Jennet Harvey

Histological grading of breast carcinomas: A study of interobserver agreement

Interobserver variation in the histological grading of breast carcinoma was investigated using the hypothesis that optimal fixation, more precise grading guidelines, some experience, the use of training and test sets, and a comparison of results with an expert group might allow higher levels of agreement. For the training sets sections from 50 consecutive cases of breast carcinoma received at the Sir Charles Gairdner Hospital (SCGH) and fixed in both B5 and buffered formal saline (BFS) were graded by consensus of three pathologists at the SCGH and independently by consensus of two pathologists at the Nottingham City Hospital (NCH) using a modified Scarff-Bloom-Richardson histological grading system with guidelines as suggested by NCH pathologists. The section quality and degree of preservation of nuclear morphology were judged by NCH pathologists to be superior for B5-fixed material. Complete agreement in grade between SCGH and NCH results was achieved for 83.3% of B5-fixed cases and 73.5% of BFS-fixed cases (P = .05) with relative disagreement rates (RDRs) of 0.15 and 0.29 and kappa statistic values of 0.73 and 0.58, respectively. Approximately 80% complete agreement was achieved for tubule formation, nuclear score, and mitotic count, with RDRs ranging from 0.19 to 0.27 and kappa values from 0.46 to 0.69. There was a consistent bias in the SCGH results toward a higher tubule score in both B5- and BFS-fixed material because of a difference in interpretation of cribriform or complex gland patterns and a consistent bias in SCGH results toward a lower nuclear size/pleomorphism score for B5 and BFS material. For the test set sections from 50 further consecutive cases of breast cancer fixed in B5 were examined using similar criteria but taking into account the sources of error shown by the training set. Approximately 80% complete agreement was again achieved for grade components and grade (RDRs, 0.18 and 0.72). Systematic bias was reduced in the test set, but no other improvement was observed. Of the tumors designated as grade I by NCH, 87.5% were called grade I tumors by SCGH in the B5 training set, 84.6% in the B5 test set, and 66.6% in the BFS training set. The levels of agreement shown in both the training and test sets were satisfactory and represented a significant improvement over our previous study, suggesting that experience and precise grading guidelines are of value. The similar levels of agreement in training and test sets suggest that reasonable results can be achieved without direct training by expert groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Histological grading in breast cancer: interobserver agreement, and relation to other prognostic factors including ploidy

Summary Sections of neoplasms from 76 female patients with primary operable carcinoma of the breast were independently assessed by 2 pathologists for histological features and assigned a grade score. Relative disagreement rates between pathologists were estimated by use of a log‐linear model and found to be similar to those reported by many other groups, but higher than that reported by acknowledged experts. Tumor grade was related to nuclear DNA content as measured by static cytometry, inversely related to oestrogen receptor status and provided some additional prognostic information but, in this small series of patients, did not correlate with short‐term survival as closely as other prognostic indicators such as ploidy, tumor size or the extent of lymph node involvement. Patients with Grade III tumors had a particularly poor prognosis, however, there were few patients allotted to Grade III (poorly differentiated tumors), and survival differences between Grades I and II were small; in short‐term followup, used alone, grading separated out only a small proportion of patients into useful prognostic groups. This preliminary study emphasizes the need for a careful approach to the use of grading of breast carcinomas in the routine histopathology laboratory. Demonstration of higher levels of interobserver agreement, or concordance with experts in the field, will be necessary before our grading can be incorporated into a prognostic index useful for patient management.

