Formalin-fixed Paraffin-embedded Tissue Blocks Research Articles

Differential long-term stability of microRNAs and RNU6B snRNA in 12\u201320 year old archived formalin-fixed paraffin-embedded specimens

BackgroundThe quantitative analysis of microRNA (miRNA) gene expression in archived formalin-fixed, paraffin embedded (FFPE) tissues has been instrumental to identifying their potential roles in cancer biology, diagnosis, and prognosis. However, it remains unclear whether miRNAs remain stable in FFPE tissues stored for long periods of time.MethodsHere we report Taqman real-time RT-PCR quantification of miR-21, miR-141, miR-221, and RNU6B small nuclear RNA (snRNA) levels from 92 radical prostatectomy specimens stored for 12–20 years in FFPE blocks. The relative stability of each transcript over time was assessed using general linear models. The correlation between transcript quantities, sample age, and RNA integrity number (RIN) were determined utilizing Spearman rank correlation.ResultsAll transcript levels linearly decreased with sample age, demonstrating a clear loss of miRNA stability and RNU6B snRNA stability over time. The most rapid rates of degradation were observed for RNU6B and miR-21, while miR-141 and miR-221 were more stable. RNA quality was not correlated with sample age or with miR-21, miR-221, or RNU6B snRNA levels. Conversely, miR-141 levels increased with RNA quality.ConclusionsMiRNA and snRNA levels gradually decreased over an eight year period in FFPE tissue blocks. Sample age was the most consistent feature associated with miRNA stability. The reference snRNA, RUN6B, was more rapidly degraded when compared to miR-141 and miR-221 miRNAs. Various miRNAs demonstrated differential rates of degradation. Quantitative miRNA studies from long-term archived FFPE tissues may therefore benefit from epidemiologic study design or statistical analysis methods that take into account differential storage-dependent transcript degradation.

The Immunoexpression of Glucocorticoid Receptors in Breast Carcinomas, Lactational Change, and Normal Breast Epithelium and Its Possible Role in Mammary Carcinogenesis.

The role of estrogen and progesterone receptors in breast cancer biology is well established. In contrast, other steroid hormones are less well studied. Glucocorticoids (GCs) are known to play a role in mammary development and differentiation; thus, it is of interest to attempt to delineate their immunoexpression across a spectrum of mammary epithelia. Aim. To delineate the distribution pattern of glucocorticoid receptors (GRs) in malignant versus nonmalignant epithelium with particular emphasis on lactational epithelium. Materials and Methods. Immunohistochemistry (IHC) for GRs was performed on archival formalin-fixed paraffin-embedded tissue blocks of 96 cases comprising 52 invasive carcinomas, 21 cases with lactational change, and 23 cases showing normal mammary tissue histology. Results. Results reveal an overexpression of GRs in mammary malignant epithelium as compared to both normal and lactational groups individually and combined. GR overexpression is significantly more pronounced in HER-2-negative cancers. Discussion. This is the first study to compare GR expression in human lactating epithelium versus malignant and normal epithelium. The article discusses the literature related to the pathobiology of GCs in the breast with special emphasis on breast cancer. Conclusion. The lactational epithelium did not show overexpression of GR, while GR was overexpressed in mammary NST (ductal) carcinoma, particularly HER-2-negative cancers.

Correlation of CD44v6 and type IV collagen immunohistochemical expression in borderline and malignant surface epithelial tumors

