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Supplementary Figure Legend from Global Levels of Specific Histone Modifications and an Epigenetic Gene Signature Predict Prostate Cancer Progression and Development

Supplementary Table 1 from Global Levels of Specific Histone Modifications and an Epigenetic Gene Signature Predict Prostate Cancer Progression and Development

Supplementary Figure 5 from Global Levels of Specific Histone Modifications and an Epigenetic Gene Signature Predict Prostate Cancer Progression and Development

Supplementary Figure 1 from Global Levels of Specific Histone Modifications and an Epigenetic Gene Signature Predict Prostate Cancer Progression and Development

Supplementary Table 2 from Global Levels of Specific Histone Modifications and an Epigenetic Gene Signature Predict Prostate Cancer Progression and Development

Supplementary Figure 3 from Global Levels of Specific Histone Modifications and an Epigenetic Gene Signature Predict Prostate Cancer Progression and Development

Background: Epigenetic alterations are common in prostate cancer, yet how these modifications contribute to carcinogenesis is poorly understood. We investigated whether specific histone modifications are prognostic for prostate cancer relapse, and whether the expression of epigenetic genes is altered in prostate tumorigenesis.Methods: Global levels of histone H3 lysine-18 acetylation (H3K18Ac) and histone H3 lysine-4 dimethylation (H3K4diMe) were assessed immunohistochemically in a prostate cancer cohort of 279 cases. Epigenetic gene expression was investigated in silico by analysis of microarray data from 23 primary prostate cancers (8 with biochemical recurrence and 15 without) and 7 metastatic lesions.Results: H3K18Ac and H3K4diMe are independent predictors of relapse-free survival, with high global levels associated with a 1.71-fold (P < 0.0001) and 1.80-fold (P = 0.006) increased risk of tumor recurrence, respectively. High levels of both histone modifications were associated with a 3-fold increased risk of relapse (P < 0.0001). Epigenetic gene expression profiling identified a candidate gene signature (DNMT3A, MBD4, MLL2, MLL3, NSD1, and SRCAP), which significantly discriminated nonmalignant from prostate tumor tissue (P = 0.0063) in an independent cohort.Conclusions: This study has established the importance of histone modifications in predicting prostate cancer relapse and has identified an epigenetic gene signature associated with prostate tumorigenesis.Impact: Our findings suggest that targeting the epigenetic enzymes specifically involved in a particular solid tumor may be a more effective approach. Moreover, testing for aberrant expression of epigenetic genes such as those identified in this study may be beneficial in predicting individual patient response to epigenetic therapies. Cancer Epidemiol Biomarkers Prev; 19(10); 2611–22. ©2010 AACR.

Supplementary Figure 4 from Global Levels of Specific Histone Modifications and an Epigenetic Gene Signature Predict Prostate Cancer Progression and Development

Supplemental Digital Content is available in the text., SP142 programmed cell death ligand 1 (PD-L1) status predicts response to atezolizumab in triple-negative breast carcinoma (TNBC). Prevalence of VENTANA PD-L1 (SP142) Assay positivity, concordance with the VENTANA PD-L1 (SP263) Assay and Dako PD-L1 IHC 22C3 pharmDx assay, and association with clinicopathologic features were assessed in 447 TNBCs. SP142 PD-L1 intraobserver and interobserver agreement was investigated in a subset of 60 TNBCs, with scores enriched around the 1% cutoff. The effect of a 1-hour training video on pretraining and posttraining scores was ascertained. At a 1% cutoff, 34.2% of tumors were SP142 PD-L1 positive. SP142 PD-L1 positivity was significantly associated with tumor-infiltrating lymphocytes (P

Premenopausal breast cancer is a complex disease with poorer outcomes compared to postmenopausal breast cancer. Despite the well-known impact of estrogen and progesterone on the biology of hormone responsive breast cancers, the effect of menstrual cycle phase on diagnosis, treatment, and survival outcomes has been under-studied. Evidence is now emerging that menstrual cycle-associated hormonal fluctuations affect expression of clinically-employed biomarkers and impact upon surgical and adjuvant treatment outcomes. As we engage in a new era of precision medicine, there is an ongoing effort to improve prediction of treatment response. For premenopausal breast cancer, this must include incorporation of menstrual cycle data into treatment recommendations. We advocate that menstrual cycle phase at the time of diagnosis and treatment be routinely recorded. This will enable establishment of robust datasets to support research on how best to incorporate menstrual cycle-associated changes in breast cancer biology into breast cancer care.

