Statistical association of basal cell keratins with metastasis-inducing proteins in a prognostically unfavorable group of sporadic breast cancers.

Two subgroups of invasive breast carcinomas have been identified with a poor prognosis in different patient cohorts: the basal-like category and the subgroup containing proteins capable of inducing metastasis in experimental rodents, the metastasis-inducing proteins (MIPs). Here we identify by immunohistochemical staining for cytokeratin CK5/6 or CK14 the basal-like subgroup in a set of 297 primary invasive breast carcinomas in which the staining profile for the MIPs S100A4, osteopontin, anterior gradient-2, and S100P has already been established. Monoclonal antibodies to CK5/6 or CK14 specifically stain 31% to 34% of the primary carcinomas. These positively stained tumors are highly significantly associated with premature death of the patient (Wilcoxon statistics, P < 0.0001), the increased relative risk being approximately 5.6-fold. Positive staining for either cytokeratin is very significantly associated with that for each of the four MIPs separately and with loss of staining for the Fanconi anemia protein FANCD2 (corrected Fisher's exact test, P < 0.0007). There is no significant correlation with the remaining tumor variables tested, including staining for the estrogen receptor α, progesterone receptor, and c-erbB-2. These results show that the basal cytokeratin-like carcinomas contain many of the MIPs and that these may arise by their selection for tumors with an inherent deficiency in the FANC/BRCA pathway of DNA repair.

Significance of the Fanconi anemia FANCD2 protein in sporadic and metastatic human breast cancer.

FANCD2, a pivotal protein in the Fanconi anemia and BRCA pathway/network, is monoubiquitylated in the nucleus in response to DNA damage. This study examines the subcellular location and relationship with prognostic factors and patient survival of FANCD2 in breast cancer. Antibodies to FANCD2 were used to immunocytochemically stain 16 benign and 20 malignant breast specimens as well as 314 primary breast carcinomas to assess its association with subcellular compartment and prognostic factors using Fisher's Exact test or with patient survival over 20 years using Wilcoxon-Gehan statistics. Immunoreactive FANCD2 was found in the nucleus and cytoplasm of all 16 benign tissues, but nuclear staining was lost from a significant 19/20 malignant carcinomas (P < 0.0001). Antibodies to FANCD2 stained the cytoplasm of 196 primary carcinomas, leaving 118 as negatively stained. Negative cytoplasmic staining was significantly associated with positive staining for the metastasis-inducing proteins S100A4, S100P, osteopontin, and AGR2 (P < or = 0.002). Survival of patients with FANCD2-negative carcinomas was significantly worse (P < 0.0001) than those with positively stained carcinomas, and only 4% were alive at the census date. Multivariate regression analysis identified negative staining for cytoplasmic FANCD2 as the most significant indicator of patient death (P = 0.001). Thus FANCD2's cytoplasmic loss in the primary carcinomas may allow the selection of cells overexpressing proteins that can induce metastases before surgery.

The metastasis-associated anterior gradient 2 protein is correlated with poor survival of breast cancer patients.

The secreted metastasis-inducing protein, human anterior gradient 2 (AGR2), has been independently reported to be associated with either a reduced or an increased survival of different groups of patients with breast cancer. We now aim to analyze the expression of AGR2 in a third completely independent group of patients using a specific AGR2 monoclonal antibody (mAb). Primary tumors from a group of 315 patients suffering from operable (stage I and II) breast cancer with 20-years follow-up were immunocytochemically stained with a specific mAb to AGR2 and associations with prognostic factors and patient survival were analyzed. The mAb specifically recognized AGR2 in Western blots, and positive staining for AGR2 was significantly associated with involved lymph nodes and staining for estrogen receptor alpha, progesterone receptor, and the metastasis-inducing proteins osteopontin, S100P, and S100A4. After 20 years of follow-up, only 26% of patients with AGR2-positive carcinomas survived compared with 96% of those with AGR2 negative carcinomas, with the highly significant difference in median survival times of 68 and >216 months, respectively (P < 0.0001). Cox's multivariate regression analysis showed that staining for AGR2 was one of the most significant independent prognostic indicators, with a corrected relative risk of 9.4. The presence of AGR2 in the primary tumor is therefore a possible prognostic indicator of poor patient outcome in breast cancer.

