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Phosphohistone H3 assessed proliferation has strong prognostic value. Lymph vessel invasion by D2-40 is also prognostic, but D2-40+ myoepithelial expression in small ducts completely filled by solid-pattern ductal carcinoma in situ can mimic lymphovascular invasion. As myoepithelial cells are also p63 positive, we have investigated whether lymph vessel invasion identified by combined D2-40/p63 is stronger prognostically than by D2-40 alone, and whether it has independent prognostic value to phosphohistone H3. In 240 operable T1–2N0M0node negative invasive breast cancer patients <71 years, phosphohistone H3 was determined by quantitative immunohistochemistry and lymph vessel invasion by D2-40/p63 double immunostaining. Correlation analysis between the clinico-pathologic factors and lymph vessel invasion, and univariate and multivariate prognostic survival analysis were performed. With median 117 (range: 12–192) months follow-up, 36 patients (15%) developed and 28 (12%) died of distant metastases. Ten of the 61 patients (16%) with cancer cells surrounded by D2-40 were p63 positive and none of these ‘false lymph vessel invasion’ recurred. D2-40+/p63− lymph vessel invasion occurred in 51/239 (21%) cases and correlated with grade, mitotic activity index, phosphohistone H3, ER, cytokeratin14, and HER2. D2-40+/p63− lymph vessel invasion was strongly prognostic, but far more in women ≥55 than those <55 years (P<0.0001 and 0.04). With multivariate analysis, phosphohistone H3 proliferation was the strongest single prognosticator. Lymph vessel invasion had additional prognostic value to phosphohistone H3 only in women ≥55. This group of patients, without/with lymph vessel invasion, had 10-year survival rates of 83 and 50%, respectively (hazard ratio-lymph vessel invasion=3.0, P=0.04; hazard ratio-phosphohistone H3=6.9, P=0.002). Where age was <55 years, only phosphohistone H3 had independent prognostic value. Combinations of other features had no additional value. In conclusion, T1–2N0M0invasive breast cancer patients ≥55 years with phosphohistone H3≥13, D2-40+/p63− defined lymph vessel invasion identifies a subgroup with a high risk of distant metastases.

Breast cancer is a heterogeneous disease. Different subgroups can be recognized on the basis of the steroid receptors, HER-2, cytokeratin expression and proliferation patterns. As a result of mRNA-profiling studies, five major groups can be recognized, of which the triple-negative and basal-like tumors have the worst prognosis. Many of these tumors have a high proliferation that has the strongest prognostic value in node negative breast cancer. In the current study we analyzed the microRNA pattern in 103 lymph node negative breast cancers and compared these profiles with different biological characteristics and clinicopathological features. Unsupervised hierarchical cluster analysis divides the patients into four main groups, of which the basal-like/triple-negative group is the most prominent (11% of all cases), the luminal A cancers containing the Her2 negative and estrogen receptor/progesterone receptor-positive tumors is the largest group (57%), and the group of luminal B (32%) is more heterogeneous and contains the Her2 positive/estrogen receptor-negative patients as well. The highest overall classification values by analysis of variance followed by cross validation (leave one sample out and reselect genes) were found for cytokeratin 5 and 6, triple-negative and estrogen receptor, with 97, 90 and 90% accuracy, respectively. MiR-106b gene is prominent in all of these signatures and correlates strongest with high proliferation. Other interesting observations are the presence of several microRNAs (miR532-5p, miR-500, miR362-5p, and miR502-3p) located at Xp11.23 in cancers with a triple-negative signature, and the upregulation of several miR-17 cluster members in estrogen receptor-negative tumors. The current study shows that estrogen receptor negativity and cytokeratin 5 and 6 expression are important, and specific biological processes in lymph node negative breast cancer, as microRNA signatures are strongest in these subgroups.

