In situ detection of HER2:HER2 and HER2:HER3 protein–protein interactions demonstrates prognostic significance in early breast cancer

HER2 overexpression/amplification is linked with poor prognosis in early breast cancer. Co-expression of HER2 and HER3 is associated with endocrine and chemotherapy resistance, driven not simply by expression but by signalling via HER2:HER3 or HER2:HER2 dimers. Proximity ligation assays (PLAs) detect protein–protein complexes at a single-molecule level and allow study of signalling pathways in situ. A cohort of 100 tumours was analyzed by PLA, IHC and FISH. HER complexes were analyzed by PLA in a further 321 tumours from the BR9601 trial comparing cyclophosphamide, methotrexate and fluorouracil (CMF) with epirubicin followed by CMF (epi-CMF). The relationships between HER dimer expression and RFS and OS were investigated, and multivariate regression analysis identified factors influencing patient prognosis. PLA successfully and reproducibly detected HER2:HER2 and HER2:HER3 protein complexes in vivo. A significant association (P < 0.00001) was identified between HER2 homodimerization and HER2 gene amplification. Following a minimum p value approach high levels of HER2:HER2 dimers were significantly associated with reduced relapse-free (RFS; hazard ratio = 1.72, 95% confidence interval 1.15–2.56, P = 0.008) and overall survival (OS HR = 1.69 95% CI = 1.09–2.62, P = 0.019). Similarly, high levels of HER2:HER3 dimers were associated with reduced RFS (HR = 2.18, 95% CI = 1.46–3.26, P = 0.00016) and OS (HR = 2.21, 95% CI = 1.41–3.47, P = 0.001). This study demonstrates that in situ detection of HER2 and HER2:3 protein:protein complexes can be performed robustly and reproducibly in clinical specimens, provides novel prognostic information and opens a significant novel opportunity to probe the clinical impact of cellular signalling processes.     

Immunohistochemical Detection of Phospho-Akt,Phospho-BAD,HER2 and Oestrogen Receptors α and β in Malaysian Breast Cancer Patients

Activation of Akt signaling pathway has been documented in various human malignancies, including breast carcinoma. The objective of this study is to determine the incidence of Akt phosphorylation in breast tumours and its relationship with expression of ER-α, ER-β, HER2, Ki-67 and phosphorylated Bcl-2 associated death domain (p-BAD). Immunohistochemical staining was performed to detect these molecules on 43 paraffin-embedded breast tumour tissues with commercially available antibodies. Eighteen (41.9%), 3 (7.0%), 23 (53.5%), 35 (81.4%), 21 (48.8%), 29 (67.4%), and 34 (81.0%) of breast tumours were positive for nuclear ER-α, nuclear ER-β, membranous HER2, cytonuclear p-Akt (Thr308), p-Akt (Ser473), p-BAD and Ki-67, respectively. ER-α expression was inversely correlated with HER2 and Ki-67 (P = 0.041 and P = 0.040, respectively). The p-Akt (Ser473) was correlated with increased level of p-BAD (Ser136) (P = 0.012). No relationship of Akt phosphorylation with HER2, ER-α or ER-β was found. The p-Akt (Ser473) immunoreactivity was significantly higher in stage IV than in stage I or II (P = 0.036 or P = 0.009). The higher Ki-67 and lower ER-α expression showed an association with patient age of <50 years (P = 0.004) and with positive nodal status (P = 0.033), respectively. Our data suggest that the Akt phosphorylation and inactivation of its downstream target, BAD may play a role in survival of breast cancer cell. This study does not support the simple model of linear HER2/PI3K/Akt pathway in breast cancer.     

αB-crystallin is a novel predictor of resistance to neoadjuvant chemotherapy in breast cancer