Induction of metastasis by S100P in a rat mammary model and its association with poor survival of breast cancer patients.

S100P, an EF-hand calcium-binding protein, has been reported to be associated with the progression of many types of cancers. Transfection of an expression vector for S100P into a benign, nonmetastatic rat mammary cell line causes a 4- to 6-fold increase in its level in all four transformant cell clones. When the resultant transformant cell lines are introduced in turn into the mammary fat pads of syngeneic Furth-Wistar rats, there is a significant 3-fold increase in local muscle invasion and a significant induction of metastasis in 64% to 75% of tumor-bearing animals. In a group of 303 breast cancer patients followed for up to 20 years, antibodies to S100P immunocytochemically stain 161 primary tumors. Survival of patients with S100P-positive carcinomas is significantly worse by about 7-fold than for those with negatively stained carcinomas. There is also a significant association between the class level of immunocytochemical staining of the carcinoma cells and decreased patient survival. Positive staining for S100P is significantly associated with that for two other metastasis-inducing proteins, S100A4 and osteopontin. Patients with tumors that stained positively for both S100P and S100A4 have a significantly reduced survival of 1.1% over patients with either S100 protein alone. Multivariate regression analysis identifies S100P, S100A4, and osteopontin as the most significant independent indicators of death in this group of patients. These results suggest that stratification of patients into groups according to expression of multiple metastasis-inducing proteins may lead to a more accurate prediction of patient survival.

Association of S100A4 and osteopontin with specific prognostic factors and survival of patients with minimally invasive breast cancer.

PURPOSE: S100A4 and the estrogen-inducible osteopontin are alone capable of inducing angiogenesis and metastasis in rodent models for breast cancer. The present study assesses the relationship of S100A4 and osteopontin with vessel density and estrogen receptor alpha (ERalpha) in primary tumors and with survival of patients to ascertain their involvement in metastatic breast cancer. EXPERIMENTAL DESIGN: Primary tumors from 312 patients treated for minimally invasive human breast cancer were immunocytochemically stained and then assessed for the significance of their association with each other using Fisher's exact test or with patient survival over 18 years of follow-up using Kaplan-Meier plots and Wilcoxon-Gehan statistics. RESULTS: Antibodies to S100A4 significantly stained endothelial cells of vessels adjacent to S100A4-staining groups of carcinoma cells, and antibodies to osteopontin significantly stained groups of carcinoma cells staining for ERalpha (P < 0.0001). There was a significant association of tumors staining for S100A4 with those with high vessel density (P = 0.021) and of tumors staining for osteopontin with those staining for ERalpha (P = 0.034). The association of staining for S100A4, osteopontin, or vessel density with patient death was significant (P < 0.0001, P = 0.005, and P = 0.014, respectively). The difference in cumulative proportion surviving between S100A4-positive patients with higher or lower vessel density increased up to about 12 years, but thereafter decreased to virtually zero after 18 years of follow-up. Patients with both S100A4-positive and osteopontin-positive primary tumors showed a statistically significant reduction in survival time over those with either one alone (P < 0.019), although in multivariate regression analysis, only staining for S100A4 was significant (P < 0.001). CONCLUSIONS: It is suggested that in human breast cancer, S100A4 exerts some of its effects through angiogenesis, and that osteopontin is dependent on ERalpha for its expression.

Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer.

