HER2 status in molecular apocrine breast cancer: associations with clinical,pathological, and molecular features

Molecular apocrine breast cancer (MABC) is a distinct subtype of breast cancer. The purpose of this study was to investigate the relationship between HER2 status and clinicopathologic characteristics of MABCs from Chinese Han cohort. A cohort of 90 MABC patients were enrolled. Immunohistochemical method was performed to analyze the molecular expression, and the human epidermal growth factor receptor 2 (HER2) amplification was verified by fluorescence in situ hybridization (FISH). By studying these 90 MABC cases, the majority of studied patients were premenopausal young women (median age 48 yr) with high grade tumors. We also found that MABCs had high positive expression rates of HER2, CK8, CD44, CD166, p53 and BRCA1, the elevated Ki-67 labeling index, and favorable prognosis. There was a significantly higher incidence of lymph node metastasis and lower CD166 positive rate in HER2-negative patients compared to HER2-positive patients (54.5% vs. 37.0%, P = 0.044 and 72.7% vs. 91.3%, P = 0.021, respectively). The CK5/6 and EGFR expression rates were significant higher in HER2-negative cases than in HER2-positive cases, suggesting that there is overlap between MABC with HER2-negative phenotype and basal-like breast cancer. In addition, HER2 positive was found to be significantly associated a poor overall survival in MABCs. In conclusion, HER2 are highly expressed, and HER2 positivity could be considered as a significant biomarker of poor prognosis in MABC. The results also suggest that a subtype tumor with distinct patterns of molecule expression depending on HER2 status presented in MABC.     

TMEM16A/ANO1 suppression improves response to antibody‐mediated targeted therapy of EGFR and HER2/ERBB2

TMEM16A, a Ca²⁺‐activated Cl^? channel, contributes to tumor growth in breast cancer and head and neck squamous cell carcinoma (HNSCC). Here, we investigated whether TMEM16A influences the response to EGFR/HER family‐targeting biological therapies. Inhibition of TMEM16A Cl^? channel activity in breast cancer cells with HER2 amplification induced a loss of viability. Cells resistant to trastuzumab, a monoclonal antibody targeting HER2, showed an increase in TMEM16A expression and heightened sensitivity to Cl^? channel inhibition. Treatment of HNSCC cells with cetuximab, a monoclonal antibody targeting EGFR, and simultaneous TMEM16A suppression led to a pronounced loss of viability. Biochemical analyses of cells subjected to TMEM16A inhibitors or expressing chloride‐deficient forms of TMEM16A provide further evidence that TMEM16A channel function may play a role in regulating EGFR/HER2 signaling. These data demonstrate that TMEM16A regulates EGFR and HER2 in growth and survival pathways. Furthermore, in the absence of TMEM16A cotargeting, tumor cells may acquire resistance to EGFR/HER inhibitors. Finally, targeting TMEM16A improves response to biological therapies targeting EGFR/HER family members.     

p16INK4a expression in basal-like breast carcinoma

BLBC represents a distinctive group of invasive breast carcinomas with specific genotype and immunopro-file. BLBC is usually defined by gene expression profiling and is currently associated with poor outcome. BLBCs are estrogen receptor (ER) negative, progesterone receptor (PgR) negative, HER2 negative, and usually show a variable expression of basal cytokeratins (CKs), EGFR and CD117. p16 ^INK4a is a tumor suppressor protein, encoded by the CDKN2A gene, which regulates cell cycle. The reported association of abnormalities in the p16/Rb pathway with increased risk of malignancy prompted us to determine the expression of p16^INK4a in a group of BLBC; the results were compared with a group of high-grade invasive carcinoma (HG-IC) of breast. Tissue microarrays (TMA) were constructed in triplicate including 18 BLBC and 18 HG-IC. All BLBC cases were ER-/PgR-/HER2-. Seventeen (94%) BLBC were CK 5/6+/CK 14+; 14 (78%) BLCB showed EGFR expression and 13 (72%) were CD117 positive. BLBCs showed a strong positive reaction with p16 ^INK4a antibody in 16 of 18 (89%) cases. Although the significance of p16 ^INK4a expression in breast cancer is not fully understood, we have shown that p16^INK4a is strongly expressed in breast cancers with basal-like phenotype. Since it is known that p16^INK4a is associated with aggressive behavior in human carcinomas, these data suggest that p16^INK4a play a role in the poor prognosis of BLBC.     