Background: the difference in expression of type IV collagen in borderline tumors and ovarian carcinomas has been studied, but the association with adhesion molecules like CD44 have not gain enough interest. Objectives: The purpose of this study is to assess the expression of CD44v6 and type IV collagen status in borderline tumors and invasive ovarian carcinomas and the correlation between them to define the role of these molecules in tumor invasion and metastasis. Type of the study: A cross sectional study Methods: The study included a total of (101) formalin-fixed paraffin-embedded ovarian tissue blocks; of which (19) cases were borderline tumors and (82) cases were overt ovarian carcinomas. Sections from each block were immunohistochemically stained for CD44v6 and type IV collagen. Results: CD44v6 was significantly correlated with FIGO stage of borderline tumors (p=0.001) and ovarian carcinoma (<0.001) and with histological grade of ovarian carcinomas (p=0.004). There was significantly higher expression of type IV collagen in borderline tumors compared to invasive carcinoma(p<0.001) this significance was also seen in correlation to age, stage and grade of the invasive carcinoma, no significant differences in other clinicopathological features were found. There was negative correlation between CD44 v6 & type IV collagen which was statistically significance (P<0.05) in carcinoma but not in borderline tumors. Conclusions: Our data suggest that observed inverse correlation of type IV collagen expression with CD44v6 positivity in surface epithelial tumors indicates that these molecules may cooperate in the invasion and progression of ovarian carcinomas.

The Prostate, Lung, Colorectal and Ovarian Cancer (PLCO) Screening Trial Pathology Tissue Resource.

Pathology tissue specimens with associated epidemiologic and clinical data are valuable for cancer research. The Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial undertook a large-scale effort to create a public resource of pathology tissues from PLCO participants who developed a cancer during the trial. Formalin-fixed paraffin-embedded tissue blocks were obtained from pathology laboratories on a loan basis for central processing of tissue microarrays, with additional free-standing tissue cores collected for nucleic acid extraction. Pathology tissue specimens were obtained for prostate cancer (n = 1,052), lung cancer (n = 434), colorectal cancer (n = 675) and adenoma (n = 658), ovarian cancer and borderline tumors (n = 212), breast cancer (n = 870), and bladder cancer (n = 204). The process of creating this resource was complex, involving multidisciplinary teams with expertise in pathology, epidemiology, information technology, project management, and specialized laboratories. Creating the PLCO tissue resource required a multistep process, including obtaining medical records and contacting pathology departments where pathology materials were stored after obtaining necessary patient consent and authorization. The potential to link tissue biomarkers to prospectively collected epidemiologic information, screening and clinical data, and matched blood or buccal samples offers valuable opportunities to study etiologic heterogeneity, mechanisms of carcinogenesis, and biomarkers for early detection and prognosis. The methods and protocols developed for this effort, and the detailed description of this resource provided here, will be useful for those seeking to use PLCO pathology tissue specimens for their research and may also inform future tissue collection efforts in other settings. Cancer Epidemiol Biomarkers Prev; 25(12); 1635-42. ©2016 AACR.

Deamination Effects in Formalin-Fixed, Paraffin-Embedded Tissue Samples in the Era of Precision Medicine

Deamination of nucleotides causes C:G>T:A changes in formalin-fixed, paraffin-embedded (FFPE) tissue samples and produces false positives during next-generation sequencing (NGS). Uracil DNA glycosylase (UDG) helps eliminate this issue, but the effect of UDG in different tissue preparation conditions has not been rigorously studied. To investigate whether UDG can reduce false-positive single-nucleotide variant (SNV) calls, we used tumor and normal tissues from gastric adenocarcinoma patients prepared using different fixation times and pH conditions. FFPE tumor blocks >10 years were also evaluated for the comparison. We performed semiconductor-based NGS to evaluate nucleotide changes and used UDG to test deamination-related effects. Sequencing quality parameters mildly worsened with prolonged fixation time, acidic pH, and delayed fixation. SNV calls and C:G>T:A changes increased after >48 hours of fixation. In both recently prepared and old FFPE tissue blocks, UDG treatment reduced deamination-induced nucleotide changes. In the recently prepared samples, both high-quality SNVs and mean target coverage were remarkably increased on treatment with UDG. However, the quality of NGS results from old-age samples varied irrespective of UDG treatment. In conclusion, based on our findings, we believe that when performing NGS on recently embedded blocks, it is important to consider that certain poorly fixed samples may be at the risk of being deaminated, which can be corrected with UDG treatment.