Introduction: The 21-gene Oncotype DX Recurrence Score is predictive of adjuvant chemotherapy benefit for women with early stage, hormone receptor (HR)-positive, HER2-negative breast cancer. In premenopausal women, fluctuations in estrogen and progesterone during the menstrual cycle can impact gene expression in these hormone-responsive cancers. However, the extent to which hormonal fluctuations affect Oncotype DX Recurrence Scores remains unclear. Aim: To determine the extent to which ovarian cycling affects the Oncotype DX 21-gene signature using paired premenopausal breast cancer samples and mouse models. Methods: To investigate menstrual variation in the Oncotype DX 21-gene signature within the same tumour, paired formalin-fixed paraffin-embedded, HR-positive invasive breast cancer samples were collected on different days of the menstrual cycle from women 50 years old (n=11). Samples were collected an average of 18 days apart and were neoadjuvant therapy naive. To determine the effect of progesterone on gene expression, HR-positive T47D breast cancer cells were xenografted into the mammary fat pad of BALB/c nude mice, treated with exogenous estrogen±progesterone (n=12, 11 respectively). Additionally, HR-positive mammary tumours were collected from naturally cycling Mmtv-Pymt mice at the estrus or diestrus phase of the ovarian cycle (n=25, 28 respectively). The 21-gene Oncotype DX signature was assessed through quantitative RT-PCR and an experimental recurrence score (RS) was calculated using the Oncotype DX Recurrence Score algorithm. Results: Increased discordance in RS was observed between paired samples collected from younger women (3.2±2.5; mean±stdev), compared to older women (2.0±1.7; p=0.04). In young women, discordance was primarily driven by variable expression of proliferative genes, compared to older women, where discordances were a result of variable expression of invasive genes. Variable concentrations of progesterone at the time of tissue collection may influence proliferative gene expression and contribute to discordant RS. In support of this, in HR-positive xenograft tumours, expression of proliferative genes Ki67 (p=0.03) and STK15 (p=0.04), and Ki67 protein expression (p=0.02), were reduced following progesterone treatment. Furthermore, in naturally cycling Mmtv-Pymt mice, mammary tumours collected at diestrus, analogous to the luteal phase of the menstrual cycle in humans, show significant differences in expression of 6 Oncotype DX signature genes (Ki67, Ccnb1, Esr1, Her2, Grb7, Bag1; p≤0.05) and a significant increase in RS (21.1±2.4; mean±SEM) compared to tumours dissected at estrus (15.5±1.9; p=0.03). Clustering analysis revealed a subgroup of Mmtv-Pymt mammary tumours collected at diestrus characterised by increased expression of proliferative (p Conclusion: Our results suggest that menstrual cycling affects the expression of genes included in the Oncotype DX 21-gene signature and influences experimental recurrence scores. Oncotype DX may be less effective for guiding chemotherapy treatment decisions for cycling premenopausal women compared to older postmenopausal women. Citation Format: Sarah M Bernhardt, Pallave Dasari, Danielle J Glynn, Lucy Woolford, Wendy Raymond, Lachlan M Moldenhauer, David Walsh, Amanda R Townsend, Timothy J Price, Wendy V Ingman. Menstrual cycling critically affects the Oncotype DX 21-gene signature: Implications for predictive biomarker assays in premenopausal women [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-12.

BACKGROUND A group of international experts in breast fine needle aspiration biopsy (FNAB) cytopathology, supported by the International Academy of Cytology (IAC), drafted a comprehensive system for reporting breast FNAB cytopathology in 2017-2018. The editorial team produced a survey to assess the international response to the proposed category structure, definitions, and management recommendations in this draft. METHODS A web-based survey of 186 questions was generated using the Qualtrics software package (Provo, Utah) supported by the Division of Information Technology at the University of Wisconsin-Madison. The survey was advertised widely-including through the IAC, American Society of Cytopathology, Japanese Society of Clinical Cytology, Papanicolaou Society of Cytopathology, and Australian Society of Cytology and to audiences at national and international meetings-and was available from April to June 2018. The data obtained from the 265 respondents was assessed by the editorial team. RESULTS The survey provided a snapshot of the current role and use of breast FNAB and the international variations. Demographic questions were followed by specific questions based on the draft category definitions and statements and focused on issues that had generated discussion among the authors, including the FNAB diagnosis of ductal carcinoma in situ. CONCLUSION The survey results strongly supported the development of the IAC Yokohama System and informed subsequent discussions among the authors regarding the final text.

Background The Oncotype DX 21-gene Recurrence Score is a genomic-based algorithm that guides adjuvant chemotherapy treatment decisions for women with early-stage, oestrogen receptor (ER)-positive breast cancer. However, there are age-related differences in chemotherapy benefit for women with intermediate Oncotype DX Recurrence Scores that are not well understood. Menstrual cycling in younger women is associated with hormonal fluctuations that might affect the expression of genomic predictive biomarkers and alter Recurrence Scores. Here, we use paired human breast cancer samples to demonstrate that the clinically employed Oncotype DX algorithm is critically affected by patient age. Methods RNA was extracted from 25 pairs of formalin-fixed paraffin-embedded, invasive ER-positive breast cancer samples that had been collected approximately 2 weeks apart. A 21-gene signature analogous to the Oncotype DX platform was assessed through quantitative real-time PCR, and experimental recurrence scores were calculated using the Oncotype DX algorithm. Results There was a significant inverse association between patient age and discordance in the recurrence score. For every 1-year decrease in age, discordance in recurrence scores between paired samples increased by 0.08 units (95% CI − 0.14, − 0.01; p = 0.017). Discordance in recurrence scores for women under the age of 50 was driven primarily by proliferation- and HER2-associated genes. Conclusion The Oncotype DX 21-gene Recurrence Score algorithm is critically affected by patient age. These findings emphasise the need for the consideration of patient age, particularly for women younger than 50, in the development and application of genomic-based algorithms for breast cancer care.