Aims αB-crystallin is an anti-apoptotic protein commonly expressed in poor prognosis basal-like breast tumors, which are largely triple (estrogen receptor (ER), progesterone receptor (PR), and HER2) negative. We examined whether αB-crystallin expression in breast cancer was associated with a poor response to neoadjuvant (preoperative) chemotherapy. Methods One hundred and twelve breast cancer patients who received neoadjuvant chemotherapy and who had post-chemotherapy tumor specimens available for analysis were included in the study. Forty-nine percent of patients were treated with doxorubicin and cyclophosphamide (AC), 37% received AC in combination with a taxane, and 14% received other regimens. Paired pre- and post-chemotherapy tumor specimens were available for 33 patients. αB-crystallin expression was determined by immunohistochemistry in tissue microarrays. Results Seventeen percent of tumors were αB-crystallin positive. αB-crystallin expression was identical in 32 of 33 cases for which both pre- and post-chemotherapy tumor tissue was available. αB-crystallin expression was associated with ER-negative (P = 0.0024) and triple negative status (P = 0.005). Overall response rates (ORR) defined as ≥50% reduction in tumor size after treatment were 53% (clinical ORR) and 61% (pathological ORR). Although tumor grade, size, ER, PR, HER2 or triple negative status was not associated with response, αB-crystallin-positive tumors had poorer overall response rates than αB-crystallin-negative tumors (clinical ORR, 21% vs. 59%, respectively, P = 0.0045; pathological ORR, 16% vs. 70%, respectively, P < 0.0001). Conclusion αB-crystallin is a novel biomarker expressed predominantly in triple negative breast tumors that identifies a subset of chemotherapy-resistant tumors, which may contribute to their poor prognosis.     

Topoisomerase II alpha protein and responsiveness of breast cancer to adjuvant chemotherapy with CEF compared to CMF in the NCIC CTG randomized MA.5 adjuvant trial

Overexpression of topoisomerase II protein (topo 2α) is postulated to be more closely associated with responsiveness to anthracycline-containing chemotherapy than human epidermal growth factor receptor type 2 (HER2) gene amplification or alterations in the topoisomerase II alpha gene (TOP2A). The authors used tissue microarrays from 477 of 710 premenopausal women with node-positive breast cancer randomized to CEF or CMF adjuvant chemotherapy in the NCIC Clinical Trials Group Mammary 5 (MA.5) trial. No significant interaction was found between treatment and continuous topo 2α level in either relapse-free (RFS) or overall survival (OS). In 136 patients (28.5%) whose tumors showed topo 2α overexpression by immunohistochemistry based on a cut-off of 13%, CEF was superior to CMF for RFS (adjusted HR 0.45; 95% CI 0.25–0.82; P = 0.009) and OS (adjusted HR 0.50; 95% CI 0.26–0.96; P = 0.04). When tumors lacked topo 2α overexpression, CEF was not superior for RFS (adjusted HR 0.88; 95% CI 0.64–1.22; P = 0.46) or OS (adjusted HR 0.95; 95% CI 0.66–1.38; P = 0.80). Interaction between topo 2α and treatment was borderline significant for RFS (P = 0.04) and OS (P = 0.05) and not substantially more significant than between TOP2A gene alteration (P _interaction = 0.09 for RFS and 0.02 for OS) or HER2 overexpression (P _interaction = 0.002 for RFS and 0.009 for OS). Topo 2α protein overexpression based on the cut-off identified in this study, TOP2A gene alterations and HER2 protein overexpression were each associated with responsiveness to anthracycline-containing chemotherapy. The topo 2α protein analysis was exploratory and will require further validation.     

Nuclear and cytoplasmic expressions of ERβ1 and ERβ2 are predictive of response to therapy and alters prognosis in familial breast cancers

Estrogen receptor (ER) α has been studied extensively in familial breast cancers but there are limited data on ERβ and its isoforms. This is an important issue since many BRCA1-associated tumours are “triple negative” and are resistant to conventional and targeted therapies. We performed an immunohistochemical study of pan-ERβ, ERβ1 and ERβ2 in a cohort of 123 familial breast carcinomas (35 BRCA1, 33 BRCA2 and 55 BRCAX) using a cut-off for positivity at 20% (Shaaban et al. in Clin Cancer Res 14:5228–5235, 2008). BRCA1 cancers were more likely to be nuclear ERα negative and nuclear pan-ERβ positive (21/32, 66%) when compared with BRCA2 (2/29, 7%) and BRCAX cancers (11/49, 22%) (both P < 0.001). For survival analysis, expression was also stratified using cut-offs defined by Bates et al. (Breast Cancer Res Treat 111:453–459, 2008) (score out of 7). Cytoplasmic ERβ2 expression correlated with shorter overall survival at 15 years regardless of cut-off used (both P < 0.046) At a cut-off score of 6 out of 7, cytoplasmic ERβ2 expression correlated with a poorer response to chemotherapy in both univariate (P = 0.011) and multivariate analyses including grade, lymph node status and chemotherapy as an interaction variable (P = 0.045, Hazard ratio 1.22, 95% CI 1.004–9.87). A similar trend was seen in a univariate analysis with a cut-off of 20% although this did not reach statistical significance (P = 0.057). Expression of nuclear ERβ1 was associated with a favourable response to endocrine therapy at 15 years regardless of cut-offs employed (both P < 0.025). However, this did not reach statistical significance in a multivariate analysis (P > 0.05). Since a significant proportion of ERα negative familial breast carcinomas are positive for nuclear ERβ1 and cytoplasmic ERβ2, the different ERβ isoforms and their intracellular location may need to be assessed, to identify patients that may benefit from hormonal and chemotherapy.     