Discordant Mutations in Paired Primary and Metastatic Endometrial Adenocarcinomas Identified by Semiconductor-Based Sequencing for Rapid Cancer Genotyping.

Although endometrial adenocarcinoma is usually treated with surgery, patients with metastatic disease have a poor prognosis. To address the need for better treatment options, molecularly targeted drug therapies are being developed. These targeted therapies rely on accurate mutational profiling of the tumor, which is most often performed on DNA from the primary tumor. Our objective was to compare mutational concordance in primary tumors with their metastases. We genotyped 11 pairs of primary and metastatic endometrial adenocarcinomas using DNA from formalin-fixed paraffin-embedded tissue blocks and semiconductor-based next-generation sequencing. Five of these cases had multiple metastases for comparison. We sequenced 37 known cancer genes that are targets for new drug therapies. A total of 62 mutations were identified in 16 of these 37 genes. The most common mutations were in PIK3CA and PTEN. Overall, there was a 53% discordance in mutations between primary tumors and their paired (33 of 62). The absence of mutations in metastases (25 of 33, 76%) compared with the primary neoplasm was more common than gain of mutations (8 of 33, 24%). There was a 15% discordance rate between paired metastases within individuals (6 of 40), which was significantly less frequent than the rate between primary tumors and their metastases (Fisher exact P value <.0001). Although the sample size is relatively small, our data suggest it may be prudent to test metastases, rather than the primary neoplasm, when using molecularly targeted drug therapies, because isolated metastases may lack mutations detected in the heterogeneous mixture of the tumor's origin.

HER-2/neu Overxpression in Esophageal Squamous Cell Carcinoma (ESCC) and Its Correlation with Patient’s Clinicopathological Features

Background: HER-2/neu overexpression has been reported in various human cancers and identified as a significant predictor of poor survival. In this study HER-2/neu overexpression and its associations with clinicopathological characteristics were evaluated in patients with esophageal squamous cell carcinoma (ESCC). Methods: This cross-sectional study was performed on 64 patients with histological diagnosis of primary ESCC who underwent surgery for curative treatment. Immunohistochemistry (IHC) was used to assess expression of HER-2/neu receptor in formalin-fixed paraffin-embedded tissue blocks. Results: The mean age of patients was 60.1 ± 1.28 years. The overall HER2 expression was observed in 51.5% of ESCC patients without considering IHC scores. HER2/neu overexpression (6%) was significantly associated with the tumor differentiation (P < 0.001). 20.3% of cases were stage I, 67.2% stage II, and 12.5% stage III. 17 patients (26.2%) had vascular invasion, 12 patients (18.8%) had neuronal invasion and 7 patients (10.9%) had invasion to margins. Nine of 12 patients with HER-2/neu over expression had thoracic tumors and only three of them had an abdominal ESCC. Conclusions: No significant correlations were found between HER2/neu overexpression and gender, age, tumor invasion, location of tumor, TNM stages and stage of tumor in patients with ESCC.

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

Human Papillomavirus Genotype Distribution in Invasive Cervical Cancer in Pakistan.

