Increased SOX2 expression in less differentiated breast carcinomas and their lymph node metastases.

AIMS: SOX2 is a key regulatory gene in embryonic stem cells. Although it has been implicated in cancer progression, its role in breast carcinoma is poorly understood. MATERIALS AND METHODS: Fifty-seven ductal carcinomas in situ (DCIS), 552 invasive breast carcinomas and 107 corresponding metastatic lymph nodes were evaluated immunohistochemically for the expression of SOX2. Its correlation with clinicopathological features, other biomarker profiles and patients' outcomes were analysed. RESULTS: SOX2 was detected in 19.0% (105 of 552) of invasive breast carcinomas and 12.3% (seven of 57) of DCIS. Expression correlated with larger tumour size (P = 0.005) and higher grade (P = 0.002). It was associated negatively with ER (P = 0.015) and PR (P = 0.046) expression, but positively with Ki67 index (P = 0.013). Interestingly, it was also associated with neuroendocrine marker expression (synpatophysin and chromogranin/synaptophysin, P = 0.048 and 0.028, respectively). Expression appeared to be independent from that of common stem cell markers, namely CD44, CD24 and aldehyde dehydrogenase 1 (ALDH1). Furthermore, a higher rate of expression was observed in metastatic lymph nodes than in the corresponding primary tumours (P = 0.034). High SOX2 expression was correlated with poor disease-free survival (log-rank=9.489, P = 0.012) and was an independent prognostic factor (HR=2.918, P = 0.015) in patients with high nodal stages. CONCLUSIONS: In summary, SOX2 expression was related to adverse breast carcinoma profile and poor outcome in selected patient groups.

Nerve growth factor receptor (NGFR): a potential marker for specific molecular subtypes of breast cancer.

AIMS: Nerve growth factor receptor (NGFR) is a transmembrane receptor for the neurotrophin family. It acts either as tumour suppressor or oncogene depending on cellular context. Its role in breast cancers remained conflicting, possibly due to the heterogeneity of breast cancer subtypes. METHODS: In this study, we have analysed NGFR expression in 602 cases of breast cancers by immunohistochemistry. Its expression was correlated with biomarker expression and different breast cancer subtypes. RESULTS: NGFR expression was found to be positively correlated with basal markers, including Ki67, Cytokeratin (CK5/6), CK14, p63, c-kit and Epidermal growth factor receptor (EGFR) , but negatively with hormonal receptors. Among different molecular subtypes, it was negatively associated with luminal A, but positively with luminal B, and basal-like breast cancer BLBC subtypes. When comparing NGFR with other basal markers in BLBC, though less sensitive, its specificity was comparable to or better than other basal markers. For luminal B cancers, NGFR showed a high specificity which was also comparable to or better than the defining markers (estrogen receptor (ER), progesterone receptor (PR), Human epidermal growth receptor 2 (HER2) and Ki-67) for the subtype. CONCLUSIONS: Overall, these findings suggested that NGFR expression could be indicative for the BLBCs or luminal B subtypes. It may represent a potential adjunct marker for these two subtypes.

Cancer stem cell markers are associated with adverse biomarker profiles and molecular subtypes of breast cancer.

Cumulative evidence has demonstrated the presence of cancer stem cells (CSC) in breast cancer and its putative role in cancer progression. Nonetheless, the clinical significance of CSC in breast cancer remains elusive. The underlying reasons could be due to the heterogeneity of breast cancer subtypes as well as different markers used to define breast CSC. In this study, three widely used markers (aldehyde dehydrogenase (ALDH)1+ and CD24-CD44+) were used to define two populations of CSC in a large cohort of breast cancers. The expressions of these markers were correlated with different clinicopathological features and the molecular subtypes. ALDH1+ breast cancers were associated with basal-like and HER2-overexpressing subtypes and the characteristics histologic features were related to these two subtypes. On the other hand, CD24-CD44+ breast cancers were associated positively with the presence of extensive in situ component, the absence of lymph node involvement, and basal markers, but negatively with HER2. CD24-CD44+ breast cancers were also positively associated with luminal B cancers. As the expression of CSC markers varied among different molecular subtypes and different clinicopathological features, it appeared that each CSC population could have distinct clinical values in different subgroups of breast cancers. For improved prognostication with CSC, combining the analysis of CSC markers would be required. Within the luminal cancers, CSC appeared to identify cancers with poor outcome. The presence of CSC populations was associated with ER-PR+ cancers and tumors expressing basal markers. Basal marker expression can complement with CSC for improved indicator for poor prognosis in luminal breast cancers. For the first time, the possible contribution of CSC to these aggressive luminal cancers was demonstrated. The association of basal features and CSC in luminal cancers also raised the possibility that luminal cancer cells may acquire basal phenotype and CSC properties together during their progression.