Prognostic value of novel biomarkers in astrocytic brain tumors: nuclear receptor co-regulators AIB1, TIF2, and PELP1 are associated with high tumor grade and worse patient prognosis

Estrogen receptors alpha (ERα) and beta (ERβ) and their co-regulatory proteins are key components of complex signaling networks that specifically regulate the growth and development of various tissues and tumors. Still, their protein expression profiles and possible role in the pathogenesis of astrocytic tumors remain largely unknown. The purpose of the present study is to evaluate the differential protein expression of ΕRα, ERβ, and their co-activators, AIB1, TIF2, and PELP1 in astrocytic tumors of World Health Organization (WHO) grade II–IV, using immunohistochemistry. Potential correlations with clinicopathological parameters and patient prognosis were also explored. ERα protein expression was undetectable while ERβ levels were significantly decreased with progression of tumor grade (P < 0.001). High expression of ERβ was an independent favorable prognostic factor on multivariate analysis (P = 0.003). Expression of AIB1, TIF2, and PELP1 was not correlated with ERβ expression and followed an opposite trend, with increasing levels in high-grade relative to low-grade tumors (P < 0.001). Univariate survival analysis revealed that high AIB1, TIF2, and PELP1 expression was associated with worse prognosis (P = 0.049, P = 0.033, and P = 0.020, respectively). ERβ and ER co-activators AIB1, TIF2, and PELP1 appear to play an important role in the pathogenesis and progression of astrocytic tumors and might have prognostic significance. The mechanisms underlying their involvement in astrocytic tumorigenesis, as well as their utility for prognostic and therapeutic purposes merit further investigation.     

Semi-quantitative evaluation of estrogen receptor expression is a strong predictive factor of pathological complete response after anthracycline-based neo-adjuvant chemotherapy in hormonal-sensitive breast cancer

Absence of hormonal receptors (HR) expression is a predictive factor of high pathologic complete response (pCR) rate after neo-adjuvant chemotherapy. However, HR-positive tumors are less chemosensitive. In the present study, we evaluated the predictive value of estrogen (ER) and progesterone (PgR) semi-quantitative expression in patients with HR-positive tumors treated uniformly with antracycline-based neoadjuvant chemotherapy without hormonal treatment. Value of HR expression as a predictive factor was then evaluated in a multivariate analysis with tumor grade, Ki67 index and HER2 expression. From January 2000 and December 2006, 177 patients with HR-positive breast ductal invasive carcinoma ≥2 cm in its largest diameter were treated with six cycles of an anthracycline-based neo-adjuvant chemotherapy. Tumor grade, ER, PgR, HER2 status and Ki67 index were determined on microbiopsy performed before chemotherapy. A semi-quantitative evaluation of ER and PgR expression by IHC was performed using the Barnes’score. pCR rate was significantly different (P < 0.001) according to the ER expression score. pCR rate was 28% for low score, 9% for medium score and 3% for high score. On the contrary, pCR rate was not significantly different (P = 0.49) according to the PgR expression score. In the multivariate analysis, ER expression score (P = 0.0002) and Ki67 index (P = 0.02) were the only predictive factors of response for HR-positive tumors. pCR after anthracycline-based chemotherapy is significantly correlated with the ER expression score.     

