Diagnostic reproducibility of tumour budding in colorectal cancer: a multicentre, multinational study using virtual microscopy.

AIMS: Despite the established prognostic relevance of tumour budding in colorectal cancer, the reproducibility of the methods reported for its assessment has not yet been determined, limiting its use and reporting in routine pathology practice. METHODS AND RESULTS: A morphometric system within telepathology was devised to evaluate the reproducibility of the various methods published for the assessment of tumour budding in colorectal cancer. Five methods were selected to evaluate the diagnostic reproducibility among 10 investigators, using haematoxylin and eosin (H&E) and AE1-3 cytokeratin-immunostained, whole-slide digital scans from 50 pT1-pT4 colorectal cancers. The overall interobserver agreement was fair for all methods, and increased to moderate for pT1 cancers. The intraobserver agreement was also fair for all methods and moderate for pT1 cancers. Agreement was dependent on the participants' experience with tumour budding reporting and performance time. Cytokeratin immunohistochemistry detected a higher percentage of tumour budding-positive cases with all methods compared to H&E-stained slides, but did not influence agreement levels. CONCLUSION: An overall fair level of diagnostic agreement for tumour budding in colorectal cancer was demonstrated, which was significantly higher in early cancer and among experienced gastrointestinal pathologists. Cytokeratin immunostaining facilitated detection of budding cancer cells, but did not result in improved interobserver agreement.

αvß3 imaging can accurately distinguish between mature teratoma and necrosis in 18F-FDG-negative residual masses after treatment of non-seminomatous testicular cancer: a preclinical study.

PURPOSE: We assessed whether imaging α(v)ß(3) integrin could distinguish mature teratoma from necrosis in human non-seminomatous germ cell tumour (NSGCT) post-chemotherapy residual masses. METHODS: Human embryonal carcinoma xenografts (six/rat) were untreated (controls) or treated to form mature teratomas with low-dose cisplatin and all-trans retinoic acid (ATRA) over a period of 8 weeks. In another group, necrosis was induced in xenografts with high-dose cisplatin plus etoposide (two cycles). (18)F-Fluorodeoxyglucose ((18)F-FDG) small animal positron emission tomography (SA PET) imaging was performed in three rats (one control and two treated for 4 and 8 weeks with cisplatin+ATRA). Imaging of α(v)ß(3) expression was performed in six rats bearing mature teratomas and two rats with necrotic lesions on a microSPECT/CT device after injection of the tracer [(99m)Tc]HYNIC-RGD [6-hydrazinonicotinic acid conjugated to cyclo(Arg-Gly-Asp-D-Phe-Lys)]. Correlative immunohistochemistry studies of human and mouse α(v)ß(3) expression were performed. RESULTS: Cisplatin+ATRA induced differentiation of the xenografts. After 8 weeks, some glandular structures and mesenchymal cells were visible; in contrast, control tumours showed undifferentiated tissues. SA PET imaging showed that mature teratoma had very low avidity for (18)F-FDG [mean standardised uptake value (SUV(mean)) = 0.48 ± 0.05] compared to untreated embryonal carcinoma (SUV(mean) = 0.92 ± 0.13) (p = 0.005). α(v)ß(3) imaging accurately distinguished mature teratoma (tumour to muscle ratio = 4.29 ± 1.57) from necrosis (tumour to muscle ratio = 1.3 ± 0.26) (p = 0.0002). Immunohistochemistry studies showed that α(v)ß(3) integrin expression was strong in the glandular structures of mature teratoma lesions and negative in host stroma. CONCLUSION: Imaging α(v)ß(3) integrin accurately distinguished mature teratoma from necrosis following cisplatin-based treatment in human NSGCT xenografts.

Stromal compartment as a survival prognostic factor in advanced ovarian carcinoma.

OBJECTIVE: We investigated the prognostic significance of stromal compartment on the overall survival of patients with advanced epithelial ovarian cancer. METHODS: We evaluated retrospectively the stroma proportion of the tumor surgical specimens of 194 patients with stages III and IV disease, using histochemical staining and fully automatic virtual slide processing. The prognostic significance of stroma proportion and clinical parameters were evaluated using log-rank test and Cox regression. RESULTS: Stroma proportion was found to be an independent prognostic factor by both univariate (P = 0.016) and multivariate analyses (hazards ratio = 1.45, P = 0.011). CONCLUSION: The present data indicate that a high stroma proportion is related to a poor prognosis of stage III and IV ovarian carcinomas.