Expression and significance of HER family receptors in neuroblastic tumors

HER receptor family plays an important role in normal embryonic development and is involved in pathogenesis and progression of some types of cancer. Neuroblastic tumors (NT) are common pediatric neoplasms with a poor outcome in a significant number of patients. The biological and prognostic role of HER family in NT is not well established. In the current study we evaluated HER1–4 receptors expression, their prognostic significance and clinicopathological correlations in a series of 103 NTs by immunohistochemical assessment of HER1–4 expression and FISH analysis of EGFR and HER2 copy number status. HER receptors are commonly expressed in NT but it was not due to EGFR or HER2 amplification. EGFR, HER2 and HER4 show correlation with tumor histology. It seems that these receptors take part in neuroblastic cell differentiation and Schwannian stroma development. EGFR and HER2 positivity are more frequently found in favorable histological risk group of tumours (P = 0.004 and P = 0.01 respectively) while high expression of HER4 is significantly more often found in patients with metastatic disease (P = 0.03). Moreover tumors with HER2 polysomy were more often found in children ≤18 months, with localized disease, and favorable histological group. Our study showed that the role of HER family members in NT biology is interrelated and complex but their expression level may present a novel prognostic factor for NT patients outcome.     

The Differences in CXCR4 Protein Expression Are Significant for the Five Molecular Subtypes of Breast Cancer

The aim was to investigate the expression of the CXCR4 protein in five molecular subtypes of breast cancer. The authors randomly selected the breast cancer paraffin-embedded specimens of the Affiliated Third Hospital of Harbin Medical University between 2007 and 2009. Details are as follows: basal-like subtype—ER (–), PR (–), C-erbB-2 (–), CK5/6 (+), n = 36; normal breast subtype—ER (–), PR (–), C-erbB-2 (–), CK5/6(–), n = 40; luminal A subtype—ER/PR (+), C-erbB-2 (–), n = 38; luminal B subtype—ER/PR (+), C-erbB-2 (+), n = 60; C-erbB-2 (+) subtype—ER (–), PR (–), C-erbB-2 (+), n = 58. Using the immunohistochemistry method, the authors detected the expression of the CXCR4 protein in the five subtypes. The CXCR4 protein expression in the basal-like subtype was the highest, and that in the luminal A subtype was the lowest. In terms of five molecular subtypes of breast cancer, the differences in CXCR4 protein expression were significant (p < .001). In terms of C-erbB-2 expression, tumor stage, and lymph node metastasis of breast cancer, the differences in CXCR4 protein expression were significant (p < .01).     

Immunomagnetic enrichment and detection of isolated tumor cells in bone marrow of patients with epithelial malignancies

The detection of isolated tumor cells (ITC) in the bone marrow of patients with epithelial malignancies is an independant prognostic factor for several entities as breast cancer, colorectal cancer or non-small lung cancer. However, with conventional immunocytology using Ficoll density gradient and APAAP staining, only a small proportion of the bone marrow samples can be scanned for cytokeratin-positive (CK+) cells. To improve detection rates, we evaluated the enrichment of ITC by magnetic activated cell sorting (MACS) compared to regularly stained cytospins. Recovery experiments with a CK+ breast cancer cell line (SKBR3) were performed to calculate the MACS enrichment rate. Bone marrow was obtained by aspiration from 20 patients with carcinomas of epithelial origin and from 17 controls. ITC were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to cytokeratin 7 and 8. MACS of SKBR3 seeded in peripheral blood revealed average recovery rates of 62% and 48% and average enrichment factors of 104-fold and 8139-fold of the CK+ cells after one and after two separations, respectively. After immunomagnetic enrichment, CK+ cells were detected in 16 of 20 (80%) cancer patients, whereas only 7 (35%) patients showed CK+ cells without magnetic enrichment (P=0.002). Ten of twelve (83%) patients with metastatic disease (stage M₁) and six of eight (75%) patients without any overt metastases (M₀) had CK+ cells in their bone marrow. None of the negative controls showed any CK+ cells. Enrichment with magnetically labeled anti cytokeratin antibodies increases the detection rate of epithelial cells in bone marrow of cancer patients compared to immunocytology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Remarkably high frequency of EGFR expression in breast carcinomas with squamous differentiation

