Background

Prognostic factors in breast cancer have exploded over the past several years. Pathologists have played a major role in identifying different histological and immunohistochemical markers that have a direct bearing on both the treatment and behavior of breast cancer.

OUTLINE

GENERAL	Activated Akt Kinase Age Angiogenesis Beta-Catenin Bilateral Cancers Biopsy effect CD105 Chromosomal abnormalities Cyclin D1 Cyclooxygenase-2 DNA Ploidy DCIS associated Epidermal Growth Factor Receptor Fibrotic focus Glutathione S-transferase Grading Hormone receptor status Ipsilateral Breast Failure (IBF) Ki-67 LCIS associated Lymph node status Lymphovascular invasion Mast Cells Microarray Gene Expression Mitotic figure count Mucin Neuroendocrine differentiation Nuclear features Oncogenes p53 Pregnancy PTEN Race Second primary breast cancer Size of tumor Skin Involvement Stromelysin-3 Topoisomerases Treatment inadequacies Tumor associated antigens Vascular endothelial growth factor
COMMONLY USED TERMS
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PROGNOSIS	CHARACTERIZATION
GENERAL BACKGROUND	The prognosis of breast cancer is influenced by a number of factors
Size of primary tumor	Tumors <1 cm with negative nodes have 98% survival at 5 yrs Tumors <2 cm with negative nodes have 96% survival
Lymph node involvement	Single most important factor in determining prognosis for early breast cancer 10 yr disease free survival is 70-80% with negative nodes One to three positive nodes 35-40% Ten or more positive nodes 10-15% Micrometastasis <0.2 cm are associated with better prognosis NOTE: 20-30% of node-negative women will have a recurrence and die within 10 yrs
Histologic type and grade of tumor	Better for tubular, papillary, colloid, medullary versus NOS types >80% of women with Grade I tumors survive 16 yrs
Estrogen and progesterone receptor status	50-85% of tumors have estrogen receptors 70% of tumors with estrogen receptors regress after hormone treatment Only 5% of receptor negative tumors respond
Proliferative rate of tumor	Higher proliferation rates have poorer prognosis High S-phase fraction by flow cytometry
Amplified oncogenes	See her2-neu
Degree of angiogenesis	High correlation with increased vessel density and metastasis
Lymphovascular invasion	Strong association with lymph node metastasis Dermal lymphatic invasion (inflammatory carcinoma) has 3 yr survival of 3-10%
GENERAL
Histopathologic types of benign breast lesions and the risk of breast cancer: case-control study. Shaaban AM, Sloane JP, West CR, Moore FR, Jarvis C, Williams EM, Foster CS. Department of Cellular and Molecular Pathology (A.M.S., J.P.S., F.R.M., C.J., C.S.F.), Department of Public Health (C.R.W.), Merseyside and Cheshire Cancer Registry Office (E.M.W.), University of Liverpool, Liverpool, U.K.	Am J Surg Pathol 2002 Apr;26(4):421-30 Abstract quote 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.
ACTIVATED Akt KINASE
Prognostic relevance of activated Akt kinase in node-negative breast cancer: a clinicopathological study of 99 cases. Schmitz KJ, Otterbach F, Callies R, Levkau B, Holscher M, Hoffmann O, Grabellus F, Kimmig R, Schmid KW, Baba HA. 1Institute of Pathology, University of Essen, Germany.	Mod Pathol. 2004 Jan;17(1):15-21 Abstract quote.   Patients with lymphnode-negative breast cancer show a 10-year tumor recurrence rate of approximately 30%. Therefore, it is important to identify high-risk patients who would benefit from further adjuvant therapy. For this purpose, we examined the activation state of two kinases important in the regulation of cell proliferation and apoptosis in a series of 99 node-negative breast cancer cases with a mean follow-up of 10 years: Akt and extracellular regulated kinase (ERK1/2). The activation of Akt and ERK1/2 was investigated by immunohistochemistry using phospho-specific antibodies. The results were correlated with HER-2/neu expression, histological grading, receptor status, overall survival (OS) as well as with cell proliferation (Ki67 immunoreactivity, mitotic count) and tumor apoptosis assessed by TUNEL staining. Activation of Akt (pAkt) but not activation of ERK1/2 (pERK1/2) correlated with HER-2/neu overexpression (P<0.05) and was related to reduced tumor apoptosis (P<0.05). No association was found between pAkt or pERK1/2 with cell proliferation assessed by Ki67 and mitotic count (MC). Survival analysis of receptor status, HER2/neu expression, histological grading, MC and pAkt immunoexpression showed a significant correlation with decreased OS, but only pAkt reached statistical significance in the multivariate Cox regression analysis (P=0.015). Activation of Akt in node-negative breast cancer may indicate aggressive tumor behavior and may constitute an independent prognostic factor of OS. The determination of pAkt status may be of value in identifying high-risk patients, who would benefit from adjuvant therapy, and gives a rationale to investigate new therapy strategies by specific inhibition of the Akt signaling pathway in breast cancer.
AGE
Pathology and heredity of breast cancer in younger women. Marcus JN, Watson P, Page DL, Lynch HT. Department of Pathology, Creighton University, Omaha, Neb.	J Natl Cancer Inst Monogr 1994;(16):23-34 Abstract quote The pathology of early-age onset breast cancer is considered here from three perspectives: 1) benign proliferative disease, 2) the cancers themselves, and 3) familial and hereditary breast cancer. Hereditary breast cancer, a subset of familial breast cancer featuring a strong autosomal dominant pedigree pattern and multiple primary cancers, has a strong predilection for younger women, accounting for about one half of breast cancers under age 30. With respect to benign proliferative disease, the increased relative risk of breast cancer associated with proliferative disease with atypia, about fourfold to fivefold for all ages, is doubled by the presence of a family history of breast cancer and amplified by young age. With respect to the carcinomas, the relative incidences of medullary carcinoma and ductal carcinoma in situ are increased in young women, while lobular and tubular carcinomas are decreased. Invasive breast cancer is higher grade and more proliferative in younger women, as measured by thymidine-labeling index, DNA flow cytometric S-phase fraction, and proliferation-associated proteins. The increased fraction of ductal carcinoma in situ and higher grade invasive cancers may help to account, respectively, for increased recurrence rates with conservative therapy, and more aggressive natural history in younger women. Familial breast cancers show trends for increased medullary type, but the effect is not independent of age. Weak associations of family history with tubular carcinoma have been reported, but data for associations with lobular carcinoma in situ and invasive lobular carcinoma are conflicting. Hereditary breast cancer as a class has higher tumor proliferation rates, an effect independent of age. Knowledge of the pathology and biomarker characteristics of BRCA1 gene-linked hereditary breast cancers, which account for a substantial fraction of breast cancers in younger women, should shed light on the nature of the responsible gene(s) and guide approaches to therapy and prophylaxis.
The histologic phenotypes of breast carcinoma occurring before age 40 years in women with and without BRCA1 or BRCA2 germline mutations: a population-based study. Armes JE, Egan AJ, Southey MC, Dite GS, McCredie MR, Giles GG, Hopper JL, Venter DJ. Molecular Pathology Laboratory, Victorian Breast Cancer Research Consortium, Peter MacCallum Cancer Institute, East Melbourne, Victoria, Australia.	Cancer 1998 Dec 1;83(11):2335-45 Abstract quote BACKGROUND: Women with breast carcinoma diagnosed before age 40 years have a greater prevalence of germline BRCA1 and BRCA2 mutations than women with breast carcinoma diagnosed at older ages. Several recognizable histologic characteristics have been identified in breast carcinoma from studies of BRCA1/2 mutation carriers who belong to multiple-case families. The authors attempted to determine whether breast carcinoma occurring before age 40 years in BRCA1 or BRCA2 mutation carriers who were not selected for family history could be distinguished histologically from one another and from breast carcinoma in women of a similar age without a germline BRCA1 or BRCA2 mutation. METHODS: The study undertook a histologic assessment of breast carcinomas diagnosed before age 40 years identified from a population-based study. RESULTS: Breast carcinoma in BRCA1 mutation carriers was associated with a distinct histologic appearance; these tumors were high grade, and had exceptionally high mean mitotic counts, a syncytial growth pattern, pushing margins, and confluent necrosis. Atypical medullary carcinoma was overrepresented in BRCA1 mutation carriers. All low grade tumors and tumors with low mitotic rates belonged to the group without BRCA1 or BRCA2 mutations. Pleomorphic lobular carcinomas and extensive intraduct carcinomas were more common in BRCA2 mutation carriers. CONCLUSIONS: Breast carcinoma occurring in women with a germline BRCA1 or BRCA2 mutation have recognizable histologic phenotypes, which may be useful in identifying individuals more likely to carry germline mutations. Histologic examination of breast carcinoma should become an important part of the evaluation of women seeking genetic testing for germline mutations in these breast carcinoma susceptibility genes.