Usefulness of automatic quantification of immunochemical staining on whole tumor sections for correlation with oncological small animal PET studies: an example with cell proliferation, glucose transporter 1 and FDG.

AIM: To highlight the use of automatic quantification of immunochemical staining on digitized images of whole tumor sections in preclinical positron emission tomography (PET) studies. MATERIALS AND METHODS: Xenografted human testicular tumors (36) were imaged with 2-deoxy-2[F-18]fluoro-D: -glucose (FDG) small animal PET (SA-PET). Tumor cell proliferation and glucose transportation were assessed with cyclin A and Glut-1 immunostaining. Tumor slides were digitized and processed with PixCyt software enabling whole slide quantification, then compared with junior and senior pathologist manual scoring. Manual and automatic quantification results were correlated to FDG uptake. RESULTS: For cyclin A, inter- and intra-observer agreement for manual scoring was 0.52 and 0.72 and concordance between senior pathologist and automatic quantification was 0.84. Correlations between Tumor/Background ratio and tumor cell proliferation assessed by automatic quantification, junior and senior pathologists were 0.75, 0.55, and 0.61, respectively. Correlation between Tumor/Background ratio and Glut-1 assessed by automatic quantification was 0.74. CONCLUSION: Automatic quantification of immunostaining is a valuable tool to overcome inter- and intra-observer variability for correlation of cell proliferation or other markers with tumor tracer uptake.

Flow cytometry in primary breast carcinomas: prognostic impact of S-phase fraction according to different analysis patterns.

BACKGROUND: The aim of the present work was to study the prognostic impact of ploidy and S-phase fraction (SPF) assessed according to recently described methods. These methods of analysis combine different ploidy groups and separate euploid (good) prognostic groups from noneuploid (bad) prognostic groups. The definition of euploidy varied according to the author; some of them even included aneuploid peaks with few events. A comparison was also drawn to the average SPF and the diploid peak SPF observed in aneuploid histograms. METHODS: From January 3, 1990 to January 7, 1999, 1,984 previously untreated, invasive breast carcinoma samples were snap-frozen and processed for FCM. The present study evaluated all nondiploid and nonmultiploid histograms, using different analysis patterns and the values of the average SPF and diploid SPF. RESULTS: SPF is a salient prognostic factor even after multivariate analysis for DFS and MFS. Using several methods of analysis of ploidy and SPF shows that the classical method of analysis involving separation of ploidy according to diploidy versus aneuploidy and analysis of SPF restricted to the aneuploid peak in nondiploid and nonmultiploid histograms is as relevant as other recently proposed patterns of analysis, and that the average SPF or the diploid SPF of aneuploid tumors does not add significant prognostic information. CONCLUSIONS: SPF is a valuable predictor of survival and can be confidently assessed in a simple way by restricting the analysis to the peak of interest (except for multiploid tumors).

Flow cytometry in primary breast carcinomas: prognostic impact of multiploidy and hypoploidy.

BACKGROUND: The aims of the present work were to study the prognostic impact of multiploidy and/or hypoploidy in breast cancers and their relation to other classic clinicopathologic prognostic factors (T, grade, receptors, and lymph node status). METHODS: From 3 January 1990 to 7 January 1999, 1984 previously untreated, invasive breast carcinoma samples were snap frozen for flow-cytometry. RESULTS: Multiploid tumors had the same prognosis as the aneuploid ones, and those with one hypoploid peak had a better prognosis than did the other aneuploid tumors. However, the presence of both multiploid and hypoploid peaks was correlated with a poor outcome, even after multivariate analysis. In this series after quality control, 93.4% of the histograms could be evaluated concerning ploidy; of these 81.6% could be assessed concerning S-phase fraction (SPF) in the entire population and 77.1% in the multiploid population. In the entire population, we performed a multivariate analysis including all relevant prognostic factors remaining after monovariate analysis by using a compound factor (proliferative activity) regrouping SPF and mitotic activity. This analysis showed that lymph node status and proliferative activity correlates with every type of survival, whereas receptor status correlates with all types of survival except recurrence free survival size, correlated with non-metastasis and overall survival. Grade and age correlated only with overall survival and vascular permeations only with disease-free survival. CONCLUSIONS: SPF is a valuable predictor of survival, can be confidently assessed in multiploid histograms, and thus improves the yield of flow cytometry. When combined with mitotic activity, the prognostic impact of SPF is the same as that of lymph node status. Tumors that are hypoploid and multiploid have a significantly worse prognosis.

