Age-related clusters and favorable immune phenotypes in breast cancer of the young.

Breast cancer (BC) patients aged below 40 years at diagnosis experience aggressive disease and poorer survival compared to women diagnosed at 40-49 years, but the age-related biology is described to little extent. Here, we explored transcriptional alterations in BC to gain better understanding of age-related tumor biology. We studied a subset of the Bergen in-house cohort (n=127, age range 26-49) and used the NanoString Breast Cancer 360 expression panel on formalin-fixed paraffin-embedded breast cancer tissue, and publicly available global breast cancer mRNA expression data (n=204, age range 22-49), to explore differentially expressed genes between the young (age <40) and older (age 40-49) patients. Unsupervised hierarchical clustering was applied to identify gene expression-based patient clusters. We applied established computational approaches to define the PAM50 subtypes, Risk of recurrence scores (ROR), and Risk groups, and to infer the proportions of 22 immune cell types from bulk gene expression profiles of patients aged <50 at breast cancer diagnosis. Differentially expressed genes and gene sets were investigated using OncoEnrichR and g:Profiler to describe functional profiles and pathway enrichment. We identified four age-related patient clusters presenting distinct characteristics of PAM50 subtypes and ROR profiles, which demonstrated independent prognostic value when adjusted for traditional clinico-pathologic variables and the known molecular subtypes. Our findings showed better survival than expected in the basal-enriched cluster 2, and in triple negative and basal-like BC. Deconvolution analyses of immunophenotypes indicated higher levels of M0 and M1 macrophages than M2 macrophages in subsets of young BC. Our approach identifies age-based patient clusters with distinct clinico-pathologic profiles, to a large extent overlapping with the PAM50 subtypes, although with independent prognostic value in multivariate survival analyses. The patient clusters provided new insight in the immune cell distribution across tumor subtypes, potentially contributing to survival differences between the clusters and the molecular subtypes and indicating age-related mechanisms improving outcome. Our study confirms the applicability of ROR as a valid prognosticator also in a young breast cancer cohort.

Brystkreft hos menn ­ histopatologi og biomarkører.

Background: Breast cancer in men accounts for around 1 % of all cases of the disease. The study aimed to identify histopathological parameters and selected biomarkers in men with breast cancer. Material and method: Retrospective study of archival material from 53 men diagnosed with breast cancer at the department of pathology, Haukeland University Hospital, in the period 1996-2020. The prevalence of the oestrogen receptor (ER), progesterone receptor (PGR) and Human Epidermal Growth Factor (HER2) biomarkers was examined. Results: Median age at time of diagnosis was 72 years. Median tumour diameter was 24 mm. Forty-nine tumours were classified histologically as invasive carcinoma of no special type (NST), 29 tumours were histologic grade 2 and 18 were grade 3. Fifty-two tumours were ER positive, 39 were PGR positive and four were HER2 positive. Twenty-five patients had lymph node metastases. Interpretation: Our findings indicate that men with breast cancer are diagnosed at an older age than women, and that men have a more advanced stage than women at the time of diagnosis. The histopathology and expression of biomarkers of breast cancer differ between men and women.

Age-related phenotypes in breast cancer: A population-based study.

Breast cancer in young (<40 years) is associated with a higher frequency of aggressive tumor types and poor prognosis. It remains unclear if there is an underlying age-related biology that contributes to the unfavorable outcome. We aim to investigate the relationship between age and breast cancer biology, with emphasis on proliferation. Clinico-pathologic information, immunohistochemical markers and follow-up data were obtained for all patients aged <50 (Bergen cohort-1; n = 355, not part of a breast screening program) and compared to previously obtained information on patients aged 50 to 69 years (Bergen cohort-2; n = 540), who participated in the Norwegian Breast Cancer Screening Program. Young breast cancer patients presented more aggressive tumor features such as hormone receptor negativity, HER2 positivity, lymph-node metastasis, the HER2-enriched and triple-negative subtypes and shorter survival. Age <40 was significantly associated with higher proliferation (by Ki67). Ki67 showed weaker prognostic value in young patients. We point to aggressive phenotypes and increased tumor cell proliferation in breast cancer of the young. Hence, tumors of young breast cancer patients may present unique biological features, also when accounting for screen/interval differences, that may open for new clinical opportunities, stratifying treatment by age.