The human epidermal growth factor receptor (EGFR) is reportedly overexpressed in 15-20% of breast carcinomas. EGFR overexpression is associated with reduced survival and is inversely correlated with expression of estrogen receptor (ER). This study assessed EGFR expression in breast carcinomas with squamous differentiation. The immunohistochemical (IHC) expression of EGFR was evaluated in 39 breast carcinomas with squamous differentiation (30 pure squamous, 6 adenosquamous, 3 carcinosarcomas) by use of the pharmDx assay (clone 2-18C9, DakoCytomation). Cases were considered positive if at least 10% of the cells showed 1+ positivity in the squamous component. Squamous differentiation was confirmed with IHC for CK5-6 (clone D5/16B4, DakoCytomation). ER, PR, and HER2 status as well as clinical information regarding treatment and outcome were correlated. As a control, a tissue microarray comprising 280 lymph node positive breast carcinomas was evaluated with the same EGFR assay. The 39 patients ranged in age from 33 to 77 years (mean 52). The tumors measured 1.3-30 cm (mean 4.8). Sentinel or full axillary lymph node dissection was performed in 28 patients. Fourteen patients had positive lymph nodes. At the time of initial diagnosis, 3 patients had distant metastasis. Follow-up was available for 16 patients (mean 45 months). Disease-free survival at 3 years was 70%. Among the 39 tumors 87% (34) were positive for EGFR (p<0.0001). Sixty-nine percent (27 of 39) showed >50% 2+ EGFR staining. EGFR-positive tumor cells (showing squamous morphology) were also found in 1 bone, 1 lung, and 8 of 11 lymph node metastases available for evaluation. All 11 lymph nodes showed squamous differentiation. All but 1 of the EGFR+ tumors examined were ER and PR negative. Six EGFR-positive tumors were HER2 positive. No statistically significant differences in HER2 status, size, lymph node status and disease-free survival were observed between EGFR+ and EGFR- cases, but the number of EGFR-negative tumors was quite small. Nine of 280 (3%) of lymph node-positive invasive carcinomas on the tissue microarray were EGFR+; review of the initial diagnostic slides failed to reveal squamous features in all but 1 of the 9 EGFR+ tumors. Breast carcinomas with squamous differentiation are a distinct subgroup of breast tumors with a very high frequency of EGFR positivity. Breast carcinomas of this type would be ideal candidates for a trial with EGFR inhibitors.     

The Influence of Tumor-Host Interactions in the Stromal Cell-Derived Factor-1/CXCR4 Ligand/Receptor Axis in Determining Metastatic Risk in Breast Cancer

