Multifactorial analysis of differences between sporadic breast cancers and cancers involving BRCA1 and BRCA2 mutations.

BACKGROUND: We have previously demonstrated that breast cancers associated with inherited BRCA1 and BRCA2 gene mutations differ from each other in their histopathologic appearances and that each of these types differs from breast cancers in patients unselected for family history (i.e., sporadic cancers). We have now conducted a more detailed examination of cytologic and architectural features of these tumors. METHODS: Specimens of tumor tissue (5-microm-thick sections) were examined independently by two pathologists, who were unaware of the case or control subject status, for the presence of cell mitosis, lymphocytic infiltration, continuous pushing margins, and solid sheets of cancer cells; cell nuclei, cell nucleoli, cell necrosis, and cell borders were also evaluated. The resulting data were combined with previously available information on tumor type and tumor grade and further evaluated by multifactorial analysis. All statistical tests are two-sided. RESULTS: Cancers associated with BRCA1 mutations exhibited higher mitotic counts (P = .001), a greater proportion of the tumor with a continuous pushing margin (P<.0001), and more lymphocytic infiltration (P = .002) than sporadic (i.e., control) cancers. Cancers associated with BRCA2 mutations exhibited a higher score for tubule formation (fewer tubules) (P = .0002), a higher proportion of the tumor perimeter with a continuous pushing margin (P<.0001), and a lower mitotic count (P = .003) than control cancers. CONCLUSIONS: Our study has identified key features of the histologic phenotypes of breast cancers in carriers of mutant BRCA1 and BRCA2 genes. This information may improve the classification of breast cancers in individuals with a family history of the disease and may ultimately aid in the clinical management of patients.

Potential pathological application of immunocytochemical methods to the detection of micrometastases.

Immunocytochemical methods can be used to demonstrate tumor products and antigens at the cellular level. This approach facilitates the classification of tumors and the detection of small metastatic tumor foci in biopsy material from various sites including the bone marrow.

The pathology of familial breast cancer: histological features of cancers in families not attributable to mutations in BRCA1 or BRCA2.

Breast cancers arising in carriers of mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, differ histologically from each other and from breast cancers unselected for a family history. However, a substantial proportion of families with multiple cases of breast cancer is not attributable to these two genes (non-BRCA1/2 families). We have now characterized the pathology of 82 breast cancers from non-BRCA1/2 families. Breast cancers in non-BRCA1/2 families were of lower grade (P = 0.0018), showed fewer mitoses (P < 0.0001), less nuclear pleomorphism (P = 0.0014), less lymphocytic infiltrate (P < 0.0001), a lesser extent of the tumor with a continuous pushing margin (P = 0.004), a lesser extent of the tumor composed of solid sheets of cells (P = 0.0047), less necrosis (P = 0.002), and wereparison with BRCA2 tumors, non-BRCA1/2 tumors were lower grade (P = 0.017) and exhibited less pleomorphism (P = 0.01) and more tubule formation (P = 0.05). In comparison with control breast cancers unselected for a family history of the disease, non-BRCA1/2 tumors were of significantly lower grade (P = 0.001), showed less pleomorphism (P = 0.0002), and had a lower mitotic count (P = 0.003). The results indicate that non-BRCA1/2 breast cancers differ histologically from both BRCA1 and BRCA2 breast cancers and are overall of lower grade. They also suggest that non-BRCA1/2 breast cancers differ from nonfamilial breast cancers, but these differences may be attributable to various types of bias.

Micrometastases in breast cancer: long-term follow-up of the first patient cohort.

"Micrometastases" can be identified in the bone marrow of patients with apparently localised breast cancer using an immunocytochemical stain for epithelial membrane antigen (EMA). Of 39 women who had marrow samples examined at the time of initial presentation (37), or with locally recurrent disease (2), 13 (33%) had samples which contained small numbers of EMA positive cells. 10 out of 23 (44%) lymph-node positive patients were marrow positive, compared to 1 out of 14 (7%) lymph node negative cases (P = 0.03). Long-term follow-up (median 9.5 years) has shown that 11 out of 13 (85%) patients with micrometastases have developed metastatic disease compared to 8 out of 26 (31%) with negative bone marrow aspirates (P less than 0.05). The small number of EMA positive cells detected in bone marrow samples probably reflects the high metastatic potential of primary or recurrent cancers rather than established microscopic deposits; it is not yet clear whether the finding of such micrometastases will act as an independent variable compared to established prognostic factors.