Prognostic Significance of Microsatellite Instability and Ki-ras Mutation Type in Stage II Colorectal Cancer

Objectives: The survival of stage II colorectal cancer (CRC) patients is approximately 70% at 5 years. Identification of the patient subgroup at high risk for tumour recurrence and death would allow more informed use of chemotherapy for this stage of disease. Several clinical and pathological factors have been reported to associate with worse survival. In the present study we investigated the prognostic significance of two major genetic alterations in CRC: microsatellite instability (MSI+) and the type of Ki-ras mutation. Methods: PCR-based molecular techniques were used to screen for MSI+ and Ki-ras mutation in 396 stage II CRC patients with an average follow-up time of 75 months. Clinicopathological information was obtained by retrospective review of pathology reports. Results: Prominent vascular invasion was identified in 19% of cases and was found to be an independent prognostic factor for poor outcome (relative risk = 2.08, 95% confidence interval: 1.22–3.57, p = 0.008). The MSI+ phenotype was found in 23% of proximal tumours and Ki-ras mutations in 38% of the overall series. Neither MSI+ nor the type of Ki-ras mutation showed prognostic significance in this cohort of stage II CRC. Conclusions: MSI+ and Ki-ras mutation type are not useful markers for the identification of high-risk stage II CRC patients. Further prospective and/or cohort studies are required to determine whether these molecular changes have predictive value for survival benefit from 5-fluorouracil-based adjuvant chemotherapy.

Population-based detection of lynch syndrome in young colorectal cancer patients using microsatellite instability as the initial test

Approximately 1–2% of colorectal cancers (CRC) arise because of germline mutations in DNA mismatch repair genes, referred to as Lynch syndrome. These tumours show microsatellite instability (MSI) and loss of expression of mismatch repair proteins. Pre‐symptomatic identification of mutation carriers has been demonstrated to improve survival; however, there is concern that many are not being identified using current practices. We evaluated population‐based MSI screening of CRC in young patients as a means of ascertaining mutation carriers. CRC diagnosed in patients aged <60 years were identified from pathology records. No prior information was available on family history of cancer. PCR techniques were used to determine MSI in the BAT‐26 mononucleotide repeat and mutation in the BRAF oncogene. Loss of MLH1, MSH2, MSH6 and PMS2 protein expression was evaluated in MSI+ tumours by immunohistochemistry. MSI+ tumours were found in 105/1,344 (7.8%) patients, of which 7 were excluded as possible Lynch syndrome because of BRAF mutation. Of the 98 “red flag” cases that were followed up, 25 were already known as mutation carriers or members of mutation carrier families. Germline test results were obtained for 35 patients and revealed that 22 showed no apparent mutation, 11 showed likely pathogenic mutations and 2 had unclassified variants. The proportion of MSI+ cases in different age groups that were estimated to be mutation carriers was 89% (<30 years), 83% (30–39), 68% (40–49) and 17% (50–59). We recommend MSI as the initial test for population‐based screening of Lynch syndrome in younger CRC patients, regardless of family history.

Detection of p53 gene mutation by rapid PCR-SSCP and its association with poor survival in breast cancer.

We examined the association between mutation of the p53 gene and survival in a large cohort of breast cancer patients. Using a rapid, non‐isotopic single‐strand conformation polymorphism (SSCP) method we screened for mutations in exons 4–10 of the p53 gene in 375 primary breast cancers from patients with a median follow‐up of 57 months. Mutations were found in 19% of tumours. Statistically significant associations were found between p53 mutation and histological grade, hormone receptor status, ploidy and S‐phase fraction. No association was found between p53 mutation and axillary lymph node involvement, histological type, tumour size, vascular invasion or patient age. In univariate survival analysis, p53 mutation was strongly associated with poor prognosis. This was maintained in the lymph node‐negative and hormone receptor‐positive patient subgroups. In multivariate analysis, p53 mutation was associated with poor survival independent of lymph node status, estrogen receptor status and S‐phase fraction. Our results demonstrate the feasibility of using a rapid and simple polymerase chain reaction‐SSCP screening procedure to detect p53 gene mutation in breast cancer for the provision of prognostic information. Int. J. Cancer 74:642–647, 1997.© 1997 Wiley‐Liss, Inc.

