Oncogenic PIK3CA mutations in lobular breast cancer progression

Infiltrating lobular breast cancer (ILBC) is a tumor‐biologically distinct breast cancer subtype. A high frequency of oncogenic PIK3CA mutations has been reported in ILBC, which may allow for targeted therapy with newly developed PI3K inhibitors. This is of particular clinical relevance for ILBC patients, who have failed to respond to current treatment regimes and suffer from tumor recurrence or dissemination. In anticipation of this therapeutic strategy, we investigated PIK3CA mutations in ILBC with special reference to late stage tumor progression. A total of 88 ILBCs from 73 patients, including primary tumors (PTs, n = 43), ipsilateral locally recurrent tumors (LRTs, n = 15), and distant organ metastases (DOMs, n = 30), were compiled on tissue microarrays. Established ILBC marker proteins were evaluated by immunohistochemistry and PIK3CA hot spot mutations in exons 9 and 20 by direct sequencing. Matched PT/LRT, PT/DOM, and DOM/DOM cases were characterized on a patient‐by‐patient basis. Following correction for redundant patient representations, mutation frequencies were compared in PTs versus LRTs or DOMs. Nearly all specimens were E‐cadherin‐negative (99%), estrogen receptor (ER)‐positive (91%), and lacked basal epithelial markers (100%), demonstrating correct ILBC classification. PIK3CA mutations were detected in 32/88 (36%) specimens. The mutation rate was similar in PTs (33%) and DOMs (26%, P = 0.769), but approximately two‐fold increased in LRTs (69%, P = 0.022). Consistently, matched PT/LRT and LRT/DOM cases showed additional PIK3CA mutations in LRTs. Intriguingly, these findings imply that PIK3CA mutations are positively selected for during ILBC progression to local recurrence but not distant metastasis, which may have clinical implications for PI3K inhibitor‐based therapy. © 2012 Wiley Periodicals, Inc.     

Comprehensive genetic and functional characterization of IPH‐926: a novel CDH1‐null tumour cell line from human lobular breast cancer

Infiltrating lobular breast cancer (ILBC) is a clinically and biologically distinct tumour entity defined by a characteristic linear cord invasion pattern and inactivation of the CDH1 tumour suppressor gene encoding for E‐cadherin. ILBCs also lack β‐catenin expression and show aberrant cytoplasmic localization of the E‐cadherin binding protein p120‐catenin. The lack of a well‐characterized ILBC cell line has hampered the functional characterization of ILBC cells in vitro. We report the establishment of a permanent ILBC cell line, named IPH‐926, which was derived from a patient with metastatic ILBC. The DNA fingerprint of IPH‐926 verified genetic identity with the patient and had no match among the human cell line collections of several international biological resource banks. IPH‐926 expressed various epithelial cell markers but lacked expression of E‐cadherin due to a previously unreported, homozygous CDH1 241ins4 frameshift mutation. Detection of the same CDH1 241ins4 mutation in archival tumour tissue of the corresponding primary ILBC proved the clonal origin of IPH‐926 from this particular tumour. IPH‐926 also lacked β‐catenin expression and showed aberrant cytoplasmic localization of p120‐catenin. Array‐CGH analysis of IPH‐926 revealed a profile of genomic imbalances that included many distinct alterations previously observed in primary ILBCs. Spectral karyotyping of IPH‐926 showed a hyperdiploid chromosome complement and numerous clonal, structural aberrations. IPH‐926 cells were anti‐cancer drug‐resistant, clonogenic in soft agar, and tumourigenic in SCID mice. In xenograft tumours, IPH‐926 cells recapitulated the linear cord invasion pattern that defines ILBCs. In summary, IPH‐926 significantly extends the biological spectrum of the established breast cancer cell lines and will facilitate functional analyses of genuine human ILBC cells in vitro and in vivo. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Racially restricted contribution of immunoglobulin Fcγ and Fcγ receptor genotypes to humoral immunity to human epidermal growth factor receptor 2 in breast cancer