Angiogenesis and invasive recurrence in ductal carcinoma in situ of the breast.

The development of an invasive recurrence following treatment for ductal carcinoma in situ (DCIS) converts a non-fatal disease to one associated with mortality. To date, no pathological or molecular features have been found to predict for the type of recurrence. Previous studies have suggested that in DCIS angiogenesis may be an important factor in determining the transformation from in situ to invasive carcinoma. We looked at 355 cases of DCIS and found that 32 had subsequently developed recurrent disease. In these 32 cases and in matched controls, periductal vascular density was determined using morphometry and anti-endothelial antibodies, von Willebrand factor (vWF) and CD34. Vascular density was related to the risk of both invasive and in situ recurrence. Normal lobules at least 2 mm away were used as controls. Differences in the phenotype of individual blood vessels was detected by performing dual staining immunofluorescence on selected cases. The microvessel density (MVD), as detected with the CD34 antibody, was higher around foci of DCIS than around normal breast lobules (P=0.001). Furthermore, it was significantly higher in cases of DCIS that recurred (P<0.0001). The findings with the vWF antibody were less clear cut and suggested a trend in decreasing MVD with increasingly aggressive disease. Dual immunofluorescence staining shows that the increase in MVD seen around DCIS is due to an increase in CD34+/vWF-blood vessels. An increase in CD34+/vWF-of blood vessels may be able to predict cases of DCIS that are at a high risk of developing a recurrence.

Consistency of histopathological reporting of breast lesions detected by screening: findings of the U.K. National External Quality Assessment (EQA) Scheme. U. K. National Coordinating Group for Breast Screening Pathology.

The aim of the scheme was to determine consistency of histopathological reporting in the United Kingdom National Breast Screening Programme. This external quality assessment scheme involved 51 sets of 12 slides which were circulated to 186-251 pathologists at intervals of 6 months for 3 years. Participants recorded their diagnoses on standard reporting forms, which were submitted to the U.K. National Cancer Screening Evaluation Unit for analysis. A high level of consistency was achieved in diagnosing major categories of breast disease including invasive carcinoma and the important borderline lesions, radial scar and ductal carcinoma in situ (DCIS), the latter exceeding a national target set prior to the onset of the scheme. Atypical hyperplasia (AH) was reported with much less consistency although, where it was the majority opinion, over 86% of diagnoses were of benign disorders and only 14% were of DCIS. Inconsistency was encountered in subtyping and measuring DCIS, the former apparently due to current uncertainties about classification and the latter to poor circumscription, variation in size in different sections and merging with zones of AH. Reporting prognostic features of invasive carcinomas was variable. Measurement of size was achieved with adequate consistency except in a small number of very poorly circumscribed tumours. Grading and subtyping were inconsistent although the latter was not specifically tested and will be the subject of future study. Members of the National Coordinating Group achieved greater uniformity than the remainder of the participants in all diagnostic categories, but both groups experienced similar types of problem. Our findings suggest that participation in the scheme improves diagnostic consistency. In conclusion, consistency in diagnosing invasive carcinoma and radial scar is excellent, and good in DCIS, but improvements are desirable in diagnosing atypical hyperplasia, classifying DCIS and reporting certain prognostic features of invasive tumours. Such improvements will require further research, the development of improved diagnostic criteria and the dissemination of clearer guidelines.

Causes of inconsistency in diagnosing and classifying intraductal proliferations of the breast. European Commission Working Group on Breast Screening Pathology.