The International Academy of Cytology (IAC) gathered together a group of cytopathologists expert in breast cytology who, working with clinicians expert in breast diagnostics and management, have developed the IAC Yokohama System for Reporting Breast Fine-Needle Aspiration Biopsy (FNAB) Cytology. The project was initiated with the first cytopathology group meeting in Yokohama at the 2016 International Congress of Cytology. This IAC Yokohama System defines five categories for reporting breast cytology, each with a clear descriptive term for the category, a definition, a risk of malignancy (ROM) and a suggested management algorithm. The key diagnostic cytopathology features of each of the lesions within each category will be presented more fully in a subsequent atlas. The System emphasizes that the crucial requirements for diagnostic breast FNAB cytology are a high standard for the performance of the FNAB and for the making of direct smears, and well-trained experienced cytopathologists to interpret the material. The performance indicators of breast FNAB, including specificity and sensitivity, negative predictive value, positive predictive value and ROM stated in this article have been derived from the recent literature. The current practice of breast FNAB has evolved with the increasing use of ultrasound guidance and rapid on-site evaluation. Two recent publications have shown a range of ROM for the insufficient/inadequate category of 2.6–4.8%, benign 1.4–2.3%, atypical 13–15.7%, suspicious of malignancy 84.6–97.1%, and malignant 99.0–100%. The management algorithm in the System provides options because there are variations in the management of breast lesions using FNAB and core-needle biopsy in those countries utilizing the “triple test” of clinical, imaging, and FNAB assessment, and also variations in the availability of CNB and imaging in low- and middle-income countries. The System will stimulate further discussion and research, particularly in the cytological diagnostic features of specific lesions within each category and in management recommendations. This will lead to continuing improvements in the care of patients with breast lesions and possible modifications to the IAC Yokohama System.

This chapter defines the Suspicious of Malignancy category, discusses the reasons why suspicious of malignancy reports occur, and why a definitive diagnosis of malignancy cannot or should not be made in certain circumstances. The category allows for the maintenance of a high positive predictive value of a malignant diagnosis. Many of the reasons for an inability to make a definitive diagnosis of malignancy are similar to those seen in the atypical category and include the specimen quality, which is largely reliant on the skill of the FNAB operator, the experience of the cytopathologist interpreting the smears, and the inherent nature of the lesion. There are overlapping cytological criteria between some breast proliferative and in situ lesions, and between in situ lesions and some malignant tumours, and distinguishing carcinoma and lymphoma also may be difficult on occasion. The chapter details the cytological features of low-grade ductal carcinoma in situ (DCIS), and discusses this controversial area to provide an approach to the diagnosis and differential diagnoses for low-grade DCIS. The management of Suspicious of Malignancy FNAB cytology cases is presented and highlights the need for further investigation by core needle or excision biopsy in all cases. Sample reports are presented.

The International Academy of Cytology (IAC) Yokohama System for Reporting Breast Fine Needle Aspiration Biopsy (FNAB) Cytology reporting system is an initiative of the IAC that seeks to provide a comprehensive, standardized approach to breast FNAB reporting to facilitate reproducible cytopathology reporting and to establish best practice guidelines. The system is evidence-based, supports a structured reporting format and will aid in maximizing the usefulness of the FNAB technique and optimizing communication with the clinical team and the patient.

This chapter provides an outline of a pattern approach to the interpretation of breast fine needle aspiration biopsy direct smears. A number of different low and medium power patterns are described that suggest certain lesions. High power is then used to assess nuclear and other features, and low and high power key features are integrated into a diagnosis or a differential diagnosis. Details of the key diagnostic cytological features are provided in the various chapters of the atlas.

The present report reviews the current problems associated with the routine use of breast fine needle aspiration biopsy (FNAB) and discusses the potential impact that the new International Academy of Cytology (IAC) Yokohama Reporting System and the use of rapid on-site evaluation (ROSE) should have on reducing these problems to optimize breast care for patients. The recently reported IAC System aims to establish the best practice guidelines for breast FNAB, emphasizing the importance of the FNAB technique and the skillful preparation of direct smears. The IAC System proposes a standardized report and established clear terminology for defined reporting categories, each of which has a risk of malignancy and is linked to management options. The FNAB techniques that will optimize the biopsy specimen and reduce poor quality smears are reviewed and the benefits of ROSE are discussed. FNAB can diagnose accurately the vast majority of breast lesions, and ROSE has been recommended whenever possible to reduce the rate of insufficient/inadequate cases and increase the number of specific benign and malignant diagnoses. ROSE performed by a cytopathologist provides a provisional diagnosis, reducing patient anxiety and facilitating management through cost-effective immediate triage and patient selection for ancillary testing. Thus, patients can be selected for immediate core needle biopsy, as required.

