Discrimination of grade 2 and 3 cervical intraepithelial neoplasia by means of analysis of water soluble proteins recovered from cervical biopsies.

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.

Maternal host responses to poly(I:C) during pregnancy leads to both dysfunctional immune profiles and altered behaviour in the offspring.

PROBLEM: Autism spectrum disorder (ASD)-like phenotypes in murine models are linked to elevated pro-inflammatory cytokine profiles caused by maternal immune activation (MIA), but whether MIA alters the immune response in the offspring remains unclear. METHOD OF STUDY: Polyinosinic:polycytidylic acid (poly:[IC]) was used to induce MIA in immunocompetent and control TLR3-deficient pregnant mice, and cytokine levels were measured in maternal and foetal organs. Furthermore, cytokines and behaviour responses were tested after challenge with lipopolysaccharide in 7-day-old and adult mice. RESULTS: MIA induced on E12 resulted in changes in the cytokine expression profile in maternal and foetal organs and correlated with TNFα and IL-18 dysregulation in immune organs and brains from neonatal mice born to MIA-induced dams. Such changes further correlated with altered behavioural responses in adulthood. CONCLUSION: MIA induced by pathogens during pregnancy can interfere with the development of the foetal immune and nervous systems leading to dysfunctional immune responses and behaviour in offspring.

Assessing the prognostic value of PAX2 and PTEN in endometrial carcinogenesis.

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.

Cervical intraepithelial neoplasia grade 3 lesions can regress.

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.

A monotonous population of elongated cells (MPECs) in colorectal adenoma indicates a high risk of metachronous cancer.

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, 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.

Prospective multicenter validation confirms the prognostic superiority of the endometrial carcinoma prognostic index in international Federation of gynecology and obstetrics stage 1 and 2 endometrial carcinoma.

PURPOSE: To validate the prognostic value of the endometrial carcinoma prognostic index (ECPI; combined myometrium invasion, flow cytometric DNA ploidy, and morphometric mean shortest nuclear axis [MSNA]) versus classic prognosticators. PATIENTS AND METHODS: Prospective multicenter ECPI analysis was conducted in 463 endometrial carcinomas with a median of 6.5 years (range, 1 to 10 years) follow-up, review of pathology features, and univariate (Kaplan-Meier) and multivariate (Cox) analyses. RESULTS: Initial routine and review diagnoses varied considerably (invasion depth, 11%; type, 20%; grade, 34%; vessel invasion, 72%); the review diagnoses were stronger prognostically. In International Federation of Gynecology and Obstetrics stage 1 (after histopathologic examination; pFIGO-1; n = 372; 38 deaths occurred as a result of disease [10.2%]), DNA ploidy was prognostic in hysterectomies (P <.00001) but not in curettages (P =.06). ECPI was a stronger prognostic indicator than other features. ECPI, MSNA, and DNA ploidy were also prognostic in pFIGO-1B and -1C subgroups. Multivariate analysis in pFIGO-1 showed that uterine MSNA < or = versus > 7.93 microm (hazard ratio [HR], 3.4) and grade (as 1 + 2 v 3; HR, 2.6) added to the ECPI (HR, 32), but only in patients with an unfavorable ECPI of > 0.87. Adjuvant radiotherapy was not an independent prognostic factor in any of the subgroups. In pFIGO-2 (n = 46), ECPI, DNA-ploidy, and age (< or = 64, > 64 years) were significant. In FIGO-3 (n = 31) and FIGO-4 (n = 14), none of the classic or other features analyzed was of prognostic value, which explains why in previous studies using different mixtures of FIGO stages, DNA ploidy prognostic results varied. CONCLUSION: In endometrial carcinoma, DNA-ploidy is prognostic in hysterectomy and not in curettage samples. The ECPI is prognostically much stronger than the classic features widely used for therapy triage in pFIGO-1 and -2.

Multivariate prognostic evaluation of the mitotic activity index and fibrotic focus in node-negative invasive breast cancers.

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.

Lack of PTEN expression in endometrial intraepithelial neoplasia is correlated with cancer progression.