The chemokine stromal cell-derived factor-1 (SDF-1) may function to attract CXCR4-expressing cancer cells to metastatic organs. We have previously demonstrated that low plasma SDF-1, a host-derived marker, increases distant metastatic risk in breast cancer. We therefore hypothesized that tumors overexpressing the SDF-1 receptor CXCR4 have an enhanced ability to metastasize in patients with low plasma SDF-1 levels. In this study, we determined the prognostic significance of activated CXCR4, or phosphorylated CXCR4 (p-CXCR4), and CXCR7, another receptor for SDF-1. Immunohistochemistry was performed on a tissue microarray built using 237 samples from the same cohort of patients for which we measured plasma SDF-1 levels. We found that the prognostic value of p-CXCR4 expression (hazard ratio or HR, 3.95; P = 0.004) was superior to total CXCR4 expression (HR, 3.20; P = 0.03). The rate of breast cancer-specific mortality was much higher in patients with both high p-CXCR4 expression and low plasma SDF-1 levels (HR, 5.96; P < 0.001) than either low plasma SDF-1 (HR, 3.59; P = 0.01) or high p-CXCR4 expression (HR, 3.83; P = 0.005) alone. The added prognostic value of low plasma SDF-1 was only effective in patients with high p-CXCR4 expression, and as such, provides clinical validation for modulation of the metastatic potential of tumor cells by an inherent host-derived metastatic risk factor.Breast cancer is the second most common cancer in women and represents a major risk to women’s lives because of the life-threatening consequences of metastatic disease (SEER Cancer Statistics Review, http://seer.cancer.gov/csr/1975_2005/; accessed September 1, 2008).¹ The process of metastasis has often been reported as a cascade of events, with emphasis placed on the tumor cell and its potential to proliferate, invade into the circulation, exit the bloodstream, and grow at the metastatic site.² However, little is known about the manner in which the host can modulate tumor progression and the propensity of the tumor to metastasize. Indeed, the role of the host was recognized over a century ago in the “seed and soil” theory, whereby the presence of a “congenial” environment of the host metastatic organ influenced the colonization of tumor cells at specific distant organs.³ More recently, a chemokine-receptor model was proposed to help explain the manner in which the host influences the homing of cancer cells to specific target organs. Muller et al. proposed that chemokines, such as stromal cell-derived factor-1 (SDF)-1, are normally overexpressed by those target organs to which breast cancer metastasizes, such as lung, liver, and bone, and serve to attract breast cancer cells that express their receptors, such as CXCR4.⁴ Various animal studies have subsequently demonstrated the functional role of CXCR4 as the prime chemokine receptor involved in distant metastasis in breast and other types of cancers.^5,6,7,8,9Several studies, including our own, have since observed an association between CXCR4 expression and distant metastasis in primary breast cancer patients.^10,11,12,13 Furthermore, we recently identified circulating levels of SDF-1 as a prognostic blood marker in a series of patients with primary breast cancers. Interestingly, circulating SDF-1 levels were found to be independent from tumor-derived SDF-1, and as such, plasma SDF-1 is the first candidate host-derived blood marker in breast cancer. We found that a low plasma SDF-1 level was predictive of distant metastasis, suggesting that low SDF-1 in the circulation may favor the extravasation of tumor cells from the circulation to the metastatic site.¹⁴ In accordance with Muller’s hypothesis, the differential concentration gradient of SDF-1, that is, low blood SDF-1 and high tissue SDF-1 at the metastatic site, may enhance the homing of CXCR4-expressing cancer cells. In this case, it would be expected that tumors with high CXCR4 expression would be especially sensitive to the SDF-1 gradient at metastatic target organs. To test this hypothesis, we determined the expression of CXCR4 in primary tumors using the same cohort of breast cancer patients in which we previously measured plasma SDF-1 levels. We determined if patients with an innate susceptibility for metastasis, associated with low levels of plasma SDF-1, demonstrated a greater risk of metastasis when their tumors expressed higher levels of CXCR4. In addition to tumor expression of CXCR4, we also measured the levels of the phosphorylated CXCR4 receptor as a means of quantifying CXCR4 activity and compared its expression in the primary tumor and metastatic lymph nodes. We also measured tumor expression of the two factors that may activate CXCR4: its ligand SDF-1, and another chemokine receptor, CXCR7, which may activate CXCR4 via heterodimerization.¹⁵ In this way, we provide a more detailed picture of metastatic risk associated with the activity of CXCR4 in the primary breast tumor and relate it with the risk of metastasis associated with low plasma SDF-1 levels.     