Fifteen to thirty percent of cases with histologically confirmed CIN2–3 in cervical biopsies regress spontaneously (ie, show CIN1 or less in the follow-up cervical cone). The balance between immune-reactive cells from the host and high-risk human papillomavirus (hrHPV) genotypes may provide a biological explanation for this phenomenon. We retrospectively studied 55 cases of CIN2–3 in a cervical biopsy with subsequent cervical cone to assess whether hrHPV genotypes (by AMPLICOR and Linear Array tests) CD4, CD8, CD25, CD138 and Foxp3 cells (by quantitative immunohistochemistry) in the cervical biopsies can predict regression (defined as CIN1 or less in the follow-up cone biopsy). Eighteen percent of the CIN2–3 cases regressed (median biopsy–cervical cone time interval: 12.0 weeks, range: 5.0–34.1 weeks). HPV-16 correlated with low CD8+and high CD25+. None of the regressing CIN2–3 lesions contained HPV-16. The regressing CIN2–3 lesions had lower numbers of stromal CD138+and higher numbers of stromal CD8+cells; higher stromal and intra-epithelial ratios of CD4+/CD25+cells; higher ratios of CD8+/CD25+cells and lower ratios of CD8+/CD4+, CD138+/Foxp3+and CD25+/Foxp3+cells in the stroma. With multivariate survival analysis, stromal CD8+cell numbers, CD4+/CD25+cell ratios and CD138+cell numbers are found to be independent regression predictors. In conclusion, in non-HPV-16 CIN2–3 lesions, assessing stromal immune cells can be a useful prognostic indicator of regression or persistence.

The proliferation factor mitotic activity index is the strongest prognostic factor in early breast cancer, but it may lack reproducibility. We analyzed the prognostic value of phosphohistone H3, a marker of cells in late G2and M phase, measuring highly standardized immunohistochemical nuclear phosphohistone H3 expression by subjective counts and digital image analysis. Expression was compared with classical clinico-pathologic prognostic variables and the mitotic activity index in 119 node-negative invasive breast cancers in patients less than 55 years old treated with adjuvant systemic chemotherapy with long-term follow-up (median 168 months). Nineteen patients (16%) developed distant metastases and 16 (13%) died. Strong phosphohistone H3 expression occurred preferentially in the peripheral growing front; counts were highly reproducible between observers (R=0.92) and highly consistent with digital image analysis (R=0.96). Phosphohistone H3 correlated (P<0.05) with tumor diameter, estrogen receptor, carcinoma grade, and mitotic activity index. Phosphohistone H3 values were systematically (80%) higher than the mitotic activity index. Receiver-operating curve analysis objectively showed that phosphohistone H3 <13 (n=53; 45% of all cases) vs phosphohistone H3≥13 (n=66; 55% of all cases) was the strongest prognostic threshold, with 20-year recurrence-free survival of distant metastases of 96 and 58%, respectively (P=0.0002, HR=9.6). Mitotic activity index was the second strongest prognostic variable (P=0.003, HR=3.9). In multivariate analysis, phosphohistone H3 <13 vs≥13 exceeded the prognostic value of the mitotic activity index. None of the other classical prognostic factors examined offered prognostic value additional to phosphohistone H3. Phosphohistone H3 is by far the strongest prognostic variable in early invasive node-negative breast cancer patients less than 55 years old with long-term follow-up.Modern Pathology (2007) 20, 1307–1315; doi:10.1038/modpathol.3800972; published online 5 October 2007

Endometrial "hyperplasia," as currently diagnosed, includes the changes caused by an abnormal hormonal state and those caused by a separate category of monoclonal premalignant disease. The appearance of the disease in these 2 functional categories is discontinuous, permitting more specific diagnosis of the condition using the terms "benign endometrial hyperplasia" and "endometrial intraepithelial neoplasia" (EIN), respectively. Benign endometrial hyperplasia involves the entire endometrial compartment and, with protracted estrogen exposure, shows the progressive development of cysts, remodeled glands, vascular thrombi, and stromal microinfarcts. They are best construed as a sequence of changes whereby the appearance at any single time point is uniquely dependent on the preceding combination and the duration of hormonal exposures. In contrast, the premalignant clone of an EIN lesion is characteristically offset from the background endometrium by its altered cytology and crowded architecture. The use of an internal standard for cytology assessment, combined with the distinctive topography of a clonal process, enables the diagnosis of EIN lesions with a long-term cancer risk 45-fold greater than that of their benign endometrial hyperplasia counterparts. The resolution of hormonal and premalignant subsets of traditional "endometrial hyperplasias" is possible using redefined diagnostic criteria, enabling patient therapy to be appropriately matched with the underlying disease mechanisms.