Background Ductal carcinoma in situ (DCIS) is commonly managed with breast-conserving surgery (BCS) and adjuvant radiotherapy. Oncoplastic BCS allows wide excision without compromising the breast's natural shape. We use ‘level one’ techniques to excise a ‘sector’ of tissue (apex at the nipple) rather than traditional ‘lumpectomy’. There are concerns that some DCIS is over-treated and radiotherapy administered unnecessarily incurring the associated cost, time and morbidity without added benefit. This study aims to determine if pure DCIS can be managed safely without relying upon adjuvant therapies with an acceptable breast conservation rate. Method A retrospective clinical study of 96 patients who underwent BCS alone for pure DCIS between 1995 and 2009. Data were collected on patient's demographics, presentation, excision margins, re-excision and final margins, size, grade and nuclear architecture of DCIS, recurrent ipsilateral breast events, contralateral breast events, date of the last follow-up with mammography and patients’ assessment of cosmetic outcome (scale 1–5: very dissatisfied, somewhat dissatisfied, somewhat satisfied, satisfied, very satisfied). Results Twelve (12.5%) had an ipsilateral recurrence. Mean follow-up was 7.6 years (median 7.4); yearly recurrence rate was 1.67%. Of the 52 patients who gave an assessment of cosmetic outcome, 46 were satisfied. Conclusion Ipsilateral recurrence rates were favourable compared with previous trials where DCIS was treated with lumpectomy and radiotherapy. Oncoplastic techniques may be used to successfully treat pure DCIS with BCS alone without adjuvant therapies.

In August 2006, the Australian government announced a decision to subsidize trastuzumab therapy for early breast cancer, to commence 6 weeks later. It was mandated that HER2 gene amplification, determined by in situ hybridization (ISH), be shown, and that the sponsor company, Roche Products Pty Ltd, should fund this testing. This announcement potentially required provision of ISH testing for HER2 for every newly diagnosed breast cancer, where previously HER2 testing had been performed by immunohistochemistry with support from a single fluorescence ISH (FISH) reference laboratory for indeterminate cases. The Australian HER2 Testing Advisory Board, an independent expert group, responded to the challenge of rapidly providing accurate nationwide ISH testing. Bright-field ISH was selected as the testing platform and a decentralized testing model, with support from a central FISH laboratory, was adopted. An implementation plan was developed addressing standards for training, accreditation, and quality assurance. Within 6 weeks, 8 pathology laboratories were accredited for ISH testing and by September 2008, 2 years after the announcement, 22 ISH testing laboratories were taking part in the national program and almost 20,000 ISH tests had been performed. This article describes the design and rapid implementation of a nationwide program of bright-field ISH as the first-line testing platform for HER2 status in early breast cancer. We believe that this model for the coordinated and large-scale implementation of a new biomarker test has wide application, given that accurate assessment of a range of novel biomarkers is being used increasingly to determine eligibility for new targeted treatment modalities.

The tests that are currently available for the measurement of overexpression of the human epidermal growth factor-2 (HER2) in breast cancer have shown considerable problems in accuracy and interlaboratory reproducibility. Although these problems are partly alleviated by the use of validated, standardised 'kits', there may be considerable cost involved in their use. Prior to testing it may therefore be an advantage to be able to predict from basic pathology data whether a cancer is likely to overexpress HER2. In this study, we have correlated pathology features of cancers with the frequency of HER2 overexpression assessed by immunohistochemistry (IHC) using HercepTest (Dako). In addition, fluorescence in situ hybridisation (FISH) has been used to re-test the equivocal cancers and interobserver variation in assessing HER2 overexpression has been examined by a slide circulation scheme. Of the 1536 cancers, 1144 (74.5%) did not overexpress HER2. Unequivocal overexpression (3+ by IHC) was seen in 186 cancers (12%) and an equivocal result (2+ by IHC ) was seen in 206 cancers (13%). Of the 156 IHC 3+ cancers for which complete data was available, 149 (95.5%) were ductal NST and 152 (97%) were histological grade 2 or 3. Only 1 of 124 infiltrating lobular carcinomas (0.8%) showed HER2 overexpression. None of the 49 'special types' of carcinoma showed HER2 overexpression. Re-testing by FISH of a proportion of the IHC 2+ cancers showed that only 25 (23%) of those assessable exhibited HER2 gene amplification, but 46 of the 47 IHC 3+ cancers (98%) were confirmed as showing gene amplification. Circulating slides for the assessment of HER2 score showed a moderate level of agreement between pathologists (kappa 0.4). As a result of this study we would advocate consideration of a triage approach to HER2 testing. Infiltrating lobular and special types of carcinoma may not need to be routinely tested at presentation nor may grade 1 NST carcinomas in which only 1.4% have been shown to overexpress HER2. Testing of these carcinomas may be performed when HER2 status is required to assist in therapeutic or other clinical/prognostic decision-making. The highest yield of HER2 overexpressing carcinomas is seen in the grade 3 NST subgroup in which 24% are positive by IHC.