Screening for defective DNA mismatch repair in stage II and III colorectal cancer patients

BACKGROUND & AIMS Colorectal cancers associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome usually present in younger patients, show loss of mismatch repair (MMR) gene expression, and exhibit microsatellite instability (MSI). About 12% of sporadic colorectal cancers also show MMR loss and MSI. The aims of this study were to evaluate MMR loss and MSI in relation to patient age, sex, tumor stage, and site in the large bowel. METHODS Tissue microarrays were created from 1020 stage II and III colorectal cancer cases and immunohistochemical staining performed to detect expression of the 2 major MMR proteins, hMLH1 and hMSH2. MSI was determined using the BAT-26 mononucleotide repeat. RESULTS Ten percent of tumors showed loss of hMLH1 expression and 1.2% showed loss of hMSH2 expression. hMLH1 loss was more frequent in women (P < .001), older patients (P = .004), earlier stage tumors (P = .0001), and proximal colon tumors ( P < .0001). In contrast, tumors showing hMSH2 loss were more frequent in younger (P < .001), male (P = .05) patients and were distributed evenly between the proximal colon and distal colon/rectum. Eleven percent of tumors were MSI+ and these showed similar age, sex, stage, and site characteristics as tumors with hMLH1 loss. Discordance between MMR loss and MSI+ was found in 24 of 983 (2.4%) tumors. Of the 231 patients aged <60 years at diagnosis, 12 (5.2%) showed loss of hMLH1 and 8 (3.5%) showed loss of hMSH2. CONCLUSIONS Routine immunohistochemical screening for MMR loss in younger colorectal cancer patients may provide a useful, first-step screening tool for the population-based detection of HNPCC.

A comparison of immunohistochemical staining for oestrogen receptor, progesterone receptor and HER-2 in breast core biopsies and subsequent excisions

Aims: To compare immunohistochemical staining for oestrogen receptor, progesterone receptor and HER‐2 between core biopsy and matched subsequent excisional specimens. Methods: One hundred consecutive core biopsy cases and subsequent excisional specimens were retrieved and immunohistochemical staining performed. Proportion and intensity of staining for hormone receptors and HercepTest score were recorded for each case in a blinded fashion by the authors. Results: Overall hormone receptor status was concordant between cores and excisions in 96.9% of cases. ER status was concordant between the core and excision in 95.8% of cases. The intensity of staining for ER was similar in both core and excision specimens. PR status was concordant in cores and excisions in 90.3% of cases. There was weaker PR staining in the excisional specimens when compared with the cores. HER‐2 status was concordant in cores and excisions in 86.6% of cases. Conclusions: Hormone receptor staining produced similar results on core and excisional specimens, although a small number of additional hormone receptor positive cases could be detected by performing staining on a previously received core in the case of a negative result on the excisional specimen. HER‐2 staining is less reproducible between cores and excisions, but the clinical significance of this observation remains to be tested.

What's New in Breast Cancer Molecular Perspectives of Cancer Development and the Role of the Oncogene c-erbB-2 in Prognosis and Disease