Few studies have assessed the burden of human papillomavirus (HPV) infection in Pakistan. We aim to provide specific information on HPV-type distribution in invasive cervical cancer (ICC) in the country. A total of 280 formalin-fixed paraffin-embedded tissue blocks were consecutively selected from Shaukat Khanum Memorial Cancer Hospital and Research Centre (Lahore, Pakistan). HPV-DNA was detected by SPF10 broad-spectrum PCR followed by DNA enzyme immunoassay and genotyping by LiPA25. HPV-DNA prevalence was 87.5% (95%CI: 83.0–91.1), with 96.1% of cases histologically classified as squamous cell carcinoma. Most of the HPV-DNA positive cases presented single infections (95.9%). HPV16 was the most common type followed by HPV18 and 45. Among HPV-DNA positive, a significantly higher contribution of HPV16/18 was detected in Pakistan (78.4%; 72.7–83.3), compared to Asia (71.6%; 69.9–73.4) and worldwide (70.8%; 69.9–71.8) and a lower contribution of HPVs31/33/45/52/58 (11.1%; 7.9–15.7 vs. 19.8%; 18.3–21.3 and 18.5%; 17.7–19.3). HPV18 or HPV45 positive ICC cases were significantly younger than cases infected by HPV16 (mean age: 43.3, 44.4, 50.5 years, respectively). A routine cervical cancer screening and HPV vaccination program does not yet exist in Pakistan; however, the country could benefit from national integrated efforts for cervical cancer prevention and control. Calculated estimations based on our results show that current HPV vaccine could potentially prevent new ICC cases.

Abstract 4935: Targeted proteomic analysis of hepatocellular carcinoma and its histologic mimickers

Abstract Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Well differentiated HCC can sometimes be extremely difficult to be distinguished from other hepatocellular mass lesions such as hepatocellular adenoma and dysplastic liver nodule due to considerable morphologic overlaps. Immunohistochemical markers may have a limited utility, especially on core biopsy. Choice of treatment depends on the extent and location of the cancer, and the overall health of the patient. For patients who are healthy enough to undergo surgery and who have early-stage cancer confined to the liver, treatment may involve hepatic resection; however many patients develop a cancer recurrence, which is the main cause of death in long-term evaluations. Recurrence rates after treatment with resection are high, highlighting the importance of finding effective adjuvant treatments and markers that aid in determining differences between hepatocellular lesions. To explore differences between hepatocellular lesions that could be indicators of tumor behavior, we used targeted proteomic analysis to assess different protein biomarkers in formalin-fixed paraffin-embedded (FFPE) HCC tumor tissue Twenty-two FFPE HCC tissue blocks were obtained and a pathologist marked a minimum 8mm2 of tumor area. Following laser microdissection, proteins were extracted using the Liquid-Tissue® process and subjected to selected reaction monitoring mass spectrometry to quantify the amounts of 30 different targeted proteins. As anticipated, well differentiated HCC, hepatocellular adenoma and dysplastic nodule expressed high rates of P-glycoprotein and the majority expressed multi-drug resistance gene protein (MDR1). Such markers may account in part for the chemotherapy refractory nature of HCC. All 22 patients expressed high levels of hENT1 and lacked expression of RRM1, indicating that gemcitabine-based therapy would be an appropriate choice. All 22 patients lacked expression of marker of sensitivity to anthracycline (TOPO2A) and the majority of patients did not express a marker of resistance to platinum therapy (ERCC1). Of the 22 patients whose tumors expressed EGFR, 5 had expression above the 75%ile and would thus be eligible for EGFR small molecule inhibitor therapy. Dysplastic liver nodule patients did not expressed significant level of EGFR. Further, multiplex-targeted proteomics discovered patients expressing cMET and IDO1, which indicate eligibility for clinical trials of targeted therapies or immunotherapies. 60% of dysplastic liver nodule patients did not expressed cMET at any level This study retrospectively evaluates HCC patients in an attempt to identify predictors of tumor behavior. Proteomic and genomic screening should be performed to identify differences between various hepatocellular lesions. Prospective evaluation of molecular and genetic profile is warranted in HCC patients to be distinguished from other hepatocellular mass lesions. Citation Format: Fabiola Cecchi, Nam Ku, Hanlin Wang, Adele Blackler, Todd Hembrough, Shahrooz Rabizadeh, Patrick Soon-Shiong, Jiaoti Huang. Targeted proteomic analysis of hepatocellular carcinoma and its histologic mimickers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4935.

Investigation into the presence of human papillomavirus in patients with obstructive sleep apnea.