Glycodelin expression associates with differential tumour phenotype and outcome in sporadic and familial non-<Emphasis Type="Italic">BRCA1</Emphasis>/<Emphasis Type="Italic">2</Emphasis> breast cancer patients

Glycodelin (encoded by PAEP gene) is a secreted lipocalin protein mainly expressed in reproductive tissues, but also in several tumour types. In the breast, glycodelin is expressed both in normal epithelial and cancerous tissue. To investigate the association of glycodelin with clinicopathological features of breast cancer and outcome of patients we evaluated the protein expression of glycodelin in a large series of breast tumours. Immunohistochemical analysis of tissue microarrays was used to study glycodelin expression on 399 sporadic and 436 familial non-BRCA1/2 tumours with strong family history. Gene expression analysis was used to define genes co-expressed with PAEP in sporadic and familial non-BRCA1/2 breast tumours. In the sporadic series, the glycodelin expression associated with low proliferation rate (P < 0.001), with a tendency towards well-differentiated tumours (grades 1 and 2, P = 0.012) and high cyclin D1 (P = 0.034) expression. However, in familial non-BRCA1/2 cases with strong family history glycodelin expression associated with a less favourable phenotype, i.e. positive lymph node status (P = 0.003) and HER2-positive tumours (P = 0.009). Moreover, the patients with glycodelin-positive tumours had an increased risk for distant metastases (P = 0.001) and in multivariate analysis glycodelin expression was an independent predictor of metastasis (hazard ratio (HR) = 2.22, 95% confidence interval (95% CI) = 1.22–4.03, P = 0.009) in familial non-BRCA1/2 breast cancer. Gene expression analysis further revealed different gene expression profiles correlating with the PAEP expression in the sporadic and familial non-BRCA1/2 breast cancers. Our findings suggest differential progression pathways in the sporadic and familial non-BRCA1/2 breast tumours expressing glycodelin.     

Shorter survival-times following adjuvant endocrine therapy in oestrogen- and progesterone-receptor positive breast cancer overexpressing HER2 and/or with an increased expression of vascular endothelial growth factor

Purpose: To investigate the possible correlation between expression of HER2 and vascular endothelial growth factor (VEGF), and to determine the predictive value of these factors in patients receiving adjuvant endocrine therapy including the group with a breast cancer (BC) positive for both oestrogen receptor (ER) and progesterone receptor (PgR). Material and methods: By enzyme immuno-sorbent assays (ELISA) tumour levels of HER2 and VEGF proteins were determined in 679 consecutive primary BC patients, median age 63 years, median follow-up time 92 months. A total of 404 patients received adjuvant endocrine therapy, mainly tamoxifen, out of them 295 had an ER and PgR positive BC. In 160 patients, HER2 status was also determined by immunohistochemistry (IHC) using the monoclonal antibody CB11. Results: Overexpression of HER2 by IHC was found in 15% of the patients. Overexpression of HER2 by ELISA correlated with HER2 by IHC (P < 0.001) and a higher VEGF expression (P = 0.004). Patients receiving adjuvant endocrine therapy with high VEGF (RFS P = 0.0087, BCCS P = 0.0012) or over-expressing HER2 (RFS P = 0.0116, BCCS P = 0.0036) had significantly shorter survival. Factors retaining statistical significance in multivariate analyses for recurrence-free survival (RFS) were nodal status (P < 0.001), tumour size (P = 0.005) and VEGF (P = 0.032) and for breast cancer corrected survival (BCCS) nodal status (P < 0.001), tumour size (P = 0.001), ER status (P = 0.022), and VEGF (P = 0.016). Both factors were significantly correlated with survival in the group with a BC positive for both ER and PgR; VEGF (RFS P = 0.0177, BCCS P = 0.0321) and HER2 (RFS P = 0.0143, BCCS P = 0.0311). In multivariate analyses, nodal status (P < 0.001) and VEGF (P = 0.021) were independent factors for RFS. Nodal status (P < 0.001) and tumour size (P = 0.016) retained independent factors for BCCS. Combined analysis identified a high-risk group (HER2 positive and high VEGF) with significantly reduced survival. Conclusion: The results from this retrospective analysis suggest that overexpression of HER2 and higher VEGF expression may add information on patient’s outcome after adjuvant endocrine therapy in ER and PgR positive BC.     

