Biomarkers of Crohn's Disease to Support the Development of New Therapeutic Interventions.

BACKGROUND: Currently, 2 coprimary end points are used by health authorities to determine the effectiveness of therapeutic interventions in patients with Crohn's disease (CD): symptomatic remission (patient-reported outcome assessment) and endoscopic remission (ileocolonoscopy). However, there is lack of accepted biomarkers to facilitate regulatory decision-making in the development of novel therapeutics for the treatment of CD. METHODS: With support from the Helmsley Charitable Trust, Critical Path Institute formed the Crohn's Disease Biomarkers preconsortium (CDBpC) with members from the pharmaceutical industry, academia, and nonprofit organizations to evaluate the CD biomarker landscape. Biomarkers were evaluated based on biological relevance, availability of biomarker assays, and clinical validation data. RESULTS: The CDBpC identified the most critical need as pharmacodynamic/response biomarkers to monitor disease activity in response to therapeutic intervention. Fecal calprotectin (FC) and serum C-reactive protein (CRP) were identified as biomarkers ready for the regulatory qualification process. A number of exploratory biomarkers and potential panels of these biomarkers was also identified for additional development. Given the different factors involved in CD and disease progression, a combination of biomarkers, including inflammatory, tissue injury, genetic, and microbiome-associated biomarkers, will likely have the most utility. CONCLUSIONS: The primary focus of the Inflammatory Bowel Disease Regulatory Science Consortium will be development of exploratory biomarkers and the qualification of FC and CRP for IBD. The Inflammatory Bowel Disease Regulatory Science Consortium, focused on tools to support IBD drug development, will operate in the precompetitive space to share data, biological samples for biomarker testing, and assay information for novel biomarkers.

Determination of Optimum Formalin Fixation Duration for Prostate Needle Biopsies for Immunohistochemistry and Quantum Dot FISH Analysis.

Prostate biopsy is the key clinical specimen for disease diagnosis. However, various conditions used during biopsy processing for histologic analysis may affect the performance of diagnostic tests, such as hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), or in situ hybridization (ISH). One such condition that may affect diagnostic test performance is fixation duration in 10% neutral buffered formalin (NBF). For example, prostate needle biopsies are often <1 mm in diameter and thus overfixed. It is important to understand the impact of tissue fixation duration on diagnostic test performance to enable optimized assay procedures. This study was designed to study the effect of 10% NBF fixation duration of prostate needle biopsy on multiplexed quantum dot (QD) ISH assay of ERG and PTEN, 2 genes commonly altered in prostate cancer. The samples were also evaluated for H&E staining and ERG and PTEN IHC. H&E staining and ERG and PTEN IHC were acceptable for all the durations of fixation tested. For QD ISH, we observed good signals with biopsy samples fixed from 4 to 120 hours. Biopsy specimens fixed between 8 and 72 hours gave the best signal as scored by the study pathologist. In a separate cohort of 18 routinely processed prostate biopsy cores, all cores were stained successfully with the QD ISH assay, and results were 100% concordant to ERG and PTEN IHC. We conclude that 8 to 72 hours duration of fixation for prostate needle biopsies in 10% NBF results in optimal QD ISH assay performance.

Polymerase chain reaction detection of HPV in squamous carcinoma of the oropharynx.

Human papillomavirus (HPV) testing is routinely performed on oropharyngeal carcinomas. We compared the Access Genetics (Minneapolis, MN) polymerase chain reaction (PCR) assay (AGPCR), DNA-DNA in situ hybridization (ISH; Ventana, Tucson, AZ), and HPV-16 E7 PCR amplification in consecutively accessioned oropharyngeal cancers. We tested 126 cases by both PCR methods; 102 were positive by either for a maximum positive rate (MPR) of 81.0%. Relative to the MPR, the sensitivities of AGPCR and E7 PCR were 90.2% and 72.5%, respectively. Of 17 AGPCR+ cases tested by ISH, 14/14 unequivocally positive/negative were concordant. All cases (97/97) positive by either PCR assay were positive for p16. There was no relationship between level of histologic differentiation and HPV status. ISH and AGPCR have comparable performance for the detection of HPV in oropharyngeal carcinomas. PCR is a suitable and economical assay that is comparable to ISH in sensitivity and may provide logistical advantages relative to ISH for assessing HPV status in oropharyngeal malignancies. However, it is imperative that appropriate sensitivity controls be in place for such assays.

Histological 'progression' from low (LSIL) to high (HSIL) squamous intraepithelial lesion is an uncommon event and an indication for quality assurance review.