It is now widely recognised that classifying ductal carcinoma in situ (DCIS) of the breast and diagnosing atypical ductal hyperplasia are associated with significant interobserver variation. Two possible reasons for this inconsistency are differences in the interpretation of specified histological features and field selection where morphology is heterogeneous. In order to investigate the relative contribution of these two factors to inconsistent interpretation of intraductal proliferations, histological sections of 32 lesions were sent to 23 European pathologists followed 3 years later by images of small parts of these sections. Kappa statistics for diagnosing hyperplasia of usual type, atypical ductal hyperplasia and ductal carcinoma in situ were 0.54, 0.35 and 0.78 for sections and 0.47, 0.29 and 0.78 for images, respectively, showing that most of the inconsistency is due to differences in morphological interpretation. Improvements can thus be expected only if diagnostic criteria or methodology are changed. In contrast, kappa for classifying DCIS by growth pattern was very low at 0.23 for sections and better at 0.47 for images, reflecting the widely recognised variation in the growth pattern of DCIS. Higher kappa statistics were obtained when any mention of an individual growth pattern was included in that category, thus allowing multiple categories per case; but kappa was still higher for images than sections. Classifying DCIS by nuclear grade gave kappa values of 0.36 for sections and 0.49 for images, indicating that intralesional heterogeneity has hitherto been underestimated as a cause of inconsistency in classifying DCIS by this method. More rigorous assessment of the proportions of the different nuclear grades present could lead to an improvement in consistency.

Histopathologic types of benign breast lesions and the risk of breast cancer: case-control study.

The hypothetical multistep model of carcinogenesis indicates that breast cancer develops via a series of intermediate hyperplastic lesions through in situ to invasive carcinoma. To identify the risk inherent within the different morphologic lesions, we have analyzed the data from 674 benign biopsy specimens comprising 120 cases who subsequently developed breast cancer and 382 controls (matched for age and date of biopsy) spanning a period up to 20 years of follow-up (mean 66.95 months). In this series we have confirmed an increased risk associated with certain types of benign breast lesions. Atypical lobular hyperplasia was the most significant risk factor for breast cancer with more unfavorable outcome in patients <50 years of age (p = 0.003) and a relative risk (RR) of 4.55 (confidence interval [CI] 1.77-11.69). Hyperplasia of usual type showed an RR of 1.53 (CL 1.10-2.13) with a statistically worse probability of survival (cancer-free time) for patients >50 years. For atypical ductal hyperplasia the RR was 2.03 (CI 0.80-1.39). Blunt duct adenosis was significantly more common in cases progressing to breast cancer compared with controls, showing an RR of 2.08 (CI 1.12-2.85). We describe in detail the criteria of morphologic changes observed in blunt duct adenosis and define, for the first time, the level of risk associated with each of its six subtypes. Improved knowledge of breast carcinogenesis will provide insight for defining high-risk groups thus resulting in improved screening and management regimens.

Enlargement of the human spleen in graft-versus-host disease.

The spleens of 49 patients who had undergone allogeneic bone marrow transplantation for leukemia were compared at autopsy to determine the pathological changes associated with graft-versus-host disease (GVHD). The only significant finding was an increase in weight of about 1.7 times that of spleens from patients without GVHD. This was not explained by differences in the patients' sex, length of survival after transplantation, presence of infection, or liver pathology. On histological examination, there was no detectable increase in congestion, siderosis, or numbers of lymphocytes, macrophages, antigen-presenting cells, blast cells, pyknotic cells, plasma cells, or hemopoietic cells to explain the increase in spleen weight. On the contrary, there was actually a reduction in CD8+ T lymphocytes. No proliferative phase of GVHD could be identified, possibly due to a lack of specimens examined less than 8 days after transplantation and to prophylactic measures undertaken to minimize GVHD. The pathogenesis of splenomegaly in human GVHD is unclear.

ICAM-1 expression on epidermal keratinocytes in cutaneous graft-versus-host disease.

An immunohistological study of epidermal keratinocytes for the intercellular adhesion molecule, ICAM-1 (CD54), was undertaken on skin biopsies from allogeneic bone marrow transplant recipients. In control biopsies from normal donors and patients prior to transplantation, staining was weak and confined to relatively few cells. After transplantation there was a significant increase in both the number of positive cells and their staining intensity in biopsies showing histological evidence of GVHD but not in those exhibiting normal appearances or epidermal abnormalities that could be attributed to cytotoxic drugs or irradiation. There was a strong positive correlation between ICAM-1 and HLA-DR expression by keratinocytes. All cases exhibiting increased ICAM-1 also exhibited an increase in HLA-DR antigens. The converse was not true, however, as 6 biopsies exhibited HLA-DR positivity without detectable increases in ICAM-1. Both ICAM-1 and HLA-DR antigen synthesis may be stimulated by local cytokine release following interactions between donor and recipient cells in the early posttransplant period. Our present findings suggest that immunostaining for ICAM-1 has little value in the early diagnosis of cutaneous GVHD but further, more detailed prospective studies would be of value.