The adhesive glycophosphoprotein (OPN) is capable of inducing metastasis in rodent models ofbreast cancer. We now show that a monoclonal antibody to rat OPN recognizes specifically human OPN using Western blotting techniques andused it to assess the prognostic significance of OPN in primary tumors of a group of 333 patients treated between 1976 and 1982 for operable stage I and stage II breast cancer. The antibody stains immunocytochemically normal breast tissue weakly but pregnant/lactating tissue and 66% of the carcinomas strongly, leaving the remaining 34% as negatively stained. In addition to the carcinoma cells, some host reactive stromal cells, macrophages, lymphocytes, and blood vessels are also stained, but these have been excluded in the following analyses. There is a significant association of staining of carcinomas for OPN with some tumor variables reported previously to be associated with patient outcome: high histological grade (P = 0.024), staining for c-erbB-3 (P < 0.001), p53 (P = 0.014), pS2 (P = 0.025), and borderline significance for progesterone receptor (P = 0.089). The association of staining for OPN with survival times of the patients has been evaluated using life tables over 14-20 years of follow-up (mean 16 years) and analyzed using generalized Wilcoxon statistics. Of the patients who have been classified as OPN-negative, 94% are alive, but only 26% of those classified as OPN-positive are alive after 19 years of follow-up. This association is highly significant (P < 0.0001); the former have a median survival of >228 months and the latter 68 months. When the patients are divided into separate classes based on the percentage of carcinoma cells staining for OPN, the five classes show a progressive decrease in survival with increasing percentage of stained carcinoma cells, and this association is also highly significant (P < 0.0001). Other tumor variables that show a significant association with patient survival times in this group of patients include nodal status, tumor size, histological grade, staining for c-erbB-2, estrogen receptor alpha, or p53. Analysis of the association of patients with carcinomas staining for OPN and their survival in subgroups defined by these tumor variables shows that positive staining for OPN in each subgroup is associated with poorer survival. There is little difference in patient survival times in the OPN-negative group of patients with or without any of the other tumor variables examined. Multivariate regression analysis for 202 patients shows that staining for OPN is most highly correlated with patients' deaths (P < 0.0001), but involved lymph nodes (P = 0.0007), fixed tumors (P = 0.0008), and staining for estrogen receptor alpha (P = 0.008) are also significant independent prognostic variables with that for c-erbB-2 being of borderline significance (P = 0.060). These results suggest that in this group of patients, the presence of the metastasis-associated protein OPN is tightly correlated with patient demise.

Hand surgery after axillary lymph node clearance for breast cancer: contra-indication to surgery?

INTRODUCTION: Breast cancer patients who have had prior axillary lymph node clearance (ALNC) can present with ipsilateral hand conditions that could easily be treated with surgical intervention. These patients are often advised to avoid interventional procedures due to risks of complications such as lymphoedema, infection and cellulitis. SUBJECTS AND METHODS: Between April and June 2009, we conducted an online survey of hand surgeons, breast surgeons and breast-care nurses to obtain their views on hand surgery after ipsilateral axillary lymph node clearance. RESULTS: The majority of hand surgeons (58%) felt there was no contra-indication to surgery in a breast cancer patient with prior ipsilateral ALNC compared to just 30% of breast surgeons and 10% of breast-care nurses. The majority of breast surgeons and breast-care nurses (70% and 89%, respectively) felt that hand surgery was a relative contra-indication compared to just 41% of hand surgeons. Postoperative lymphoedema was the commonest cited reason for avoiding surgery. The majority of hand surgeons (79%) and nearly two-thirds of breast surgeons (57%) would use a tourniquet during surgery if it was normal practice. CONCLUSIONS: A review of the published literature does not support the notion that these patients experience increased complications; therefore, we recommend the advice given to breast cancer patients regarding ipsilateral surgery be re-evaluated.

Audit of blood transfusion in elective breast cancer surgery--do we need to group and save pre-operatively?

INTRODUCTION: Current guidelines on blood ordering in our hospital require all patients undergoing elective breast cancer surgery to have blood grouped, screened and saved as an part of a pre-operative assessment. The aim of this audit was to assess the need for, and cost effectiveness of, this approach in elective breast cancer surgery. PATIENTS AND METHODS: Retrospective data collection was undertaken for a 2-year period using the theatre booking system. As a result, 497 consecutive elective breast surgery operations including mastectomies, wide local excisions and breast reconstruction procedures were identified for analysis. Using the hospital blood bank computer system, we established the blood group and save or cross-match status as well as the pre- and postoperative haemoglobin results and blood transfusion related data for each of the patients identified. RESULTS: Of the 497 patients, 438 (88.1%) had blood sent for group-and-save. Of the total 497 patients identified, only 19 (3.82%) patients received a blood transfusion. From the 447 patients undergoing simple mastectomy or wide local excisions alone, 9 patients (1.81%) required transfusion. Fifty patients underwent an immediate reconstruction procedure of whom 10 (20%) required a transfusion. CONCLUSIONS: This study demonstrates that reconstruction is more likely to be associated with the need for a postoperative transfusion. However, in the context of all breast surgery, blood transfusion is rarely requested. Given this, the time and cost involved in processing a group-and-save pre-operatively is not justified.