The objective of this study was to compare the accuracy of disease progression prediction of the molecular genetics and morphometry‐based Endometrial Intraepithelial Neoplasia (EIN) and World Health Organization 1994 (WHO94) classification systems in patients with endometrial hyperplasias.A multicenter, multivariate analysis was conducted on 477 patients with endometrial hyperplasia who were required to have a 1‐year minimum disease‐free interval from the time of the index biopsy (1–18 years of follow‐up). The results from that analysis were compared with the results from 197 patients who had < 1 year of follow‐up.Twenty‐four of 477 hyperplasias (5.0%) progressed to malignant disease over an average of 4 years (maximum, 10 years). According to the WHO94 classification, 16 of 123 atypical hyperplasias (13%) and 8 of 354 nonatypical hyperplasias (2.3%) progressed (hazard ratio [HR] = 7). Twenty‐two of 118 EINs (19%) and 2 of 359 non‐EINs (0.6%) progressed (HR = 45). EIN was prognostic within each WHO94 subcategory. Progression rates were 3% in simple hyperplasias, 22% in complex hyperplasias, 17% in simple atypical hyperplasias, and 38% in complex atypical hyperplasias with EIN, compared with progression rates of 0.0–2.0% in all hyperplasias if EIN was absent. EIN detected precancerous lesions (sensitivity, 92%) better than WHO94 atypical hyperplasias collectively (67%) or complex atypical hyperplasias alone (46%). In a Cox regression analysis, EIN was the strongest prognostic index of future endometrial carcinoma. The same was true for patients with < 1 year of follow‐up (HR for EIN, atypical hyperplasia, and complex atypical hyperplasia: 58, 7, and 8, respectively).The EIN classification system predicted disease progression more accurately than the WHO94 classification and identified many women with benign changes that would have been regarded as high risk according to the WHO94 classification system. Cancer 2005. © 2005 American Cancer Society.

Three different cancers predominantly occur at the gastro‐oesophageal junction: squamous cell carcinomas of the distal oesophagus, adenocarcinomas of the distal oesophagus (Barrett's carcinomas), and adenocarcinomas of the gastric cardia. The aim of the present study was to investigate how, and to what extent, Barrett's carcinoma differs from adenocarcinoma of the gastric cardia on the one hand and squamous cell carcinoma of the distal oesophagus on the other, with respect to chromosomal aberrations and related gene expression. The present study analysed 14 squamous cell carcinomas, 24 Barrett's carcinomas, and 16 carcinomas of the gastric cardia. Comparative genomic hybridization revealed chromosomal abnormalities in all cases. Typical chromosomal aberrations for the squamous cell carcinoma type were gains at 3q and 11q13, and losses at 3p, 4q, 9p, 11q, and 13q. In contrast, typical copy number changes for both cardiac and Barrett's adenocarcinomas were gains at 2q, 7p, and 13q, and losses at 17p. High‐level amplification occurred in all three groups, but its frequency in the cardiac carcinomas was lower than in the other two groups. In conclusion, squamous cell carcinomas are characterized by chromosomal aberrations which are distinct from those seen in carcinomas of the gastric cardia and in Barrett's adenocarcinomas. With respect to Barrett's cancer, the chromosomal aberrations more closely reflect the adenocarcinoma phenotype than the squamous origin of the epithelium. Copyright © 2002 John Wiley & Sons, Ltd.

We assessed the reproducibility and prognostic variability of grade and lamina propria invasion in stages Ta, T1 urothelial carcinoma of the bladder.