HLA-DR expression in epidermal keratinocytes after allogeneic bone marrow transplantation. Relationship to histology, rash, marrow purging, and systemic graft-versus-host disease.

HLA-DR expression on epidermal keratinocytes was studied in leukemic recipients of allogeneic marrow in order to clarify its relationship to GVHD, investigate its diagnostic value, and gain insight into the pathogenetic mechanisms. Using frozen-section immunohistological techniques, positive keratinocytes were encountered in a small minority of normal donors and in a few recipients prior to transplantation. In patients receiving marrow unpurged of T lymphocytes, keratinocyte HLA-DR staining was found in the majority of patients with, and in about one third of those without, histological evidence of GVHD. Positivity was strongly related to the presence of a rash and was more likely to be found if the interval between the onset of the rash and biopsy exceeded 24 hr. There was a strong association between the presence of positive cells and the subsequent development of systemic GVHD, indicating that staining for HLA-DR on keratinocytes may be a useful adjunct to conventional morphological analysis in the interpretation of post-transplant skin biopsies. Positivity was not observed in patients who received marrow depleted of T lymphocytes, indicating a crucial role for these cells in stimulating keratinocyte HLA-DR expression. Sequential studies, however, showed that keratinocyte positivity preceded lymphocytic infiltration of the epidermis.

Chimerism in skin of bone marrow transplant recipients.

Skin biopsies from 3 patients receiving one-haplotype-matched bone marrow grafts have provided a unique opportunity to demonstrate the presence of donor cells in situ using immunohistological techniques and a monoclonal antibody directed against an epitope common to HLA-A2 and HLA-A28 antigens. The infiltrating cells were also analyzed in consecutive tissue sections with a panel of monoclonal antibodies to human leukocyte antigens, T cells, and epidermal Langerhans cells. Most of the infiltrating cells were shown to be T lymphocytes of donor origin, regardless of whether the histological changes were consistant with graft-versus-host disease (GVHD) or were eczematous. Donor T cells were also shown to colonize histologically normal skin soon after transplantation. Epidermal keratinocytes, dermal endothelium, and adnexal structures did not express the donor HLA type (i.e., were host derived) but the origin of the epidermal Langerhans cells could not clearly be established. The data show that donor cells preferentially migrate to certain sites in skin after transplantation and are not always associated with GVHD.

Prediction of local recurrence of ductal carcinoma in situ of the breast using five histological classifications: a comparative study with long follow-up.

The increased detection of ductal carcinoma in situ (DCIS) by mammographic screening and the more widespread use of breast-conserving surgery have led to a search for histological features associated with the risk of recurrence. In a case control study of 141 patients with long follow-up, we compared the ability of five morphological classifications to predict recurrence after local excision. A significant correlation was not found between recurrence and growth pattern when a traditional classification based on architecture was used nor with necrosis when a scheme based principally on this feature was employed. A correlation was, however, found between recurrence and "differentiation" as defined by nuclear features and cell polarization in a classification recently formulated by the European Pathologists Working Group (EPWG), but this failed to reach statistical significance at the 5% level. A stronger and statistically significant correlation was found between nuclear grade as defined by the EPWG and recurrence when cell polarization was disregarded, using the classification currently employed by the UK National Health Service and European Commission-funded Breast Screening Programmes. This was attributable to a small number of recurring cases being downgraded as a consequence of exhibiting polarized cells. A significant correlation between histology and recurrence was also observed using the Van Nuys classification, which is based on nuclear grade and necrosis. Whether the tumor recurred as in situ or invasive carcinoma was unrelated to histological classification, as was the time course over which it occurred. These findings strongly support the use of nuclear grade to identify cases of DCIS at high risk of recurrence after local excision, but further work is necessary to determine whether nuclear grade or necrosis is more appropriate to subdivide the non-high-grade cases.

neu overexpression correlates with extent of disease in large cell ductal carcinoma in situ of the breast.

