In order to avoid the consequences of over- and under-treatment of endometrial hyperplasia, diagnostic accuracy and progression risk assessment must be improved. The aim of this study was to assess whether PAX2 or PTEN expression could predict progression-free survival in endometrial intraepithelial neoplasia (EIN) and endometrial endometrioid carcinoma (EEC). Immunohistochemistry for detection of PAX2 and PTEN was performed on 348 endometrial samples; 75 proliferative endometrium (PE), 36 EIN and 237 EEC. Cases classified as PTEN null (1 or more glands negatively stained) were more prevalent in EEC than in PE and EIN (64% EEC vs 11% PE/EIN). A progressive decrease in PAX2 expression was observed from PE to EIN to EEC. Long-term clinical follow-up (6-310 months, median: 126) was available for 62 PE cases, all 36 EIN cases and 178 EEC cases. No patients with PE demonstrated progression to EIN or EEC. Progression of disease was observed in 10 (28%) EIN patients. These patients had significantly lower PAX2 expression than those that regressed (P = 0.005). Progression-free survival analysis revealed that EIN patients with a high-risk PAX2 expression score (H-score ≤75) had a higher probability of progression of disease in comparison to those with a low-risk score (H-score >75). PAX2 expression was not prognostic in EEC nor was PTEN status of prognostic value in either EIN or EEC. PAX2 expression analysis by means of H-score has prognostic potential for the identification of high-risk progression cases in EIN but needs to be validated in a larger cohort.

Regression of cervical intraepithelial neoplasia (CIN) 2-3 to CIN 1 or less is associated with immune response as demonstrated by immunohistochemistry in formaldehyde-fixed paraffin-embedded (FFPE) biopsies. Proteomic analysis of water-soluble proteins in supernatants of biopsy samples with LC-MS (LTQ-Orbitrap) was used to identify proteins predictive of CIN2-3 lesions regression. CIN2-3 in the biopsies and persistence (CIN2-3) or regression (≤CIN1) in follow-up cone biopsies was validated histologically by two experienced pathologists. In a learning set of 20 CIN2-3 (10 regressions and 10 persistence cases), supernatants were depleted of seven high abundance proteins prior to unidimensional LC-MS/MS protein analysis. Mean protein concentration was 0.81 mg/mL (range: 0.55-1.14). Multivariate statistical methods were used to identify proteins that were able to discriminate between regressive and persistent CIN2-3. The findings were validated in an independent test set of 20 CIN2-3 (10 regressions and 10 persistence cases). Multistep identification criteria identified 165 proteins. In the learning set, zinc finger protein 441 and phospholipase D6 independently discriminated between regressive and persistent CIN2-3 lesions and correctly classified all 20 patients. Nine regression and all persistence cases were correctly classified in the validation set. Zinc finger protein 441 and phospholipase D6 in supernatant samples detected by LTQ-Orbitrap can predict regression of CIN2-3.

European treatment guidelines of non–muscle-invasive urothelial carcinoma of the urinary bladder are strongly dependent on grade, but grading reproducibility is wanting. Protocolized proliferation features such as Mitotic Activity Index (MAI), Ki-67, and phosphohistone H3 are prognostic and reproducible. The objective of this population-based study was to compare proliferation biomarkers with each other and with World Health Organization (WHO) 1973/2004 grades with regard to prediction of stage progression. A total of 193 primary non–muscle-invasive urothelial carcinomas were analyzed using WHO73/04 grades and measurement of the proliferation markers mentioned above. Sensitivities, specificities, and positive and negative predictive values with confidence intervals (CIs) were estimated with regard to progression prediction. Kaplan-Meier survival curves were made, and the hazard ratio and Harrell's C-index with 95% CIs, P values, and adjusted C-index for stage progression or not of WHO73, WHO04, and the proliferation markers were calculated. The median follow-up time was 75 months (range, 1-127). A total of 111 patients (52%) experienced recurrence within 5 years, and 14 patients (7%) progressed. High values of MAI predicted stage progression with a positive predictive value of 0.22 (95% CI, 0.12-0.37). The positive predictive value of Ki-67 and phosphohistone H3 were 0.15 (both 95% CIs, 0.07-0.29) and comparable to that of the WHO04. The prognostic value of MAI was strongest, exceeding that of the other proliferation markers and the WHO grading systems. In conclusion, in non–muscle-invasive urinary bladder urothelial carcinomas, proliferation biomarkers have prognostic value, possibly exceeding that of the WHO classifications.

Cervical intraepithelial neoplasia (CIN), a frequently encountered disease caused by Human Papilloma Virus (HPV) is often diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. Since it is known that this procedure strongly affects the water-soluble proteins contained in the cervical tissue we decided to investigate whether a water-soluble protein-saving biopsy processing method can be used to support the diagnosis of normal and CIN.