Keratin expression in breast cancers.

Cytokeratin (CK) immunohistochemistry can play an important role in breast carcinoma evaluation. We evaluated the expression of a panel of commonly used CKs in a large cohort of breast cancers and assessed its correlation with other biomarkers and breast cancer subtypes. Expression of CK7, CK8, CK18 and CK19 was observed in more than 90 % of all breast carcinomas in this study, confirming their efficacy in immunohistochemical identification of breast cancer. A combination of CK8 and CK7 gave the highest sensitivity for detection of a minute number of breast cancer cells. Expression of other CKs, including CK5/6, CK14 and CK20, correlated positively with high tumour grade. The expression of CK5/6 and CK14 in a significant number of high-grade tumours raised concern regarding the use of absence of their expression to identify breast carcinoma. For identification of the basal subtype, CK5/6 gave a higher detection rate than CK14. CK20 expression was found more frequently than reported in previous studies, might constitute an indicator of poor prognosis and may be associated with the molecular apocrine subtype. This study highlights the diagnostic and prognostic relevance of the unique CK expression patterns in breast cancer.

Involvement of α- and ß-catenins and E-cadherin in the development of mammary phyllodes tumours.

AIMS: Phyllodes tumours (PT) are rare but clinically important fibroepithelial tumours of the breast. ß-Catenin, a key component in Wnt signalling, has been shown to be important in the development of PT. It also functions as a component of the cadherin complex, which may therefore be implicated in PT pathogenesis. By assessing stromal α-catenin, ß-catenin and E-cadherin expression in 158 PT cases using immunohistochemistry and examining associations with clinicopathological features, we aimed to determine the role of these proteins in PT pathogenesis. METHODS AND RESULTS: Cytoplasmic ß-catenin correlated with α-catenin expression. A significantly higher expression of both markers was observed in borderline than in benign PT (P = 0.003 and <0.001, respectively), but a lower level was found in malignant PT. Cytoplasmic E-cadherin expression was significantly higher in borderline and malignant than in benign PT (P = 0.001 and 0.012, respectively), but was not correlated with other markers. Both E-cadherin and α-catenin showed stronger correlations with histological parameters than ß-catenin. α-Catenin showed a significant correlation with recurrence (P = 0.005 and 0.016, respectively). CONCLUSION: α- and ß-catenins may be important in the early stages of PT development, while E-cadherin may be required for malignant development. The correlation of α-catenin expression with tumour recurrence may be relevant in predicting PT behaviour.

Relationship between columnar cell changes and low-grade carcinoma in situ of the breast--a cytogenetic study.

Columnar cell lesions of the breast include columnar cell changes without atypia and columnar cell changes with atypia. The latter frequently coexist and share molecular changes with low-grade carcinoma in situ and invasive carcinoma, suggesting that columnar cell changes may be precursors to progression of low-grade advanced lesions. In this study, we assessed chromosomal aberrations at 16q, hallmark for low-grade lesions, in columnar cell changes with or without atypia and their adjacent carcinoma in situ by fluorescent in situ hybridization using 3 region-specific probes spanning the entire chromosomal arm. The results were correlated with the histomorphological features of the corresponding lesions. Forty-four percent of low-grade carcinoma in situ and 31% of high-grade carcinoma in situ were associated with columnar cell changes with atypia, suggesting a link between columnar cell changes with atypia and low-grade carcinoma in situ. For the genetic aberrations, heterozygous deletion of 16q was present in 56% of low-grade carcinoma in situ but only in 19% of high-grade carcinoma in situ. Conversely, aneuploidy was found mostly in high-grade carcinoma in situ (88%). Twenty percent of columnar cell changes with atypia but none of the columnar cell changes without atypia showed heterozygous deletion of 16q. Interestingly, the same changes in 16q were observed in the columnar cell changes and their associated low-grade carcinoma in situ lesions. These findings demonstrated a genetic commonality between columnar cell changes with atypia and low-grade carcinoma in situ and substantiated the precursor role of columnar cell changes with atypia for low-grade carcinoma in situ but not high-grade carcinoma in situ of the breast.

αB-crystallin is a useful marker for triple negative and basal breast cancers.