Combined confocal laser scan microscopy (CLSM) and Fourier analysis (FA) by non‐pathologists of dermal collagen bundle orientation recently gave results superior to subjective evaluation by experts. According to Good Laboratory Practice (GLP) criteria, combined CLSM/FA has not yet been adequately tested to replace current collagen evaluation, but this will not take long. Non‐pathologists (clinicians) will then have taken over a laboratory test historically belonging to pathology. A general trend in this direction may develop, because pathologists seem not always to care enough about clinical significance, reproducibility and prognostic value, and new demands for innovative methods. Quantitative image analysis (QIA) and molecular methods are reproducible, inexpensive, and easy to perform; they often have greater value than classical evaluations and their cost–benefit ratio is good. However, their acceptance is not as widespread as one would expect and theoretical reasons which have been advanced do not provide a satisfactory explanation. A formal implementation study was therefore performed, in which an attempt was made to modernize a classical pathology laboratory. An external customer satisfaction investigation showed that 96% of the clinicians were ‘very satisfied’ (the highest rating possible) with the completed innovations, contrasting with low satisfaction at the beginning. Lack of primary innovative leadership among pathologists was judged to be the dominant cause preventing implementation. Pathologists should focus on carefully reacting to new clinical needs, using GLP criteria. Reproducibility and predictive accuracy should be major themes in any pathology practice. Copyright © 2002 John Wiley & Sons, Ltd.

Confocal laser scanning microscopy (CLSM) presents the opportunity to perform three-dimensional (3D) DNA content measurements on intact cells in thick histological sections. So far, these measurements have been performed manually, which is quite time-consuming. In this study, an intuitive contour-based segmentation algorithm for automatic 3D CLSM image cytometry of nuclei in thick histological sections is presented. To evaluate the segmentation algorithm, we measured the DNA content and volume of human liver and breast cancer nuclei in 3D CLSM images. A high percentage of nuclei could be segmented fully automatically (e.g., human liver, 92%). Comparison with (time-consuming) interactive measurements on the same CLSM images showed that the results were well correlated (liver, r = 1.00; breast, r = 0.92). Automatic 3D CLSM image cytometry enables measurement of volume and DNA content of large numbers of nuclei in thick histological sections within an acceptable time. This makes large-scale studies feasible, whereby the advantages of CLSM can be exploited fully. The intuitive modular segmentation algorithm presented in this study detects and separates overlapping objects, also in two-dimensional (2D) space. Therefore, this algorithm may also be suitable for other applications. Cytometry 49:12–21, 2002. © 2002 Wiley-Liss, Inc.

Despite intensive therapy, the survival of children with medulloblastoma remains disappointing. Moreover, children who survive are affected by serious long-term sequelae of treatment that impair their quality of life. In search of chromosomal aberrations indicative of sites involved in oncogenic transformation and in an attempt to find reliable prognostic markers, the authors analyzed 15 medulloblastomas by comparative genomic hybridization. All neoplasms showed chromosomal abnormalities. The most frequent losses were 17p (7/15 tumors), 8p and 11p (6/15), 10p, 11q, 16q, and 20q (5/15), and 20p (4/15). Gains were recurrently found at 7q (10/15 tumors), 17q and 18q (9/15 tumors), 7p and 13q (7/15), 18p (6/15), and 1q, 4q, 6q, and 9p (5/15 tumors). Four tumors showed loss of 17p together with gain of 17q, suggesting an isochromosome 17q. High-level amplifications were seen at 1p34, 5p15, 13q34, and 18p11 (one tumor each), and at 2p15 in two tumors, one of which was proven to be N-Myc amplification. The overall pattern of alterations found in this study confirms the findings of other studies and adds two novel regions with chromosomal gains, at 13q and 18q. Previous reports on the relation between 17q gain and survival could not be confirmed, whereas amplification of N-mycor L-mycseems to indicate poor clinical outcome.