FOXA1 is an independent prognostic marker for ER-positive breast cancer

Forkhead box protein A1 (FOXA1) is a “pioneer factor” that plays a role in controlling nearly 50% of estrogen receptor target genes. FOXA1 expression correlates with estrogen receptor (ER)-positivity especially in luminal subtype A breast cancers. The aim of this study was to investigate the precise role of FOXA1 in breast cancer using a large population-based cohort. Nuclear expression of FOXA1 was analyzed in a tissue microarray of 4,444 invasive breast cancer cases using immunohistochemistry and correlated with clinicopathologic variables using previously described methods and cutoff points. The entire cohort was equally divided into a training and validation set. All survival analyses were performed using a previously defined cutoff (3) for validation. Additional X-tile analysis performed to analyze prognostic effects of low and high FOXA1 levels identified 24 as a cutoff. Bonferroni–Holmes test was used as appropriate. FOXA1 expression significantly correlated positively with markers of good prognosis or ER-positivity, and negatively with tumor size, tumor grade, nodal status, Ki67, HER2 expression, and basal subtype (each P value <0.0001). In both survival analyses, FOXA1 was a significant predictor of breast cancer-specific survival (P < 0.0001) and relapse-free survival (P < 0.0001). FOXA1 was also an independent predictor of breast cancer-specific survival at 10 years using both cutoffs. Among the ER-positive subgroup treated with tamoxifen, FOXA1 was an independent prognostic marker using the 24 cutoff (P = 0.030). FOXA1 is a significant marker of good prognosis in breast cancer; it also identifies a subset of ER-positive tamoxifen treated patients at low risk of recurrence.     

Pattern of metastatic spread in triple-negative breast cancer

Purpose The prognosis of women with triple-negative breast cancers (defined as cancers that are estrogen receptor-negative, progesterone receptor-negative and HER2/neu negative) is poor, compared to women with other subtypes of breast cancer. It is proposed that the underlying difference in recurrence rates may be explained in part by different routes of metastatic spread. Experimental design We studied a cohort of 1608 patients diagnosed with breast cancer, diagnosed between January 1987 and December 1997 at Women’s College Hospital in Toronto. Triple-negative breast cancers were defined as those that were estrogen receptor-negative, progesterone receptor-negative and HER2/neu-negative. We compared the incidence rates of metastatic spread to bone and to other (non-bone) organs in women with triple-negative and other forms of breast cancer. Results Of the 1,608 patients, 180 (11.2%) had triple-negative breast cancer. The 1608 women were followed for a median of 9.0 years (range 0.1–19 years). Compared to other patients, those with triple-negative breast cancer had an increased likelihood of distant recurrence over the study period (adjusted hazard ratio (HR) 1.9; 95% CI: 1.5–2.5, P < 0.0001). The relatively poor prognosis was apparent in the five years after diagnosis (HR 2.9; 95% CI: 2.1–3.9; P = 0.0001) but not thereafter (HR 0.5; 95% CI: 0.2–1.1; P = 0.07). In particular, women with triple-negative breast cancer were four times more likely to experience a visceral metastasis within five years of diagnosis than those with other types of cancer (HR 4.0; 95% CI: 2.7–5.9; P < 0.0001). The rates of bone metastases were comparable for triple-negative and for other forms of cancer in this time period (HR 0.8; 95% CI: 0.4–1.6 P = 0.5). Conclusions The excess risk of distant recurrence in triple-negative breast cancers, versus other forms of cancer, is attributable in large part to an excess of visceral metastases in the first five years following diagnosis.     

The prognostic role of circulating tumor cells (CTCs) detected by RT-PCR in breast cancer: a meta-analysis of published literature