AIMS: Basal-like breast cancers (BLBCs), a breast cancer subtype with triple-negative status, pose significant problems in clinical management because of their aggressive behaviour. Recently, an association between αΒ-crystallin expression and BLBCs has been suggested, and we therefore investigated whether αΒ-crystallin could be a putative marker allowing BLBCs to be identified more accurately. METHODS AND RESULTS: We evaluated the expression of αB-crystallin and other biomarkers in 395 cases of breast carcinoma by immunohistochemistry, analysed the correlation of their expression with different breast cancer subtypes, and compared their sensitivity as well as specificity in identifying BLBCs. αΒ-crystallin expression was found to be correlated positively with basal markers and histological subtypes associated with BLBCs. A significant positive correlation of αΒ-crystallin expression was also found with triple-negative breast cancers (TNBC) (C = 0.409, P < 0.001) and BLBCs (C = 0.393, P < 0.001). Comparing αΒ-crystallin with other basal markers, only αΒ-crystallin demonstrated both high sensitivity (48.6%) and specificity (93.8%) as a TNBC marker. All other markers showed either a lower sensitivity of <40% or a lower specificity of <90%. αΒ-crystallin also demonstrated a high specificity (92.9%) and an even higher sensitivity (56.5%) for BLBCs. CONCLUSIONS: The findings indicated that αB-crystallin was a highly sensitive and specific marker for TNBCs and BLBCs.

Biopsy sampling of breast lesions: comparison of core needle- and vacuum-assisted breast biopsies.

Needle biopsy is now the initial investigation of choice for the pre-operative diagnosis of breast lesions. This includes core needle biopsy (CNB) and vacuum-assisted biopsy (VAB) with or without radiologic assistance. The performance indices of both of these biopsy techniques were evaluated. In a large cohort of patients with breast lesions including 464 cases (285 CNB and 179 VAB), with confirmed outcomes, the diagnostic accuracy was compared using parameters including quantitation of the sampling based on the total number of cores taken, cores containing breast parenchyma, and cores with lesion; and non-epithelial changes including necrosis and calcification. CNB showed a 99% PPV, 94% NPV, 96% sensitivity, and 99% specificity, whereas VAB demonstrated a 100% PPV, 100% NPV, 100% sensitivity, and 100% specificity. The correct diagnosis in CNB was proportional to the number of cores extracted, whereas accuracy of VAB was independent of the total number of cores taken. There was a positive correlation between the presence of calcification and malignancy in CNB, but not detected under VAB. CNB and VAB were equally efficient in palpable lesions, in detecting necrosis, and calcification. Large calcification was found to be associated with malignancy in both CNB and VAB. In non-palpable lesions, VAB was more effective in the detection of calcification. The diagnostic accuracy of VAB appeared to be independent of number of cores sampled, whereas CNB required a minimum of 3-4 cores to achieve high diagnostic accuracy.

Increased alpha-B-crystallin expression in mammary metaplastic carcinomas.

AIMS: Mammary metaplastic carcinoma is a rare breast carcinoma, and may present diagnostic difficulty. Alpha-B-crystallin has been recently reported to be expressed in basal-like and metaplastic carcinomas. METHODS AND RESULTS: Thirty-three metaplastic carcinomas, 44 conventional high-grade carcinomas and 28 mesenchymal spindle cell neoplasms as controls were assessed for their expression of αB-crystallin and conventional basal-like phenotypic markers CK5/6, CK14, p63, c-kit and epidermal growth factor receptor (EGFR) by immunohistochemistry. Alpha-B-crystallin staining was positive in 68% of the metaplastic carcinomas with cytoplasmic staining in all tumour cell components. CK5/6, CK14, p63, c-kit and EGFR stained 43%, 68%, 45%, 21% and 25% of the metaplastic carcinomas, respectively. Combining these markers, 84% of the metaplastic carcinomas expressed either αB-crystallin or CK14. In comparison, only 14% (six cases) of conventional high-grade carcinoma and 7% (two cases) of mesenchymal spindle cell neoplasm expressed αB-crystallin; all but one of these carcinomas were ER/PR/HER2 triple-negative. CONCLUSIONS: Using αB-crystallin for diagnosis of metaplastic carcinoma gives a 68% sensitivity, 88% specificity, 74% positive predictive value, 85% negative predictive value and 78% accuracy. The sensitivity is enhanced to 84% with combinations of αB-crystallin/CK14. Alpha-B-crystallin may be used as an adjunct marker in the diagnosis of metaplastic carcinoma.

Granulomatous mastitis: the histological differentials.