[Primary thyroid lymphomas: clinicopathologic study of 30 cases and review of the literature]. / Lymphomes thyroïdiens primitifs: étude clinico-pathologique de 30 cas et revue de la littérature.

During a both retrospective and prospective study of thyroid cancers treated in the Basse Normandie between 1960 and 1999, we have identified 32 patients with thyroid lymphoma. The correct diagnosis was made initially in 69% of all cases. In the other cases, the diagnosis was secondarily corrected after review of the pathological material. According to the REAL classification, 7 (21%) corresponded to low grade MALT lymphomas, 2 to low grade lymphomas, 10 to high grade MALT Lymphomas and 10 (31%) to high grade lymphomas, one plasmocytoma and two unclassified lymphomas. According to the Ann Arbor classification, stage was IE for 56%, IIE for 19%, IIIE for 3% and 9% for IV. Median survival was 28 months with a mean at 61 months. 20 patients died (62%), 12 from the lymphoma and 8 from intercurrent causes. The overall survival at 5 years was 36% (9 5% CI 16 54%). A comparison of our results with those of the literature was performed.

Standardization of whole slide image morphologic assessment with definition of a new application: Digital slide dynamic morphometry.

BACKGROUND: In histopathology, the quantitative assessment of various morphologic features is based on methods originally conceived on specific areas observed through the microscope used. Failure to reproduce the same reference field of view using a different microscope will change the score assessed. Visualization of a digital slide on a screen through a dedicated viewer allows selection of the magnification. However, the field of view is rectangular, unlike the circular field of optical microscopy. In addition, the size of the selected area is not evident, and must be calculated. MATERIALS AND METHODS: A digital slide morphometric system was conceived to reproduce the various methods published for assessing tumor budding in colorectal cancer. Eighteen international experts in colorectal cancer were invited to participate in a web-based study by assessing tumor budding with five different methods in 100 digital slides. RESULTS: The specific areas to be tested by each method were marked by colored circles. The areas were grouped in a target-like pattern and then saved as an .xml file. When a digital slide was opened, the .xml file was imported in order to perform the measurements. Since the morphometric tool is composed of layers that can be freely moved on top of the digital slide, the technique was named digital slide dynamic morphometry. Twelve investigators completed the task, the majority of them performing the multiple evaluations of each of the cases in less than 12 minutes. CONCLUSIONS: Digital slide dynamic morphometry has various potential applications and might be a useful tool for the assessment of histologic parameters originally conceived for optical microscopy that need to be quantified.

Expression of p53 family members and CD44 in oral squamous cell carcinoma (OSCC) in relation to tumorigenesis.

Oral squamous cell carcinomas (OSCCs) are described as the result of a multistep tumorigenesis process. In order to develop useful diagnosis of pre-malignant lesions, expression of p53 family members and the cancer stem cell (CSCs) marker, CD44v6, were studied in histologically normal oral epithelium, precancerous lesions and succeeding invasive OSCCs. p53 was expressed focally in normal epithelium adjacent to tumors, while expression was high in intra-epithelial neoplasia and moderate in OSCC. p63 nuclear staining was important in basal and suprabasal layers of histologically normal oral mucosa and in immature compartments of premalignant lesions and cancer. In epithelium without neoplasia, intense p73 staining was observed in the basal layer, while focal expression was present in suprabasal layers. Most immature dysplastic areas showed either high or moderate staining, whereas those in OSCCs expressed low and moderate p73 level expression. CD44v6 was only expressed in poorly differentiated areas of epithelium, altered or not. p53, p63 and p73 positive stainings were statistically related in intra-epithelial neoplasia to tumours. Analysis of TP53 mutations in 17% of tumours principally revealed G>A and A>G transitions. No relation was observed between this mutational profile and different immunostainings. In conclusion, our results support that immunostaining of p53 family members might be helpful in diagnosis and monitoring of high-risk pre-malignant lesions of oral epithelium. The combination of staining patterns of p63, p73alpha and CD44v6 enabled us to isolate phenotypic undifferentiated or transient amplifying areas, reflecting the immaturity of the tumour cell lineage. While CD44v6 expression is an interesting marker of such epithelial cells, it is not specific enough to be useful alone and other phenotypic markers are needed.