The human papillomavirus (HPV) is known to infect the tissues of the oropharynx as demonstrated in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC). HPV has also been shown to induce benign lymphoid hypertrophy. We sought to investigate an association between obstructive sleep apnea (OSA) and the presence of HPV in palatine and lingual tonsillar oropharyngeal tissue. Case series with chart review. This retrospective laboratory-based study of oropharyngeal tissue from patients with OSA included patients >18 years old who underwent surgical treatment for OSA at a single institution between January 2012 and May 2014. Surgical specimens of adequate size were analyzed for HPV6, 11, and 16 using real-time quantitative polymerase chain reaction from DNA extracted from formalin-fixed paraffin-embedded tissue blocks. Student t test, Pearson χ2 test, and linear logistic regression were used to assess comparisons of body mass index (BMI), apnea-hypopnea index (AHI), age, and gender between HPV-positive and HPV-negative groups. Of 99 cases included in the study, six were positive for HPV: two with HPV16 and four with HPV6. BMI, AHI, age, and gender showed no significant differences between the HPV-positive and HPV-negative groups. Logistic regression to predict HPV positivity accounting for each variable and multivariate analysis were not statistically significant. Our study did not show HPV to have a statistically significant association with OSA. None of the covariates analyzed (BMI, AHI, gender, age) predicted HPV positivity in surgically resected oropharyngeal tissue from OSA patients. 4 Laryngoscope, 127:1231-1234, 2017.

Benign and Malignant Brenner Tumors Show an Absence of TERT Promoter Mutations That Are Commonly Present in Urothelial Carcinoma.

Brenner tumors are uncommon ovarian neoplasms, which have morphologic and immunophenotypical features of transitional cell (urothelial) differentiation. The origin of Brenner tumors is perplexing, but they are believed to arise from transitional cell metaplasia occurring within the ovary and/or fallopian tube, although it is controversial whether this metaplasia is truly along transitional cell lines. Recently, TERT promoter mutations have been identified in urothelial carcinoma (UC) with high frequency (approximately 70%), and the current literature suggests a potential diagnostic and/or prognostic role of these mutations in UC. Molecular evidence supporting that Brenner tumors represent neoplasms exhibiting transitional cell differentiation is scant. To explore this further, we investigated a series of 19 Brenner tumors of the ovary (15 benign and 4 malignant) for the presence of TERT promoter mutations after genomic DNA extraction from formalin-fixed paraffin-embedded tissue blocks and standard polymerase chain reaction sequencing. TERT promoter mutations were not identified in any of the cases (0/19). The absence of TERT promoter mutations in Brenner tumors suggests that despite the morphologic and some immunophenotypical resemblance to non-neoplastic and neoplastic transitional epithelium, Brenner tumors may exhibit a molecularly distinct pathogenesis. The findings also may portend diagnostic utility in rare cases wherein it is difficult to distinguish a primary malignant Brenner tumor of the ovary from metastatic UC.

Image-guided Coring for Large-scale Studies in Molecular Pathology.

Sampling of formalin-fixed paraffin-embedded (FFPE) tissue blocks is a critical initial step in molecular pathology. Image-guided coring (IGC) is a new method for using digital pathology images to guide tissue block coring for molecular analyses. The goal of our study is to evaluate the use of IGC for both tissue-based and nucleic acid-based projects in molecular pathology. First, we used IGC to construct a tissue microarray (TMA); second, we used IGC for FFPE block sampling followed by RNA extraction; and third, we assessed the correlation between nuclear counts quantitated from the IGC images and RNA yields. We used IGC to construct a TMA containing 198 normal and breast cancer cores. Histopathologic analysis showed high accuracy for obtaining tumor and normal breast tissue. Next, we used IGC to obtain normal and tumor breast samples before RNA extraction. We selected a random subset of tumor and normal samples to perform computational image analysis to quantify nuclear density, and we built regression models to estimate RNA yields from nuclear count, age of the block, and core diameter. Number of nuclei and core diameter were the strongest predictors of RNA yields in both normal and tumor tissue. IGC is an effective method for sampling FFPE tissue blocks for TMA construction and nucleic acid extraction. We identify significant associations between quantitative nuclear counts obtained from IGC images and RNA yields, suggesting that the integration of computational image analysis with IGC may be an effective approach for tumor sampling in large-scale molecular studies.