The prognostic significance of circulating tumor cells (CTCs) in patients with breast cancer is controversial. We performed a meta-analysis of published literature to assess whether the detection of CTCs in patients diagnosed with primary breast cancer can be used as a prognostic factor. We searched Medline, Science Citation Index, and Embase databases as well as reference lists of relevant articles (including review articles) for studies that assessed the prognostic relevance of tumor cell detection in the peripheral blood (PB). A total of 24 eligible studies with 4,013 cases and 1,333 controls were included. Meta-analyses were performed using a random-effects model, using the hazard ratio (HR) and 95% confidence intervals (95% CIs) as effect measures. The positive detection of CTCs in patients was significantly associated with poor overall survival (OS) (HR = 3.00 [95% CI 2.29–3.94], n = 17, P < 0.0001) and recurrence-free survival (RFS) (HR = 2.67 [95% CI 2.09–3.42], n = 22, P < 0.0001). CTC-positive breast cancers were significantly associated with high histological grade (HR = 1.21 [95% CI 1.09–1.35], n = 34, P < 0.0001), tumor size (>2 cm) (HR = 1.12 [95% CI 1.02–1.22], n = 31, P = 0.01). and nodal status (≥1) (HR = 1.10 [95% CI 1.00–1.21], n = 32, P = 0.037), but cytokeratin-19 (CK-19) mRNA-positive CTCs were not associated with these clinicopathological parameters of breast cancer. Furthermore, the presence of CTCs was not associated with estrogen receptor (ER) negativity, progesterone receptor (PR) negativity, or human epidermal growth factor receptor type 2 (HER2) positivity. Detection of CTCs in the PB indicates poor prognosis in patients with primary breast cancer. Larger clinical studies are required to further evaluate the role of these markers in clinical practice.     

HER2 expression and efficacy of preoperative paclitaxel/FAC chemotherapy in breast cancer

Purpose We examined the correlation between HER2 expression and pathologic complete response (pCR) to paclitaxel/FAC (T/FAC) preoperative chemotherapy in breast cancer. Patients and Methods Retrospective analysis of data including 534 patients treated with preoperative T/FAC was performed. Gene expression results were available from two datasets of 132 and 286 patients, and were used to examine the co-expression of HER2 and topoisomerase II α (TOP2A) and microtubule associated protein tau (MAP-Tau). Results Of the 534 patients, 105 (20%) had HER2-overexpressing breast cancer. The pCR rates were 33% and 15% for patients with HER2+ and HER2- tumors (P < 0.001). The 5-year relapse-free survival rates were 94% and 70% in HER2+ tumors with and without pCR (P = 0.009). HER2 overexpression (odds ratio 2.3, 95%CI: 1.3–3.9, P = 0.004), estrogen receptor (ER) status, grade and weekly schedule of paclitaxel were each significantly and independently associated with pCR in multivariate analysis. When patients were stratified by ER status, the pCR rates were 50% for HER2+/ER−, 30% for HER2−/ER−, 19% for HER2+/ER+, and 6% for HER2−/ER+ tumors. HER2 overexpression was associated with lower expression of MAP-tau (P = 0.001 and P < 0.001) and higher expression of TOP2A mRNAs (P = 0.048 and P = 0.001) in patients with ER+ disease. ER− cancers had low MAP-tau expression regardless of HER-status. Conclusion HER2 overexpression is associated with higher rate of pCR to preoperative T/FAC chemotherapy regardless of ER status. HER2 overexpression also correlates with increased TOP2A and decreased MAP-tau expression in ER-positive cancers.     

High frequency of HIF-1α overexpression in BRCA1 related breast cancer

Hypoxia is a hallmark of cancer. Hypoxia inducible factor-1α (HIF-1α) is the key regulator of the hypoxia response. HIF-1α is overexpressed during sporadic breast carcinogenesis and correlated with poor prognosis. Little is known on the role of HIF-1α in hereditary breast carcinogenesis. A recent study suggests a role for BRCA1 in HIF-1α regulation. We therefore examined the expression of HIF-1α in BRCA1 related breast cancers. By immunohistochemistry we studied expression of HIF-1α and some of its downstream targets in 30 hereditary invasive breast cancers in comparison with 200 sporadic controls. HIF-1α overexpression was significantly more frequent in BRCA1 related breast cancers (27/30, 90%) than in sporadic controls (88/200, 44%) (P < 0.0001). 19/30 (63%) of BRCA1 tumors showed perinecrotic (hypoxia induced) and 8/30 (27%) a diffuse HIF-1α overexpression pattern, the latter more likely related to genetic alterations in oncogenes and tumor suppressor genes. In contrast, sporadic breast cancer HIF-1 expressing tumors showed an inverse ratio of perinecrotic/diffuse expression (31 vs. 69%, P = 0.0002). Glut-1 and CAIX, downstream HIF1 targets, were expressed in 27/30 (90%) and 15/21 (71%) of hereditary cases versus 54/183 (29%) and 24/183 (13%) in sporadic cases. p300 levels, necessary for HIF-1 downstream activation, were significantly higher in hereditary cases (20/21, 95%) compared to sporadic cases (91/183, 50%, P = 0.0001). In conclusion, in BRCA1 germline mutation related breast cancer, functional HIF-1α overexpression is seen at a much higher frequency than in sporadic breast cancer, mostly hypoxia induced. This points to an important role of hypoxia and its key regulator HIF-1α in hereditary breast carcinogenesis.     