Background Cervical intraepithelial neoplasia (CIN) grades 2 and 3 are usually grouped and treated in the same way as "high grade", in spite of their different risk to cancer progression and spontaneous regression rates. CIN2-3 is usually diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. This procedure virtually eliminates the availability of water-soluble proteins which could have diagnostic and prognostic value. Aim To investigate whether a water-soluble protein-saving biopsy processing method followed by a proteomic analysis of supernatant samples using LC-MS/MS (LTQ Orbitrap) can be used to distinguish between CIN2 and CIN3. Methods Fresh cervical punch biopsies from 20 women were incubated in RPMI1640 medium for 24 hours at 4°C for protein extraction and subsequently subjected to standard FFPE processing. P16 and Ki67-supported histologic consensus review CIN grade (CIN2, n = 10, CIN3, n = 10) was assessed by independent gynecological pathologists. The biopsy supernatants were depleted of 7 high abundance proteins prior to uni-dimensional LC-MS/MS analysis for protein identifications. Results The age of the patients ranged from 25-40 years (median 29.7), and mean protein concentration was 0.81 mg/ml (range 0.55 - 1.14). After application of multistep identification criteria, 114 proteins were identified, including proteins like vimentin, actin, transthyretin, apolipoprotein A-1, Heat Shock protein beta 1, vitamin D binding protein and different cytokeratins. The identified proteins are annotated to metabolic processes (36%), signal transduction (27%), cell cycle processes (15%) and trafficking/transport (9%). Using binary logistic regression, Cytokeratin 2 was found to have the strongest independent discriminatory power resulting in 90% overall correct classification. Conclusions 114 proteins were identified in supernatants from fresh cervical biopsies and many differed between CIN2 and 3. Cytokeratin 2 is the strongest discriminator with 90% overall correct classifications.

Appropriate stratification tools for targeted surveillance after resection for colorectal cancer (CRC) are lacking. The objective of the current study was to investigate the effect of microsatellite instability (MSI) and DNA ploidy on surveillance after surgery.

Accurate, long-term risk predictors for colorectal cancer development in patients with sporadic adenomas are lacking. We sought to validate biomarkers predictive of metachronous colorectal cancer (mCRC) in patients with sporadic colorectal adenomas, using 374 consecutive patients from a large defined population.

Androgen receptor (AR) expression is prognostic for breast cancer. Techniques to determine AR levels include the dextran-coated charcoal method (charc-AR), which measures AR levels in both normal and cancer cells; immunohistochemical analysis (1HC-AR) of whole sections (WS), which targets cancer cells but relies on subjective assay interpretation; and IHC of tissue microarray (TMA) samples, an approach that can be inaccurate when AR heterogeneity is present. This study compared charc-AR (n=151) with quantitative IHC analysis (n=138) of WS and TMA samples (1.7 mm cylinder samples; 2.3 mm). Charc-AR results correlated poorly with IHC-WS and IHC-TMA results due to the presence of AR-positive noncancer cells. IHC-WS revealed intertumor and intratumor AR heterogeneity. Consequently, the IHC-WS and IHC-TMA results were similar for tumors in which the percentage of AR-positive WS samples was >80% (31% of tumors), but differed in other tumors due to TMA false negatives (33%). Computer simulation was used to determine the optimal number and size of TMA samples for reliable results, and showed that five to eight 1.7 mm samples, or up to sixty-four 0.6 mm samples, were needed for reliable AR determination for most tumors. Thus, AR determination by charc-AR is inaccurate due to evaluation of both normal and cancer cells. IHC is more sensitive and specific than charc-AR in WS samples, but not when applied to single or a few TMA samples.

Up to 30% of cervical intraepithelial grades 2-3 (CIN2-3) lesions regress, but some believe that "regression" is due to "curative" punch biopsies. If this is true, CIN2-3 in the resection margins of the biopsies would be associated with more frequent "persistent" CIN2-3. If, however, immunology-related regression exists, regression would increase with increasing biopsy-cone interval. In 61 punch biopsies diagnosed as CIN3 at careful review by two independent gynaecological pathologists, CIN3 in the resection margins and duration of the biopsy-cone interval was evaluated in relation to CIN2-3-or-not in the cones (again after independent review by expert pathologists). 10 of 61 (16%) patients with CIN3 showed CIN1 or less in the follow-up cones. CIN3-or-not in the resection margins, size of the lesion in the punch biopsy, and presence or absence of CIN2-3 in the cones were not correlated with regression-or-not. However, the number of cones without CIN2-3 increased with longer biopsy-cone interval, 5% in patients with a punch-cone biopsy interval under 9 weeks and 38%> or =9 weeks (p<0.001). These results favour the hypothesis that CIN3 can regress, and do not support the "curative punch biopsy" theory.