BACKGROUND: The management of granulomatous mastitis depends on the causative factor, and accurate diagnosis in distinguishing between idiopathic granulomatous mastitis (IGM) and tuberculous mastitis (TBM) is indispensable. This is particularly problematic in the cases of granulomatous mastitis in which the microbiological studies are negative. In this study, in a large cohort, the histological features for IGM and TBM were compared. METHODS: The histopathology files from the two participating hospitals were searched for cases of granulomatous inflammation of the breast over an 8-year period. The parameters assessed included age of patient, lesional size, systemic and local symptoms, and histological findings of inflammatory cells, granulomas, necrosis, multinucleated giant cells, fibrosis and calcifications. RESULTS: 29 cases of IGM and 33 cases of TBM were included in this study. A significant difference was seen between the two groups with regard to patient age (t=2.52, p<0.05) and lesional size (t=-5.56, p<0.01). TBM occurred in a significantly younger population, and demonstrated larger lesional sizes than IGM. There was no difference between the number of cases showing mass, local and systemic symptoms. Comparing the different histological features, the TBM group showed significantly more fibrosis, eosinophils and necrosis, whereas the IGM group showed significantly more plasma cells. Taking all the cases together as one group to evaluate the relationship between the histological parameters, there was significant positive correlation between eosinophils and fibrosis (r(s)=0.39, p<0.01), and negative correlation between vague and well-formed granulomas (r(s)=-0.38, p<0.01). CONCLUSION: TBM was more likely to occur in younger patients, with a larger clinical mass at presentation. Histologically, TBM tends to show more eosinophils and necrosis, and IGM is associated with more plasma cells. The characteristics of the granulomas and giant cells were not distinguishing features.

Correlation of biomarkers in head and neck squamous cell carcinoma.

OBJECTIVE: To evaluate the relationship of functional magnetic resonance imaging (MRI) parameters, including choline/creatine ratio (Cho/Cr) and apparent diffusion coefficient (ADC) with protein expression of 10 common tumor and prognostic markers in head and neck squamous cell carcinoma. STUDY DESIGN: Cross-sectional study. SETTING: University hospital. SUBJECTS AND METHODS: The Cho/Cr and ADC obtained from 74 patients with head and neck squamous cell carcinoma were correlated with the expression level of the 10 protein markers as determined by immunohistochemistry. RESULTS: Cho/Cr showed significant positive correlations with cyclooxygenase 2 in primary tumors (r = 0.714), and epidermal growth factor receptor in metastatic cervical lymph nodes (r = 0.522). ADC showed significant (r = -0.591) negative correlation with human epidermal growth factor receptor 2 in metastatic cervical lymph nodes. CONCLUSION: There are relationships between protein and functional MRI markers. Future research in this direction may improve our understanding of the cancer micro-environment.

Apoptotic and necrotic effects of tumour necrosis factor-alpha potentiated with hyperthermia on L929 and tumour necrosis factor-alpha-resistant L929.

PURPOSE: The cytotoxic effect of the combination treatment of TNF-alpha and hyperthermia on L929 and TNF-alpha-resistant L929 (rL929) cells was investigated. MATERIALS AND METHODS: L929 cells were treated with TNF-alpha (5 ng/mL), heating at 43 degrees C or the combination of TNF-alpha and heating. The cells were harvested at different time within the 24-hour period. The viability and the type of cell death of the harvested cells were examined. RESULTS: When L929 cells were treated with a combination of TNF-alpha and heating the cells died quickly and apoptosis increased to an overwhelming extent, especially in the group pre-treated with TNF-alpha for 1 h prior to heating. Although rL929 cells were resistant to TNF-alpha alone, the cells became sensitive to TNF-alpha treatment when combined with heating. Similar to the L929 cell, the cells also died rapidly and exhibited apoptosis to a higher extent. Using an Annexin-V-FITC kit and flow cytometer, we found that both necrosis and apoptosis occurred. Agarose gel electrophoresis of DNA extracted from treated cells showed that the DNA fragments were multiples of approximately 200 bp. Furthermore, by studying the kinetics of cell death and apoptosis, we found that the loss of cell membrane integrity preceded the DNA fragmentation in both L929 and rL929 cells. CONCLUSION: The results suggested that hyperthermia may enhance the necrotic and apoptotic effects of TNF-alpha on some tumour cells and overcome the resistance of some tumour cells to TNF-alpha.

Atypia in fine needle aspirates of breast lesions.