Image analysis was used to investigate the prognostic significance of immunostaining for oestrogen receptor (ER), p53 tumour-suppressor protein and tumour cell proliferation (MIB-1) in a random cohort of 200 primary breast cancer patients with between 4 and 7 years of clinical follow-up. Image measurements of the percentage of immunopositive cancer cell nuclei (% positive nuclear area) were recorded for the above tumour features for each patient. Assessment of relative risk using Cox's univariate analysis indicated that tumour size, number of cancer-involved nodes, MIB-1 and ER % positive nuclear area were significantly associated with breast cancer disease outcome, i.e., relapse-free survival and overall survival. In multivariate analysis, tumour size, number of involved nodes, ER and MIB-1 % positive nuclear area were retained as independent predictors of prognosis, depending on the image measurement cut-point used. A prognostic model, which can be used without reference to nodal involvement, was constructed for tumour size, ER cut-point of 30% positive nuclear area and MIB-1 cut-point of 10% positive nuclear area. Kaplan-Meier analysis of this image-based prognostic index identified 4 risk groups with predicted 5-year overall survival rates of 93%, 83%, 76.7% and 61.5%. We conclude that image measurements of ER and proliferative rate can be combined with tumour size to construct a prognostic index which reliably predicts disease outcome in primary breast cancer without knowledge of the nodal status of the patient. Int. J. Cancer (Pred. Oncol.) 84:203–208, 1999. © 1999 Wiley-Liss, Inc.

The clinical significance of vimentin intermediate filament (VIF) expression was studied in relation to other established prognostic parameters in primary breast cancer. Archival tumour samples embedded in paraffin were examined by immuno-histochemistry with monoclonal antibodies (MAbs) to VIF, p53 protein and cell proliferation marker MIB-I. The vimentin staining pattern was heterogeneous, but in vimentin-positive areas > 80% of the tumour cells were positive. There was no association between vimentin expression and tumour size or the number of axillary lymph nodes involved. Vimentin expression was significantly associated with high-grade tumours, absence of hormone receptors, increased p53 expression and high tumour proliferation fraction as estimated by MIB-I count. Despite these associations with several recognised features of tumour aggressiveness, vimentin expression was not associated with increase in risk of relapse or death from breast cancer.

Background: Epigenetic alterations are common in prostate cancer, yet how these modifications contribute to carcinogenesis is poorly understood. We investigated whether specific histone modifications are prognostic for prostate cancer relapse, and whether the expression of epigenetic genes is altered in prostate tumorigenesis. Methods: Global levels of histone H3 lysine-18 acetylation (H3K18Ac) and histone H3 lysine-4 dimethylation (H3K4diMe) were assessed immunohistochemically in a prostate cancer cohort of 279 cases. Epigenetic gene expression was investigated in silico by analysis of microarray data from 23 primary prostate cancers (8 with biochemical recurrence and 15 without) and 7 metastatic lesions. Results: H3K18Ac and H3K4diMe are independent predictors of relapse-free survival, with high global levels associated with a 1.71-fold (P < 0.0001) and 1.80-fold (P = 0.006) increased risk of tumor recurrence, respectively. High levels of both histone modifications were associated with a 3-fold increased risk of relapse (P < 0.0001). Epigenetic gene expression profiling identified a candidate gene signature (DNMT3A, MBD4, MLL2, MLL3, NSD1, and SRCAP), which significantly discriminated nonmalignant from prostate tumor tissue (P = 0.0063) in an independent cohort. Conclusions: This study has established the importance of histone modifications in predicting prostate cancer relapse and has identified an epigenetic gene signature associated with prostate tumorigenesis. Impact: Our findings suggest that targeting the epigenetic enzymes specifically involved in a particular solid tumor may be a more effective approach. Moreover, testing for aberrant expression of epigenetic genes such as those identified in this study may be beneficial in predicting individual patient response to epigenetic therapies. Cancer Epidemiol Biomarkers Prev; 19(10); 2611–22. ©2010 AACR.

Summary This paper describes immunostaining of consecutive sections from 15 cases of fibrocystic change of the breast (including 2 examples of intraductal papilloma), 4 ductal carcinomas-in-situ and 17 invasive carcinomas (4 tubular, 1 papillary, 2 lobular and 10 infiltrating ductal, NOS) with antisera to components of the basement membrane (BM), type IV collagen and laminin, and with the muscle antibodies actin and muscle-specific actin. A simple digestion technique was developed to improve the clarity of BM staining with these antibodies. The BM stains facilitated identification of small invasive foci through breaks in the BM in 2 of the cases which had been reported as pure intraductal carcinoma. Tubular carcinomas were surrounded by abnormal, fragmented, and focally discontinuous BM, a feature which could be used to distinguish this well-differentiated breast carcinoma sub-type from sclerosing adenosis, in which individual acini were invariably surrounded by a continuous BM. BM staining emphasized the fibrovascular core of intraductal papillomas, whereas the BM layer was absent in intraductal, cytologically malignant, papillary projections. Similarly, myoepithelial cells, stained with antisera to muscle actins, were identified in a continuous layer surrounding benign epithelial proliferations. These immuno-histochemical staining techniques may thus assist the diagnostic histopathologist in differentiating between benign epithelial proliferations of the breast and well-differentiated invasive breast carcinoma, and in identifying foci of microinvasive carcinoma.