An accurate assessment of 'progression' from a low (LSIL) to high (HSIL) grade squamous intraepithelial lesion (cervical intraepithelial neoplasia (CIN)2 or CIN3) of the cervix is critical to ascertaining HSIL outcome risk, the value of predictive biomarkers, and the need for excisional therapy. We obtained biopsy outcome data on a series of initially diagnosed LSIL to assess this risk. Consecutive biopsy diagnoses of LSIL were obtained from the archives, and the frequency of HSIL biopsy outcomes were ascertained by record and histological review. Then, a 'numerical severity score' was recorded for each diagnosis: LSIL (1-2), CIN2 (3-4) and CIN3 (5-6) with lower and higher values corresponding to the degree (low vs high) of histological severity within each category, respectively. Of 264 initial LSILs, 29 (11%) were reported with an HSIL outcome. However, histological review of 21 of these HSILs confirmed only 8 (38%) HSIL diagnoses by review with the numerical severity score: three cases scored as 5, three cases scored as 4, and two cases scored as 3; the remaining 13 cases were assigned a numerical severity score of 1 or 2. P16 immunostains of corresponding previous and subsequent biopsies were discordant in 4 of 12 cases (33%). In a blind review of a randomly selected series of HSILs from the same practice, HSIL was significantly more likely to be confirmed on re-review (10 of 13 (77%), P=0.024). These findings show that confirmed HSIL outcomes (on review) following an LSIL biopsy are infrequent ( approximately 3%). A diagnosis of HSIL following an LSIL should always be reviewed, as this diagnostic pairing may more likely be associated with a diagnostic error.

P16 immunostaining patterns in microglandular hyperplasia of the cervix and their significance.

P16 immunostaining is an important adjunct in the differential diagnosis of difficult squamous and glandular intraepithelial lesions of the cervix. However, unexpected staining of epithelium other than the target lesion can pose a problem in the interpretation. This study examined a common entity in the cervix, microglandular hyperplasia (MGH), that is associated with proliferations of both columnar and squamous epithelial cells-and ascertained the frequency of p16 staining, its pattern, and relationship to human papillomavirus. Fifty-seven cases of MGH were analyzed; 25 scored strongly immunopositive (44%). In 18, staining of the superficial columnar epithelium was patchy, involving 10% to 20% of cells on the surface; in 4 cases, 30% to 40% of cells; and in another 3, over 50% of the cells in a given area were strongly positive. Staining involved both nucleus and cytoplasm of columnar cells. P16 positivity did not colocalize with either cyclin E or MIB-1. Adjacent non-MGH-related columnar epithelium scored negative for p16. Of 25 p16-positive columnar epithelia analyzed, all were human papillomavirus -negative. In conclusion, benign columnar epithelium in the setting of MGH can be expected to stain strongly for p16. Practitioners should be aware of this when evaluating diagnostically difficult squamous or glandular epithelial changes occurring in the setting of MGH or when interpreting cytologic preparations stained with p16.

Intraepithelial carcinoma of the fimbria and pelvic serous carcinoma: Evidence for a causal relationship.

Proposed origins of pelvic serous carcinoma include the ovary, fallopian tube, and peritoneum. Prophylactic salpingo-oophorectomies in BRCA+ women have recently identified the fimbria as a site of origin for early serous carcinoma (tubal intraepithelial carcinoma or TIC). We explored the relationship of TIC to pelvic serous carcinomas in consecutive cases with complete adnexal exam (SEE-FIM protocol). Cases positive (group A) or negative (group B) for endosalpinx (including fimbria) involvement, were subclassified as tubal, ovarian, or primary peritoneal in origin. Coexisting TIC was recorded in group A when present and p53 mutation status was determined in 5 cases. Of 55 evaluable cases, 41 (75%) were in group A; including tubal (n = 5), peritoneal (n = 6), and ovarian (n = 30) carcinomas. Foci of TIC were identified in 5 of 5, 4 of 6, and 20 of 30, respectively. Ninety-three percent of TICs involved the fimbriae. Five of 5 TICs and concurrent ovarian carcinomas contained identical p53 mutations. Thirteen of 14 cases in group B were classified as primary ovarian carcinomas, 10 with features supporting an origin in the ovary. Overall, 71% and 48% of "ovarian" serous carcinomas had endosalpinx involvement or TIC. TIC coexists with all forms of pelvic serous carcinoma and is a plausible origin for many of these tumors. Further studies are needed to elucidate the etiologic significance of TIC in pelvic serous carcinoma, reevaluate the criteria for tubal, peritoneal, and ovarian serous carcinoma, and define the role of the distal tube in pelvic serous carcinogenesis.