Breast cancer in young women: clinicopathologic correlation. Bertheau P, Steinberg SM, Cowan K, Merino MJ. Laboratory of Surgical Pathology, Saint-Louis Hospital, Paris, France.	Semin Diagn Pathol 1999 Aug;16(3):248-56 Abstract quote It has been suggested that early-onset breast carcinomas may be different from those that occur in older women. The clinicopathologic characteristics of 191 young female patients (under 40 years of age) diagnosed with breast carcinoma (BC) were studied. Clinical history, staging, treatment and outcome were reviewed. Histology was assessed for tumor subtypes, invasive and in situ components, nuclear and histologic grades and lymph node status. Adjacent nontumoral breast tissue was evaluated. Clinically, 11 patients were stage 0, 21 stage I, 94 stage II, 38 stage III, 6 stage IV, and in 21 no information was obtained. Sixty five percent of patients had positive lymph nodes at diagnosis; 102 patients (54%) relapsed at a median of 29 months after diagnosis. Histologically, 180 cases were infiltrating BC, 150 ductal (83%), 19 lobular (11%) and 11 of special types (6%); 11 cases were ductal carcinoma in situ. We found no cases of medullary carcinoma. High nuclear grade and vascular invasions were frequent (68% and 67%, respectively) even in patients who remained disease-free at least 5 years after diagnosis (61% and 60%, respectively). Our study demonstrates that the histologic types of early-onset breast cancer are not different from other BC. However, BC in young women is often associated with histologic features of high-grade malignancy even in patients with better survival. Our results suggest that BCs in young women are different from those that occur in older women.
ANGIOGENESIS
Lymphangiogenesis does not occur in breast cancer. Agarwal B, Saxena R, Morimiya A, Mehrotra S, Badve S. From the Department of Pathology, Indiana University School of Medicine, Indianapolis, IN.	Am J Surg Pathol. 2005 Nov;29(11):1449-55. Abstract quote   Breast cancer metastasis predominantly occurs via lymphatic vessels. However, the study of lymphatic vessels and lymphangiogenesis has been hampered by lack of specific markers. Recently, antibodies directed against M2A (D2-40), Podoplanin, and Prox-1 that specifically mark lymphatic vessels in paraffin-embedded sections have become available. These were used to study lymphangiogenesis in archival paraffin sections of normal breast (n = 23), fibrocystic disease (n = 7), ductal carcinoma in situ (n = 32), invasive ductal carcinoma (n = 50), and invasive lobular carcinoma (n = 5). In addition, endothelial proliferation in lymphatic vessels was analyzed by dual-color immunohistochemistry with D2-40 and proliferating cell nuclear antigen (PCNA). Expression of D2-40, Prox-1, and Podoplanin was seen in lymphatic vessels but not in blood vessels. Lymphatic vessels were seen in the peritumoral area and as "entrapped" intratumoral vessels adjacent to preexisting normal lobules and ducts. Unlike angiogenesis, there was no increase of lymphatic vessel density in association with neoplastic transformation. On the contrary, a marked reduction in intratumoral lymphatic vessel density was seen in comparison to normal breast tissue, fibrocystic disease, and ductal carcinoma in situ (P = 0.0001). There was an increase in peritumoral lymphatic vessel density as compared with normal breast (P = 0.0001). However, the endothelial cells in the "entrapped" or the peritumoral lymphatic vessels did not show any expression of PCNA indicating minimal or no proliferative activity. This was in contrast to the strong expression seen in adjacent tumor cells and blood vessel endothelial cells. Thus, lymphangiogenesis was not evident when studied by lymphatic vessel density or by lymph vessel endothelial proliferation.
Long-term prognostic significance of neoangiogenesis in breast carcinomas: comparison of Tie-2/Tek, CD105, and CD31 immunocytochemical expression. Dales JP, Garcia S, Carpentier S, Andrac L, Ramuz O, Lavaut MN, Allasia C, Bonnier P, Charpin C. Department of Pathology, Centre Hospitalier et Universitaire Nord, Marseille, France.	Hum Pathol. 2004 Feb;35(2):176-83 Abstract quote. The immunocytochemical detection of Tie-2/Tek, CD105, and CD31 was assessed in a large series (n = 905) of breast carcinomas on frozen sections. Results were correlated with patients' long-term outcome (median, 11.7 years) to define the respective prognostic significance of these markers. Univariate (Kaplan-Meier) analysis demonstrated that higher expression of CD31 (P = 0.032), CD105 (P = 0.001), and Tie-2/Tek (P = 0.025) correlated with poorer survival. However, only greater CD105 expression could significantly (P = 0.035) identify node-negative patients with poorer survival. Moreover, in multivariate analysis, CD105 and Tie-2/Tek, but not CD31, expression proved to be independent significant prognostic indicators. Marked expression of CD31 (P = 0.024), CD105 (P = 0.001), and Tie-2/Tek (P = 0.01) also correlated with higher risk of metastases in node-negative patients. It is concluded that CD105 immunoexpression in breast carcinomas is an independent prognostic indicator in node-negative patients, better in terms of overall survival than Tie-2/Tek and CD31. Also, Tie-2/Tek expression proved more sensitive than CD31 expression in terms of prognostic significance. Compared with CD31, CD105 and Tie-2/Tek have more clinical relevance for patient monitoring and also can serve as targets for specific therapy, such as CD105 immunotoxins or Tie-2/Tek pathway blockade, as recently suggested in experimental studies.
BETA CATENIN
Study of phospho-beta-catenin subcellular distribution in invasive breast carcinomas in relation to their phenotype and the clinical outcome. Nakopoulou L, Mylona E, Papadaki I, Kavantzas N, Giannopoulou I, Markaki S, Keramopoulos A. 1Department of Pathology, Attikon Hospital, Athens, Greece.	Mod Pathol. 2006 Apr;19(4):556-63. Abstract quote   beta-Catenin has a crucial role in cell-cell adhesion as well as a signaling role as a member of the Wnt pathway. The aim of this study was to examine the clinicopathological and prognostic value of phosphorylated beta-catenin, as well as its relation to the tumors' phenotype, in breast cancer. Immunohistochemistry was applied on 141 paraffin-embedded breast tissue specimens for the detection of phospho-beta-catenin, ER, PR, c-erbB-2, p53, Ki-67, bcl-2, uPAR and TIMP-1. For each case, a phospho-beta-catenin index was determined by image analysis. Phospho-beta-catenin staining was detected in the cytoplasm and the nucleus of the malignant cells. Cytoplasmic phospho-beta-catenin was statistically higher in carcinomas of smaller tumor size (P=0.030), lower stage (P=0.026), decreased Ki-67 and high c-erbB-2 immunoreactivity (P=0.052 and P=0.037, respectively). Nuclear phospho-beta-catenin showed a parallel correlation with ER and ERbeta (P=0.022 and P=0.043, respectively), bcl-2 (P=0.042), uPAR in cancer cells (P=0.041) and TIMP-1, although the correlation was borderline (P=0.066). Cytoplasmic phospho-beta-catenin was found to be independently correlated with prolonged disease-free and overall survival (P=0.046 and P=0.002, respectively), whereas nuclear localization was correlated with a shortened overall survival (P=0.046). In conclusion, phospho-beta-catenin may have a different involvement in invasive breast carcinomas, according to its subcellular distribution. Nuclear localization seems to be related to an aggressive tumor phenotype, negatively affecting patients' overall survival, whereas cytoplasmic localization is associated with a favorable tumor phenotype and a longer disease-free and overall survival.
BILATERAL CANCERS
Bilateral carcinomas of the breast with local recurrence: analysis of genetic relationship of the tumors. Regitnig P, Ploner F, Maderbacher M, Lax SF. Department of Pathology, Division of Medical Oncology, Medical University of Graz, Austria.	Mod Pathol. 2004 May;17(5):597-602. Abstract quote   Local recurrence of bilateral breast carcinomas is rare, but of biological interest, since it is unclear as to which tumor the local recurrence is related to, the ipsilateral or the contralateral, or whether it is an independent neoplasm. The aim of this study was to investigate the genetic relationship of bilateral breast carcinomas to each other and to their local recurrences. Eight cases of bilateral breast carcinomas, five with and three without local recurrence were analyzed using a microsatellite assay for 13 microsatellite loci. The presence of loss of heterozygosity and microsatellite instability in the various tumors was used for clonal analysis. All eight bilateral breast carcinomas showed divergent alterations in at least two microsatellite loci, which ruled out a genetic relationship. Four of five local recurrences were genetically related to the ipsilateral tumor and unrelated to the contralateral tumor. Only one local recurrence that occurred 11.8 years after the surgery of an infiltrative lobular carcinoma simultaneously with distant metastases was genetically related to the contralateral breast carcinoma. Although the number of cases in our study is limited, there is evidence that local recurrence of bilateral breast carcinoma frequently arises from the ipsilateral tumor.