Analysis of a bone metastasis gene expression signature in patients with bone metastasis from solid tumors

Bone is a major target for metastases in the most frequent solid tumors, which result in severe complications and are a major cause of pain. A bone metastasis gene expression signature was identified using human breast cancer cells in a mouse model. The bone metastasis-related genes encode secretory and cell surface proteins implicated in bone-homing (CXCR4), angiogenesis (CTGF and FGF5), invasion (MMP-1 and ADAMTS1), and osteoclast recruitment (IL11). This signature superimposes on the 70-gene poor prognosis gene expression signature for breast cancer, and only ADAMTS1, CTGF and IL11 were found to be overexpressed in human primary breast cancers with bone relapse. We analyzed the expression of the six bone metastasis-related genes in bone metastases from patients with different types of solid tumors, to assess its relevance in human clinical samples. MMP-1, CXCR4, FGF5 and CTGF were found to be overexpressed in tumor cells of human bone metastases when compared to a human normal epithelial cell line. All the analyzed genes were overexpressed in the tumor cells of breast cancer bone metastases when compared to normal breast tissue. We did not detect any differences between the expression of these genes in bone metastases from breast cancer or from other types of solid tumors. Importantly, there was a significant correlation between the expressions of IL11/CTGF, IL11/ADAMTS1, CTGF/CXCR4, CTGF/ADAMTS1, and MMP-1/ADAMTS1, supporting the cooperative function of these proteins in the bone microenvironment, and the potential functional role of these genes in the establishment of bone metastases in vivo.     

Biomarker profile in breast carcinomas presenting with bone metastasis

Bone is the most preferred site for metastatic dissemination in breast cancer. The purpose of this study was to examine the expression of a set of antibodies that could serve as predictive biomarkers associated with breast cancer metastasis in a subset of sixteen (16) breast cancer patients who developed bone metastasis. The clinical and pathologic data were obtained, and tissue microarrays were constructed. Tissue microarray slides were stained for TFF-1, CXRC4, MMP1, PTHrP, HER2, CD44, FGFR3 and IL-11. The expression rates were compared between the metastatic breast cancer to bone (MBC-B) group and a group of sixty-four (64) primary breast cancer (PBC). The results demonstrated that MBC-B group patients were more likely to be HER2 positive (P = 0.016). There was no significant difference on estrogen receptor or progesterone receptor expression between MBC-B group and PBC group (P > 0.05). There was a high expression of CXCR4, MMP-1, CD44, TFF-1, PTHrP, FGFR3 and IL-11, in both, PBC and MBC-B, and no significant differences between the groups were identified. We found that tumors associated with bone metastasis tended to be larger than 2 cm. The high morbidity associated to metastatic breast cancer prompts the identification of predictive biomarkers of relapse of breast tumors to categorize patients at high risk of bone metastasis and serve as targeted therapy.     

Co-expression of uPAR and CXCR4 promotes tumor growth and metastasis in small cell lung cancer

Urokinase-type plasminogen activator receptor (uPAR) and C-X-C-chemokine receptor-4 (CXCR4) are considered as key molecules in invasion and metastasis of several cancers via extracellular matrix degeneration and assist tumor metastasis to specific sites by chemotaxis. However, the combined effect of uPAR and CXCR4 on small cell lung cancer (SCLC), the most aggressive type of lung cancer, is not clear. In this study, we detected the expression of uPAR and CXCR4 in SCLC tissue samples (n = 50) by immunohistochemistry. The tumors with high expression of both uPAR and CXCR4 (12/50) had larger size, higher lymph node (LN) metastasis and worse prognosis of patients than those with low expression of uPAR and CXCR4 (38/50) (P < 0.05). We further identified and isolated the both uPAR and CXCR4 positive expression subpopulation cells (uPAR⁺CXCR4⁺ cells) from the SCLC cell line H446 by flow cytometry. The uPAR⁺CXCR4⁺ cancer cells showed a higher invasive and migrating capacity in the transwell and wound healing assays compared with other subpopulation cells (P < 0.05). uPAR⁺CXCR4⁺ cells injected subcutaneously in nude mice markedly increased tumor growth and induced lung metastasis, while other subpopulation cells did not. In conclusion, these data suggest that uPAR and CXCR4 co-expression predicts worse prognosis of SCLC patients. uPAR⁺CXCR4⁺ cells promote the tumor growth and play a potential role in metastasis of SCLC.     