Objective.Endometrioid endometrial carcinoma is caused by a combination of mutational events and hormonal factors. We used large-scale messenger RNA expression analysis to discover genes that distinguish neoplastic transformation and examine the patterns of tumor expression of those genes which are normally regulated during the menstrual cycle.

Women with usual ductal hyperplasia have a relative risk of 1.6–1.9 of subsequent breast cancer development. This slightly increased risk is generally not considered sufficiently high to justify (chemo)preventive therapy. It is therefore important to identify high-risk ductal hyperplastic lesions that would benefit from such a treatment. Nuclear morphometric features have been shown in previous work to be useful for objectively describing morphologic features associated with high risk in (pre)invasive breast lesions. The aim of this study was to evaluate whether such morphometric features can also predict subsequent invasive cancer development in patients with the common pattern of usual ductal hyperplasia or a normal breast biopsy. The present case-control study included 423 women with normal breast biopsies (n = 89) or biopsies containing usual ductal hyperplasia (n = 334). Of these 423 women, 132 developed invasive breast cancer during follow-up (mean 16.7 ± 7.0 years). On the original hematoxylin and eosin-stained sections, nuclear morphometry was performed with a digitizing video overlay system, and mitotic and apoptotic indices were assessed. Patients with mean nuclear feature values for area, perimeter, diameter or longest axis above the 75^th percentile had 1.6–1.7 times the breast cancer risk of women with mean nuclear feature values below this value. Pairwise combinations of these features yielded slightly higher cancer risks for the fourth quartile patients, with the highest risk (1.9) for patients with SD of nuclear area and perimeter values above the 75^th percentile. The number of apoptotic or mitotic cells had no prognostic value for patients with apparently normal tissue or usual ductal hyperplasia. Our results give a first indication that normal breast tissue or usual ductal hyperplasia harbor nuclear morphologic changes that, when assessed by morphometry, may be used to predict breast cancer development. It is worthwhile studying this further in independent groups of patients with long-term follow-up. © 2001 Wiley-Liss, Inc.

There is increasing evidence that there are different progression routes leading to invasive breast cancer, depending on histology and differentiation grade. The aim of this study was to determine alterations in the expression of proteins involved in proliferation and apoptosis in non‐invasive and invasive ductal breast lesions. Immunohistochemistry was performed on 106 usual ductal hyperplasias (UDH), 61 DCIS lesions and 53 invasive ductal breast carcinomas. Increased proliferation (Ki67), overexpression of cyclin D1, HER‐2/neu, p21 and p53, and decreased expression of bcl‐2 and p27 could already be found in UDH. Significant differences between UDH and DCIS lesions were found for only one protein when UDH was compared with well‐differentiated DCIS (p27), for three proteins when compared with intermediately differentiated DCIS (p21, cyclin D1, Ki‐67), and for all proteins when compared with poorly‐differentiated DCIS. Comparing DCIS with invasive lesions of same differentiation grade, proliferation was elevated in the invasive lesions. Altered expression of the other proteins was in general only slightly increased in the invasive lesion compared with DCIS. The number of proteins with altered expression per lesion was highest in poorly‐differentiated lesions and was comparable between DCIS and invasive cancer of the same differentiation grade. In conclusion, the biggest changes in expression of these proliferation and apoptosis related proteins appear to occur during the transition from hyperplasia to DCIS; they probably play a minor role in the transition from DCIS to invasive breast lesion of same differentiation grade. Well‐differentiated in situand invasive breast lesions share many of the aberrations in expression of these proteins, as do poorly‐differentiated in situand invasive lesions. However, there are many differences between the well and poorly‐differentiated lesions. This further supports the existence of different progression routes leading to breast cancer. Copyright © 2001 John Wiley & Sons, Ltd.