Deparaffinization of formalin-fixed paraffin-embedded tissue blocks using hot water instead of xylene

This study aimed to deparaffinize formalin-fixed paraffin-embedded (FFPE) tissues using hot water instead of xylene and measuring the quantity and quality of the extracted DNA from the respective tissues. To deparaffinize the tissue sections with hot water, small sections were exposed to 90 °C distilled sterile water. After 25 FFPE tissue samples were deparaffinized with the hot water method, DNA was then extracted. The mean of optical density and the ratio of absorbance of the DNA solution were 220.01 ± 36.1 ng/μl and 1.65 ± 0.1, respectively. Polymerase chain reaction (PCR) analysis of the toll-like receptor 4(TLR4) gene showed that the method can be used as a tool for different applications.

High prevalence of TERT promoter mutations in primary squamous cell carcinoma of the urinary bladder

TERT promoter mutations (TERT-mut) are detectable in the majority of urothelial carcinomas. The detection of TERT-mut in urine is under investigation as a potential urine-based molecular-screening assay for bladder cancer. A small but significant number of bladder carcinomas are pure squamous cell carcinoma. We sought to assess the incidence of TERT-mut in squamous cell carcinoma of the urinary bladder. A retrospective search of the institutional pathology archives yielded 15 cystectomy specimens performed for squamous cell carcinoma (2000–2014). Histologic slides were reviewed by a senior urologic pathologist to confirm the diagnosis and select a representative formalin-fixed paraffin-embedded tissue block for mutational analysis. All cases yielded adequate material for DNA analysis. Sequencing for TERT-mut was performed using previously described SafeSeq technique. We detected TERT-mut in 12/15 (80%) of bladder squamous cell carcinomas. TERT promoter mutations, commonly found in conventional urothelial carcinoma, are also highly prevalent in urinary bladder squamous cell carcinoma suggesting a common tumorigenesis and potential utility as a molecular urine-based-screening assay.

Expression of Notch 1 receptor associated with tumor aggressiveness in papillary thyroid carcinoma.

AimThe aim of this study was to assess if the expression of Notch 1 receptor is associated with tumor aggressiveness in papillary thyroid carcinomas (PTCs).Patients and methodsBy searching the electronic medical record system of Xin Hua Hospital, all cases of PTC patients who underwent thyroidectomy in the hospital between 2013 and 2014 were retrieved. Then, the cases of patients who had a history of any other malignancy or whose thyroid tumor specimen was not available for assay were rejected. Finally, 68 cases of PTC patients were obtained. Formalin-fixed paraffin-embedded tissue blocks of these patients were studied by immunohistochemistry to learn the expression of Notch 1 receptor. Meanwhile, the clinical data of these patients including sex, age, size of the tumor, presence of node metastasis or distant metastasis, and presence of capsule invasion and tumor multicentricity were collected. Pearson’s chi-square test or Fisher’s exact test was used for measuring statistical differences in categorical variables. All the statistical tests were two-sided. A P-value <0.05 was considered to be statistically significant.ResultsA total of 19 male and 49 female PTC patients with a mean age of 44.8±13.6 years (range 18–78 years) were studied. Notch 1 receptor expression was found in 15/68 (22%) samples of PTC. The expression of Notch 1 receptor was significantly associated with tumor size (P=0.021), distant metastasis (P=0.008), capsule invasion (P=0.001), tumor multicentricity (P=0.018), and age (P=0.033). However, the expression of Notch 1 receptor was not significantly correlated with node metastasis (P=0.096) and sex (P=0.901).ConclusionThe expression of Notch 1 receptor is associated with tumor aggressiveness in PTC, and may be used as a molecular marker of tumor invasiveness in PTC. PTC patients who show positive expression of Notch 1 receptor may benefit from radioiodine remnant ablation.