Hypoxia induced responses are reflected in the stromal proteome of breast cancer.

Cancers are often associated with hypoxia and metabolic reprogramming, resulting in enhanced tumor progression. Here, we aim to study breast cancer hypoxia responses, focusing on secreted proteins from low-grade (luminal-like) and high-grade (basal-like) cell lines before and after hypoxia. We examine the overlap between proteomics data from secretome analysis and laser microdissected human breast cancer stroma, and we identify a 33-protein stromal-based hypoxia profile (33P) capturing differences between luminal-like and basal-like tumors. The 33P signature is associated with metabolic differences and other adaptations following hypoxia. We observe that mRNA values for 33P predict patient survival independently of molecular subtypes and basic prognostic factors, also among low-grade luminal-like tumors. We find a significant prognostic interaction between 33P and radiation therapy.

Vessel size as a marker of survival in estrogen receptor positive breast cancer.

PURPOSE: Angiogenesis is crucial for tumor growth and is one of the hallmarks of cancer. In this study, we analyzed microvessel density, vessel median size, and perivascular a-SMA expression as prognostic biomarkers in breast cancer. METHODS: Dual IHC staining was performed where alpha-SMA antibodies were used together with antibodies against the endothelial cell marker CD34. Digital images of stainings were analyzed to extract quantitative data on vessel density, vessel size, and perivascular alpha-SMA status. RESULTS: The analyses in the discovery cohort (n = 108) revealed a statistically significant relationship between large vessel size and shorter disease-specific survival (p = 0.007, log-rank test; p = 0.01, HR 3.1; 95% CI 1.3-7.4, Cox-regression analyses). Subset analyses indicated that the survival association of vessel size was strengthened in ER + breast cancer. To consolidate these findings, additional analyses were performed on a validation cohort (n = 267) where an association between large vessel size and reduced survival was also detected in ER + breast cancer (p = 0.016, log-rank test; p = 0.02; HR 2.3, 95% CI 1.1-4.7, Cox-regression analyses). CONCLUSION: Alpha-SMA/CD34 dual-IHC staining revealed breast cancer heterogeneity regarding vessel size, vessel density, and perivascular a-SMA status. Large vessel size was linked to shorter survival in ER + breast cancer.

CD47 and CD68 expression in breast cancer is associated with tumor-infiltrating lymphocytes, blood vessel invasion, detection mode, and prognosis.

CD47 expressed on tumor cells binds to signal regulatory protein alpha on macrophages, initiating inhibition of phagocytosis. We investigated the relationships between tumor expression of CD47 and CD68 macrophage content, subsets of tumor-infiltrating lymphocytes (TILs), and vascular invasion in breast cancer. A population-based series of 282 cases (200 screen detected and 82 interval patients) from the Norwegian Breast Cancer Screening Program was examined. Immunohistochemical staining for CD47 and CD68 was evaluated on tissue microarray (TMA) slides. For CD47 evaluation, a staining index was used. CD68 tumor-associated macrophages were counted and dichotomized. TIL subsets (CD45, CD3, CD4, CD8, and FOXP3) were counted and dichotomized using immunohistochemistry on TMA slides. Vascular invasion (both lymphatic and blood vessel) was determined on whole tissue slides. High CD47 tumor cell expression or high counts of CD68 macrophages were significantly associated with elevated levels of all TIL subsets (p < 0.02), CD163 macrophages (p < 0.001), blood vessel invasion (CD31 positive) (p < 0.01), and high tumor cell Ki67 (p < 0.004). High CD47 expression was associated with ER negativity (p < 0.001), HER2 positive status (p = 0.03), and interval-detected tumors (p = 0.03). Combined high expression of CD47-CD68 was associated with a shorter recurrence-free survival (RFS) by multivariate analysis (hazard ratio [HR]: 2.37, p = 0.018), adjusting for tumor diameter, histologic grade, lymph node status, and molecular subtype. Patients with luminal A tumors showed a shorter RFS for CD47-CD68 high cases by multivariate assessment (HR: 5.73, p = 0.004). This study demonstrates an association of concurrent high CD47 tumor cell expression and high CD68 macrophage counts with various TIL subsets, blood vessel invasion (CD31 positive), other aggressive tumor features, and interval-presenting breast cancer. Our findings suggest a link between CD47, tumor immune response, and blood vessel invasion (CD31 positive). Combined high expression of CD47-CD68 was an independent prognostic factor associated with poor prognosis in all cases, as well as in the luminal A category.