We tested the hypothesis that PTEN inactivation may stratify cancer progression risk among putative endometrial hyperplasias, classified prognostically by means of the morphometric D score (DS). The DS, calculated from 3 morphometric variables measured in routine hematoxylin-eosin-stained endometrial biopsy slides, is the most sensitive and specific method of endometrial cancer risk prediction currently available. Clinical outcomes of 103 women with endometrial hyperplasia on biopsy were tallied according to the DS. Seven (7/103; 7%) patients with carcinoma during follow-up were all distributed within the high-risk prognostic group (ie, DS <1 = endometrial intraepithelial neoplasia [EIN]) (7/21; 33% progression). None of the 82 cases with a DS higher than 1 progressed. All cases that progressed were PTEN null, indicating that this genotype is capable of further stratifying cancer progression risk in hyperplasias irrespective of histological categorization. However, only 16% of the PTEN-null cases progressed. When PTEN expression pattern was combined with EIN, the prognostic power was greatly increased (specificity from 63% for PTEN and 85% for EIN to 93% when combined; positive predictive value from 16% and 33% to 50%). We conclude that loss of PTEN expression is the first biomarker in EIN that increases the accuracy of the prognostic DS to predict cancer progression risk. Unless endometrial hyperplasias are stratified by histological morphometric D-Score, PTEN has a low positive predictive value.

Predictive testing of early CIN behaviour by molecular biomarkers.

Each year, 330,000 new Cervical Intraepithelial Neoplasias(CIN) occur in the European Union (EU) of which 120,000 are early CIN where grade (1, 2) indicates the progression-risk to CIN-3 and therefore determines the treatment choice. However, the Positive Predictive Value (PPV) of CIN grade to predict progression is low (10% and 20% for CIN-1 and -2 respectively, 16% on average) resulting in an enormous number of over-treatments indicating worrisome grade reproducibility.Certain molecular biomarkers such as Ki-67 have a higher PPV (30%, an improvement of 14%), which in Europe alone could improve treatment for many thousands of women per year with considerable cost reduction for the health care system. The quantitative Ki-67 prognostic model has been validated in independent retrospective and prospective studies from different laboratories. Moreover, the PPV of Ki-67 alone can be improved by additional molecular biomarkers (retinoblastoma protein = Rb, cytokeratins= CK-14/-13). Combined Ki67-Rb allows a 2-tiered progression-risk subgroup assignment as very low ( approximately 0% progression, 71% of all CIN-I/II patients)and high risk (48% progression risk, incidence 32%), leaving a small (7% of all) prognostically undetermined group (17% progression). Additional CK-14 and -13 analysis can sub-classify the high-risk in an intermediate and very high risk subgroup(with 40% and 100% progression risks respectively).Thus, molecular biomarkers are potentially important determinators of early CIN lesion behaviour. Important factors for widespread acceptance of molecular biomarkers are (1) market penetration by user-friendly equipment, (2) (inter)national keeping of GLP conditions (reproducibility, independent validation), requiring customer-driven industrial efforts,governmental measures, and additional PPV improvement to further reduce over-treatment.

Prognostic comparison of proliferation markers and World Health Organization 1973/2004 grades in urothelial carcinomas of the urinary bladder.

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.

Prediction of spontaneous regression of cervical intraepithelial neoplasia lesions grades 2 and 3 by proteomic analysis.

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.

A protein profile study to discriminate CIN lesions from normal cervical epithelium.

BACKGROUND: 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. METHODS: Cervical punch biopsies from 55 women were incubated for 24 h at 4°C in RPMI1640 medium for protein analysis prior to usual FFPE processing and p16 and Ki67-supported histologic consensus diagnosis was assessed. The biopsy supernatants were subjected to surface-enhanced laser desorption-ionization time of flight mass spectrometry (SELDI-TOF MS) for identifying differentially expressed proteins. Binary logistic regression and classification and regression trees (CART) were used to develop a classification model. RESULTS: The age of the patients ranged from 26 to 40 years (median 29.7). The consensus diagnoses were normal cervical tissue (n = 10) and CIN2-3 (n = 45). The mean protein concentration was 1.00 and 1.09 mg/ml in the normal and CIN2-3 group, respectively. The peak detection and clustering process resulted in 40 protein peaks. Many of these peaks differed between the two groups, but only three had independent discriminating power. The overall classification results were 88%. CONCLUSIONS: Water-soluble proteins sampled from punch biopsies are promising to assist the diagnosis of normal and CIN2-3.