High grade primary breast lymphoma: is it a different clinical entity?

Primary lymphoma of the breast (PBL) is a rare neoplasm, its outcome remains unclear compared to other lymphomas. We performed a retrospective study of 19 cases of high grade PBL. There were 17 Diffuse large B cell lymphoma (DLBCL) and 2 follicular and diffuse grade 3 lymphomas. Four patients were treated with local treatment only, 15 received chemotherapy including 11 treated with CHOP or ACVBP regimens followed by involved field radiotherapy. The actuarial survival for the whole population was 38%. Three of the 4 patients treated only with a local treatment died of their lymphoma. Three patients progressed on therapy and 5 relapsed in the first year of follow-up including 2 central nervous system recurrences. Among the 11 patients treated with chemotherapy, 2 died of their lymphoma. The overall survival of this subgroup was 73% (median follow-up of 57 months). We observed, like others in the literature, a better prognosis for lymphomas co-expressing Bcl6 and CD 10. The treatment should be based on the same modalities, but including a CNS prophylaxis even if poor prognosis factors are lacking. A radical mastectomy increases the risk of treatment failure and has to be avoided.

Proliferative activity in primary breast carcinomas is a salient prognostic factor.

BACKGROUND: The goal of the current study was to investigate the prognostic impact of proliferative activity, together with the other classic clinicopathologic prognostic factors (tumor size, tumor grade, receptor status, ploidy, and lymph node status), in breast carcinoma by counting mitoses and evaluating S phase fraction (SPF) in fresh and frozen tumor samples. METHODS: From March 1, 1990, to July 1, 1999, a total of 1984 previously untreated invasive breast carcinoma samples were snap-frozen for flow cytometry. RESULTS: After multivariate analysis incorporating all classic prognostic factors, SPF combined with mitotic activity (i.e., proliferative activity) remained the sole prognostic factor in the lymph node-negative group; proliferative activity was accompanied by tumor size as a prognostic factor in patients with lymph node-positive disease and by lymph node status, lymphatic invasion, and receptor status in the overall population. The predictive value of proliferative activity was superior to that of the reference standards (classic prognostic predictors according to the guidelines of our institution [common oncology practice] and the St. Gallen classification). A review of the literature, focusing on series in which fresh material was used, allowed us to demonstrate that there is widespread agreement regarding the correlation between SPF and prognosis, even after multivariate analysis. CONCLUSIONS: S phase fraction is a valuable predictor of survival and can confidently be assessed in approximately 80% of cases. In conjunction with mitotic activity, SPF should become a prognostic factor that is used in daily practice by oncologists for the management of breast carcinoma.

Flow cytometry and quantitative immunohistochemical study of cell cycle regulation proteins in invasive breast carcinoma: prognostic significance.

BACKGROUND: Between January 11, 1991 and January 8, 1992, 104 patients with previously untreated, invasive, primitive breast carcinoma were admitted to the authors' hospital. METHODS: For each patient, flow cytometry DNA analyses on frozen samples and on immunohistochemical staining were performed, including Ki-67, cyclin A, p53, and p21(waf1) (p21), with assessment of the percentages of positive nuclei were assessed. Correlations with classic clinicopathologic data and survival (overall, metastasis free, or recurrence free) and a multivariate analysis were performed. RESULTS: After a multivariate analysis according to a Cox model that was stratified by age, tumor size, tumor grade, lymph node status, and receptor status, among the factors studied, the presence of p21 was the unique remaining prognostic factor for patients with invasive breast carcinoma. Because of the lack of a correlation between p21 and proliferative factors (Ki-67, S-phase, and cyclin A), the authors combined p21 with those markers and found that, for the different combinations, after statistical analysis, only p21 combined with S-phase or with cyclin A and lymph node status were salient survival prognostic factors. CONCLUSIONS: Immunohistochemical study of proteins involved in the cell cycle and assessment of proliferative activity using flow cytometric DNA analysis aided the authors in singling out correlations of cyclin A and S-phase, along with p21, with metastasis free survival and overall survival in patients with invasive breast carcinoma. These promising results will require confirmation in a larger series of patients.