Abstract P5-17-09: Biomarkers to distinguish hazardous from harmless ductal carcinoma in situ (DCIS) of the breast

Abstract Background. The incidence of DCIS has increased since the introduction of population-based screening. This has not resulted in a decrease in invasive breast cancer incidence, implying overdiagnosis exists. All women with DCIS are still intensively treated, by surgery, radiotherapy, and/or hormonal treatment, although only a minority will develop a subsequent invasive breast cancer. As we cannot discriminate such hazardous from harmless DCIS lesions, accurate prognostic biomarkers are urgently needed. In the current study we aim to identify molecular markers for DCIS aggressiveness, using a large population-based cohort. Patients and methods. We used a population-based, nation-wide cohort consisting of 10,090 women treated for primary DCIS between 1989 and 2004 with a median follow-up time of 10.7 years. Within this cohort, a case-control study was set up to analyse which markers are associated with progression to invasive breast cancer. Formalin-fixed paraffin embedded (FFPE) tissue blocks were retrieved from 1580 DCIS patients who were treated by breast conserving surgery without radiotherapy (316 DCIS patients with a subsequent ipsilateral invasive breast cancer (iiBC): i.e. the "cases"; and 1264 DCIS patients without subsequent invasive breast cancer: i.e. the "controls"). A first study using this population-based cohort will involve immunohistochemistry (IHC) on 200 "cases" and 500 "controls" for an 8-marker IHC panel (ER, PR, HER2, Ki67, p16, p53, COX-2, and Annexin A1). Molecular subtypes of the DCIS and invasive breast cancer lesions will be determined and intra-individual heterogeneity will be assessed. IHC marker expression will be both compared between "cases" and " controls" as well as between DCIS lesions and its subsequent invasive breast cancer. In a second study, DNA and RNA will be isolated from these specimens, using laser microdissection, and extensive molecular profiling will be performed. Results. We have collected FFPE tissue blocks of 287 "cases" and 1149 "controls" (86% of requested material) from 56 participating hospitals. At present, the specimens of 223 "cases" (matched DCIS and iiBC specimen) and 103 "controls" have been centrally revised for extensive morphological characteristics. Only a small part (14%) of the specimens had to be excluded from the study population. IHC staining of the tissue specimens, using the 8-marker IHC panel is ongoing. Conclusion. Within a nation-wide cohort of 10,090 patients diagnosed with primary DCIS, we were able to collect tissue material of a representative case-control series of 200 "cases" with subsequent invasive breast cancer and 500 invasive breast cancer-free "controls". This is the first time such a large unique, unbiased DCIS series, with long-term follow-up is analysed integrating clinical, histological, and immunohistochemical data. The results will be presented at SABCS 2015. Citation Format: Visser L, Elshof L, Groen E, van de Vijver K, Lips E, de Maaker M, Nieboer F, Schaapveld M, Rutgers E, Wesseling J. Biomarkers to distinguish hazardous from harmless ductal carcinoma in situ (DCIS) of the breast. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-17-09.

Abstract P1-08-02: CYP2D6 gene variants and effectiveness of adjuvant tamoxifen in breast cancer: A population-based case-control study