Multicentricity and bilaterality in invasive breast carcinoma. Lesser ML, Rosen PP, Kinne DW.	Surgery 1982 Feb;91(2):234-40 Abstract quote Multicentricity and bilaterality are well-established characteristics of breast carcinoma, but little is known about the relationship of these variables with each other. This question was explored by analyzing the data pertaining to 880 women with invasive breast carcinoma. Patients with multicentric carcinoma had bilateral disease more often than those whose carcinoma was apparently limited to a single quadrant. Among women who had lobular carcinoma in situ coexisting with infiltrating duct carcinoma or infiltrating lobular carcinoma, bilaterality and multicentricity were significantly more common than they were among patients whose only lesion was infiltrating duct or medullary cancer. Other variables associated with bilaterality and multicentricity were degree of ductal differentiation, tumor size, nodal status, type of tumor margin, intensity of lymphoid infiltrate, and menstrual status. Age at diagnosis and estrogen receptors were related to bilaterality but not to multicentricity. The following variables proved to be unrelated to bilaterality and multicentricity: family history of breast carcinoma, height, weight, and parity. The data obtained in this study tend to support a conclusion that multicentricity and bilaterality are manifestations of similar factors involved in the neoplastic transformation of mammary gland epithelium leading to the development of breast cancer.
Women with breast cancer: histologic findings in the contralateral breast. Roubidoux MA, Helvie MA, Wilson TE, Lai NE, Paramagul C. Department of Radiology, University of Michigan Medical Center, Ann Arbor 48109-0326, USA.	Radiology 1997 Jun;203(3):691-4 Abstract quote PURPOSE: To investigate contralateral breast biopsy histologic findings in women with breast cancer. MATERIALS AND METHODS: Histologic findings in 237 patients with breast cancer who underwent contralateral breast biopsy for clinically or mammographically detected abnormalities were retrospectively reviewed. Malignant findings were categorized by histologic type. Benign findings were categorized by risk of breast cancer. Comparison was made with mammographically guided breast biopsy results in 1,294 patients without breast cancer. RESULTS: Of the 237 patients, 168 (70.9%) had either malignancy or high-risk histologic findings. One hundred thirty-nine patients (58.6%) had malignant findings; 98 (41.4%) had benign findings. Of the 98 with benign findings, 29 (30%) had high-risk histologic findings. Thirty (33%) of the 91 patients with invasive cancer had invasive lobular carcinoma. Forty-seven (45.6%) of the 103 patients with malignant lesions at mammographically guided biopsies had ductal carcinoma in situ alone. CONCLUSION: Compared with biopsy in women without breast cancer, contralateral biopsy in women with breast cancer was more likely to show malignancy, invasive lobular carcinoma, or ductal carcinoma in situ alone (P < .001) or to show high-risk histologic benign findings (P < .001). Mammographic and clinical findings in the contralateral breast should be regarded as more suspicious than those in patients without known breast cancer.
A case-control study of unilateral and bilateral breast carcinoma patients. Newman LA, Sahin AA, Bondy ML, Mirza NQ, Vlastos GS, Whitman GJ, Brown H, Buchholz TA, Lee MH, Singletary SE. Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.	Cancer 2001 May 15;91(10):1845-53 Abstract quote BACKGROUND: Women with unilateral breast carcinoma are at increased risk for developing contralateral disease. The clinical significance of bilateral breast carcinoma has not been fully defined, and the subset of patients who may benefit from medical or surgical risk-reduction intervention has not yet been characterized. The purpose of this study was to evaluate risk factors and outcomes for bilateral breast carcinoma. METHODS: A subject group of 70 bilateral breast carcinoma patients (62% metachronous) was matched by age and survival interval with a control group of 70 unilateral breast carcinoma patients. Median follow-up was 103 months. RESULTS: Eighty-two percent of the unilateral patients and 80% of the bilateral patients had Stage I or II disease at diagnosis. Median age at presentation was 53 years. In the bilateral group, the contralateral cancer was diagnosed at the same or earlier stage than the first cancer in 87% of cases. Bilateral patients were significantly more likely to have multicentric disease and to have a positive family history for breast carcinoma compared with the unilateral group. There were no significant differences regarding history of exogenous hormone exposure, lobular histology, hormone-receptor status, or HER-2/neu expression. Five-year disease-free survival was 94% for the unilateral breast carcinoma patients and 91% for the bilateral breast carcinoma patients (P = 0.16). CONCLUSIONS: Survival for patients with bilateral breast carcinoma is similar to that of patients with unilateral disease; however, prophylactic risk-reduction intervention for the contralateral breast should be considered in patients who have multicentric unilateral disease or a positive family history for breast carcinoma.
BIOPSY, PRIOR
Effect of time interval on residual disease in breast cancer. Wiley EL, Diaz LK, Badve S, Morrow M.	Am J Surg Pathol 2003 Feb;27(2):194-8 Abstract quote The histologic responses of breast tissue to injury are limited. Needle core biopsies of the breast are associated with displacement of tumor cells, and the incidence of tumor displacement decreases as the time interval between needle core biopsy and subsequent excision increases. This suggests that displaced tumor cells are destroyed by reparative processes induced by tissue injury. Residual tumor in a lumpectomy site may also be subjected to the same destructive processes associated with tissue repair. A total of 259 consecutive cases of infiltrating ductal carcinoma with margin-positive lumpectomies and their associated reexcision specimens obtained over a 7-year period were analyzed for the presence, type, and quantity of residual disease. The overall incidence of residual disease was 69%. Residual infiltrating ductal carcinoma was present in 35% of cases, and residual ductal carcinoma in situ was present in 50%. An increased time interval between lumpectomy and reexcision was associated with a decreased incidence of residual infiltrating carcinoma (p <0.0043); this decrease was not found associated with ductal carcinoma in situ. These findings suggest that the host response to injury may destroy residual infiltrating carcinoma cells in some margin-positive cases.
BRAIN METASTASES
Breast Cancers With Brain Metastases are More Likely to be Estrogen Receptor Negative, Express the Basal Cytokeratin CK5/6, and Overexpress HER2 or EGFR. Hicks DG, Short SM, Prescott NL, Tarr SM, Coleman KA, Yoder BJ, Crowe JP, Choueiri TK, Dawson AE, Budd GT, Tubbs RR, Casey G, Weil RJ. *Clinical and Anatomic Pathology daggerBreast Center double daggerTaussig Cancer Center section signLerner Research Institute paragraph signBrain Tumor Institute, Cleveland Clinic Foundation, Cleveland, OH 44195.	Am J Surg Pathol. 2006 Sep;30(9):1097-1104. Abstract quote Brain metastases (BM) from breast cancer are associated with significant morbidity and mortality. In the current study, we have examined a cohort of breast cancer patients who went on to develop BM for clinical-pathologic features and predictive markers that identify this high-risk subgroup of patients at the time of diagnosis. The primary tumors from 55 patients who developed BM were used to construct a tissue microarray. The clinical and pathologic features were recorded and the tissue microarray was stained for estrogen receptor, human epidermal growth factor receptor 2, cytokeratin 5/6, and epidermal growth factor receptor by immunohistochemistry . This cohort of patients was compared against a group of 254 patients who remain free of metastases (67 mo mean follow-up), and another cohort of 40 patients who developed mixed visceral and bone metastatic disease without brain recurrence over a similar period of time. Breast cancer patients who went on to develop BM were more likely to be <50 years old (P<0.001), and the primary tumors were more likely to be estrogen receptor negative (P<0.001) and high grade (P=0.002). The primary tumors were also more likely to express cytokeratin 5/6 (P<0.001) and epidermal growth factor receptor (P=0.001), and to overexpress human epidermal growth factor receptor 2 (P=0.001). The data presented above suggest a profile for breast cancer patients at increased risk for developing BM. Predictive factors to help identify patients with metastatic breast cancer who are at an increased risk for developing central nervous system recurrence might allow for screening of this population for early detection and treatment or for the development of targeted strategies for prevention.
CD105
CD105 Expression Is a Marker of High Metastatic Risk and Poor Outcome in Breast Carcinomas Correlations Between Immunohistochemical Analysis and Long-Term Follow-up in a Series of 929 Patients Jean-Philippe Dales, MD Stephane Garcia, MD Pascal Bonnier, MD Florence Duffaud, MD Lucile Andrac-Meyer, MD Olivier Ramuz, MD Marie-Noëlle Lavaut, MD Claude Allasia, PhD Colette Charpin, MD	Am J Clin Pathol 2003;119:374-380 Abstract quote CD105 (endoglin) is expressed significantly in activated endothelial cells in culture and in tumor microvessels. Quantification of CD105 immunocytochemical expression that may be clinically relevant has not been accurately evaluated. We studied CD105 expression on frozen tissue sections by using immunohistochemical assays in a series of 929 patients and correlated the findings with long-term follow-up (median, 11.3 years). Univariate (Kaplan-Meier) analysis showed that the number of CD105+ microvessels (cutoff, 15 vessels) correlated significantly with poor overall survival among all patients (P = .001). This correlation was less significant in node-negative patients (P = .035). Marked CD105 expression also correlated with a high risk for metastasis among all patients (P = .006) and among node-negative patients (P = .001). Multivariate analysis (Cox model) identified CD105 immunodetection as an independent prognostic indicator. Our results suggest that immunohistochemical expression of CD105 has practical clinical relevance for identifying node-negative patients with a poor prognosis. Moreover, immunodetection of CD105 also may be considered a potential tool for selecting patients who could benefit from specific antiangiogenic therapy, using anti-CD105 conjugates.