Tumour‐associated antigen human epidermal growth factor receptor 2 (HER2) is over‐expressed in 25–30% of breast cancer patients and is associated with poor prognosis. Naturally occurring anti‐HER2 antibody responses have been described in patients with HER2 over‐expressing tumours. There is significant interindividual variability in antibody responsiveness, but the host genetic factors responsible for this variability are poorly understood. The aim of the present investigation was to determine whether immunoglobulin genetic markers [GM (genetic determinants of γ chains)] and Fcγ receptor (FcγR) alleles contribute to the magnitude of natural antibody responsiveness to HER2 in patients with breast cancer. A total of 855 breast cancer patients from Japan and Brazil were genotyped for several GM and FcγR alleles. They were also characterized for immunoglobulin (Ig)G antibodies to HER2. In white subjects (n = 263), GM 23‐carriers had higher levels of anti‐HER2 antibodies than non‐carriers of this allele (p = 0·004). At the GM 5/21 locus, the homozygotes for the GM 5 allele had higher levels of anti‐HER2 antibodies than the other two genotypes (P = 0·0067). In black subjects (n = 42), FcγRIIa‐histidine/histidine homozygotes and FcγRIIIa‐phenylalanine/valine heterozygotes were associated with high antibody responses (P = 0·0071 and 0·0275, respectively). FcγR genotypes in white subjects and GM genotypes in black subjects were not associated with anti‐HER2 antibody responses. No significant associations were found in other study groups. These racially restricted contributions of GM and FcγR genotypes to humoral immunity to HER2 have potential implications for immunotherapy of breast cancer.     

HER2 overexpression and amplification as a potential therapeutic target in colorectal cancer: analysis of 3256 patients enrolled in the QUASAR,FOCUS and PICCOLO colorectal cancer trials

HER2 overexpression/amplification is linked to trastuzumab response in breast/gastric cancers. One suggested anti‐EGFR resistance mechanism in colorectal cancer (CRC) is aberrant MEK–AKT pathway activation through HER2 up‐regulation. We assessed HER2‐amplification/overexpression in stage II–III and IV CRC patients, assessing relationships to KRAS/BRAF and outcome. Pathological material was obtained from 1914 patients in the QUASAR stage II–III trial and 1342 patients in stage IV trials (FOCUS and PICCOLO). Tissue microarrays were created for HER2 immunohistochemistry. HER2‐amplification was assessed using FISH and copy number variation. KRAS/BRAF mutation status was assessed by pyrosequencing. Progression‐free survival (PFS) and overall survival (OS) data were obtained for FOCUS/PICCOLO and recurrence and mortality for QUASAR; 29/1342 (2.2%) stage IV and 25/1914 (1.3%) stage II–III tumours showed HER2 protein overexpression. Of the HER2‐overexpressing cases, 27/28 (96.4%) stage IV tumours and 20/24 (83.3%) stage II–III tumours demonstrated HER2 amplification by FISH; 41/47 (87.2%) also showed copy number gains. HER2‐overexpression was associated with KRAS/BRAF wild‐type (WT) status at all stages: in 5.2% WT versus 1.0% mutated tumours (p < 0.0001) in stage IV and 2.1% versus 0.2% in stage II–III tumours (p = 0.01), respectively. HER2 was not associated with OS or PFS. At stage II–III, there was no significant correlation between HER2 overexpression and 5FU/FA response. A higher proportion of HER2‐overexpressing cases experienced recurrence, but the difference was not significant. HER2‐amplification/overexpression is identifiable by immunohistochemistry, occurring infrequently in stage II–III CRC, rising in stage IV and further in KRAS/BRAF WT tumours. The value of HER2‐targeted therapy in patients with HER2‐amplified CRC must be tested in a clinical trial. © 2015 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Validity of immunohistochemistry method in predicting HER‐2 gene status and association of clinicopathological variables with it in invasive breast cancer patients