Tumor-associated lymphocytes and macrophages are related to stromal elastosis and vascular invasion in breast cancer.

The tumor microenvironment plays a critical role in breast cancer progression. Here, we investigated tumor-infiltrating lymphocytes (TILs) and associations with macrophage numbers, tumor stromal elastosis, vascular invasion, and tumor detection mode. We performed a population-based retrospective study using data from The Norwegian Breast Cancer Screening Program in Vestfold County (2004-2009), including 200 screen-detected and 82 interval cancers. The number of TILs (CD45+, CD3+, CD4+, CD8+, and FOXP3+) and tumor-associated macrophages (CD163+) was counted using immunohistochemistry on tissue microarray slides. Lymphatic and blood vessel invasion (LVI and BVI) were recorded using D2-40 and CD31 staining, and the amount of elastosis (high/low) was determined on regular HE-stained slides. High numbers of all TIL subsets were associated with LVI (p ≤ 0.04 for all), and high counts of several TIL subgroups (CD8+, CD45+, and FOXP3+) were associated with BVI (p ≤ 0.04 for all). Increased levels of all TIL subsets, except CD4+, were associated with estrogen receptor-negative tumors (p < 0.001) and high tumor cell proliferation by Ki67 (p < 0.001). Furthermore, high levels of all TIL subsets were associated with high macrophage counts (p < 0.001) and low-grade stromal elastosis (p ≤ 0.02). High counts of CD3+, CD8+, and FOXP3+ TILs were associated with interval detected tumors (p ≤ 0.04 for all). Finally, in the luminal A subgroup, high levels of CD3+ and FOXP3+ TILs were associated with shorter recurrence-free survival, and high counts of FOXP3+ were linked to reduced breast cancer-specific survival. In conclusion, higher levels of different TIL subsets were associated with stromal features such as high macrophage counts (CD163+), presence of vascular invasion, absence of stromal elastosis, as well as increased tumor cell proliferation and interval detection mode. Our findings support a link between immune cells and vascular invasion in more aggressive breast cancer. Notably, presence of TIL subsets showed prognostic value within the luminal A category.

Fibulin-2 expression associates with vascular invasion and patient survival in breast cancer.

Stromal elastosis is related to good prognosis in breast cancer and fibulin-2 helps to stabilize elastic fibers in basement membranes. Here, we examined the level of perivascular fibulin-2 expression in relation to elastosis content, vascular invasion, molecular subtypes, tumour detection mode, and patient prognosis in breast cancer. We performed a population based retrospective study of invasive breast cancers from the Norwegian Breast Screening Program (Vestfold County, 2004-2009) including 200 screen-detected and 82 interval cancers. Perivascular fibulin-2 staining was semi-quantitatively graded based on immunohistochemistry (1-3) and dichotomized as high expression (grade 2-3) and low expression (grade 1). Elastosis content was graded on a 4-tiered scale and dichotomized as high (score 3) and low (score 0-2) expression, whereas lymphatic (LVI) and blood vessel invasion (BVI) were recorded as absent or present by immunohistochemistry. High perivascular fibulin-2 expression was strongly related to stromal elastosis (p<0.001), and inversely associated with BVI and LVI (p<0.001 for both). High fibulin-2 was associated with luminal breast cancer subgroups (p<0.001) and inversely with interval cancers compared with screen-detected tumours (p<0.001). By univariate analysis, low perivascular fibulin-2 was associated with reduced recurrence-free survival (p = 0.002) and disease specific survival (p = 0.019). Low perivascular fibulin-2 expression was strongly related to vascular invasion, low stromal elastosis, non-luminal breast cancer subtypes, interval presentation, and adverse prognosis.

Baseline microvessel density predicts response to neoadjuvant bevacizumab treatment of locally advanced breast cancer.