CHROMOSOMAL ABNORMALITIES
Simultaneous chromosome 1q gain and 16q loss is associated with steroid receptor presence and low proliferation in breast carcinoma. Farabegoli F, Hermsen MA, Ceccarelli C, Santini D, Weiss MM, Meijer GA, van Diest PJ. Department of Experimental Pathology, University of Bologna, Bologna, Italy.	Mod Pathol. 2004 Apr;17(4):449-55. Abstract quote We applied comparative genomic hybridization (CGH) to 46 breast carcinoma samples, collected from 1993 to 1995, in order to detect chromosome 1q gains and 16q losses and to define whether samples showing both these alterations had distinct biopathologic features and different clinical outcome. A total of 22 samples (48%) had simultaneous chromosome 1q gain and 16q loss, which was always associated with other genetic changes. In total, 23 samples had various chromosome imbalances (including chromosome 1q gain independent of chromosome 16q loss and vice versa) and one sample did not show detectable alterations. Samples having chromosome 1q gain/16q loss were compared to the other samples with regard to neoplasm size, lymph-node status, histologic and nuclear grade, estrogen and progesterone receptor presence, Ki-67, pRB, Cyclin D1, Cyclin A, p53, p21 and p27 expression as detected by immunohistochemistry. The samples showing chromosome 1q gain/16q loss had high steroid hormone receptor expression (P=0.02), low cell growth fraction (Ki-67, P=0.03) and high p27 expression (P<0.001). No statistical correlation with disease-free survival and overall survival or response to hormonal therapy was found. We conclude that simultaneous chromosome 1q gain/16q loss is a frequent event in invasive breast cancer, which occurs in a subset of both intermediate- and high-grade breast carcinomas. Although the final chromosome 1q and 16q imbalances might have originated from different chromosome alterations in low- and high-grade samples, the gene-dosage effect might be important in conferring peculiar biopathologic characteristics to this subset of samples. The cytogenetic and molecular mechanisms underlying these chromosome changes deserve further investigations.
Aneuploidy of chromosome 20 in invasive breast cancer correlates with poor outcome. Nakopoulou L, Tsirmpa I, Giannopoulou I, Trafalis D, Katsarou S, Davaris P. Department of Pathology, Medical School, The National and Kapodistrian University of Athens, Greece.	Cancer Genet Cytogenet 2002 Apr 15;134(2):127-32 Abstract quote Breast carcinoma is a genetically and phenotypically heterogeneous disease and is frequently associated with nonrandom chromosomal alterations. The aim of this study was to investigate the numerical aberrations of chromosome 20 in breast cancer. The observed chromosome-specific numerical abnormalities were evaluated along with the established clinicopathological parameters, the immunohistochemical expression of ER, PR, p53, c-erbB-2, Ki-67 and patients' survival. Nonisotopic in situ hybridization was applied to interphase cell nuclei on paraffin embedded tissue sections. Polysomy of chromosome 20 was the prevalent alteration in 45 of 50 (90%), monosomy in 2 of 50 (4%) and disomy in 3 of 50 (6%) cases. Invasive ductal carcinomas displayed a higher percentage of polysomy than lobular ones. A statistical significant association was demonstrated between Ki-67 immunohistochemical expression and polysomy of chromosome 20. Disomy was inversely correlated with Ki-67, while monosomy was suggestively associated with PR positive expression. Among the patients, those with the highest levels of polysomy showed the worst survival. In conclusion, the gain of chromosome 20 is the prevalent aberration in patients with breast carcinomas and may be useful prognostic marker in breast cancer.
CYCLIN D1
Bio-pathologic Characteristics Related to Chromosome 11 Aneusomy and Cyclin D1 Gene Status in Surgically Resected Stage I and II Breast Cancer: Identification of an Adverse Prognostic Profile. Mottolese M, Orlandi G, Sperduti I, Merola R, Buglioni S, Di Benedetto A, Pinnaro P, Perracchio L, Venturo I, Cognetti F, Cianciulli A. Departments of *Pathology daggerClinical Pathology (Cytogenetic Unit) double daggerBiostatistic Unit section signRadiotherapy paragraph signOncology B parallelOncology A, Regina Elena Cancer Institute, Rome, Italy.	Am J Surg Pathol. 2007 Feb;31(2):247-254. Abstract quote We aimed at developing a more detailed understanding of cyclin D1 in early stage human breast cancer and defining the biologic profiles with different prognostic value correlating cyclin D1 gene amplification and chromosome 11 aneusomy with bio-pathologic variables of known clinical importance. Cyclin D1 gene amplification and chromosome 11 aneusomy were investigated using fluorescence in situ hybridization whereas cyclin D1, PgR, HER-2, Bcl2, p53, and Ki-67 expressions were analyzed by immunohistochemistry in 121 stage I or II breast cancer patients uniformly treated with cyclophosphamide/metotrexate/5-fluorouracil-based chemotherapy. Cyclin D1 was amplified in 6.6% and overexpressed in 32.2% of cases. Amplification was not associated with any selected bio-pathologic variables, whereas the chromosome 11 aneusomy level significantly increased in tumors with higher histologic grade (P<0.01), higher tumor size (P<0.003), p53 nuclear accumulation (P<0.04), and ERalpha negativity (P<0.049). Multiple correspondence analysis showed 4 different biologic tumor profiles. The first, characterized by high Ki-67 score, p53+, cyclin D1+, HER-2+, aneusomy level>30%, ratio (cyclin D1 gene/CEP11)>2, was associated with tumor relapse defining the most unfavorable biologic profile. Kaplan-Meier's method showed significantly shorter disease-free survival in patients with at least 3 variables positive out of the 6 detected by multiple correspondence analysis. In multivariate analysis, the identified biologic profile emerged as the only significant prognostic indicator. Our findings are of particular clinical interest for early stage breast cancer patients, because the assessment of biologic factors predictive of tumor aggressiveness may influence postoperative therapeutic strategies.
CYCLOOXYGENASE-2
Overexpression of cyclooxygenase-2 is associated with breast carcinoma and its poor prognostic factors. Shim JY, An HJ, Lee YH, Kim SK, Lee KP, Lee KS. Department of Pathology, Bundang CHA Hospital, Pochon CHA University, Sungnam, Korea.	Mod Pathol. 2003 Dec;16(12):1199-204. Abstract quote   Cyclooxygenase is the rate-limiting enzyme that catalyzes the conversion of arachidonic acid to prostaglandins. The inducible form, cyclooxygenase-2, is known to be overexpressed in various human cancers including the colon, stomach, and urinary bladder. In this study, we evaluated the overexpression of cyclooxygenase-2 in 64 cases of breast cancer and correlated the results with clinicopathologic parameters. Immunohistochemical staining for cyclooxygenase-2 demonstrated positivity of the tumor cells in 46 of 64 cases (72%). Cyclooxygenase-2 overexpression was significantly correlated with larger tumor size and advanced clinical stage. Cyclooxygenase-2 overexpression tended to be more frequently observed in cases with presence of lymph node metastasis and in cases without expression of estrogen and progesterone; however, there was no significant correlation statistically. Nuclear and histologic grade were not well correlated with cyclooxygenase-2 overexpression. When ductal carcinoma in situ was considered separately, 32 of 42 cases (76%) were positive for cyclooxygenase-2. We conclude that cyclooxygenase-2 is up-regulated in a high proportion of breast cancers. The overexpression of cyclooxygenase-2 was associated with larger tumor size and advanced clinical stage, although lymph node status, estrogen and progesterone expression, and nuclear and histologic grade were not significantly correlated. Therefore, cyclooxygenase-2 overexpression may be a feature of the aggressive phenotype and may be useful as a prognostic indicator in breast cancer.
DNA PLOIDY
DNA ploidy, S-phase, and steroid receptors in more than 127,000 breast cancer patients. Wenger CR, Beardslee S, Owens MA, Pounds G, Oldaker T, Vendely P, Pandian MR, Harrington D, Clark GM, McGuire WL. University of Texas Health Science Center, Department of Medicine/Medical Oncology, San Antonio 78284-7884.	Breast Cancer Res Treat 1993 Oct;28(1):9-20 Abstract quote Several potential prognostic factors are available today for patients with breast cancer, and many more are being identified and studied. To evaluate the clinical utility of these factors, it will be necessary to measure them on a large number of patients, and then follow these patients so that multivariate survival analyses can be performed. The Oncology Research Network was established in 1986 by the University of Texas Health Science Center at San Antonio and Nichols Institute Reference Laboratories in order to evaluate the clinical utility of new prognostic factors for patients with primary breast cancer. The first generation of prognostic factors included steroid receptors, along with DNA ploidy and S-phase fraction determined by flow cytometry. Currently, laboratory results have been obtained from more than 127,000 patients, and follow-up information is available on a subset of more than 25,000 of these patients. S-phase fraction was related to the ploidy status of the tumor. An increased incidence of aneuploidy and higher S-phase fractions were found in estrogen and progesterone receptor negative tumors, tumors from patients with positive axillary lymph nodes, tumors greater than 2 cm in diameter, and patients younger than 35 years of age. Preliminary survival analyses suggest that S-phase fraction and DNA ploidy, in combination with other prognostic factors, are powerful predictors of early disease relapse. The Oncology Research Network provides an important resource for examining the clinical significance of new laboratory assays and for expediting improvements in existing laboratory techniques.