Human epidermal growth factor receptor‐2 is an important and prognostic factors and one of the most targeted proteins in breast cancer's therapy. There is no globally accepted method for determining its status. Here, we aimed to evaluate the immunohistochemistry method validity in predicting HER‐2 status by Fluorescence in situ hybridization method and investigate some clinicopathological variables association with HER‐2 amplification. A total of 190 HER‐2 2+ and 3+ by immunohistochemistry (IHC) invasive breast cancer cases were enrolled in this study. Fluorescence in situ hybridization (FISH) was performed for these cases using FDA criteria and the association between clinicopathological variables and HER‐2 status evaluated. Study consisted of 190 invasive breast cancer patients (160 HER‐2 2+ and 30 HER‐2 3+). HER‐2 FISH amplification according to FDA criteria was found 27.5% (44/160 patients) in HER‐2 2+ patients and 83.3% (25/30 patients) in HER‐2 3+ patients. Tumors with HER‐2 amplification were more likely to be ER‐negative (51.0% vs 31.2%, p = 0.013) and PR‐negative (52.9% vs 27.0%, p < 0.001). This study showed that immunohistochemistry is not a good method for evaluating HER‐2 status and decision‐making about trastuzumab therapy even with 3+ score patients. However, this result may not be too strong for IHC 3+ cases due to the limited number of these patients in this study.     

Amplified in breast cancer 1 expression in breast cancer

Aims: The amplified in breast cancer 1 (AIB1), steroid receptor co‐activator family member, acts as an oestrogen receptor (ER) co‐activator. Acting with HER‐2, it is thought to play a role in endocrine resistance by facilitating ER–growth factor crosstalk. The aim was to analyse AIB1 expression by immunohistochemistry and study its correlations with other prognostic variables in breast cancer and its effect on survival. Methods: A tissue microarray comprising tumours from 438 patients with 15.4 years’ median follow‐up was used. Interpretable AIB1 expression obtained in 395 patients was analysed along with other prognostic factors in breast cancer. Results: AIB1 expression scores ranged from 0 to 30; positive AIB1 expression (score > 14) was seen in 146/395 breast cancers; it correlated negatively with ER (P = 0.003) and progesterone receptor (PR) (P = 0.007), and positively with HER‐2 (P = 0.005) and tumour grade (P = 0.014). It did not correlate with nodal status (P = 0.437). Among ER+ patients, AIB1 expression showed a trend towards loss of PR expression (29% versus 20%; P = 0.14). AIB1 did not predict survival on univariate or multivariate analysis. Conclusions: AIB1 expression correlates with HER‐2 expression in breast cancer and shows a trend of association with loss of PR expression in ER+ tumours. Our study supports the postulated role of AIB1 in ER–growth factor interactions.     

The expression and clinical significance of ribophorin II (RPN2) in human breast cancer

Ribophorin II (RPN2), part of the N‐oligosaccharyltransferase complex, is highly expressed in breast cancer stem cells and is associated with tumor metastasis through interaction with mutant p53. The clinicopathological implication of RPN2 expression is undetermined. We examined immunohistochemically the expression levels of RPN2 and p53 in primary breast cancer tissues surgically resected from 218 patients. The correlations of RPN2 expression with the intrinsic subtype defined by hormone receptors (HRs) and HER2, clinicopathological parameters, p53 expression, and patients’ clinical outcomes were examined. RPN2 was positive in 139 (64%), and the incidence of RPN2 expression was higher in the triple‐negative breast cancer (TNBC) (HR‐/HER2‐) (65%) and HER2‐enriched (HR‐/HER2+) subtype (95%) than in the luminal A‐like (HR+/HER2‐) subtype (58%) (P = 0.0009). RPN2 expression was also correlated with p53 nuclear accumulation (P = 0.04). The RPN2‐positive/p53‐positive patient group showed significantly poorer prognosis than the RPN2‐negative group for disease‐free survival (P = 0.05) and for overall survival (P = 0.02). By multivariate analyses, the combination of RPN2 and p53 was not an independent prognostic factor. RPN2 expression was correlated with clinically aggressive features of breast cancer. These data support the further clinical application of anti‐RPN2 therapy and the development of personalized medicine.     