Exon 8 amplification of epidermal growth factor receptor (EGFR) in invasive breast carcinomas

Souka E, Alexiadis E, Theohari I, Giannopoulou I, Papadimitriou C & Nakopoulou L
(2012) Histopathology  61, 644–651 Exon 8 amplification of epidermal growth factor receptor (EGFR) in invasive breast carcinomas Aims: The rate of EGFR amplification in breast cancer ranges between 0% and 15%. Recent studies have focused on the amplification status of cytosine–adenine (CA) repeats in intron 1 of the gene and correlated it with increased EGFR protein. The aim of this study was to investigate, for the first time, the significance of coding exon 8 amplification of EGFR in invasive breast cancer. Methods and results: We investigated, by means of real‐time polymerase chain reaction (PCR), the amplification status of exon 8 of the EGFR gene in 148 paraffin‐embedded tissue sections, 115 with invasive breast carcinoma and 33 controls. Immunohistochemistry was utilized to detect EGFR and human epidermal growth factor receptor 2 (HER2) expression. Univariate and multivariate statistical analyses were performed for the evaluation of our results. EGFR amplification was observed in 7.8% of the patients, and EGFR was immunodetected in 9.6%. EGFR amplification was correlated positively with EGFR expression (P < 0.0001), HER2 expression (P = 0.023), coexpression of EGFR/HER2 (P < 0.0001) and nuclear grade (P = 0.047), and inversely with ER protein expression (P = 0.047). Conclusions: It appears that amplification of the coding sequence exon 8 exhibits similar biological behaviour to amplification of the regulatory sequence in intron 1, leading to elevated levels of EGFR protein.     

Epidermal growth factor receptor gene amplification and protein overexpression in basal-like carcinoma of the breast

Shao M‐M, Zhang F, Meng G, Wang X‐X, Xu H, Yu X‐W, Chen L‐Y & Tse G M
(2011) Histopathology 59 , 264–273 Epidermal growth factor receptor gene amplification and protein overexpression in basal‐like carcinoma of the breast Aims: Epidermal growth factor receptor (EGFR) is frequently expressed in basal‐like breast cancer (BLBC). The aim of this study was to evaluate their correlation as detected by immunohistochemistry (IHC) or fluorescence in‐situ hybridization (FISH). Methods and results: IHC for oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER) 2, cytokeratin (CK) 5/6 and EGFR, and FISH for EGFR amplification, were performed in 59 cases of BLBC. EGFR IHC results were scored semiquantitatively, and compared with its gene amplification status. ER, PR and HER2 were negative in all cases, whereas 35 and 55 cases were positive for CK5/6 and EGFR. For EGFR IHC, 20, 11, 11 and 17 cases showed a negative, a low, an intermediate or a high staining level, respectively, and seven cases showed gene amplification by FISH, with two, 19, 11 and 20 cases showing balanced monosony, disomy, trisomy, and polysomy respectively. Immunohistochemical expression in gene‐amplified tumours was significantly higher than in those without amplification, including balanced polysomy tumours. EGFR immunohistochemical expression also correlated with the EGFR/chromosome 7 ratio. High sensitivity (86%) and negative predictive value (98%) were achieved with high‐level immunohistochemical expression as a cut‐off to predict gene amplification. Conclusions: High‐level EGFR immunohistochemical expression correlated with and predicted EGFR amplification, and may be used as a screening method to exclude gene amplification.     