DCIS ASSOCIATED (EXTENSIVE INTRADUCTAL COMPONENT)
An assessment of extensive intraductal component as a risk factor for local recurrence after breast-conserving therapy. Jacquemier J, Kurtz JM, Amalric R, Brandone H, Ayme Y, Spitalier JM. Department of Anatomic Pathology, Marseille Cancer Institute, France.	Br J Cancer 1990 Jun;61(6):873-6 Abstract quote The influence of extensive intraductal component (EIC) on local recurrence risk was studied for 496 patients with stage I-II infiltrating ductal cancers treated by conservative surgery and irradiation. EIC was diagnosed in 65 of 231 (28%) premenopausal and 41 of 265 (15.5%) post-menopausal patients. Local recurrence risk was markedly increased in EIC+ patients (5-year actuarial risk 18% versus 8% without EIC, P less than 0.001), but this effect appeared limited to premenopausal patients. Local recurrence risk increased with increasing degree of EIC. EIC with more than 50% intraductal carcinoma was more prevalent in patients younger than 40, perhaps accounting to some degree for the higher local recurrence rates observed in younger patients. The presence of EIC had no influence on overall survival, on median time to local recurrence, or on short-term survival after local failure. The usefulness of EIC as a risk factor for local recurrence is discussed.
Intraductal carcinoma associated with invasive carcinoma of the breast. A comparison of the two lesions with implications for intraductal carcinoma classification systems. Goldstein NS, Murphy T. Department of Anatomic Pathology, William Beaumont Hospital, Royal Oak, Michigan 48073, USA.	Am J Clin Pathol 1996 Sep;106(3):312-8 Abstract quote Intraductal carcinoma (DCIS) is a useful marker for predicting which women will develop a recurrent breast malignancy. The authors examined 150 consecutive, mammographically detected, T1 invasive carcinomas associated with DCIS to study the DCIS and compare it to its associated invasive carcinoma. Intraductal carcinoma nuclear grades were assigned to each duct on a scale of 1 to 3. The percentage of DCIS ducts that were involved by each grade was quantitated into quartiles for cases with more than one DCIS nuclear grade. The predominant architectural pattern corresponding to each DCIS nuclear grade was recorded. Ninety-two percent of the 150 invasive carcinomas were of ductal type, 4% were tubular, and the remainder were various other subtypes. Nine percent of the DCIS cases were nuclear grade 1. The remaining 91% of cases were almost evenly distributed between mixed DCIS nuclear grades 1 and 2 (19%), pure DCIS nuclear grade 2 (24%), mixed DCIS nuclear grade 2 to 3 (25%), and pure DCIS nuclear grade 3 (22%). Two percent of cases were a mixture of DCIS nuclear grades 1 and 3 or 1, 2, and 3. All pure DCIS nuclear grade 1 or mixed 1 and 2 were associated with well or moderately differentiated invasive carcinomas, whereas the majority (61%) of the pure DCIS nuclear grade 3 cases were associated with poorly differentiated invasive carcinomas. There was no relation between the DCIS architectural pattern and the invasive carcinoma grade. In general, the DCIS nuclear grade correlates with the grade of the invasive carcinoma. Unlike DCIS architecture, nuclear grade heterogeneity within DCIS associated with invasive carcinoma is minimal. DCIS classification systems based on nuclear grade have merit because there is little variation in nuclear grade within a given patient's lesion.
Extensive and predominant in situ component in breast carcinoma: their influence on treatment results after breast-conserving therapy. Sinn HP, Anton HW, Magener A, von Fournier D, Bastert G, Otto HF. Department of Pathology, University of Heidelberg, Germany.	Eur J Cancer 1998 Apr;34(5):646-53 Abstract quote Intramammary tumour recurrence is one of the most important problems in breast-conserving therapy. We reviewed a series of 957 patients treated with breast-conserving therapy for primary invasive breast carcinomas between 1 January 1985 and 31 December 1992 at the University of Heidelberg. All histological slides were re-evaluated for risk factors with special emphasis on the extent and subclassification of the in situ tumour and the margin status. Six parameters were identified as significant risk factors for intramammary recurrence in the univariate analysis, including extensive or predominant in situ component (EIC, with at least twice the greatest dimension of the invasive tumour component), histological grade, angioinvasion, lobular tumour type, involved resection margin and lymph node status. The presence of an EIC was statistically correlated with low tumour grade, tumour at the resection margins and in re-excision specimens and with multifocal tumour invasion. Multivariate logistic regression analysis revealed that EIC (relative risk (RR) = 1.9), tumour grade (RR = 1.76), angioinvasion (RR = 1.34), lobular tumour type (RR = 1.65) and young age (< or = 40 years, RR = 1.39) were independent predictors of local recurrence. When combining these factors in a linear model, the simultaneous presence of at least two of the five risk factors predicted a 5-year risk of intramammary recurrence of 20.9% compared with a risk of only 1-5% when none or one of these risk factors were identifiable. We conclude that the risk of subsequent intramammary recurrence after breast-conserving therapy can be estimated from a scoring system that includes four histological risk factors and the patient's age.
EPIDERMAL GROWTH FACTOR RECEPTOR
Clinical Value of Epidermal Growth Factor Receptor Expression in Primary Breast Cancer. Rampaul RS, Pinder SE, Nicholson RI, Gullick WJ, Robertson JF, Ellis IO. From the *Nottingham Breast Institute, University of Nottingham, Nottingham City Hospital, NHS Trust, Nottingham, U.K.; daggerHistopathology Department, Addenbrooke's NHS Trust, Cambridge, UK; double daggerCardiff University, Cardiff, UK; and section signSchool of Biological Sciences, University of Kent, Kent, UK.	Adv Anat Pathol. 2005 Sep;12(5):271-273. Abstract quote   EGFR expression in primary breast cancer has been extensively investigated for its prognostic and predictive value. However overall there is no consensus on its potential to guide such prognostication. This is largely because of the great heterogeneity in study designs and methods used to assay the EGFR protein. The impetus to standardize such studies is much needed as there are now several tyrosine kinase inhibitors directed against the EGF receptor and phase II trials are showing significant promise.
EGFR gene amplification in breast cancer: correlation with epidermal growth factor receptor mRNA and protein expression and HER-2 status and absence of EGFR-activating mutations. Bhargava R, Gerald WL, Li AR, Pan Q, Lal P, Ladanyi M, Chen B. 1Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.	Mod Pathol. 2005 Aug;18(8):1027-33. Abstract quote   The human epidermal growth factor receptor (HER) family of receptor tyrosine kinase has been extensively studied in breast cancer; however, systematic studies of EGFR gene amplification and protein overexpression in breast carcinoma are lacking. We studied EGFR gene amplification by chromogenic in situ hybridization (CISH) and protein expression by immunohistochemistry in 175 breast carcinomas, using tissue microarrays. Tumors with >5 EGFR gene copies per nucleus were interpreted as positive for gene amplification. Protein overexpression was scored according to standardized criteria originally developed for HER-2. EGFR mRNA levels, as measured by Affymetrix U133 Gene Chip microarray hybridization, were available in 63 of these tumors. HER-2 gene amplification by fluorescence in situ hybridization (FISH) and protein overexpression by immunohistochemistry were also studied. EGFR gene amplification (copy number range: 7-18; median: 12) was detected in 11/175 (6%) tumors, and protein overexpression was found in 13/175 (7%) tumors. Of the 11 tumors, 10 (91%) with gene amplification also showed EGFR protein overexpression (2+ or 3+ by immunohistochemistry). The EGFR mRNA level, based on Affymetrix U133 chip hybridization data, was increased relative to other breast cancer samples in three of the five tumors showing gene amplification. Exons 19 and 21 of EGFR, the sites of hotspot mutations in lung adenocarcinomas, were screened in the 11 EGFR-amplified tumors but no mutations were found. Three of these 11 tumors also showed HER-2 overexpression and gene amplification. Approximately 6% of breast carcinomas show EGFR amplification with EGFR protein overexpression and may be candidates for trials of EGFR-targeted antibodies or small inhibitory molecules.
FIBROTIC FOCUS
Fibrotic focus in invasive ductal carcinoma: an indicator of high tumor aggressiveness. Hasebe T, Tsuda H, Hirohashi S, Shimosato Y, Iwai M, Imoto S, Mukai K. Pathology Division, National Cancer Center Research Institute, East, Chiba, Japan.	Jpn J Cancer Res 1996 Apr;87(4):385-94 Abstract quote Histological examination of invasive ductal carcinoma of the breast often demonstrates the presence of an extensive central fibrotic focus (FF). The clinicopathological significance of the FF, or scar, in primary invasive ductal carcinoma is still ambiguous. One hundred and fifty-three cases of invasive ductal carcinoma (IDC) were classified into two groups, those with and those without FF. The differences in frequency of immunohistochemically detected overexpression of c-erbB-2 protein and nuclear accumulation of p53 protein, and the labeling index of proliferating cell nuclear antigen (PCNA), as well as histopathological parameters were compared between these two groups. IDCs smaller than 50 mm with FF showed a higher frequency of high-grade tumors, a higher frequency of lymph node metastasis, and a significantly higher frequency of c-erbB-2 protein overexpression than those without FF. In tumors of 20 mm or less, the incidence of nuclear accumulation of p53 protein was significantly higher in tumors with than those without FF. Tumors with FF showed a significantly higher PCNA labeling index than those without FF, regardless of tumor size. The present results indicate that the presence of FF is an important clinicopathological parameter associated with a higher degree of malignancy in IDCs, especially those smaller than 50 mm. Therefore, dividing IDCs into those with and those without FF appears to be meaningful clinicopathologically.