Immunohistochemical analysis of RTKs expression identified HER3 as a prognostic indicator of gastric cancer

Standard treatment in Japan for the 13th Japanese Gastric Cancer Association stage II/III advanced gastric cancer is postoperative adjuvant S‐1 administration after curative surgery. High expression of receptor type tyrosine kinases (RTKs) has repeatedly represented poor prognosis for cancers. However it has not been demonstrated whether RTKs have prognostic relevance for stage II/III gastric cancer with standard treatment. Tumor tissues were obtained from 167 stage II/III advanced gastric cancer patients who underwent curative surgery and received postoperative S‐1 chemotherapy from 2000 to 2010. Expression of the RTKs including EGFR, HER2, HER3, IGF‐1R, and EphA2 was analyzed using immunohistochemistry (IHC). Analysis using a multivariate proportional hazard model identified the most significant RTKs that represented independent prognostic relevance. When tumor HER3 expression was classified into IHC 1+/2+ (n = 98) and IHC 0 (n = 69), the cumulative 5‐year Relapse Free Survival (5y‐RFS) was 56.5 and 82.9%, respectively (P = 0.0034). Significant prognostic relevance was similarly confirmed for IGF‐1R (P = 0.014), and EGFR (P = 0.030), but not for EphA2 or HER2 expression. Intriguingly, HER3 expression was closely correlated with IGF‐1R (P < 0.0001, R = 0.41), and EphA2 (P < 0.0001, R = 0.34) expression. Multivariate proportional hazard model analysis identified HER3 (IHC 1+/2+) (HR; 1.53, 95% CI, 1.11–2.16, P = 0.0078) as the sole RTK that was a poor prognostic factor independent of stage. Of the 53 patients who recurred, 40 patients (75.5%) were HER3‐positive. Thus, of the RTKs studied, HER3 was the only RTK identified as an independent prognostic indicator of stage II/III advanced gastric cancer with standard treatment.     

A randomised study of the effects of letrozole and anastrozole on oestrogen receptor positive breast cancers in postmenopausal women

Introduction Changes in proliferation as measured by Ki67 occur within 14 days of starting treatment with an aromatase inhibitor and these changes have been shown to be predictors of long term outcome. This study aimed to compare changes in proliferation following 14 days of treatment with anastrozole and letrozole. Methods Two hundred and six women with 209 estrogen receptor (ER) positive operable breast cancers (three bilateral) were randomly allocated to receive either 14 days treatment with 2.5 mg of letrozole or 1 mg of anastrozole prior to surgery. Changes in expression of estrogen (ER) and progesterone receptors (PgR) as assessed by ALLRED scores and proliferation as assessed by Ki67 were analysed. The HER2 status of each tumour was also assessed using a combination of the Hercept test and FISH. Results Both letrozole and anastrozole reduced ER expression (ALLRED score) by a mean of 0.32 (0.20–0.44), P < 0.001 and PgR fell by a mean of 2.54 (2.20–2.89) P < 0.0001. Letrozole reduced proliferation from a geometric mean of 6.37% to 0.81%, P < 0.0001 and anastrozole reduced proliferation from 5.81% to 0.77%, P < 0.0001. There was no differences between drugs in the fall in ER, PgR or proliferation. Both letrozole and anastrozole produced significant falls in proliferation in both HER2 positive and HER2 negative cancers, all P < 0.001. Discussion 14 days of both letrozole and anastrozole reduces proliferation, ER and PgR expression. No significant difference between these two drugs was identified. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

HER3 expression in patients with primary colorectal cancer and corresponding lymph node metastases related to clinical outcome