A subset of breast cancer patients benefits from preoperative bevacizumab and chemotherapy, but validated predictive biomarkers are lacking. Here, we aimed to evaluate tissue-based angiogenesis markers for potential predictive value regarding response to neoadjuvant bevacizumab treatment in breast cancer. In this randomized 1:1 phase II clinical trial, 132 patients with large or locally advanced HER2-negative tumors received chemotherapy ± bevacizumab. Dual Factor VIII/Ki-67 immunohistochemical staining was performed on core needle biopsies at baseline and week 12. Microvessel density (MVD), proliferative microvessel density (pMVD; Factor VIII/Ki-67 co-expression), glomeruloid microvascular proliferation (GMP), and a gene expression angiogenesis signature score, were studied in relation to pathologic complete response (pCR), clinico-pathologic features and intrinsic molecular subtype. We found that high baseline MVD (by median) significantly predicted pCR in the bevacizumab-arm (odds ratio 4.9, P = 0.012). High pMVD, presence of GMP, and the angiogenesis signature score did not predict pCR, but were associated with basal-like (P ≤ 0.009) and triple negative phenotypes (P ≤ 0.041). pMVD and GMP did also associate with high-grade tumors (P ≤ 0.048). To conclude, high baseline MVD significantly predicted response to bevacizumab treatment. In contrast, pMVD, GMP, and the angiogenesis signature score, did not predict response, but associated with aggressive tumor features, including basal-like and triple-negative phenotypes.

Stathmin expression associates with vascular and immune responses in aggressive breast cancer subgroups.

Studies indicate that stathmin expression associates with PI3K activation in breast cancer, suggesting stathmin as a marker for targetable patient subgroups. Here we assessed stathmin in relation to tumour proliferation, vascular and immune responses, BRCA1 germline status, basal-like differentiation, clinico-pathologic features, and survival. Immunohistochemical staining was performed on breast cancers from two series (cohort 1, n = 187; cohort 2, n = 198), and mass spectrometry data from 24 cases and 12 breast cancer cell lines was examined for proteomic profiles. Open databases were also explored (TCGA, METABRIC, Oslo2 Landscape cohort, Cancer Cell Line Encyclopedia). High stathmin expression associated with tumour proliferation, p53 status, basal-like differentiation, BRCA1 genotype, and high-grade histology. These patterns were confirmed using mRNA data. Stathmin mRNA further associated with tumour angiogenesis, immune responses and reduced survival. By logistic regression, stathmin protein independently predicted a BRCA1 genotype (OR 10.0, p = 0.015) among ER negative tumours. Cell line analysis (Connectivity Map) implied PI3K inhibition in tumours with high stathmin. Altogether, our findings indicate that stathmin might be involved in the regulation of tumour angiogenesis and immune responses in breast cancer, in addition to tumour proliferation. Cell data point to potential effects of PI3K inhibition in tumours with high stathmin expression.

MRPS23 amplification and gene expression in breast cancer; association with proliferation and the non-basal subtypes.

PURPOSE: MRPS23 is recognized as a driver of proliferation in luminal breast cancer. The aims of the present study were to describe MRPS23 copy number change in breast cancer, and to assess associations between MRPS23 copy number change and molecular subtype, proliferation and prognosis, and between MRPS23 gene expression and molecular subtype and prognosis. METHODS: Using fluorescence in situ hybridization (FISH), we examined MRPS23 and centromere 17 copy number in 590 formalin-fixed, paraffin-embedded primary tumours and 144 corresponding lymph node metastases from a cohort of Norwegian breast cancer patients. Furthermore, we analysed MRPS23 gene expression data in 1971 primary breast cancer tumours from the METABRIC dataset. We used Pearson's χ2 test to assess associations between MRPS23 copy number and molecular subtype and proliferation, and between MRPS23 expression and molecular subtype. We studied prognosis by estimating hazard ratios and cumulative incidence of death from breast cancer according to MRPS23 copy number and MRPS23 expression status. RESULTS: We found MRPS23 amplification (mean MRPS23 copy number ≥ 6 and/or MRPS23/chromosome 17 ratio ≥ 2) in 8% of primary tumours. Copy number increase associated with non-basal subtypes and higher tumour cell proliferation (Ki67). Higher MRPS23 expression associated with the Luminal B subtype. We found no significant association between MRPS23 amplification or MRSP23 gene expression, and prognosis. CONCLUSION: Amplification of MRPS23 is associated with higher proliferation and non-basal subtypes in breast cancer. High MRPS23 expression is associated with the Luminal B subtype.