The repertoire of somatic genetic alterations of acinic cell carcinomas of the breast: an exploratory,hypothesis‐generating study

Acinic cell carcinoma (ACC) of the breast is a rare form of triple‐negative (that is, oestrogen receptor‐negative, progesterone receptor‐negative, HER2‐negative) salivary gland‐type tumour displaying serous acinar differentiation. Despite its triple‐negative phenotype, breast ACCs are reported to have an indolent clinical behaviour. Here, we sought to define whether ACCs have a mutational repertoire distinct from that of other triple‐negative breast cancers (TNBCs). DNA was extracted from microdissected formalin‐fixed, paraffin‐embedded sections of tumour and normal tissue from two pure and six mixed breast ACCs. Each tumour component of the mixed cases was microdissected separately. Tumour and normal samples were subjected to targeted capture massively parallel sequencing targeting all exons of 254 genes, including genes most frequently mutated in breast cancer and related to DNA repair. Selected somatic mutations were validated by targeted amplicon resequencing and Sanger sequencing. Akin to other forms of TNBC, the most frequently mutated gene found in breast ACCs was TP53 (one pure and six mixed cases). Additional somatic mutations affecting breast cancer‐related genes found in ACCs included PIK3CA, MTOR, CTNNB1, BRCA1, ERBB4, ERBB3, INPP4B, and FGFR2. Copy number alteration analysis revealed complex patterns of gains and losses similar to those of common forms of TNBCs. Of the mixed cases analysed, identical somatic mutations were found in the acinic and the high‐grade non‐acinic components in two out of four cases analysed, providing evidence of their clonal relatedness. In conclusion, breast ACCs display the hallmark somatic genetic alterations found in high‐grade forms of TNBC, including complex patterns of gene copy number alterations and recurrent TP53 mutations. Furthermore, we provide circumstantial genetic evidence to suggest that ACCs may constitute the substrate for the development of more aggressive forms of triple‐negative disease. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

HER2/neu (c‐erbB‐2) gene amplification and protein expression are rare in uterine cervical neoplasia: a tissue microarray study of 814 archival specimens

Published studies have reported widely variable incidence of HER2/neu (c‐erbB‐2) protein expression and HER2/neu (c‐erbB‐2) gene amplification in cervical carcinoma. We examined tissue microarrays (TMAs) constructed from 814 formaldehyde‐fixed paraffin‐embedded archival specimens of cervical intraepithelial neoplasia (CIN)1 (n = 262), CIN2 (n = 230), CIN3 (n = 186) and invasive carcinoma (n = 136), for HER2/neu protein expression by immunohistochemistry (IHC) and for HER2/neu gene amplification by chromogenic in situ hybridization (CISH). We found moderate or strong immunohistochemical positivity for HER2/neu in 64 of 814 specimens (7.9%). Using CISH, polysomy of the HER2/neu gene was detected in 87 cases (10.7%), low/borderline amplification in five cases (0.6%) and true amplification in four cases (0.5%). The correlation between IHC and CISH was statistically significant in CIN2, CIN3 and invasive cervical carcinoma specimens. When present, Her‐2/neu positivity is more commonly seen in higher grades of cervical dysplasia and in carcinoma. However, this large TMA study shows that HER2/neu oncoprotein expression and HER2/neu gene amplification overall are uncommon events in cervical neoplasia. This provides compelling evidence that HER2/neu plays no major role in the development and progression of cervical neoplasia.     

Familial concordance of breast cancer pathology as an indicator of genotype in multiple‐case families