AimTo evaluate the expression and prognostic value of the epidermal growth factor receptor HER3 in patients with primary colorectal cancer (CRC) and corresponding lymph node metastases.Patient and methodsHER3 expression was analysed immunohistochemically (IHC) in primary tumours and in corresponding lymph node metastases from 236 patients with stage II and III CRC. In 58 primary tumours, fluorescence in situ hybridisation (FISH) detection was performed.ResultsHER3 was detected at high frequency in the cell membrane. Seventy percent of the primary tumours had a high HER3 expression compared to 75% in the lymph node metastases. HER3 expression in the primary tumour was an independent prognostic factor for overall survival in the entire group of patients (p = 0.026) and in the subgroup of patients with colon cancer stage II (p = 0.030). A high HER3 expression in the primary tumour was associated with worse clinical outcome. The expression of HER3 was homogenous within the primary tumour (r = 0.9, p < 0.0001) and correlated with the HER3 expression in corresponding lymph node metastases (r = 0.6, p < 0.0001). No gene amplification with respect to HER3 was seen in primary tumours using FISH analysis.ConclusionA high HER3 expression was found in 70% of the primary CRC tumours and in 75% of the corresponding lymph node metastases. HER3 expression in the tumour was an independent prognostic factor, where a high HER3 expression was associated with worse clinical outcome. There was a correlation in HER3 expression between primary tumour and corresponding lymph node metastases.     

Expression of the forkhead transcription factor FOXP1 is associated with that of estrogen receptorβ in primary invasive breast carcinomas

We previously identified a correlation between estrogen receptor alpha (ERα) and the candidate tumour suppressor gene Forkhead Box P1 (FOXP1), whose nuclear protein expression in breast tumours was associated with improved patient survival. However, the expression pattern of FOXP1 in normal breast tissue is more reminiscent of the second receptor, ERβ, which has an emerging role as a tumour suppressor in breast cancer and critically may underlie the ability of some ERα-negative tumours to respond to tamoxifen. In a series of 283 breast cancers, in which ERα-positive tumours were treated with tamoxifen, the nuclear expression of ERβ correlated significantly with ERα (p = 0.004), low-tumour grade (p = 0.008) and nuclear FOXP1 (p = 0.01). High-grade tumours exhibited significantly more cytoplasmic ERβ than the low-grade tumours (p = 0.006). Regression analysis demonstrated that FOXP1 expression was most closely related to nuclear ERβ (p = 0.021). Neither, nuclear or cytoplasmic ERβ expression demonstrated prognostic significance. FOXP1 is not estrogen regulated and silencing FOXP1 expression, using siRNA, did not affect ERα, ERβ or progesterone receptor expression, suggesting ER and FOXP1 co-expression may reflect a common regulatory mechanism.     

Microcephalin is a new novel prognostic indicator in breast cancer associated with BRCA1 inactivation

The authors have investigated the expression of the microcephalin (MCPH1) protein to evaluate its prognostic importance in breast cancer. Microcephalin is a damage response protein involved in the regulation of BRCA1 and BRCA2. BRCA1 mutations are often associated with basal-like breast cancer, which are also often negative for oestrogen receptor (ER), progesterone receptor (PR) and HER2. MCPH1 immunohistochemistry was performed on 319 breast cancers prepared as tissue microarray and correlated with pathology, survival, ER, PR, HER2, EGFR, CK5/6, CK14 and BRCA1 expression. After performing continuous data analysis, mean microcephalin expression decreased with increasing grade (P < 0.006). Mean microcephalin expression was lower in ER/PR negative (P < 0.001) and triple negative cancers (P < 0.004). Conversely, an association with HER2-positive cancers was also identified (P < 0.034). Reduced microcephalin also correlated with reduced nuclear BRCA1 staining (P < 0.001). No association was identified with basal markers. After dichotomising the data into low and high microcephalin expression, reduced expression was identified in 29% (93/319) of breast cancers. An association with low expression was identified in invasive ductal carcinomas with breast cancer-specific survival (BCSS) (P = 0.052). Multivariate analysis of ductal carcinomas showed that microcephalin, together with lymph node involvement and tumour size were independent predictors of BCSS (P = 0.037). Microcephalin expression is reduced in 29% of breast cancers, particularly in higher grade tumours and BRCA1-negative cases. Microcephalin is an independent predictor of BCSS in invasive ductal breast cancer patients and may prove to be a useful biomarker for the identification of aggressive breast cancers.