Abstract Tamoxifen, a cornerstone of adjuvant therapy for hormone-receptor-positive breast cancer, is metabolized to the active metabolite endoxifen through enzymatic activity of cytochrome P450 2D6. CYP2D6 has numerous alleles that affect metabolizing phenotype. Among women who take tamoxifen, those homozygous for inactive alleles (poor metabolizers) have lower levels of serum endoxifen than those with two functional alleles (extensive metabolizers). Several studies have reported increased risk of breast cancer recurrence or death in women homozygous for CYP2D6 inactive alleles, but others have found no association between CYP2D6 function and outcome. We explored this question in the large member population of the Kaiser Permanente Northwest (KPNW) integrated health plan. We conducted a population-based case-control study to evaluate the hypothesis that, after adjuvant tamoxifen treatment for breast cancer, women with CYP2D6 genotypes associated with poor metabolism of tamoxifen have an elevated risk of breast cancer recurrence compared to women with CYP2D6 genotypes associated with extensive metabolism of tamoxifen. We further hypothesized that women with CYP2D6 genotypes associated with intermediate metabolism of tamoxifen are at intermediate risk of recurrence. Study subjects were women who were diagnosed from 1980 to 2011 with hormone-receptor positive breast cancer, who received at least 180 days of adjuvant tamoxifen treatment, and for whom stored formalin-fixed paraffin-embedded (FFPE) normal tissue was available for laboratory analysis. Cases (358) were women with breast cancer recurrence recorded in the KPNW Tumor Registry and validated by medical record review. Randomly selected controls (833), without recurrent breast cancer, were matched to cases on tumor stage, diagnosis year, diagnosis age, race/ethnicity, and patterns of health plan membership. We collected data from medical records and from pharmacy, laboratory, tumor registry, and membership health plan databases. The Oregon Health &amp; Science University Molecular Genetics Laboratory extracted genomic DNA from stored FFPE tissue blocks and performed allelic discrimination assays and pyrosequencing to accurately determine CYP2D6 variant status for the alleles, *3, *4, *5, *10, *17, and *41. All assays have been completed and study subjects have been categorized according to CYP2D6 metabolizer phenotype (poor, intermediate, extensive) and activity score (0-2). Based on the ethnicities in our study population, the CYP2D6 allele frequencies are in Hardy-Weinberg equilibrium, and the frequencies of the predicted metabolizer phenotypes also fall within the expected range. Using multivariable logistic regression analysis, we will assess CYP2D6 functional status and activity score in relation to breast cancer recurrence, taking into account factors that may alter the association, including tamoxifen dose and duration of use, as well as concomitant medications that alter the activity of the CYP2D6 enzyme. Results will be available by 12/1/2015. Citation Format: Weinmann S, Richert–Boe K, Goddard K, Chen C, Punj S, Schwarzkopf D, Kalter M, Richards CS. CYP2D6 gene variants and effectiveness of adjuvant tamoxifen in breast cancer: A population-based case-control study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-08-02.

Formalin fixed paraffin embedded (FFPE) material is amenable to HPV detection by the Xpert® HPV assay

BackgroundThe Xpert® HPV Assay (Cepheid®, Sunnyvale, USA) is a rapid, cartridge-based HPV test validated for use on cervical cytology samples. However, there is an increasing demand for HPV annotation of formalin fixed paraffin embedded (FFPE) material. ObjectivesThe aim of this study was to determine the suitability of the Xpert HPV assay for the detection of nucleic acid (NA) derived from FFPE samples. Study designA total of 88, 10μm sections derived from FFPE tissue blocks were assessed, 74 originated from oropharyngeal squamous cell carcinomas (OPSCC) and 14 from a range of other sites. All had previously been tested with a sensitive Luminex® based assay with a component also tested with p16 immunohistochemistry (IHC). NA was extracted from samples using the easyMag® platform and after dilution was added directly to the Xpert cartridge.Agreement between assays was assessed. ResultsOverall agreement between the assays was 92%; with a Kappa for HR-HPV detection of 0.833 (95% CI 0.725–0.953). In the 50 samples that had been annotated for p16 status overall agreement between the Xpert assay and the p16 IHC was 90%. ConclusionsThese data indicate that FFPE material is amenable to HPV detection by the Xpert assay. To our knowledge, this is the first study to interrogate the use of the Xpert® HPV assay for this application.