The heterogeneity of multiple case breast cancer families that do not carry mutations in BRCA1 or BRCA2 (non‐BRCA1/2 families) poses a challenge to the identification of breast cancer susceptibility genes. The aim of this study was to determine whether intrafamilial concordance in breast cancer pathology could identify subgroups of non‐BRCA1/2 families with consistent genotypic features. Invasive breast cancers were reviewed from 84 individuals belonging to 30 multiple‐case families; BRCA1 (n = 9), BRCA2 (n = 10), and non‐BRCA1/2 (n = 11). Hierarchical cluster analysis based on histopathology and age at first diagnosis was then used to specify three subgroups designated Clusters 1–3. The genomic features of non‐BRCA1/2 families were examined by genome wide linkage and FGFR2 SNP genotyping, according to whether they showed cluster‐concordant or cluster‐mixed familial pathology. The majority of pathogenic BRCA1 mutation carriers (80%) fell into a single cluster. In contrast pathogenic BRCA2 mutation carriers were distributed across all three clusters and within families, cluster groups were also generally mixed. Most non‐BRCA1/2 mutation carriers belonged to Cluster 3 (71%). Genome wide linkage data from five non‐BRCA1/2 Cluster 3‐concordant families were compared with four mixed cluster non‐BRCA1/2 families. This revealed a number of distinct linkage peaks, including some regions previously associated with breast cancer susceptibility. The distribution of low risk alleles in FGFR2 was not different between these two subgroups (P = 0.237). The pattern of breast cancer pathology concordance amongst family members may assist the investigation of breast cancer susceptibility in multiple case families. © 2010 Wiley‐Liss, Inc.     

Heterogeneous amplification of ERBB2 in primary lesions is responsible for the discordant ERBB2 status of primary and metastatic lesions in gastric carcinoma

Kim M A, Lee H‐J, Yang H‐K, Bang Y‐J & Kim W H
(2011) Histopathology 59 , 822–831 Heterogeneous amplification of ERBB2 in primary lesions is responsible for the discordant ERBB2 status of primary and metastatic lesions in gastric carcinoma Aims: To determine the extent of HER2 homogeneity/heterogeneity in primary versus metastatic gastric carcinoma (GC). Materials and results: The human epidermal growth factor receptor 2 (HER2) status in primary and metastatic lesions was evaluated by immunohistochemistry (IHC) and fluorescence in‐situ hybridization (FISH). Four separate cohorts consisting of primary GC alone or primary GC paired with metastatic lesions were examined. In the FISH analysis of 325 primary GCs, eight cases (2.5%) showed amplification with a heterogeneous pattern, whereas 27 cases (8.3%) showed amplification with a homogeneous pattern, and in this cohort the discordant:concordant FISH ratio based on examination of three different areas in each primary lesion was 0.30:1. FISH testing using 250 paired primary and metastatic lesions revealed seven cases (2.8%) with discordant amplification. In metastatic disease positive conversion occurred in six cases (2.4%), whereas negative conversion happened in one case (0.4%). The discordant:concordant ratio of primary versus secondary lesions was 0.23:1. When the seven discordant cases were re‐evaluated using whole sections of primary GCs, six showed a heterogeneous pattern of amplification. Conclusions: These findings suggest that the discordant HER2 amplification observed in metastatic lesions is explained substantially by heterogeneity within primary tumours.     

Gene‐panel testing of breast and ovarian cancer patients identifies a recurrent RAD51C duplication

Gene‐panel sequencing allows comprehensive analysis of multiple genes simultaneously and is now routinely used in clinical mutation testing of high‐risk breast and ovarian cancer patients. However, only BRCA1 and BRCA2 are often analyzed also for large genomic changes. Here, we have analyzed 10 clinically relevant susceptibility genes in 95 breast or ovarian cancer patients with gene‐panel sequencing including also copy number variants (CNV) analysis for genomic changes. We identified 12 different pathogenic BRCA1, BRCA2, TP53, PTEN, CHEK2, or RAD51C mutations in 18 of 95 patients (19%). BRCA1/2 mutations were observed in 8 patients (8.4%) and CHEK2 protein‐truncating mutations in 7 patients (7.4%). In addition, we identified a novel duplication encompassing most of the RAD51C gene. We further genotyped the duplication in breast or ovarian cancer families (n = 1149), in unselected breast (n = 1729) and ovarian cancer cohorts (n = 553), and in population controls (n = 1273). Seven additional duplication carries were observed among cases but none among controls. The duplication associated with ovarian cancer risk (3/590 of all ovarian cancer patients, 0.5%, P = .032 compared with controls) and was found to represent a large fraction of all identified RAD51C mutations in the Finnish population. Our data emphasizes the importance of comprehensive mutation analysis including CNV detection in all the relevant genes.     