Fibrotic focus in invasive ductal carcinoma of the breast: a histopathological prognostic parameter for tumor recurrence and tumor death within three years after the initial operation. Hasebe T, Tsuda H, Tsubono Y, Imoto S, Mukai K. Pathology Division, National Cancer Center Research Institute, East, Tokyo.	Jpn J Cancer Res 1997 Jun;88(6):590-9 Abstract quote We investigated whether the presence of a fibrotic focus (FF) in the primary lesion and in lymph node metastasis is a good predictor of early tumor recurrence or death in patients with invasive ductal carcinoma (IDC). Multivariate relative risk (RR) of tumor recurrence and death according to the presence of FF in the primary tumor was estimated using the Cox proportional hazards regression model with adjustment for other prognostic factors (histologic grade, T classification, nodal status, tumor necrosis, DNA ploidy, c-erbB-2 protein expression, p53 protein expression, and labeling index of proliferating cell nuclear antigen). For the evaluation of the metastatic status in the axillary lymph nodes, RR of multivariate analysis was adjusted for the presence of FF in the metastatic tumor and the number of lymph nodes involved (1-3 and > 3). The presence of FF increased the RR of tumor recurrence significantly for the cases in all stages, and especially for those in stages I and II (RR = 6.9, P < 0.05 and RR = 25.0, P < 0.005, respectively). All cases that died of disease had FF. Among IDCs with FF, 24 cases had FF in lymph node metastasis. Significantly higher RRs of tumor recurrence and death were observed in cases with FF in lymph node metastasis than in those without it (RR = 2.0, P < 0.001 and RR = 5.9, P < 0.05, respectively). It was suggested that the presence of FF is an important predictor of early tumor recurrence or death in patients with IDCs. The presence of FF in lymph node metastatic lesions is also a significant prognostic parameter.
New prognostic histological parameter of invasive ductal carcinoma of the breast: clinicopathological significance of fibrotic focus. Hasebe T, Mukai K, Tsuda H, Ochiai A. Pathology Division, National Cancer Center Research Institute East, Chiba, Japan.	Pathol Int 2000 Apr;50(4):263-72 Abstract quote Immunohistochemistry, DNA ploidy analysis and molecular genetics have made it possible to predict the outcome of breast cancer more precisely than routine histological examination alone. However, in routine practice, it is difficult to incorporate these methodologies in all cases. If certain histological parameters can accurately predict the outcome of patients with breast cancer, they would be more practical for routine use. We showed that the presence of fibrotic focus (FF) in invasive ductal carcinoma (IDC) is closely associated with c-erbB-2 or p53 protein expression, high proliferative activity, and high angiogenesis of the tumors. Furthermore, multivariate analyses with well-known prognostic parameters for IDC demonstrated that the presence of FF is the most useful independent parameter to predict IDC patient outcome. In addition, our data suggested that the interaction between tumor cells and stromal fibroblasts may play an important role in the formation of FF in IDC based on growth factor and growth factor receptor protein expression in the tumor cells and fibroblasts forming FF. Based on the results of our clinicopathological studies, we propose a new prognostic classification scheme for the prediction of IDC patient outcome, which consists of FF, nuclear atypia, and fat invasion. This classification has superior predicting power to existing prognostic classifications.
Prognostic significance of fibrotic focus in invasive ductal carcinoma of the breast: a prospective observational study. Hasebe T, Sasaki S, Imoto S, Mukai K, Yokose T, Ochiai A. Pathology Division (TH, TY, AO).	Mod Pathol 2002 May;15(5):502-16 Abstract quote We have already reported invasive ductal carcinomas (IDCs) with fibrotic focus (FF) to be associated with significantly poorer survival than IDCs without FF. The purpose of this study was to prospectively investigate the effect of the presence of FF on the outcomes of 439 patients with IDCs to confirm the prognostic significance of FF, by the multivariate analysis, employing the Cox proportional hazard regression model, as compared with well-known clinicopathological parameters. We also precisely evaluated the prognostic significance of FF from the viewpoint of FF characteristics. The present study demonstrated that the presence of FF is a very useful parameter predicting tumor recurrence (TR), as well as initial distant organ metastasis (IDOM), in lymph node-negative IDCs (P =.024 and P =.026) and in IDCs positive for either or both estrogen receptor (ER) or progesterone receptor (PR) (P =.007 and P =.015), respectively. In addition, FF of >8 mm in diameter was found to be an independent prognostic parameter for TR and IDOM in lymph node-negative patients and patients with IDC positive for either or both ER or PR (P =.005 and P =.018). We conclude that the presence of FF is a very important histologic prognostic parameter for patients with IDCs of the breast.
GLUTATHIONE S-TRANSFERASE
Prognostic significance of glutathione S-transferase-pi in invasive breast cancer. Huang J, Tan PH, Thiyagarajan J, Bay BH. Department of Medical Oncology, National Cancer Centre, Singapore.	Mod Pathol. 2003 Jun;16(6):558-65. Abstract quote Glutathione S-transferase pi (GST-pi), a Phase II detoxification enzyme, has recently been implicated in protection against apoptosis. Expression of GST-pi and Bcl-2 protein, an established apoptosis marker, was analyzed by immunohistochemistry in 116 cases of infiltrative ductal breast carcinomas in Singapore women. The markers were correlated with apoptosis detected by the TUNEL method and clinico-pathological parameters. There were 67 (58%) GST-pi-positive breast tumors and 43 (37%) Bcl-2-positive tumors. In a large proportion of GST-pi-positive/Bcl-2-positive tumors, there was a distinct accumulation of the GST-pi enzyme within the nucleus of cancer cells when examined by double immunofluorescence labeling under confocal microscopy. GST-pi immunoreactivity was not significantly correlated with any of the traditional histologic factors known to influence prognosis, whereas Bcl-2 overexpression was associated with reduced size of primary tumor (P =.021) and positive estrogen receptor status (P =.001). Univariate analysis revealed that GST-pi-positive, Bcl-2-positive, and lower histological grade tumors had decreased levels of apoptosis (P =.024, P =.011, and P =.029, respectively). However, multivariate analysis showed that histological grade and Bcl-2, but not GST-pi, immunoreactivity were correlated with apoptotic status. The Kaplan-Meier disease-free survival curves showed a significant difference between GST-pi-positive and GST-pi-negative breast cancer cases (P =.002). Disease-free survival in patients with GST-pi-positive tumors was also worse than that in patients with GST-pi-negative tumors in the group who had adjuvant chemotherapy (P =.04). In patients who were lymph node positive, GST-pi immunopositivity was found to influence disease-free survival. Recurrence of tumors was also significantly affected by GST-pi immunoreactivity (relative risk of 8.1). The findings indicate that GST-pi-positive tumors are more aggressive and have a poorer prognosis than do corresponding GST-pi-negative breast cancers.
GRADING
Breast carcinoma malignancy grading by Bloom-Richardson system vs proliferation index: reproducibility of grade and advantages of proliferation index. Meyer JS, Alvarez C, Milikowski C, Olson N, Russo I, Russo J, Glass A, Zehnbauer BA, Lister K, Parwaresch R. 1St Luke's Hospital, Chesterfield, MO, USA.	Mod Pathol. 2005 Aug;18(8):1067-78. Abstract quote   Questions of reproducibility and efficacy of histologic malignancy grading relative to alternative proliferation index measurements for outcome prediction remain unanswered. Microsections of specimens from the Cooperative Breast Cancer Tissue Resource (CBCTR) were evaluated by seven pathologists for reproducibility of grade and classification. Nuclear figure classification was assessed using photographs. Grade was assigned by the Bloom-Richardson method, Nottingham modification. Proliferation index was evaluated prospectively by deoxyribose nucleic acid precursor uptake with thymidine (autoradiographic) or bromodeoxyuridine (immunohistochemical) labeling index using fresh tissue from 631 node-negative breast cancer patients accessioned at St Luke's Hospital. A modified Nottingham-Bloom-Richardson grade was derived from histopathologic data. Median post-treatment observation was 6.4 years. Agreement on classification of nuclear figures (N=43) was less than good by kappa statistic (kappa=0.38). Grade was moderately reproducible in four trials (N=10,10,19, 10) with CBCTR specimens (kappa=0.50-0.59). Of components of Bloom-Richardson grade, agreement was least for nuclear pleomorphism (kappa=0.37-0.50), best for tubularity (kappa=0.57-0.83), and intermediate for mitotic count (kappa=0.45-0.64). Bloom-Richardson grade was a univariate predictor of prognosis in node-negative St Luke's patients, and was improved when mitotic count was replaced by labeling index (low, mid, or high). When labeling index was added to a multivariate model containing tumor size and vessel invasion, grade was no longer a significant predictor of tumor-specific relapse-free or overall survival. Mitotic index predicted best when intervals were lowered to 0-2, 3-10, and >10 mitotic figures per ten 0.18 mm(2) high-power fields. We conclude that Nottingham-Bloom-Richardson grades remain only modestly reproducible. Prognostically useful components of grade are mitotic index and tubularity. The Nottingham-Bloom-Richardson system can be improved by lowering cutoffs for mitotic index and by counting 20-30 rather than 10 high-power fields. Measurement of proliferation index by immunohistochemically detectable markers will probably give superior prognostic results in comparison to grade.