In a retrospective study of ductal carcinoma in situ (DCIS) of the breast, the expression of the neu oncogene was determined immunohistochemically in 76 women treated by local excision or mastectomy. The histopathological features, including the extent of the lesion, histological subtype, cell type, and number of mitoses, were related to neu overexpression. Immunopositivity was found only in DCIS of large cell type, where it correlated with extent of disease but not with mitotic rate. Our findings, together with previous experimental evidence, suggest that this relationship is a consequence of the effect of the neu protein on cell motility.

Consistency achieved by 23 European pathologists in categorizing ductal carcinoma in situ of the breast using five classifications. European Commission Working Group on Breast Screening Pathology.

The increased detection of ductal carcinoma in situ (DCIS) by mammographic screening, the greater use of breast-conserving surgery, and the recognition that certain histological subtypes are associated with a greater risk of local recurrence has led to the formulation of several new classifications of DCIS in recent years. There are, however, no data concerning the degree of consistency with which these schemes can be applied by reasonable numbers of pathologists. Thirty-three cases of DCIS were thus examined by a working group of 23 European pathologists who categorized them using five recently published classifications: (1) that of the European Pathologists' Working Group based on differentiation (a combination of nuclear grade and cell polarization) with categories of poorly, intermediately, and well differentiated; (2) one based entirely on nuclear grade with categories of high, intermediate, and low, currently in use in the UK national and EC-funded breast screening programs; (3) the same classification in which only two categories, high nuclear grade and other, were used; (4) the Van Nuys system in which lesions are divided into high grade, non-high grade with necrosis and non-high grade without necrosis; and (5) a two-category classification based entirely on the presence or absence of comedo necrosis. Of the three systems with three categories, Van Nuys gave the highest overall kappa statistic of 0.42. Others gave similar values of 0.37 and 0.35 showing that assessing cell polarization in addition to nuclear grade neither improves nor worsens consistency. In all three systems, the middle category was associated with the lowest value for kappa. Of the two systems with two categories, that based on nuclear grade gave the highest overall kappa of 0.46 and that based on comedo necrosis the lowest of 0.34. The most robust histological features were thus high- and low-grade nuclei and necrosis as long as the latter did not involve the recognition of a comedo growth pattern. These values probably represent the maximum achievable, at least by reasonable numbers of pathologists in everyday practice. They are better than those previously reported for classification based entirely on architecture, but further improvement is needed.

An immunohistological study of gamma/delta lymphocytes in human cutaneous graft-versus-host disease.

The proportion of T lymphocytes bearing the gamma/delta and alpha/beta T cell antigen receptors was determined in histological sections of skin from normal subjects and patients suffering from acute graft-versus-host disease (GVHD) using antibodies to the delta and beta chains. The proportion of gamma/delta cells in GVHD was very low (4.3% of CD3+ cells) but significantly higher than in skin from normal donors (0.8%) and marrow recipients without GVHD (0%). The significance of this is not clear and we were unable to determine whether it represented selective localization of these cells to the skin or simply reflected peripheral blood values in the early post-transplant period in some patients with GVHD. Localization of gamma/delta cells to the epidermis was not seen; all were identified in the dermis particularly in the perivascular zone. Although it is not possible to exclude a role for gamma/delta cells in initiating GVHD, the present study suggests that they do not play an important role in producing the epidermal damage that characterizes this condition.

Adhesion molecule expression in human hepatic graft-versus-host disease.