Microsatellite instability and DNA ploidy in colorectal cancer: potential implications for patients undergoing systematic surveillance after resection.

BACKGROUND: 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. METHODS: The authors evaluated 186 consecutive, population-based patients with stage I through III CRC who underwent surgery with curative intent and who entered a systematic surveillance program. MSI was analyzed with polymerase chain reaction for 5 known quasimonomorphic markers (BAT-26, BAT-25, NR-21, NR-24, and NR-27), and DNA ploidy was analyzed with automated cytometry. Recurrence, recurrence-free survival (RFS), and disease-specific survival (DSS) were evaluated by univariate and multivariate statistical tests. RESULTS: Patients with MSI (20%) were significantly younger than patients without MSI (median age, 61 years vs 67 years; P=.016). Proximal location (adjusted odds ratio [AOR], 5.4; 95% confidence interval [95% CI], 2.1-14.1 [P=.001]), large tumor size (>or=5 cm: AOR, 3.5; 95% CI, 1.3-9.6 [P=.015]), and poor tumor differentiation (AOR, 6.6; 95% CI, 2-21.8 [P=.002]) were associated with MSI. MSI conveyed an increased risk for locoregional recurrence (OR, 2.9; 95% CI, 1.2-7 [P=.016]), with a trend toward a shorter time to recurrence (P=.060). Neither MSI status nor DNA ploidy predicted distant metastasis, RFS, or DSS. Lymph node status was the best predictor of distant spread (AOR, 3.9; 95% CI, 2-7.9 [P<.001]) and DSS (hazard ratio, 4.9; 95% CI, 2.6-9 [P<.001]). CONCLUSIONS: Patients who had microsatellite instable tumors were at increased risk for locoregional recurrence, whereas lymph node status was the best predictor of distant metastasis. Clinical surveillance and choice of modality (ie, endoscopy vs radiologic imaging) may be improved when patients are stratified according to these cancer features.

Metachronous cancer development in patients with sporadic colorectal adenomas-multivariate risk model with independent and combined value of hTERT and survivin.

BACKGROUND AND AIMS: 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. MATERIALS AND METHODS: Risk evaluation was performed for patient and adenoma risk factors (morphometric longest nuclear axis and immunohistochemical markers survivin, human telomerase reverse transcriptase (hTERT), beta-catenin, p16INK4a, p21CIP1, and cyclin D1). Diagnostic accuracy was assessed by receiver-operating characteristics curve analysis, and uni- and multivariate survival analysis was performed. RESULTS/FINDINGS: Of the 374 patients, 26 (7%) developed mCRC with a median of 5.6 years (range 2-19) from index adenoma. Independent risk factors included age greater than or equal to 60 years, proximal location, multiplicity (greater than or equal to three adenomas), and high-grade neoplasia, with high-grade intraepithelial neoplasia and proximal location as the strongest on multivariate analysis (hazard ratio [HR] of 4.1 and 5.2, respectively; both p< 0.05). The molecular markers hTERT (HR 11.3, 95% confidence interval [CI] 3.9-33.1; p < 0.001) and survivin (HR 7.0, 95% CI 2.4-20.5; p < 0.001) were independent predictors for mCRC, and proximal location (4 of 16 = 25% with mCRC) was the only clinical one. The value of hTERT and survivin were retained in the validation set. Survivin and hTERT together yielded high mCRC risk when both were positive (15 of 51 = 29%; HR 14.3, 5.6-36.5), modest with one positive (survivin 4 of 90 = 4.4%; hTERT 4 of 60 = 6.7%), and no risk with both negative (0 of 144 = 0%). INTERPRETATION/CONCLUSION: hTERT and survivin are the best risk predictors for long-term, mCRC development in patients with sporadic colorectal adenomas.