HORMONE RECEPTOR STATUS
Progesterone receptor by immunohistochemistry and clinical outcome in breast cancer: a validation study. Mohsin SK, Weiss H, Havighurst T, Clark GM, Berardo M, Roanh le D, To TV, Zho Q, Love RR, Allred DC. [1] 1The Breast Center, Baylor College of Medicine, Houston, TX, USA [2] 2Department of Pathology, The Methodist Hospital, Houston, TX, USA.	Mod Pathol. 2004 Dec;17(12):1545-54. Abstract quote   Progesterone receptor is a surrogate marker of estrogen receptor activity in breast cancer and its utility in helping predict clinical outcome has been established using biochemical assays. However, most laboratories worldwide have switched to immunohistochemistry to assess progesterone receptor, but unfortunately no validated immunohistochemical assay exists for progesterone receptor. The purpose of this study was to develop and validate an immunohistochemical assay for progesterone receptor in breast cancer. The assay was based on monoclonal antibody 1294 (DakoCytomation) and slides were scored microscopically using the 'Allred score' on a scale of 0-8. The assay was compared to ligand-binding assay in 1235 breast cancers, and a subset (n=362) that received only hormonal therapy was used to define a cutoff for progesterone receptor-positive. Clinical utility was validated in an independent set of samples (n=423) from a clinical trial randomizing premenopausal breast cancer patients to tamoxifen+oophorectomy vs observation following surgery. A cutoff of >2 (corresponding to >1% positive cells) dichotomized patients with significantly better or worse clinical outcome (P=0.0014). Progesterone receptor by immunohistochemistry provided significantly better results than progesterone receptor by ligand-binding assay in predicting clinical outcome. In the clinical trial, a positive result in univariate analyses was associated with significantly improved disease-free and overall survival both in untreated (hazard ratios/P=0.656/0.060 and 0.479/0.005, respectively) and hormonally treated patients (hazard ratios/P=0.529/0.017 and 0.451/0.007, respectively). Positive progesterone receptor remained significant for improved disease-free and overall survival (hazard ratios/P=0.666/0.038 and 0.549/0.007, respectively) in multivariate analyses including the standard variables of tumor size, nodal status, treatment, histological grade, and HER-2/neu status. Estrogen and progesterone receptors are codependent variables and progesterone receptor was a weaker predictor of response to endocrine therapy than estrogen receptor when both were included in multivariate analysis. This is the first comprehensive study assessing the clinical usefulness of progesterone receptor by immunohistochemistry in archival tissue in breast cancer. Progesterone receptor assessed by immunohistochemistry provides useful information about clinical outcome and it is better than progesterone receptor measured by ligand-binding assay.
Androgen Receptor Expression in Estrogen Receptor–Negative Breast Cancer Immunohistochemical, Clinical, and Prognostic Associations S. Nicholas Agoff, MD, Paul E. Swanson, MD, Hannah Linden, MD, Stephen E. Hawes, PhD, and Thomas J. Lawton, MD	Am J Clin Pathol 2003;120:725-731 Abstract quote We sought to determine the prevalence of androgen receptor (AR) expression in a predominantly estrogen receptor (ER)-negative subset of breast cancers and delineate the immunohistochemical and clinical associations, including whether AR expression has prognostic significance in ER-negative tumors. We identified 69 ER-negative and 19 ER-positive breast cancer cases with concurrent immunohistochemical prognostic panels (ER, PR, HER-2/neu, Ki-67, and p53); immunohistochemical analysis was performed for AR using standard techniques. Clinical data were extracted from medical records. c 2 tests were used to assess associations between variables. AR was found in 49% (34/69) of ER-negative and 89% (17/19) of ER-positive cases. In ER-negative tumors, AR was associated with increased age ( P = .02), postmenopausal status ( P < .001), tumor grade ( P = .03), tumor size ( P = .03), and HER-2/neu overexpression ( P = .003). In ER-positive tumors, AR was associated with progesterone receptor expression ( P < .03). In univariate analysis of ER-negative tumors, patients with AR-positive tumors had significantly better disease-free survival ( P = .049). AR is expressed in a significant number of ER-negative cases and shows significant associations with important clinical and pathologic prognostic factors.
Morphologic spectrum of estrogen receptor-negative breast carcinoma. Scawn R, Shousha S. Department of Histopathology, Charing Cross Hospital and Imperial College of Science, Technology and Medicine, London, United Kingdom.	Arch Pathol Lab Med 2002 Mar;126(3):325-30 Abstract quote Context.-Estrogen receptor (ER)-negative breast carcinomas are a heterogeneous group of breast cancers that are generally thought to be aggressive. Objective.-To determine the morphologic and immunohistochemical spectrum of a consecutive series of ER-negative breast carcinomas, in an attempt to understand the pathogenesis and behavior of these lesions. Design.-Seventy-four consecutive cases of ER-negative invasive carcinomas were studied. Hematoxylin-eosin-stained sections were reviewed, and new sections were stained for c-erbB-2, p53, vimentin, and androgen and prolactin receptors. The findings were correlated with the axillary lymph node status as a measure of tumor aggressiveness. Setting.-The histopathology department of a tertiary referral teaching hospital. Results.-The tumors included 50 (68%) invasive ductal carcinomas, 21 (28%) medullary/atypical medullary carcinomas, and 1 each of invasive lobular, apocrine, and papillary carcinoma. Some of the invasive ductal cases had distinctive features that are described in this report. Maximum tumor diameter varied between 5 and 100 mm. Sixty tumors (81%) were grade 3, 13 (18%) were grade 2, and 1 (1%) was grade 1. Of the 60 cases in which the axillary node status was known, 34 (57%) had metastases, and 26 did not. Tumors associated with positive nodes were significantly larger than those associated with negative nodes (37.2 vs 17.8 mm, P <.001). A higher percentage of node-negative tumors were c-erbB-2 positive (42% vs 21%, P <.05). There were no differences between the 2 groups with regard to histologic type, tumor grade, or the expression of p53, vimentin, or androgen or prolactin receptors Conclusions.-Many ER-negative breast carcinomas have distinctive microscopic features. Not all ER-negative tumors are aggressive, as judged by the absence of lymph node metastases in 43% of cases in this series. Tumor size is the most important indicator for the likelihood of the presence of lymph node metastases. The wide range of tumor sizes encountered in this series suggests that the ER status of a tumor is determined early in its natural history and supports the existence of 2 separate pathways for the development of ER-negative and ER-positive breast carcinomas.
HYPER-CHOLESTEROLEMIA
Hypercholesterolemia impairs angiogenesis in patients with breast carcinoma and, therefore, lowers the risk of metastases. Ozdemir BH, Akcali Z, Haberal M. Department of Pathology, Baskent University Faculty of Medicine, Ankara, Turkey.	Am J Clin Pathol. 2004 Nov;122(5):696-703. Abstract quote   Our aim was to study the effect of hypercholesterolemia on angiogenesis induced by breast carcinoma. Of 51 patients with invasive ductal carcinoma, 28 had hypercholesterolemia and 23 had normocholesterolemia. The intratumoral microvessel density (MVD) was evaluated by using anti-CD31 antibody. The expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) on endothelial and tumor cells was examined and graded semiquantitatively. Patients with normocholesterolemia had a higher MVD (76.4 +/- 8.2) than those with hypercholesterolemia (54.6 +/- 5.1) (P < .01). The risks of recurrence and distant metastasis were higher in patients with normocholesterolemia than in patients with hypercholesterolemia (P < .01). Patients with hypercholesterolemia showed lower expression of endothelial VEGF and bFGF than patients with normocholesterolemia (P < .05 and P < .01, respectively). In addition, tumoral bFGF and VEGF expression showed negative correlation with the presence of hypercholesterolemia (P < .01). We suggest that hypercholesterolemia impairs angiogenesis by suppressing endothelial and tumoral bFGF and VEGF expression and, therefore, lowers the risk of metastases in cases of invasive breast carcinoma.