Integrin α11ß1 is expressed in breast cancer stroma and associates with aggressive tumor phenotypes.

Cancer-associated fibroblasts are essential modifiers of the tumor microenvironment. The collagen-binding integrin α11ß1 has been proposed to be upregulated in a pro-tumorigenic subtype of cancer-associated fibroblasts. Here, we analyzed the expression and clinical relevance of integrin α11ß1 in a large breast cancer series using a novel antibody against the human integrin α11 chain. Several novel monoclonal antibodies against the integrin α11 subunit were tested for use on formalin-fixed paraffin-embedded tissues, and Ab 210F4B6A4 was eventually selected to investigate the immunohistochemical expression in 392 breast cancers using whole sections. mRNA data from METABRIC and co-expression patterns of integrin α11 in relation to αSMA and cytokeratin-14 were also investigated. Integrin α11 was expressed to varying degrees in spindle-shaped cells in the stroma of 99% of invasive breast carcinomas. Integrin α11 co-localized with αSMA in stromal cells, and with αSMA and cytokeratin-14 in breast myoepithelium. High stromal integrin α11 expression (66% of cases) was associated with aggressive breast cancer features such as high histologic grade, increased tumor cell proliferation, ER negativity, HER2 positivity, and triple-negative phenotype, but was not associated with breast cancer specific survival at protein or mRNA levels. In conclusion, high stromal integrin α11 expression was associated with aggressive breast cancer phenotypes.

PIK3CA Amplification Associates with Aggressive Phenotype but Not Markers of AKT-MTOR Signaling in Endometrial Carcinoma.

PURPOSE: Amplification of PIK3CA, encoding the PI3K catalytic subunit alpha, is common in uterine corpus endometrial carcinoma (UCEC) and linked to an aggressive phenotype. However, it is unclear whether PIK3CA amplification acts via PI3K activation. We investigated the association between PIK3CA amplification, markers of PI3K activity, and prognosis in a large cohort of UCEC specimens. EXPERIMENTAL DESIGN: UCECs from 591 clinically annotated patients including 83 tumors with matching metastasis (n = 188) were analyzed by FISH to determine PIK3CA copy-number status. These data were integrated with mRNA and protein expression and clinicopathologic data. Results were verified in The Cancer Genome Atlas dataset. RESULTS: PIK3CA amplifications were associated with disease-specific mortality and with other markers of aggressive disease. PIK3CA amplifications were also associated with other amplifications characteristic of the serous-like somatic copy-number alteration (SCNA)-high subgroup of UCEC. Tumors with PIK3CA amplification also demonstrated an increase in phospho-p70S6K but had decreased levels of activated phospho-AKT1-3 as assessed by Reverse Phase Protein Arrays and an mRNA signature of MTOR inhibition. CONCLUSIONS: PIK3CA amplification is a strong prognostic marker and a potential marker for the aggressive SCNA-high subgroup of UCEC. Although PIK3CA amplification associates with some surrogate measures of increased PI3K activity, markers for AKT1-3 and MTOR signaling are decreased, suggesting that this signaling is not a predominant pathway to promote cancer growth of aggressive serous-like UCEC. Moreover, these associations may reflect features of the SCNA-high subgroup of UCEC rather than effects of PIK3CA amplification itself.

Identification of highly connected and differentially expressed gene subnetworks in metastasizing endometrial cancer.

We have identified nine highly connected and differentially expressed gene subnetworks between aggressive primary tumors and metastatic lesions in endometrial carcinomas. We implemented a novel pipeline combining gene set and network approaches, which here allows integration of protein-protein interactions and gene expression data. The resulting subnetworks are significantly associated with disease progression across tumor stages from complex atypical hyperplasia, primary tumors to metastatic lesions. The nine subnetworks include genes related to metastasizing features such as epithelial-mesenchymal transition (EMT), hypoxia and cell proliferation. TCF4 and TWIST2 were found as central genes in the subnetwork related to EMT. Two of the identified subnetworks display statistically significant association to patient survival, which were further supported by an independent validation in the data from The Cancer Genome Atlas data collection. The first subnetwork contains genes related to cell proliferation and cell cycle, while the second contains genes involved in hypoxia such as HIF1A and EGLN3. Our findings provide a promising context to elucidate the biological mechanisms of metastasis, suggest potential prognostic markers and further identify therapeutic targets. The pipeline R source code is freely available, including permutation tests to assess statistical significance of the identified subnetworks.