Male breast cancer is a rare disease. Atypical ductal hyperplasia (ADH) in men is much rarer, and bilateral involvement is exceptional. A 20-year-old male presented with bilateral gynaecomastia who underwent subcutaneous mastectomies and histopathology revealed bilateral ADH. At 24 months, completion mastectomies were performed on both sides. The residual breast tissue revealed ADH similar to the initial specimen. ADH in women increases the risk of breast cancer by four to five times. To our knowledge, this is the first case report of bilateral ADH in a gynaecomastia specimen.

Purpose: The purpose is to determine whether the levels of expression of extracellular matrix components in peritumoral stroma are predictive of disease outcome for women with node-negative breast cancer. Experimental Design: Tumor tissue from 86 patients with node-negative breast cancer was examined by immunohistochemical staining for the expression of versican, chondroitin sulfate (CS), tenascin, and hyaluronan (HA). With the exception of HA, the expression of the extracellular matrix components was measured by video image analysis. Statistical correlation of the immunohistochemical data with clinicopathological characteristics and disease outcome was performed. Results: All of the extracellular matrix components were present in the peritumoral stroma of the entire study cohort. In contrast, immunoreactivity within the cancer cell was observed in 82% of tumors for HA, 12% for CS, and 4% for tenascin; no immunostaining of cancer cells for versican was observed for any of the tumors. Cox regression and Kaplan-Meier analyses indicated that elevated expression of stromal versican predicted increased risk and rate of relapse in this cohort. Elevated expression of tenascin was predictive of increased risk and rate of death only. Although neither CS nor HA were predictive of disease outcome in this cohort, tumor size was predictive of increased risk and rate of both relapse and survival. Conclusions: Elevated expression within peritumoral stromal matrix of versican and tenascin was predictive of relapse-free and overall survival, respectively, in women with node-negative breast cancer.

Determination of meaningful prognostic indicesremains a high priority for women diagnosed with node-negative primary breast cancer. Currently, 30% of these women relapse, and there is no reliable means of predicting this group of patients. This study investigates whether the level of expression of versican, an anticell adhesive proteoglycan, in the peritumoral stromal tissue of women with node-negative, primary breast cancer predicts relapse-free survival. This study also examines whether breast cancer cells regulate the secretion of versican by mammary fibroblasts.Immunoreactive versican was measured in breast cancer tissue sections of 58 node-negative patients by video image analysis. Primary isolates of mammary fibroblasts were cultured in medium conditioned by the breast cancer cell lines ZR-75-1, MCF-7, BT-20, and MB231. Changes in versican secretion were measured by immunoblotting and enhanced chemiluminescence.Cox analyses indicated that peritumoral versican level was the sole predictor of relapse-free survival. The relapse rate in patients with low versican levels was lower than in patients with high versican levels (Kaplan-Meier: 83% relapse free at 5 years for versican mean integrated absorbance14 versus 33% foror = 14, P = 0.0006). Accumulation of versican in medium of mammary fibroblasts was increased after culture in conditioned medium from breast cancer cell lines.Relapse in women with node-negative breast cancer is related to the level of versican deposited in peritumoral stroma by mammary fibroblasts. Versican secretion appears to be regulated by breast cancer cell mediators. Neoplastic remodeling of extracellular matrix through increased versican deposition may facilitate local invasion and metastasis.

Summary There is a continuing controversy regarding the value of estimating degree of intra-tumor vascularity to predict prognosis in breast cancer. In order to resolve this controversy, primary tumors from a cohort of 519 women with breast cancer were analysed to determine whether association exists between degree of vascutarity and prognosis. Tumor vascularity was estimated by immunohistochemistry using a monoclonal antibody to the antigen CD31. The tumor area showing the highest degree of vascularity was chosen to score the number of microvessels per unit area. Issues such as the reproducibility of the microvascularity score and its association with tumor parameters including size, histological grade and hormone receptor levels were investigated. Although previously agreed criteria were used, consensus between two pathologists' estimations of the degree of vascularity was only moderate. There was no statistically significant association between tumor vascularity score and other currently established parameters of prognosis. After a median follow up of 71 months for axiltary node negative patients, there was no association between tumor vascularity score and increased risk of relapse or death from breast cancer. In axillary node positive patients, tumor vascularity score was associated with increased risk of relapse and death from breast cancer. This association was not however independent of other established parameters of prognosis.

In Saccharomyces cerevisiae, the RAD50 gene is required for repair of X-ray and MMS-induced DNA damage during vegetative growth, and for synaptonemal complex formation and genetic recombination during meiosis. We show below that the RAD50 gene encodes major and minor transcripts of 4.2 and 4.6 kb in length which differ primarily at their 5′ ends. Steady-state levels of both RAD50 transcripts increase coordinately during meiosis, reaching maximal levels midway through meiotic prophase, about 3 or 4 h after transfer of cells to sporulation medium. The 5′ ends of the major RAD50 transcript in both meiotic and vegetative cells map to the same cluster of sites approximately 20 by upstream of the amino-terminal ATG of the RAD50 coding sequence. We conclude that the increased RAD50 transcript level observed during meiosis does not reflect utilization of a new promoter. In contrast, steady-state levels of Rad50 protein do not increase during meiosis. Thus, changes in RAD50 transcript levels are not neccessarily accompanied by commensurate changes in Rad50 protein levels. Possible explanations are considered.