The assessment of HER2 status in breast cancer: the past,the present,and the future

Humanized monoclonal anti‐human growth factor receptor 2 (HER2) antibody trastuzumab was approved for HER2 positive breast cancer patient treatment 11 years after the demonstration of HER2 gene amplification associated with the HER2 protein overexpression in breast cancer in 1987. HER2 positive status of breast cancer patients is assessed by HER2 gene amplification with in situ hybridization (ISH) and/or HER2 protein overexpression with immunohistochemistry (IHC). Because the discordance between quantitative HER2 ISH and subjective, semi‐quantitative HER2 IHC assay results is a well‐recognized issue of HER2 testing, we developed an assay combining HER2 ISH and HER2 IHC assays (HER2 gene‐protein assay; HER2 GPA) as one test on the same tissue section. HER2 GPA allows pathologists to score the HER2 gene and HER2 protein status simultaneously at the individual cell level. The possibility that HER2 GPA may become the next generation of HER2 testing is discussed, particularly for cases in which it is difficult to assess the HER2 status of breast cancer patients due to the HER2 heterogeneity.     

Observer prediction of HER2 amplification in HercepTest 2+ breast cancers as a potential audit instrument

Going J J
(2011) Histopathology 59 , 333–335 Observer prediction of HER2 amplification in HercepTest 2+ breast cancers as a potential audit instrument Aims: Correct assignment of human epidermal growth factor receptor 2 (HER2) status in breast cancer is important. Indeterminate (2+) HER2 immunohistochemistry (IHC) is usually resolved by FISH for HER2 gene amplification. It was hypothesised that predicting HER2 amplification in IHC 2+ cases could improve audit of HER2 IHC and its interpretation. Methods and results: One observer (J. J. G.) interpreted 4343 assessable HercepTests on consecutive breast cancers from the West of Scotland over 45 consecutive months during 2007–2010, with 2+ cases classified prospectively as ‘probably amplified’, ‘possibly amplified’ or ‘probably not amplified’ prior to FISH. A HER2 to chromosome 17 FISH ratio >2 was taken to define HER2 amplification. There were 265 3+ cases (6.1%) and 883 2+ cases (20.3%). Of 187 ‘probably amplified’ 2+ cases, 166 (88.8%) were amplified, as were 90 (37.8%) of 238 ‘possibly amplified’ 2+ cases. Of 458 ‘probably not amplified’ but still 2+ cases, 59 (12.8%) were in fact amplified (overall χ² 333.89, df 2, P < 0.0001). In total, 580 of 4343 (13.4%) cancers were HER2‐positive (265 3+ by IHC and 315 2+ and HER2 amplified). Conclusions: Breast cancers HER2‐indeterminate (2+) by HercepTest IHC can be strongly separated into those probably HER2 amplified, a core indeterminate group and those probably not HER2 amplified. The percentage of HER2 amplified cases in each category is proposed as an instrument for comparison of HER2 IHC and its interpretation between laboratories and observers.     

Targeted next‐generation sequencing enables reliable detection of HER2 (ERBB2) status in breast cancer and provides ancillary information of clinical relevance

HER2‐positive breast cancers are a heterogeneous group of tumors, which share amplification and overexpression of HER2. In routine diagnostics, the HER2 (ERBB2) status is currently assessed by immunohistochemistry (IHC) and in situ hybridization (ISH). Data on targeted next‐generation sequencing (NGS) approaches that could be used to determine the HER2 status are sparse. Employing two breast cancer‐related gene panels, we performed targeted NGS of 41 FFPE breast cancers for which full pathological work‐up including ISH and IHC results was available. Selected cases were analyzed by qPCR. Of the 41 cases, the HER2 status of the 4 HER2‐positive and 6 HER2‐negative tumors was independently detected by our NGS approach achieving a concordance rate of 100%. The remaining 31 cases were equivocal HER2 cases by IHC of which 5 showed amplification of HER2 by ISH. Our NGS approach classified all non‐amplified cases correctly as HER2 negative and corroborated all but one of the 5 cases with amplified HER2 as detected by ISH. For the overall cohort, concordance between the gold standard and NGS was 97.6% (sensitivity 88.9% and specificity 100%). Additionally, we observed mutations in PIK3CA (44%), HER2 (8%), and CDH1 (6%) among others. Amplifications were found in CCND1 (12%), followed by MYC (10%) and EGFR (2%) and deletions in CDKN2A (10%), MAP2K4 and PIK3R1 (2% each). We here show that targeted NGS data can be used to interrogate the HER2 status with high specificity and high concordance with gold standard methods. Moreover, this approach identifies additional genetic events that may be clinically exploitable. © 2016 Wiley Periodicals, Inc.     