BACKGROUND: The atypical category is controversial in fine needle aspiration cytology (FNAC) of the breast; most are benign, but a significant number are malignant. To date, no morphological criterion has been found to be consistent in predicting malignancy. AIMS: To evaluate specific cytological parameters and assess their usefulness in predicting histological outcome in a cohort of atypical breast FNAC, in order to establish a set of objective criteria in defining 'high risk' atypical breast FNAC. METHODS: A retrospective review of 98 cases of atypical breast FNAC with histological correlation was undertaken. The cytological preparations were evaluated for cellularity, percentage of epithelial cell cluster and single epithelial cells, nuclear atypia, nucleus:cytoplasm ratio, percentage of bipolar nuclei, and the presence of stromal fragments, histiocytes and necrosis. RESULTS: 66 of 98 cases (67.35%) showed benign histology and 32 cases (32.65%) showed malignant histology. Compared with the malignant group, the benign group had significantly lower patient age (p=0.05), higher bipolar nuclei (p<0.0001), less degree of nuclear pleomorphism (p<0.0001), lower nucleus:cytoplasm ratio (p<0.0001), lower cellularity (p=0.05) and less necrosis (p<0.001). There was no difference in the percentage of epithelial clusters and single cells, or the presence of stromal fragments and histiocytes. CONCLUSIONS: The presence of nuclear pleomorphism, high nucleus:cytoplasm ratio, epithelial cell atypia, low number of bipolar nuclei and necrosis are useful parameters to predict malignancy in atypical FNAC of the breast. Assessment of these factors in atypical FNAC may be helpful in predicting cancer risk and subsequent management decision making.

Breast cancer in the elderly: a histological assessment.

AIMS: To understand the correlation between the expression status of different biological markers in breast cancers in the elderly. METHODS AND RESULTS: Three hundred and ninety-seven cases were evaluated for expression of hormone receptors [oestrogen receptors (ER) alpha and beta, progesterone receptor (PR)], basal markers [p63, cytokeratin (CK) 5/6 and CK14] and others (HER2/neu, synaptophysin and chromogranin). The expression rates were 60, 29, 25, 6, 14, 8, 28, 17 and 5%, respectively, for these markers. The expression of ER alpha and beta, PR, synaptophysin and chromogranin at any level correlated with low nuclear or tumour grades, whereas the expression of HER2/neu, CK5/6 and CK14 at any level correlated with high nuclear grade. By using hierarchical clustering, groups of HER2, luminal and basal types were identified. In addition, a neuroendocrine group was also identified, being characterized by expression of synaptophysin, chromogranin, ER and PR, but not HER2/neu, and other basal cytokeratins. This group was associated with lower nuclear grade, and hence better prognosis. CONCLUSIONS: Breast cancer in the elderly shows similar molecular groupings as other breast cancers, with an additional neuroendocrine group that is associated with a favourable biological marker profile.

Vulvar basal cell carcinoma in China: a 13-year review.

OBJECTIVE: We conducted a 12-year retrospective review of vulvar basal cell carcinoma (BCC) in a Chinese population. STUDY DESIGN: Medical records and histopathologic reports were examined from 5 major Hospitals in Hong Kong to list all patients diagnosed with vulvar BCC. Clinical data and histologic materials were reviewed. RESULTS: Sixteen vulvar BCCs were diagnosed. Most of them were pigmented. They were removed by simple excision or wide local excision. All the carcinomas were identified in the reticular dermis. The predominant histologic pattern was nodular, which may be mistaken as adenoid cystic carcinoma. CONCLUSION: The high proportion of pigmented vulvar BCCs suggested that biopsy should be performed for any pigmented lesion in a Chinese patient. The BCCs are superficial and tissue-preserving treatment approach is recommended. The tumor depth estimation is difficult and intraoperative frozen section consultation may be helpful. Formal histopathologic assessment should be used to reach an objective diagnosis.

Increased epidermal growth factor receptor (EGFR) expression in malignant mammary phyllodes tumors.

Mammary phyllodes tumors are uncommon stromal-epithelial neoplasms, and are divided into benign, borderline malignant and frankly malignant groups on the basis of their histological features. Accumulating evidence shows that epidermal growth factor receptor (EGFR) is involved in the pathogenesis and progression of many malignancies. This study investigated 453 phyllodes tumors (296 benign, 98 borderline, 59 malignant) for EGFR expression using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for gene amplification. The staining was correlated to tumor margin status, degree of malignancy, stromal cellularity, mitotic activity, nuclear pleomorphism and stromal overgrowth. Cases with strong positive IHC staining were selected for FISH. The overall positive rate for EGFR was 16.2% (48/296), 30.6% (30/98) and 56% (33/59) for benign, borderline malignant and frankly malignant phyllodes tumors, respectively. FISH demonstrated egfr gene amplification in 8% of immunohistochemically positive cases. The results of this study provide strong evidence that EGFR overexpression is involved in the pathogenesis of phyllodes tumors, although gene amplification may not be the major underlying mechanism for overexpression.