IPSILATERAL BREAST FAILURE
Molecular Clonality Relationships in Initial Carcinomas, Ipsilateral Breast Failures, and Distant Metastases in Patients Treated With Breast-Conserving Therapy Evidence Suggesting That Some Distant Metastases Are Derived From Ipsilateral Breast Failures and That Metastases Can Metastasize Neal S. Goldstein, MD, etal.	Am J Clin Pathol 2005;124:49-57 Abstract quote We studied the clonality relationships in invasive breast carcinomas, ipsilateral breast failures (IBFs), and distant metastases (DMs) using a polymerase chain reaction–loss of heterozygosity (LOH) clonality assay to determine whether IBFs can be the source of DMs. Six cases of initial carcinomas, IBFs, and DMs were identified. Carcinoma DNA was extracted from paraffin blocks and analyzed with 20 markers. In 2 cases, the LOH pattern suggested the DM directly resulted from the IBF. In 2 cases, the initial carcinoma, IBF, and DM were one progressive, genetically unstable process. Separate subclones in the initial carcinoma gave rise to the IBF and DM in 1 case, and the DM derived from a second IBF in 1 case. The relationships of initial carcinomas, IBFs, and DMs are complex. DMs seem to be the direct result of IBFs in some cases. Some carcinomas seem to be composed of subclones with different and unrelated IBF and DM potential.
Molecular Clonality Determination of Ipsilateral Recurrence of Invasive Breast Carcinomas After Breast-Conserving Therapy Comparison With Clinical and Biologic Factors Neal S. Goldstein, MD, etal.	Am J Clin Pathol 2005;123:679-689 Abstract quote We established clonality relationships between invasive ipsilateral breast failures (IBFs; local recurrences) and initial invasive carcinomas using a molecular polymerase chain reaction loss of heterozygosity (LOH) assay for 26 patients treated with breast-conserving therapy for invasive carcinoma with no distant metastases (DMs) before the IBF. LOH was ± 50% allelic loss. Eighteen IBFs (69%) were related clonally to initial carcinomas; 8 (31%) were clonally distinct, second primary carcinomas. IBFs and initial invasive carcinomas were morphologically similar in 6 (75%) of 8 clonally different cases. Clinical IBF classification and molecular assay results differed in 11 cases (42%). The mean intervals to IBF were 4.7 years in related and 8.7 years in different cases (P = .013). In 6 patients, DMs developed; 5 had related IBFs. In related IBF cases, the mean increase in fractional allelic loss (FAL) of IBFs associated with DMs was 18.9% compared with 7.6% in cases unassociated with DMs (P = .004). Molecular assays can accurately establish the clonality of most IBFs. Morphologic comparison and clinical IBF classification are unreliable methods of determining clonality. Clonally related IBFs occurred sooner than clonally different IBFs. Patients with clonally related IBFs are the main pool in which DMs occur. Not all clonally related IBFs have the same DM association; those with large FAL gains were associated with DMs. Molecular clonality assays may provide a reliable means of identifying patients who might benefit from systemic chemotherapy at the time of IBF.
Ki-67
Immunohistochemical detection of Ki67 in breast cancer correlates with transcriptional regulation of genes related to apoptosis and cell death. Tan PH, Bay BH, Yip G, Selvarajan S, Tan P, Wu J, Lee CH, Li KB. 1Department of Pathology, Singapore General Hospital, Singapore.	Mod Pathol. 2005 Mar;18(3):374-81. Abstract quote   Ki67 is a nuclear protein that is tightly linked to the cell cycle. It is a marker of cell proliferation and has been used to stratify good and poor prognostic categories in invasive breast cancer. Its correlation with gene expression patterns has not been fully elucidated. In this study, Ki67 immunohistochemistry using the MIB-1 antibody was performed on sections cut from 21 formalin-fixed, paraffin-embedded invasive breast cancers. Scoring was determined as nil (no immunostaining), low (10% or less immunopositivity) or high (>10% immunoreactive cells) respectively. The relationship of Ki67 immunohistochemical detection with clinicopathologic parameters was evaluated. Using Affymetrix U133A GeneChips, expression profiles for these tumors were generated and correlated with Ki67 immunohistochemical findings. Analysis of variance was used to define genes that were differentially regulated between the groups. Real-time polymerase chain reaction (PCR) was used to confirm the presence of a downregulated gene. Our results showed high, low and nil Ki67 immunostaining in nine (43%), six (28.5%) and six (28.5%) invasive breast cancers respectively, with increased Ki67 protein expression correlating with high histologic grade (P=0.02), mitotic score (P=0.001) and estrogen receptor immunonegativity (P=0.002). Expression profiling trends of the Ki67 gene mirrored the observed proportions of immunostained cells when the Ki67 immunoscore was >10%. Genes related to apoptosis and cell death (bcl2, MAP2K4, TNF10) were noted to be downregulated in tumors that disclosed >40% Ki67 immunostaining (P<0.001). Downregulation of the bcl2 gene was confirmed at the RNA level by real-time RT-PCR. Differential regulation of these genes, especially bcl2, may contribute to the biological nature of clinically more aggressive and highly proliferative breast cancers.
LOBULAR CARCINOMA IN SITU
The relation between the presence and extent of lobular carcinoma in situ and the risk of local recurrence for patients with infiltrating carcinoma of the breast treated with conservative surgery and radiation therapy Abner AL, Connolly JL, Recht A, Bornstein B, Nixon A, Hetelekidis S, Silver B, Harris JR, Schnitt SJ. Joint Center for Radiation Therapy, Harvard Medical School, Boston, MA 02215, USA	Cancer 2000 Mar 1;88(5):1072-7 Abstract quote BACKGROUND: When found in an otherwise benign biopsy, lobular carcinoma in situ (LCIS) has been associated with an increased risk of development of a subsequent invasive breast carcinoma. However, the association between LCIS and the risk of subsequent local recurrence in patients with infiltrating carcinoma treated with conservative surgery and radiation therapy has received relatively little attention. METHODS: Between 1968 and 1986, 1625 patients with clinical Stage I-II invasive breast carcinoma were treated at the Joint Center for Radiation Therapy at Harvard Medical School with breast-conserving surgery (CS) and radiation therapy (RT) to a total dose to the primary site of > or =60 grays. Analysis was limited to 1181 patients with infiltrating ductal carcinoma, infiltrating lobular carcinoma, or infiltrating carcinoma with mixed ductal and lobular features who, on review of their histologic slides, had sufficient normal tissue adjacent to the tumor to evaluate for the presence of LCIS and also had a minimum potential follow-up time of 8 years. The median follow-up time was 161 months. RESULTS: One hundred thirty-seven patients (12%) had LCIS either within the tumor or in the macroscopically normal adjacent tissue. The 8-year crude risk of recurrence was not significantly increased for patients with LCIS associated with invasive ductal, invasive lobular, or mixed ductal and lobular carcinoma. Among the 119 patients with associated LCIS adjacent to the tumor, the 8-year rate of local recurrence was 13%, compared with 12% for the 1062 patients without associated LCIS. For the 70 patients with moderate or marked LCIS adjacent to the tumor, the 8-year rate of local recurrence was 13%. The extent of LCIS did not affect the risk of recurrence. The risks of contralateral disease and of distant failure were similarly not affected by the presence or extent of LCIS. CONCLUSIONS: Breast-conserving therapy involving limited surgery and radiation therapy is an appropriate method of treating patients with invasive breast carcinoma with or without associated LCIS. Neither the presence nor the extent of LCIS should influence management decisions regarding patients with invasive breast carcinoma.
Lobular carcinoma in situ increases the risk of local recurrence in selected patients with stages I and II breast carcinoma treated with conservative surgery and radiation. Sasson AR, Fowble B, Hanlon AL, Torosian MH, Freedman G, Boraas M, Sigurdson ER, Hoffman JP, Eisenberg BL, Patchefsky A. Department of Surgical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19104, USA	Cancer 2001 May 15;91(10):1862-9 Abstract quote BACKGROUND: Lobular carcinoma in situ (LCIS) is a known risk factor for the development of invasive breast carcinoma. However, little is known regarding the impact of LCIS in association with an invasive carcinoma on the risk of an ipsilateral breast tumor recurrence (IBTR) in patients who are treated with conservative surgery (CS) and radiation therapy (RT). The purpose of this study was to examine the influence of LCIS on the local recurrence rate in patients with early stage breast carcinoma after breast-conserving therapy. METHODS: Between 1979 and 1995, 1274 patients with Stage I or Stage II invasive breast carcinoma were treated with CS and RT. The median follow-up time was 6.3 years. RESULTS: LCIS was present in 65 of 1274 patients (5%) in the study population. LCIS was more likely to be associated with an invasive lobular carcinoma (30 of 59 patients; 51%) than with invasive ductal carcinoma (26 of 1125 patients; 2%). Ipsilateral breast tumor recurrence (IBTR) occurred in 57 of 1209 patients (5%) without LCIS compared with 10 of 65 patients (15%) with LCIS (P = 0.001). The 10-year cumulative incidence rate of IBTR was 6% in women without LCIS compared with 29% in women with LCIS (P = 0.0003). In both groups, the majority of recurrences were invasive. The 10-year cumulative incidence rate of IBTR in patients who received tamoxifen was 8% when LCIS was present compared with 6% when LCIS was absent (P = 0.46). Subsets of patients in which the presence of LCIS was associated with an increased risk of breast recurrence included tumor size < 2 cm (T1), age < 50 years, invasive ductal carcinoma, negative lymph node status, and the absence of any adjuvant systemic treatment (chemotherapy or hormonal therapy) (P < 0.001). LCIS margin status,