Significance of BRCA1 expression in breast and ovarian cancer patients with brain metastasis – A multicentre study

PurposeCerebral metastases develop in 10–30% of patients with breast cancer (BC) and in around 3.3 to 4% of patients with ovarian cancer (OC). The aim of the multicenter study is to investigate the correlation between the expression of estrogen alpha receptors (ERα), progesterone receptors (PR), human epidermal growth factor receptor 2 (HER2), stromal cell-derived factor 1 (SDF1) and its receptor C-X-C chemokine receptor type 4 (CXCR4), breast cancer metastasis suppressor 1 (BRMS1), astrocyte elevated gene 1 (AEG1), depending on the status of BRCA1 protein, in patients suffering from OC and BC with brain metastases.Patients and methodsThe analysis included 51 patients: 29 with BC and 22 with OC, in whom brain metastases were disclosed.ResultsIn most patients (65.5% of BC patients and 68.2% of patients with OC tumors) BRCA1 protein loss was found. No correlation was disclosed between the levels of ERα, PR receptors, HER2, SDF1, CXCR4, AEG1, BRMS1 and BRCA1 status, patient age, stage of disease advancement, grade of histological maturity of the cells, presence of metastases to lymph nodes. A statistically significant correlation was disclosed between the negative expression of PR receptors and a high expression of CXCR4 in patients with BC. High values of the AEG1 protein (linked to metastases) were detected alongside a high expression of BRMS1 (a suppressor of metastases).ConclusionsPatients with BC and OC and brain metastases have a frequent loss of BRCA1 expression. The role of ERα, PR, HER2, SDF1, CXCR4, AEG1, BRMS1 in metastatic process needs further studies.     

Comparative analysis of tumorbiology and CD133 positivity in primary and recurrent pancreatic ductal adenocarcinoma

In over 70% of the cases, patients with curative surgery and adjuvant chemotherapy for pancreatic ductal adenocarcinoma (PDAC) develop recurrent tumors. The cancer stem cell (CSC) hypothesis suggests that CSCs are chemoresistant and enriched in recurrent tumors. This study analyzes tumorbiology, expression of the metastasis-promoting CXCR4 and actinin-4, and of the CSC marker CD133 in primary and recurrent PDAC. Twenty-six patients underwent resection for primary and recurrent PDAC and most developed tumor recurrence within 2 years. In 81% the histologic tumor grade was unchanged. Immunohistochemistry could be performed with 15 pairs of primary and recurrent PDAC. The mean Ki-67 proliferation index increased (P = 0.06). About 30% of tumor cells were positive for CXCR4 and almost all tumor cells expressed actinin-4, but there were neither significant changes in the expression levels in recurrent PDAC, nor specifically enhanced levels in metastases. The prominent CD133 pattern was an apical membrane staining of inflammatorily altered, non-neoplastic ductal structures equally observed in primary and recurrent PDAC. The membrane CD133 positivity was consistently absent in neoplastic PDAC cells. Cytoplasmic CD133 positivity was extremely rare (0.85 and 0.34 cells/cm² in primary and recurrent PDAC, respectively; P = 0.07). Tumor grade is mainly unchanged and the expression of CXCR4, actinin-4 and CD133 are not enhanced in recurrent PDAC. The apical membrane CD133 positivity of normal and inflammatorily altered ductal structures and its lack in tumor cells bring the role of CD133 as a specific CSC marker in PDAC into question.     

ADAMTS1 and MMP1 proteolytically engage EGF-like ligands in an osteolytic signaling cascade for bone metastasis