Up to 30% of cervical intraepithelial grades 2–3 (CIN2–3) lesions regress, but some believe that “regression” is due to “curative” punch biopsies. If this is true, CIN2–3 in the resection margins of the biopsies would be associated with more frequent “persistent” CIN2–3. If, however, immunology-related regression exists, regression would increase with increasing biopsy-cone interval. In 61 punch biopsies diagnosed as CIN3 at careful review by two independent gynaecological pathologists, CIN3 in the resection margins and duration of the biopsy-cone interval was evaluated in relation to CIN2–3-or-not in the cones (again after independent review by expert pathologists). 10 of 61 (16%) patients with CIN3 showed CIN1 or less in the follow-up cones. CIN3-or-not in the resection margins, size of the lesion in the punch biopsy, and presence or absence of CIN2–3 in the cones were not correlated with regression-or-not. However, the number of cones without CIN2–3 increased with longer biopsy-cone interval, 5% in patients with a punch-cone biopsy interval under 9 weeks and 38%≥9 weeks (p<0.001). These results favour the hypothesis that CIN3 can regress, and do not support the “curative punch biopsy” theory.

Accurate predictors for metachronous colorectal cancer (CRC) development after polypectomy are lacking. We evaluated the prognostic value of classical clinicopathologic features and a monotonous population of elongated cells (MPECs) in colorectal adenomas from 171 consecutively selected population-based patients with long-term follow-up. Quantitative image analysis, and univariate and multivariate regression analysis were applied. Ten of 171 adenomas (5.8%) developed metachronous CRC (defined as >24 mo interval and >5 cm from the index adenoma to the cancer). Median follow-up of adenomas with metachronous CRC was 68.4 and without cancer 149.7 months (range: 25 to 192 and 25 to 256, respectively). The most prognostic classical features were the localization of the marker adenoma as proximal (ie, in the cecum through transverse colon) versus distal from the transverse colon [P=0.0003, hazard ratio (HR)=8] and the number of polyps found during colonoscopy (≤2 vs.>2, P=0.002, HR=6). Quantitative features of the MPECs included the longest nuclear axis and variance of the number of nuclei with 2 neighbors (higher and lower in cancer cases, respectively). Of the 171 adenomas, 50 (29%) had MPECs, of which 9 (18%) patients developed metachronous CRC at follow-up, contrasting 1/121 (0.8%) without MPECs (P=0.0003, HR=23). MPECs occurred in both low-grade and high-grade dysplasia, and in tubular and (tubulo) villous adenomas. MPECs had the strongest predictive value for metachronous CRC development. Adenomas proximally located had additional value but only if they were MPEC positive (which only occurred in 5 adenomas, 3 of which (60%) developed cancer). Having more than 2 polyps also had additional prognostic value but only in MPEC-negative adenomas [10 cases; 1 (10%) developed cancer]. Dysplasia grade and histologic growth pattern had no additional value. Thus, colorectal adenomas with subsequent metachronous cancer development can be identified more accurately with MPECs than with classical prognostic factors.

We validated with univariate and multivariate (Cox) analysis, the prognostic value of the mitotic activity index (MAI), the fibrotic focus (FF) and other prognosticators in 448 patients with lymph node-negative (LN-) invasive breast cancer <55 years without adjuvant systemic treatment (72.5 months median follow-up, range 4-119). Of these patients, 24.8% developed distant and 1.6% loco-regional recurrence. FF showed excellent inter-observer reproducibility (kappa = 0.93). Strong prognosticators were MAI, grade, nuclear atypia, FF and the St. Gallen criterion (SG). The subgroup with excellent survival selected by SG was only 16% of all patients, implying over-treatment of more than 70% of all LN- patients when using SG as adjuvant therapy selection criterion. If MAI <10, 13% showed distant metastases, contrasting with 41% if MAI > or = 10. FF was prognostic in the ductal and mixed ductal cancers, but not in the lobular and other subtype cancers. Patients with invasive (mixed) ductal cancers with FF absent, FF < 1/3 or FF > 1/3 of the tumour area, had distant metastasis rates of 17%, 35% and 48%; in MAI < 10 and FF absent, FF < 1/3 or FF > 1/3, metastasis rates were 11%, 13% and 42% and if MAI > or = 10, metastasis rates were 31%, 48% and 50%, respectively. In the 12 patients with MAI < 10 and a large FF > 1/3, event-free survival was similar to patients with MAI > or = 10. With multiple regression MAI < 10 versus > or = 10 is the strongest prognosticator (also stronger than the SG). The FF may be important as it has additional prognostic value to the MAI in the small subgroup of invasive ductal or mixed-ductal breast cancer patients with combined MAI < 10 and an FF > 1/3 of the tumour area.