Deblender: a semi-/unsupervised multi-operational computational method for complete deconvolution of expression data from heterogeneous samples.

BACKGROUND: Towards discovering robust cancer biomarkers, it is imperative to unravel the cellular heterogeneity of patient samples and comprehend the interactions between cancer cells and the various cell types in the tumor microenvironment. The first generation of 'partial' computational deconvolution methods required prior information either on the cell/tissue type proportions or the cell/tissue type-specific expression signatures and the number of involved cell/tissue types. The second generation of 'complete' approaches allowed estimating both of the cell/tissue type proportions and cell/tissue type-specific expression profiles directly from the mixed gene expression data, based on known (or automatically identified) cell/tissue type-specific marker genes. RESULTS: We present Deblender, a flexible complete deconvolution tool operating in semi-/unsupervised mode based on the user's access to known marker gene lists and information about cell/tissue composition. In case of no prior knowledge, global gene expression variability is used in clustering the mixed data to substitute marker sets with cluster sets. In addition, we integrate a model selection criterion to predict the number of constituent cell/tissue types. Moreover, we provide a tailored algorithmic scheme to estimate mixture proportions for realistic experimental cases where the number of involved cell/tissue types exceeds the number of mixed samples. We assess the performance of Deblender and a set of state-of-the-art existing tools on a comprehensive set of benchmark and patient cancer mixture expression datasets (including TCGA). CONCLUSION: Our results corroborate that Deblender can be a valuable tool to improve understanding of gene expression datasets with implications for prediction and clinical utilization. Deblender is implemented in MATLAB and is available from ( https://github.com/kondim1983/Deblender/ ).

Transient elevation of anti-transglutaminase and anti-endomysium antibodies in Giardia infection.

Markers of celiac disease (CeD) may be elevated in various conditions of intestinal inflammation or autoimmune disease. Recent reports argue that intestinal infection may induce development of CeD in susceptible individuals. Serum anti-tissue transglutaminase (tTG) and anti-endomysium antibodies (EMA) have been proposed in previous reports to be helpful in differentiating between giardiasis and CeD. In this report, we describe eight cases with elevated CeD serological markers and pathological duodenal histology during, or shortly after, Giardia infection. We present follow-up clinical and serological findings to determine which of these that were diagnosed with CeD. Serum levels of tTGand EMA did not discriminate well between patients where CeD was excluded, and those who were later diagnosed with CeD. The value of these serological CeD markers is discussed in relation to CeD diagnosis in cases with chronic or recent giardiasis.

Tumor-associated macrophages are strongly related to vascular invasion, non-luminal subtypes, and interval breast cancer.

Tumor-associated macrophages (TAM) resemble M2 macrophages, promote tumor invasion and show strong expression of CD163 in breast cancer. We here investigated the association between CD163-positive macrophages and vascular invasion, molecular subgroups, mode of detection, and patient outcome. We performed a population-based, retrospective study of invasive breast cancer from the Norwegian Breast Cancer Screening Programme in Vestfold County (2004-2009), including 200 screen-detected and 82 interval cancers. Immunohistochemically CD163-positive macrophages were counted in the most active areas (hotspots) and dichotomized as high (upper quartile) and low counts. Lymphatic vessel involvement (LVI) and blood vessel invasion (BVI) were recorded separately based on immunohistochemical staining (D2-40 and CD31 antibodies). High levels of CD163-positive macrophages were associated with BVI and lymphatic involvement as well as interval cancer detection when compared to screening-detected tumors. In addition, the presence of high CD163+ TAM levels was more often observed in HER2-positive, basal-like and Triple-negative breast cancers and was associated with several features of aggressive tumors. In survival analyses, cases with combined high CD163 counts and BVI showed a significantly reduced recurrence-free survival (RFS) and disease-specific survival (DSS) (P < .001 for both) compared with all other cases. The presence of CD163-positive, tumor-associated macrophages is strongly related to aggressive features of breast cancer such as vessel invasion, detection between screening intervals, non-luminal molecular subgroups and reduced survival.

C77G in PTPRC (CD45) is no risk allele for ovarian cancer, but associated with less aggressive disease.