Androgen receptor determination in breast cancer: a comparison of the dextran-coated charcoal method and quantitative immunohistochemical analysis.

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.

Evaluation of prospective, routine application of Ki-67 immunoquantitation in early CIN for assessment of short-term progression risk.

OBJECTIVE: To prospectively validate, in early cervical intraepithelial neoplasia (CIN), routine assessment of a previously developed prognostic Ki-67 immunoquantitative progression-risk model. STUDY DESIGN: Two hundred sixty-six consecutive cervical biopsies taken for an abnormal cytologic smear were routinely diagnosed by experienced pathologists as CIN. Ki-67 immunoquantitation was performed routinely by 3 technicians blinded to clinical and pathologic information. Progression of CIN 1-2 to CIN 3 in histologic follow-up biopsies was used as the intermediate end point. RESULTS: In 58 (22%) biopsies, technical shortcomings prevented Ki-67 immunoquantitation, and in 22 biopsies no follow-up was available. The routine diagnosis in the 186 remaining biopsies was CIN 1 = 24, CIN 2 = 56 and CIN 3 = 106. In 52 marker biopsies with expert review diagnosis of CIN 1-2 and adequate follow-up, histologic biopsies revealed CIN 3 in 9 (17%) cases: 9 of 34 (26%) of Ki-67 high-risk and 0 of 18 (0%) of Ki-67 low-risk lesions (log rank = 5.0, P = .03). Routine CIN grade (1 or 2) was not prognostic (P = .65). Eleven (55%) of 20 CIN 1 and 7 of 32 (22%) CIN 2 cases were Ki-67 low risk and none progressed, contrasting with 4 of 9 (44%) progressions of Ki-67 high risk CIN 1s and 5 of 25 (20%) high risk CIN 2s. Expert CIN grades were stronger prognostically than routine CIN grade, but Ki-67 was still stronger. CONCLUSION: Routine Ki-67 immunoquantitative progression prediction in CIN 1-2 is more predictive of CIN 3 in follow-up than are routine and review CIN grades.

DNA cytometric features in biopsies of TaT1 urothelial cell cancer predict recurrence and stage progression more accurately than stage, grade, or treatment modality.

OBJECTIVES: To compare retrospectively the predictive value for recurrence and stage progression of DNA ploidy and S-phase fraction by flow cytometry and highly automated ultrafast image cytometry (ICM) in biopsies of TaT1 urothelial cell carcinomas (UCCs) of the urinary bladder with stage, grade, other pathologic features, and treatment. METHODS: Three experienced pathologists reviewed the stage and grade of 228 UCCs; 193 (85%) consensus cases were analyzed further. We had enough material for single-cell suspensions for both flow cytometry and ICM in 183 cases (94.8%). The 2001 European Society for Analytical Cellular Pathology standards for DNA ICM were followed. The predictive value of DNA features, classic prognosticators (stage, grade, carcinoma in situ, multicentricity), and treatment modality for recurrence and stage progression were analyzed with univariate (Kaplan-Meier) survival and multivariate (Cox model) regression analysis. Ta and T1 cases were analyzed separately. RESULTS: Of the 228 cases, 88 (51.5%) recurred and 13 (7.6%) progressed. On univariate analysis, most of the DNA features studied were statistically significant. Treatment modality and grade were only prognostic for progression (not for recurrence) and only in Ta cases. On multivariate analysis, DNA ICM features performed best; the strongest recurrence predictor for Ta UCC was a DNA index (DI) of 1.0 versus all others, and for T1 UCC, a DI of less than 1.3 versus 1.3 or greater. The best stage progression predictor for Ta UCCs was a DI of 1.0 plus an S-phase fraction of less than 10%, and for T1 UCCs, a DI of less than 1.3 versus 1.3 or greater. With multivariate analysis, sex, age, grade, carcinoma in situ, multicentricity, and treatment modality were excluded once the DNA ICM features were selected. CONCLUSIONS: DNA image cytometric features predict recurrence and stage progression in TaT1 UCC biopsies more accurately than classic prognostic factors, independent of treatment modality.