This paper describes an improved immunohistochemical method for demonstrating oestrogen receptor (OR) protein in paraffin-embedded sections of tissue fixed for 1.5 h in formalin. Thirty-two cases of infiltrating ductal breast carcinoma were stained with a monoclonal anti-OR antibody (H222), using a standard streptavidin-biotin method, following pretreatment with pronase. OR counts in paraffin sections were compared with those of frozen sections and with cytosolic values determined by a dextran-coated charcoal method. Twenty-seven of the carcinomas were OR-positive in paraffin sections. There was concordance between the paraffin section and the frozen section-determined receptor status in 30 cases (94 per cent) and a strong correlation was observed (r = 0.76; P less than 0.0001). Similarly, OR counts in paraffin sections correlated with cytosolic OR values (r = 0.60; P less than 0.001) and there was concordance in 97 per cent of cases. The percentage of positively-stained tumour cells in paraffin sections ranged from 0 to 94 per cent with staining intensities comparable to those seen in frozen sections. Staining of paraffin sections identified more OR-positive tumours than either frozen section staining or cytosolic assay. This study validates immunohistochemical OR analysis in formalin-fixed, paraffin-embedded breast carcinomas using a commercial anti-OR antibody.

Immunohistochemical staining for estrogen receptor (ER) was performed on :12 infiltrating duct carcinomas of the breast using formalin-fixed, paraffin-embedded sections, monoclonal anti-ER (H222, Abbott Laboratories) and a strepavldin-biotin peroxidase method. Results of ER staining in paraffin sections were compared with those obtained on frozen sections and cytosolic ER levels obtained by a dextran-coated method on the same cases. Twenty-seven of the tumours were positive for ER in paraffin sections with a concordence of 94% between staining in paraffin sections and In frozen tissue (r = 0.76; p

Measurements of the growth fraction (GF) and oestrogen receptor (OR) status were performed by immunohistochemical staining, using the monoclonal antibodies Ki-67 and anti-OR, in consecutive cryostat sections of biopsies from 74 women with primary breast cancer, and 12 women with benign breast disease. A significant inverse correlation was identified between the GF and OR status (r = -0.452, P less than 0.0001), but a group of OR-positive tumours with high GFs was also observed. There was a strong positive correlation between immunocytochemically determined OR percentages and values obtained with a cytosolic OR biochemical assay for the 70 cases in which both techniques were performed (r = 0.801, P less than 0.0001). A significant relationship was also revealed between the histological grade of infiltrating ductal carcinomas and their associated GFs (r = 0.622, P less than 0.0001). No correlation was found between the GF and the number of positive axillary nodes. Our findings suggest that GF and OR status are useful independent prognostic parameters in breast cancer, but the former probably has an overriding influence. Both parameters may be useful guides for selection of appropriate endocrine and/or adjuvant chemotherapy.

We describe here a new variant of transposon Tn 10 especially adapted for transposon analysis of cloned yeast genes; it can equally well be used for analysis of prokaryotic genes. We have applied this element to analysis of the LEU2, RAD50, and CDC48 genes of Saccharomyces cerevisiae. This transposon, nicknamed mini-Tn 10-LUK, contains a lacZ gene without efficient transcription or translation start signals, an intact URA3 gene, and a kanR determinant. The lacZ gene can be activated by appropriate insertion of the element into an actively expressed gene. Other yeast genes can easily be substituted for URA3 in the available constructs. The mini-Tn 10-LUK system has several important advantages. (1) Transposition events occur in Escherichia coli at high frequency and into many different sites in yeast DNA. It is easy to obtain enough insertions to sensitively define the functional limits of a gene. (2) Transposon insertions can be obtained in a single step by standard transposon procedures and can be screened immediately for phenotype either in yeast or in E. coli. (3) The LacZ phenotypes of the insertion mutations provide a good circumstantial indication of the orientation of the target gene. (4) Under favorable circumstances, usable lacZ protein fusions are created. (5) Transposon insertion mutations obtained by this method directly facilitate additional genetic, functional, physical and DNA sequence analysis of the gene or region of interest.

Several synthetic random polymers of tyrosine containing glutamic acid, alanine, and lysine in various proportion served as substrates for tyrosine-specific protein kinases. The Km values for these substrates were much lower than for small polypeptides such as angiotensin. For the protein kinase coded by Fujinami virus, the best substrates (with the lowest Km) were polymers containing glutamic acid, alanine, and 8 to 10% tyrosine; for the insulin receptor protein kinase, the best substrate was a polymer containing 80% glutamic acid and 20% tyrosine. These polymers serve as inexpensive and tyrosine-specific substrates that can be used even with crude extracts and analyzed by the convenient filter paper assay. Several synthetic polymers with ordered sequences were found to be potent inhibitors of these tyrosine-specific protein kinases.