Oestrogen receptor‐β1 but not oestrogen receptor‐βcx is of prognostic value in apocrine carcinoma of the breast

Apocrine carcinoma of the breast, which frequently expresses oestrogen receptor‐β (ER‐β) in the absence of ER‐α and only infrequently is treated endocrinologically, gives an opportunity to investigate the clinicopathological role of ER‐β in breast cancer independent of ER‐α expression or tamoxifen treatment. Several isotypes of ER‐β, ER‐β1–5 etc., have been identified thus far; however, the clinicopathological importance of each ER‐β isotype in breast cancer is still uncertain. Here we aimed to clarify the clinicopathological importance of ER‐β1 and ER‐βcx (ER‐β2) in apocrine carcinomas, immunohistochemically examining expressions of ER‐β1 and ER‐βcx in 47 apocrine carcinomas. Positivity for ER‐β1 and ER‐βcx was observed in 41 (87%) and 18 (38%) of 47 cases, respectively. ER‐β1 positivity was related to smaller tumor size (P=0.0359), lower histological grade (P=0.0322), and higher disease‐free survival (P<0.0001), whereas ER‐βcx status was related to none of these parameters. ER‐β1 positivity was also associated with favorable clinical outcome in 24 so‐called triple‐negative (ER‐α‐negative/PR‐negative/HER2‐negative) apocrine carcinomas. ER‐β1 itself, independent of ER‐α expression and tamoxifen treatment, seems to have a tumor‐suppressive effect, at least in apocrine carcinomas. Further study of ER‐β1 is desired to optimize breast cancer treatment.     

Molecular characterization of mucinous ovarian tumours supports a stratified treatment approach with HER2 targeting in 19% of carcinomas

Mucinous ovarian carcinomas (MCs) typically do not respond to current conventional therapy. We have previously demonstrated amplification of HER2 in 6 of 33 (18.2%) mucinous ovarian carcinomas (MCs) and presented anecdotal evidence of response with HER2‐targeted treatment in a small series of women with recurrent HER2‐amplified (HER2+) MC. Here, we explore HER2 amplification and KRAS mutation status in an independent cohort of 189 MCs and 199 mucinous borderline ovarian tumours (MBOTs) and their association to clinicopathological features. HER2 status was assessed by immunohistochemistry (IHC), FISH, and CISH, and interpreted per ASCO/CAP guidelines, with intratumoural heterogeneity assessment on full sections, where available. KRAS mutation testing was performed with Sanger sequencing. Stage and grade were associated with recurrence on both univariate and multivariate analysis (p < 0.001). Assessment of HER2 status revealed overexpression/amplification of HER2 in 29/154 (18.8%) MCs and 11/176 (6.2%) MBOTs. There was excellent agreement between IHC, FISH, and CISH assessment of HER2 status (perfect concordance of HER2 0 or 1+ IHC with non‐amplified status, and 3+ IHC with amplified status). KRAS mutations were seen in 31/71 (43.6%) MCs and 26/33 (78.8%) MBOTs, and were near mutually exclusive of HER2 amplification. In the 189 MC cases, a total of 54 recurrences and 59 deaths (53 of progressive disease) were observed. Within MCs, either HER2 amplification/overexpression or KRAS mutation was associated with decreased likelihood of disease recurrence (p = 0.019) or death (p = 0.0041) when compared to cases with neither feature. Intratumoural heterogeneity was noted in 26% of HER2‐overexpressing cases. These data support the stratification of MCs for the testing of new treatments, with HER2‐targeted therapy as a viable option for HER2+ advanced or recurrent disease. Further research is required to delineate the molecular and clinical features of the ～34% of MC cases with neither HER2 amplification nor KRAS mutations.     