Squamous cell carcinomas of the head and neck generally exhibit complex karyotypes. To gain better knowledge of the changes in the subgroup of laryngeal and pharyngeal squamous cell carcinoma, chromosomal gains and losses were investigated in 42 predominantly late‐stage tumours, using comparative genomic hybridization. On average, 11.2 gains and 6.8 losses were found. Gains were detected in high frequencies at 1q, 3q, 5p, 7q, 8q, 11q13, 17q, and 18p, and losses at 3p, 4p, 5q, 11qter, and 18q. Neither the number nor the type of abnormalities, nor the occurrence of specific chromosome changes, was found to be related to DNA ploidy, tumour stage, or degree of differentiation. Apart from low‐level gains, many high‐level amplifications were identified, in particular 3q24‐qter (15 cases). Other regions recurrently involved were 11q13 (7 cases), 18p (5 cases), 18q11.2 (4 cases), and 8q23–24 and 11q14–22 (3 cases). Many of these amplified regions have not been reported before. Over half of all loci harbour genes coding for growth factors and growth factor receptors, suggesting an important role for such genes in squamous cell tumourigenesis and in the progression of late‐stage tumours. Copyright © 2001 John Wiley & Sons, Ltd.

Purpose. To assess the relation between growth factors, growth-factor receptors, p53, bcl-2 and bax expression, and the rate of apoptosis in invasive breast cancer patients. Materials and methods. Tumors from 45 patients were assessed by immunohistochemistry for expression patterns of five growth factors and their receptor platelet-derived growth factor A chain (PDGF-AA) and PDGF-receptor alpha (PDGFαR), PDGF-BB and PDGFβR, transforming growth-factor alpha (TGFα) and its receptor-epidermal growth factor receptor (EGFR) and vascular-endothelial growth factor (VEGF) and its receptors vascular-endothelial growth factor receptor I (FLT-1) and vascular-endothelial growth factor receptor II (FLK-1/KDR), two growth-inhibiting factors; transforming-growth factor beta 1 (TGFβ1) and TGFβ2 and their receptor couple TGFβ receptor I (TGFβR-I) and TGFβR-II, and basic-fibroblast growth factor (bFGF). Besides, the expression patterns of the bcl-2, bax and p53 gene products were investigated. Expression patterns were correlated to the number of apoptotic cells assessed by light microscopy. Results. PDGF-BB and bFGF showed a positive correlation with the AI (p=0.006 and p=0.030, respectively). EGFR expression was associated with a high number of apoptotic cells but did not reach significance (p=0.10). None of the other individual growth factors, growth-inhibiting factors or receptors showed a significant relation with the AI. The presence of a possible auto- or paracrine loop of the TGFα/EGFR combination was associated with a high number of apoptotic cells but did not reach significance (p=0.20). PDGF-AA, bFGF and EGFR expression showed a significant relation to p53 overexpression. TGFβ2 expression showed an inverse correlation with p53 overexpression. Conclusion. We found an association between several growth factors and growth-factor receptors with number of apoptotic cells. This underlines the importance of growth factors and their receptors not just in proliferation, but also, directly and/or indirectly, in regulating the rate of apoptosis in invasive breast cancer. Growth factors and their receptors may therefore be useful as targets of anti-cancer therapy by inducing apoptosis or increasing the sensitivity of cells for chemo- or hormonal therapy induced apoptosis.

Fanconi anemia (FA) is an autosomal recessive syndrome with a marked predisposition to malignancies, in particular acute myeloid leukemia and squamous cell carcinoma of the oral cavity. We examined oral squamous cell carcinoma tissue from two FA patients (FA-A and FA-C) by comparative genomic hybridization. Both tumors, which were negative for human papilloma as well as Epstein-Barr viral sequences, showed multiple alterations with a high proportion of whole-arm chromosomal gains and losses. This combination of features as well as the sites involved in chromosomal breakage are very similar to what is typically observed in non-FA oral tumors. These results suggest that the process leading to early occurrence of oral cancer in FA patients follows a similar pathway as in non-FA cancer patients, which would support a caretaker function for FA genes in the protection against oral carcinogenesis. Since FA patients are uniquely hypersensitive to DNA cross-linking agents, while oral cancer in the general population is thought to be environmentally induced, these results also suggest that environmental DNA cross-linkers may be causally involved in oral carcinogenesis.