An immunohistological study of the distribution of three cellular adhesion molecules, ICAM-1, VCAM-1 and ELAM-1, was undertaken on normal liver and liver biopsies taken from allogeneic bone marrow transplant (BMT) recipients. In normal controls, ICAM-1 was seen on vascular endothelium and sinusoidal lining cells, and VCAM-1 on Kuppfer cells and dendritic macrophages in portal tracts. ELAM-1 staining was virtually absent. Biopsies from BMT recipients with histological evidence of hepatic graft-versus-host disease (GVHD) showed ICAM-1 expression on damaged bile duct epithelium in only one of five cases, in contrast to four of five showing epithelial HLA-DR expression. Increased numbers of VCAM-1 positive portal tract macrophages were seen in GVHD and also in non-GVHD pathology. No increase in vascular endothelial expression of VCAM-1 or ELAM-1 was seen. These findings contrast with previous studies on other target sites for GVHD, namely skin and gastrointestinal tract, where the expression of all three molecules is increased on various cells. Although the lack of adhesion molecule expression in the liver in GVHD in this study may be related to the timing of biopsies or immunosuppressive therapy, it is likely to represent to some extent variation in cell and molecular changes occurring in the different tissues affected by GVHD.

Cyclosporine-induced graft-versus-host disease following autologous bone marrow transplantation in acute myeloid leukaemia.

Cyclosporine was used to induce graft-versus-host disease (GVHD) in patients with acute myeloid leukaemia (AML) receiving autologous bone marrow transplantation (ABMT). Nine consecutive patients with AML in remission were conditioned with either busulphan and cyclophosphamide or melphalan and total body irradiation (TBI) followed by ABMT. Cyclosporine, 1 mg/kg daily from days 1-28 was administered intravenously in four patients and orally in five. Acute GVHD of the skin, confirmed by histological and immunological criteria occurred in three patients, 4-28 days after ABMT and lasted 8-18 days. Incidence and severity of GVHD were independent of cyclosporine levels. Three patients subsequently relapsed, of whom two had had evidence of GVHD. All of these patients were in second remission at time of graft, and therefore poor risk. The potential of cyclosporine to induce GVHD in the AML autograft setting is demonstrated, although the significance of this observation in terms of an antileukaemia effect needs clarification.

A prospective study of cellular and immunologic changes in skin of allogeneic bone marrow recipients. Relationship to clinical and histologic features of acute graft-versus-host disease.

The inflammation-associated molecules intercellular adhesion molecule (ICAM)-1, endothelial lymphocyte adhesion molecule (ELAM)-1, vascular cell adhesion molecule (VCAM)-1, human leukocyte antigen (HLA)-DR, interleukin (IL)-2R (CD25), CD34, alpha-1-antichymotrypsin (alpha 1-ACT), and L1 antigen were studied in skin from marrow recipients to determine the timing and distribution of their expression in relation to the clinical and histologic evolution of graft-versus-host disease (GvHD). Four phases were recognized: 1. pretransplant with no immunohistologic change; 2. posttransplant with no evidence of GvHD when dermal alpha 1-ACT + macrophages were increased; 3. posttransplant with clinical, but not histologic, evidence of GvHD with increased keratinocyte HLA-DR and ICAM-1 expression and increased numbers of VCAM-1+ dermal cells; and 4. posttransplant with clinical and histologic evidence of GvHD characterized by an infiltrate of CD25+ T cells, L1+, alpha 1-ACT+ and VCAM-1+ macrophages, L1 antigen expression on keratinocytes accompanied by further increases in HLA-DR and ICAM-1, and increased endothelial ELAM-1 staining with a reciprocal decrease in CD34. A sequential accumulation of cellular and molecular changes, therefore, occurs in the evolution of acute GvHD, and immunostaining for HLA-DR, ICAM-1, and VCAM-1 may be helpful in diagnosing early disease.

Immunohistological study of distribution of gamma/delta lymphocytes after allogeneic bone marrow transplantation.

The distribution of T lymphocytes expressing the gamma/delta form of the T cell receptor was studied in the liver, intestine, and major lymphoid organs, after bone marrow transplantation (BMT), including cases of graft versus host disease (GvHD). The number of gamma/delta as a proportion of the total number of CD3 positive cells did not differ from that found in normal tissues; the higher percentage normally found in the intestinal epithelium and splenic red pulp was maintained. This, and the results of a previous study undertaken on the skin, provide no evidence that gamma/delta T cells have a particularly important role in T cell regeneration after marrow transplantation or in the pathogenesis of the epithelial lesions associated with GvHD.