Microglandular adenosis associated with triple‐negative breast cancer is a neoplastic lesion of triple‐negative phenotype harbouring TP53 somatic mutations

Microglandular adenosis (MGA) is a rare proliferative lesion of the breast composed of small glands lacking myoepithelial cells and lined by S100‐positive, oestrogen receptor (ER)‐negative, progesterone receptor (PR)‐negative, and HER2‐negative epithelial cells. There is evidence to suggest that MGA may constitute a non‐obligate precursor of triple‐negative breast cancer (TNBC). We sought to define the genomic landscape of pure MGA and of MGA, atypical MGA (AMGA) and associated TNBCs, and to determine whether synchronous MGA, AMGA, and TNBCs would be clonally related. Two pure MGAs and eight cases of MGA and/or AMGA associated with in situ or invasive TNBC were collected, microdissected, and subjected to massively parallel sequencing targeting all coding regions of 236 genes recurrently mutated in breast cancer or related to DNA repair. Pure MGAs lacked clonal non‐synonymous somatic mutations and displayed limited copy number alterations (CNAs); conversely, all MGAs (n = 7) and AMGAs (n = 3) associated with TNBC harboured at least one somatic non‐synonymous mutation (range 3–14 and 1–10, respectively). In all cases where TNBCs were analyzed, identical TP53 mutations and similar patterns of gene CNAs were found in the MGA and/or AMGA and in the associated TNBC. In the MGA/AMGA associated with TNBC lacking TP53 mutations, somatic mutations affecting PI3K pathway‐related genes (eg PTEN, PIK3CA, and INPP4B) and tyrosine kinase receptor signalling‐related genes (eg ERBB3 and FGFR2) were identified. At diagnosis, MGAs associated with TNBC were found to display subclonal populations, and clonal shifts in the progression from MGA to AMGA and/or to TNBC were observed. Our results demonstrate the heterogeneity of MGAs, and that MGAs associated with TNBC, but not necessarily pure MGAs, are genetically advanced, clonal, and neoplastic lesions harbouring recurrent mutations in TP53 and/or other cancer genes, supporting the notion that a subset of MGAs and AMGAs may constitute non‐obligate precursors of TNBCs. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Announcements

Mohammed Z M A, Going J J, McMillan D C, Orange C, Mallon E, Doughty J C & Edwards J
(2012) Histopathology  61, 675–684 Comparison of visual and automated assessment of HER2 status and their impact on outcome in primary operable invasive ductal breast cancer Aims: To compare visual and computerized image analysis of HER2 immunohistochemistry (IHC) with fluorescence in‐situ hybridization (FISH) for HER2 status, and to examine the relationships with outcome in patients with primary operable invasive ductal breast cancer. Methods and results: Tissue microarrays for 431 breast cancer patients were used to compare different approaches to the assessment of HER2 status. The cores were scored visually and with the Slidepath Tissue IA system, using the NICE‐approved scoring system for the HercepTest, as well as by FISH. The agreement between visual and image analysis of HER2 IHC was excellent [interclass correlation coefficient (ICCC) = 0.95, r_s = 0.90, r = 0.91, k = 0.81, and P < 0.001]. The agreement of HER2 FISH with visual and image analysis of HER2 IHC was also excellent (ICCC = 0.95 and ICCC = 0.92, respectively). Univariate survival analysis showed equivalent associations of visual and image analysis of HER2 and HER2 FISH with both recurrence‐free survival (all P < 0.01) and cancer‐specific survival (all P < 0.05) in patients with invasive ductal breast cancer. Conclusions: Computerized image analysis of HER2 IHC gives results comparable to those obtained with visual assessment, with possible advantages in diagnostic pathology.