The estrogen receptor (ER) content of breast carcinoma is generally accepted as valuable in predicting clinical outcome and tumour response to hormonal manipulation. We applied a new immunocytochemical assay for estrogen receptors (Abbott ERICA Monoclonal) to 20 breast tumours, and examined the efficacy of 16 fixation procedures before immunoperoxidase staining of frozen sections and imprint preparations. Our findings indicate that the fixatives of choice are periodatelysine-paraformaldehyde at 22 degrees C, or 10% formalin followed by acetone at -10 degrees C. These fixation procedures are simpler, less time-consuming, and provide superior staining, tumour cytomorphology and higher ER values than the 3-reagent sequence recommended by Abbott Laboratories. There was a significant correlation between the ER scores in the frozen sections and the imprints. Positive ER cytosol results correlated with the staining index in the frozen sections, and the ER scores in the imprints. We conclude that imprints are suitable preparations for ER analysis by the immunoperoxidase technique, particularly for small tumour specimens.

This report describes a positive relationship between vimentin expression in infiltrating ductal breast carcinoma, and high tumour growth fraction. Vimentin expression is potentially a predictor of aggressive behaviour, and such carcinomas may benefit from early adjuvant therapy. Eighty-four malignant breast neoplasms were stained with monoclonal anti-vimentin and anti-cytokeratin antibodies. The tumour growth fractions were determined by immunostaining cryostat sections with the Ki-67 antibody. Seven (9.2 per cent) of 76 infiltrating ductal carcinomas co-expressed cytokeratin and vimentin intermediate filaments in more than 50 per cent of neoplastic cells. In each case, the corresponding Ki-67 count was much greater than 40 per cent, significantly higher than the mean growth fraction for all tumours examined (P less than 0.0001). Vimentin immunoreactivity was also positively related to the histological grade of the ductal carcinomas (P less than 0.002) and inversely related to tumour ER count (P less than 0.0002) and patient age (P less than 0.01). No relationship was observed between vimentin positivity and either the presence of axillary nodal metastases or primary tumour size.

An argyrophil stain for nucleolar organizer regions (NORs) has recently been applied to paraffin sections of human tissues. This report describes a positive relationship between the mean numbers of AgNOR sites per nucleus and tumor growth fraction, as determined by immunostaining with the monoclonal antibody Ki-67, in 83 malignant breast tumors (P less than .01). This relationship supports recent suggestions that the NOR count may reflect cell synthetic activity and hence, proliferation. AgNOR counts correlated inversely with immunocytochemically assessed estrogen receptor content (P less than .002), but there was no relationship between the AgNOR count and primary tumor size, histologic grade, axillary node status, or patient age. A significant difference (P less than .00001) was found between the AgNOR counts in 64 benign breast lesions (mean, 2.05) and 85 malignant breast neoplasms (mean, 5.46). The limitations of the silver staining technique and the problems of reproducibility in AgNOR counting are detailed.

Until recently, [3H]-thymidine incorporation, DNA analysis by flow cytometry, and cell doubling times have been the main methods of studying tumour cell kinetics. All these techniques are laborious, expensive, and difficult to perform in a routine diagnostic laboratory. This study examined fresh frozen sections from 31 prostatic biopsy specimens with the hybridoma antibody Ki-67, a marker of proliferating cells, using a modified avidin-biotin-peroxidase complex technique. The percentage of glandular cells decorated by this antibody, representing the growth fraction, was determined for both benign and malignant samples. Benign prostatic glands showed an average Ki-67 score of 4 per cent, significantly less than the 16.3 per cent mean growth fraction found in prostatic carcinomas. There was a significant correlation between the tumour growth fraction as assessed by Ki-67 staining, and the histological grade. A positive correlation was also found between the Ki-67 score and the intensity of staining, and a definite trend was noted between the Ki-67 score and the tumour clinical stage. Ki-67 promises to be a useful marker in determining the prognosis of prostatic cancer.

This paper identifies another neoplasm of epithelial origin which may express vimentin in addition to cytokeratins, thereby adding to the expanding list of tumours which demonstrate intermediate filaments (IFs) other than those of their reputed cell of origin. Twenty examples of benign breast disease and 66 carcinomas were examined for vimentin and cytokeratin IFs using an avidin–biotin–peroxidase complex technique. Co-expression of these IF proteins was found in 35 per cent of cases of benign breast tissue and in 60 per cent of the carcinomas. In 8 (16 per cent) of 50 cases of infiltrating ductal carcinoma, vimentin and cytokeratin immunostaining was observed in more than 60 per cent of the tumour cells. These carcinomas were predominantly of a high histological grade. In benign breast disease and well-differentiated carcinoma, vimentin was distributed in the basal and perinuclear regions of the cells, with sparing of the apical portions. In those cases in which large numbers of tumour cells expressed vimentin, cytoplasmic staining was diffuse, and often exhibited distinctive perinuclear and subplasmalemmal accentuation. We propose that a knowledge of the list of carcinomas which may co-express vimentin and cytokeratin IFs might be helpful in the assessment of undifferentiated tumours and metastatic deposits.