The pan lymphocyte marker CD45 exists in various isoforms arising from alternative splicing of the exons 4, 5 and 6. While naïve T cells express CD45RA translated from an mRNA containing exon 4, exons 4-6 are spliced out to encode the shorter CD45R0 in antigen-experienced effector/memory T cells. The SNP C77G (rs17612648) is located in exon 4 and blocks the exon's differential splicing from the pre-mRNA, enforcing expression of CD45RA. Several studies have linked C77G to autoimmune diseases but lack of validation in other cohorts has left its role elusive. An incidental finding in an ovarian cancer patient cohort from West Norway (Bergen region, n = 312), suggested that the frequency of C77G was higher among ovarian cancer patients than in healthy Norwegians (n = 1,357) (3.0% vs. 1.8% allele frequency). However, this finding could not be validated in a larger patient cohort from South-East Norway (Oslo region, n = 1,198) with 1.2% allele frequency. Hence, C77G is not associated with ovarian cancer in the Norwegian population. However, its frequency was increased in patients with FIGO stage II, endometrioid histology or an age at diagnosis of 60 years or older indicating a possible association with a less aggressive cancer type.

Expression of Nestin associates with BRCA1 mutations, a basal-like phenotype and aggressive breast cancer.

We here examined whether Nestin, by protein and mRNA levels, could be a predictor of BRCA1 related breast cancer, a basal-like phenotype, and aggressive tumours. Immunohistochemical staining of Nestin was done in independent breast cancer hospital cohorts (Series I-V, total 1257 cases). Also, TCGA proteomic data (n = 103), mRNA microarray data from TCGA (n = 520), METABRIC (n = 1992), and 6 open access breast cancer datasets (n = 1908) were analysed. Patients with Nestin protein expression in tumour cells more often had BRCA1 germline mutations (OR 8.7, p < 0.0005, Series III), especially among younger patients (<40 years at diagnosis) (OR 16.5, p = 0.003). Nestin protein positivity, observed in 9-28% of our hospital cases (Series I-IV), was independently associated with reduced breast cancer specific survival (HR = 2.0, p = 0.035) and was consistently related to basal-like differentiation (by Cytokeratin 5, OR 8.7-13.8, p < 0.0005; P-cadherin OR 7.0-8.9, p < 0.0005; EGFR staining, OR 3.7-8.2, p ≤ 0.05). Nestin mRNA correlated significantly with Nestin protein expression (ρ = 0.6, p < 0.0005), and high levels were seen in the basal-like intrinsic subtype. Gene expression signalling pathways linked to high Nestin were explored, and revealed associations with stem-like tumour features. In summary, Nestin was strongly associated with germline BRCA1 related breast cancer, a basal-like phenotype, reduced survival, and stemness characteristics.

Extra-nodal extension is a significant prognostic factor in lymph node positive breast cancer.

Presence of lymph node (LN) metastasis is a strong prognostic factor in breast cancer, whereas the importance of extra-nodal extension and other nodal tumor features have not yet been fully recognized. Here, we examined microscopic features of lymph node metastases and their prognostic value in a population-based cohort of node positive breast cancer (n = 218), as part of the prospective Norwegian Breast Cancer Screening Program NBCSP (1996-2009). Sections were reviewed for the largest metastatic tumor diameter (TD-MET), nodal afferent and efferent vascular invasion (AVI and EVI), extra-nodal extension (ENE), number of ENE foci, as well as circumferential (CD-ENE) and perpendicular (PD-ENE) diameter of extra-nodal growth. Number of positive lymph nodes, EVI, and PD-ENE were significantly increased with larger primary tumor (PT) diameter. Univariate survival analysis showed that several features of nodal metastases were associated with disease-free (DFS) or breast cancer specific survival (BCSS). Multivariate analysis demonstrated an independent prognostic value of PD-ENE (with 3 mm as cut-off value) in predicting DFS and BCSS, along with number of positive nodes and histologic grade of the primary tumor (for DFS: P = 0.01, P = 0.02, P = 0.01, respectively; for BCSS: P = 0.02, P = 0.008, P = 0.02, respectively). To conclude, the extent of ENE by its perpendicular diameter was independently prognostic and should be considered in line with nodal tumor burden in treatment decisions of node positive breast cancer.