Bone metastasis is mediated by complex interactions between tumor cells and resident stromal cells in the bone microenvironment. The functions of metalloproteinases in organ-specific metastasis remain poorly defined despite their well-appreciated role in matrix degradation and tumor invasion. Here, we show a mechanism whereby two distinct metalloproteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS1) and matrix metalloproteinase-1 (MMP1), orchestrate a paracrine signaling cascade to modulate the bone microenvironment in favor of osteoclastogenesis and bone metastasis. Proteolytic release of membrane-bound epidermal growth factor (EGF)-like growth factors, including Amphiregulin (AREG), heparin-binding EGF (HB-EGF), and transforming growth factor α (TGFα) from tumor cells suppress the expression of osteoprotegerin (OPG) in osteoblasts and subsequently potentiate osteoclast differentiation. EGF receptor (EGFR) inhibitors block osteolytic bone metastasis by targeting EGFR signaling in bone stromal cells. Furthermore, elevated MMP1 and ADAMTS1 expression is associated with increased risk of bone metastasis in breast cancer patients. This study established MMP1 and ADAMTS1 in tumor cells, as well as EGFR signaling in osteoblasts, as promising therapeutic targets for inhibiting bone metastasis of breast cancer.     

Breast cancer micrometastases: Different interactions of carcinoma cells with normal and cancer patients' bone marrow stromata

The apparently dormant breast cancer micrometastases in haemopoietic marrow are correlated with distant metastatic carcinoma dissemination. We studied in vitro interactions of carcinoma cells with adjacent stromata, using connective tissue cell cultures from breast and bone marrow samples of normal donors, comparing them to the pericancerous breast tissue and bone marrows of 12 selected patients with invasive breast carcinomas. Cancer cells were detected by immunocytochemistry and RT-PCR in all the bone marrows and in most blood samples of the studied patients. We monitored the growth and interaction of carcinoma MCF-7 cells with the stromata. The normal breast stroma sustained typical massive cancer growth. The pericancerous breast stroma induced the invasive mesenchymal pattern of growth. Normal bone marrow stroma induced the same conversion and was highly adhesive, retaining the cells in the stroma, but carcinoma patients' bone marrow stromata underwent low adhesive interactions with cancer cells, releasing them potentially into the circulation. The semi-quantitative RT-PCR indicated an enhanced expression of the hepatocyte growth factor and its receptor c-met in breast and bone marrow stromata of cancer patients. The input of cancer cells into the normal bone marrow may induce modifications of the local microenvironment, favourable for growth and release of carcinoma cells into the systemic circulation, which correlate with the poor prognosis of patients with bone marrow micrometastases. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular classification predicts survival for breast cancer patients in Vietnam: a single institutional retrospective analysis

Background: The Bhagarva surrogate molecular subtype definitions classify invasive breast cancer into seven the different subgroups based on immunohistochemical (IHC) criteria according to expression levels of markers as ER, PR, HER2, EGFR and/or basal cytokeratin (CK5/6) which are different in prognosis and responsiveness to adjuvant therapy. Purpose: The present study aimed to classify primary breast cancers and directly compares the prognostic significance of the intrinsic subtypes. Methods: The current study was conducted on 522 breast cancer patients who had surgery, but had not received neoadjuvant chemotherapy, from 2011 to 2014. The clinicopathologic characteristics were recorded. IHC staining was performed for ER, PR, HER2, Ki67, CK5/6, EGFR and D2-40 markers. All breast cancer patients were stratified according to Bhagarva criteria. The followed-up patients’ survival was analyzed by using Kaplan-Meier and Log-Rank models. Results: The luminal A (LUMA) was observed at the highest rate (32.5%). Non-basal-like triple negative phenotype (TNB-) and Luminal A HER2-Hybrid (LAHH) were the least common (3.3% in both). LUMA and luminal B (LUMB) were significantly associated with better prognostic features compared to HER2, basal-like triple negative phenotype (TNB+) and TNB-. Statistically significant differences were demonstrated between overall survival (OS), disease-free survival (DFS) and molecular subtypes (P<0.05), of which LUMB and LUMA had the highest rate of OS and DFS being 97.2 and 93.7%; and 97.2 and 90.5%, respectively. Conversely, HER2 revealed the worst prognosis with the lowest prevalence of OS and DFS (72.5 and 69.9%, respectively). Conclusion: The molecular subtypes had a distinct OS and DFS. The intrinsic stratification displayed inversely to clinicopathological features in breast cancer.