study was to investigate the role of COX-2 in the carcinogenesis of ESCC by an immortalized esophageal epithelial cell line. Methods: Human papillomavirus type 16 (HPV16)–E6/E7 and human telomerase reverse transcriptase (hTERT) transfection were used for immortalization of esophageal epithelial cells. COX-2-specific RNA interference was used for the inhibition of COX-2 expression. Results: An immortalized esophageal epithelial cell line, NE6-E6E7/hTERT, was established, which had high proliferation activity but failed to induce colony formation in soft agar. COX-2 expression was upregulated in the early process of immortalization, while COX-2 small interfering RNA (siRNA) decreased the Bcl-2 expression, increased the expression of Bax, and induced cell-cycle arrest at the G0/G1 phase in NE6-E6E7/hTERT cells. Expressions of p53, cyclinD1, and the ratio of hyperphosphorylated-RB/hypophosphorylated-RB were progressively increased after E6E7 and the subsequent hTERT transfections. These changes were accompanied by the alteration of COX-2 expression, but could be reversed by COX-2 siRNA (P

Digital transcript subtraction (DTS) was developed to subtract in silico known human sequences from expression library data sets, leaving candidate nonhuman sequences for further analysis. This approach requires precise discrimination between human and nonhuman cDNA sequences. Database comparisons show high likelihood that small viral sequences can be successfully distinguished from human sequences. DTS analysis of 9,026 20-bp tags from an expression library of BCBL-1 cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) resolved all but three candidate sequences. Two of these sequences belonged to KSHV transcripts, and the third belonged to an unannotated human expression sequence tag. Overall, 0.24% of transcripts from this cell line were of viral origin. DTS analysis of 241,122 expression tags from three squamous cell conjunctival carcinomas revealed that only 21 sequences did not align with sequences from human databases. All 21 candidates amplify human transcripts and have secondary evidence for being of human origin. This analysis shows that it is unlikely that distinguishable viral transcripts are present in conjunctival carcinomas at 20 transcripts per million or higher, which is the equivalent of approximately 4 transcripts per cell. DTS is a simple screening method to discover novel viral nucleic acids. It provides, for the first time, quantitative evidence against some classes of viral etiology when no viral transcripts are found, thereby reducing the uncertainty involved in new pathogen discovery.

Nasopharyngeal carcinoma (NPC) is a common disease in Hong Kong and southern provinces of China. EBV infection is believed to play a critical role in the development of NPC. Previous studies on the transformation mechanism of EBV genes were mostly performed in either NPC or nonnasopharyngeal epithelial cells which may not be representative of premalignant nasopharyngeal epithelial cells. Establishment of a representative cell system would greatly facilitate the elucidation of the role of EBV infection in the development of NPC. Using telomerase alone, we were able to establish an immortalized nasopharyngeal epithelial cell line from primary nonmalignant nasopharyngeal biopsies. The telomerase-immortalized nasopharyngeal epithelial cells are largely diploid in karyotype. Interestingly, this newly immortalized nasopharyngeal epithelial cell line, referred as NP460hTert, harbors genetic alterations previously identified in premalignant and malignant nasopharyngeal epithelial cells. These include inactivation of p16 by homozygous deletion of the p16(INK4A) locus and downregulation of RASSF1A expression. The deletion of the p16(INK4A) locus appears to be the most crucial event for the immortalization of nasopharyngeal epithelial cells by telomerase and precedes RASSF1A downregulation. In addition, detailed analysis of the cytogenetic changes by conventional cytogenetics, spectral karyotyping (SKY) and array-based CGH revealed a gain of a 17q21-q25 fragment on 11p15 chromosome in all NP460hTert cells which occurred before deletion of the p16(INK4A) locus. Gain of 17q has been previously reported in NPC. In addition, activation of NF-kappaB was observed in immortalized NP460hTert cells at the later population doublings, and may play a role in the survival of immortalized NP epithelial cells. Id1 which is commonly expressed in various human cancers, including NPC, was also upregulated in the immortalized NP460hTert cells. Thus, the establishment of an immortalized nasopharyngeal epithelial cell line harboring common genetic alterations present in premalignant and cancerous nasopharyngeal epithelial cells may provide a valuable cell system to examine for early events involved in NPC carcinogenesis, particularly in elucidating the role of EBV infection in NPC development.

OBJECTIVE: Research into the biology of human trophoblast invasion has been hampered by a lack of in vitro models. The aim of this study was to establish and characterize a human extravillous trophoblast cell line from the first-trimester placenta. METHODS: Human papillomavirus type 16 (HPV16) E6/E7 genes were stably expressed in primary cultures of first-trimester placenta via a retroviral vector (pLXSN-E6/E7). Several clones were characterized for extravillous trophoblastic properties by reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry. The activities of matrix metalloproteinase (MMP)-2 and MMP-9 were examined with gelatin zymography. One clone (TEV-1), which retains all the established criteria for extravillous trophoblasts, was used in microarray analysis with Stanford Human cDNA chip (41, 421 cDNA features) to examine the differential gene expression after treatment of transforming growth factor beta 1 (TGF1). The responsive gene to TGF1 treatment was confirmed by quantitative real-time PCR. RESULTS: The clonal TEV-1 has been passaged for more than 105 population doublings with no sign of senescence, the activation of telomerase at early passages, and a near-diploid karyotype. TEV-1 cells expressed cytokeratin 7, HLA-G (a histocompatibility antigen, class IB), and CD9 (the cluster of differentiation antigen 9), and secreted active MMP-2 and MMP-9. TGF1 treatment altered the gene expression profile of TEV-1 cells with a marked up-regulation of insulin-like growth factor binding protein 3 (IGFBP3), which was confirmed by quantitative real-time PCR. In addition, the TEV-1 was nontumorigenic when injected into nude mice and unable to form colonies in soft agar. CONCLUSION: Phenotypic and biologic characteristics of TEV-1 were shown as the properties of extravillous trophoblasts; thus, the TEV-1 cell line may be used as a cell model in extravillous trophoblast studies. (J Soc Gynecol Investig 2005;12:e21–e32) Copyright © 2005 by the Society for Gynecologic Investigation.

Extravillous cytotrophoblast (EVCT) cultures from the normal placentas of three pregnant women were transfected by HPVE6E7. Sequential cytogenetic and molecular analyses were performed to delineate genetic events that may be critical for cell immortalization. One line, PE1-E6E7, was immortalized successfully, whereas 2 other lines, PE3-E6E7 and PE4-E6E7, could not be maintained beyond crisis. Before crisis, the majority of cells in all lines were karyotypically normal. During the early stages of crisis, there was progressive telomere shortening. Most cells were karyotypically abnormal, with extreme cytogenetic divergence and a predominance of telomeric association and dicentric chromosomes affecting many chromosomes. At the later stages of crisis, the karyotype became more convergent with a drastic decrease in nonclonal aberrations. In PE1-E6E7, after crisis the karyotype was complex, with frequent centromeric rearrangements in the form of isochromosomes and whole-arm translocations. There were unbalanced structural aberrations and numerical changes, including loss of chromosome 13, that could be traced throughout the evolution of the line. These findings support the concept that immortalization is a relatively rare and nonrandom event that occurs only in cells that have acquired the necessary or critical genetic alterations. Telomeric dysfunction may be an important mechanism leading to the acquisition of complex karyotypical aberrations.