The oncogene c-erbB-2 is frequently amplified in human breast carcinoma. The c-erbB-2 gene is present as a single copy in normal cells, and has been mapped to chromosome 17 in the region 17q 12-21.32. c-erbB-2 encodes a transmembrane glycoprotein known as p185. The intracellular component of p185 has tyrosine kinase activity; the extracellular domain has a structure resembling a growth factor receptor. c-erbB-2 amplification, p185 overexpression and levels of transcribed c-erbB-2 specific messenger RNA have been studied in a large number of breast carcinomas using a variety of techniques. In general, overexpression of p185 oncoprotein reflects various levels of DNA amplification, though in some cases amplification can be detected in the absence of overexpression of p185 and similarly overexpression of p185 can be present without detectable levels of c-erbB-2 amplification. This findings suggests that multiple mechanisms may be responsible for overexpression. c-erbB-2 amplification and/or overexpression occurs in almost all cases of high grade duct carcinoma in-situ, but has been reported in only 10%-40% of infiltrating duct carcinoma. c-erbB-2 amplification or overexpression occurs rarely in invasive lobular carcinoma, and has not been detected in ductal or lobular epithelial hyperplasia, or in atypical ductal or atypical lobular hyperplasia. It is generally believed that c-erbB-2 amplification/overexpression is an important independent prognostic indicator in breast carcinoma, identifying a subset of patients with poor prognosis tumours, particularly if axillary node metasases are present. However, many unanswered questions remain regarding c-erbB-2 and its role in breast cancer development and progression. The causes of c-erbB-2 amplification are unknown. There is no evidence of mutations in the human gene which might cause amplification or overexpression. The significance of the differences in levels of c-erbB-2 amplification/overexpression in in-situ duct carcinoma and associated invasive duct carcinoma has not been established. Amplification or overexpression have not been reported in atypical duct hyperplasia, a proposed precursor of duct carcinoma in-situ, yet overexpression occurs almost always in high grade duct carcinoma in-situ. c-erbB-2 may play a critical role in the development of a clonal in-situ, proliferation of high histological grade, yet does not obviously influence the acquisition of an invasive phenotype. We would postulated that this instability in amplification/overexpression is of biological significance, and if better understood may aid in the study of progression of human breast carcinoma.

C-ERBB-2 AMPLIFICATION AND OVEREXPRESSION IN BREAST CANCER : EVALUATION AND COMPARISON OF SOUTHERN BLOT, SLOT BLOT, ELISA AND IMMUNOHISTOCHEMISTRY

&NA; The oncogene c‐erbB‐2 has been shown to be amplified in 17‐30% of breast cancers, with similar levels of overexpression of the oncogene product p185, a transmembrane growth factor receptor glycoprotein. Amplification of c‐erbB‐2 is now generally considered to be a significant prognostic indicator in patients with breast cancer. A series of 74 consecutive breast carcinomas were analysed for c‐erbB‐2 amplification and p185 overexpression. The procedures of Southern blotting and slot blot were used for the analysis of oncogene amplification, while immunoperoxidase (IPOX) staining and enzyme‐linked immunosorbent assay (ELISA) were used for the analysis of p185 overexpression. Detection of c‐erbB‐2 oncogene amplification by both the conventional Southern blotting technique and by the slot blot technique showed complete accord, with the amplified c‐erbB‐2 oncogene being detected in 14 of the 74 patients (18.9%). The c‐erbB‐2 oncoprotein, as measured by IPOX and ELISA, was found to be overexpressed in 21% and 19% of patients, respectively. Comparison was made between the results attained by all four methods, and further comparison of the techniques was made from the point of view of ease of use, expense and ease of introduction into routine diagnostic laboratories. Immunocytochemistry in combination with slot blotting procedures were considered to be the most cost effective methods for evaluation of overexpression and amplification in routine pathology laboratories.

Comparison of vascularity and angiogenesis in primary invasive mammary carcinomas and in their respective axillary lymph node metastases.

It is well established that the ability of a neoplasm to induce a blood supply from a pre-existing circulation (angiogenesis) is a major factor in tumour growth, invasion and metastasis. However, the angiogenic potential of metastases and their subsequent growth have not been extensively studied. The question arises: can metastatic clones induce the same level of angiogenesis as in the primary neoplasm they emanated from? In this study it is hypothesised that in the same patient the level of vascularity and angiogenesis is the same in both the primary invasive ductal carcinoma and in the axillary lymph node metastasis at the time of surgery, according to Kerbels theory of clonal-dominance. To directly address the hypothesis, morphological measures of the established blood/lymphatic circulation (vascularity) as well as estimates of angiogenesis (endothelial cell proliferation) were measured in primary tumours and directly compared to the same parameters in the corresponding lymph node metastasis in a case by case basis (n=17). The results demonstrate varying associations between the level of vascularity and angiogenesis between matched individual tumours and their metastatic lymph nodal deposits. It is possible that either variations in the angiogenic characteristics of the metastasising clone or local or systemic promoters or inhibitors of angiogenesis influence tumour angiogenesis at the different sites.