Neuroendocrine Breast Carcinomas Share Prognostic Factors with Gastroenteropancreatic Neuroendocrine Tumors: A Putative Prognostic Role of Menin, p27, and SSTR-2A.

OBJECTIVES: Due to the rarity of breast carcinomas with neuroendocrine features (NEBC), the knowledge on their biology is very limited but the identification of their biology and prognostic factors is essential to evaluate both pathogenesis and possible targeted treatment options. We assessed the expression of the well-characterized prognostic factors of gastroenteropancreatic neuroendocrine tumors (GEP-NET) in NEBC. METHODS: We assessed the immunohistochemical expression of neuron-specific enolase (NSE), thymidylate synthase (TS), p27, CD56, menin, and somatostatin receptor type 2A (SSTR-2A) in a series of 36 NEBC and 45 invasive ductal carcinomas (IDC). RESULTS: Nuclear and cytoplasmic TS, nuclear and cytoplasmic NSE, and nuclear p27 had significant overexpression in NEBC compared with IDC (for all, p < 0.01). In NEBC, cytoplasmic SSTR-2A expression was associated with excellent distant disease-free survival (p = 0.013), cytoplasmic menin expression with poorer relapse-free survival (p = 0.022), and nuclear p27 with longer breast cancer-specific survival (p = 0.022). CONCLUSIONS: There is a striking similarity in GEP-NET and NEBC regarding prognostic factors. GEP-NET and NEBC also appear to show similar expression patterns of the studied markers, while there are notable differences compared to IDC. Due to the wide expression of SSTR-2A, the treatment option with somatostatin analogs in NEBC should be evaluated.

USP28 Deficiency Promotes Breast and Liver Carcinogenesis as well as Tumor Angiogenesis in a HIF-independent Manner.

Recent studies suggest that the ubiquitin-specific protease USP28 plays an important role in cellular repair and tissue remodeling, which implies that it has a direct role in carcinogenesis. The carcinogenic potential of USP28 was investigated in a comprehensive manner using patients, animal models, and cell culture. The findings demonstrate that overexpression of USP28 correlates with a better survival in patients with invasive ductal breast carcinoma. Mouse xenograft experiments with USP28-deficient breast cancer cells also support this view. Furthermore, lack of USP28 promotes a more malignant state of breast cancer cells, indicated by an epithelial-to-mesenchymal (EMT) transition, elevated proliferation, migration, and angiogenesis as well as a decreased adhesion. In addition to breast cancer, lack of USP28 in mice promoted an earlier onset and a more severe tumor formation in a chemical-induced liver cancer model. Mechanistically, the angio- and carcinogenic processes driven by the lack of USP28 appeared to be independent of HIF-1α, p53, and 53BP1.Implications: The findings of this study are not limited to one particular type of cancer but are rather applicable for carcinogenesis in a more general manner. The obtained data support the view that USP28 is involved in tumor suppression and has the potential to be a prognostic marker. Mol Cancer Res; 16(6); 1000-12. ©2018 AACR.

High-level cytoplasmic claudin 3 expression is an independent predictor of poor survival in triple-negative breast cancer.

BACKGROUND: The subtype of claudin-low breast cancer can be reliably determined only by gene-expression profiling. Attempts have been made to develop immunohistochemical surrogates, which nearly always focus on membranous claudin expression. METHODS: We assessed the immunohistochemical expression of both membranous and cytoplasmic claudins 3, 4 and 7 in a series of 197 non-metastatic breast cancers, enriched with triple-negative breast cancers (TNBCs; 60%). The expression of epithelial-to-mesenchymal transition-regulating transcription factors Sip1, Zeb1 and vimentin had previously been determined in the same material. RESULTS: In multivariate analysis, strong cytoplasmic claudin 3 expression was associated with poor relapse-free survival (RFS), disease-free survival, distant disease-free survival, breast cancer-specific survival and overall survival among TNBC patients (for RFS, RR 5.202, 95% CI 1.210-22.369, p = 0.027, vs. T-class, RR 0.663, 95% CI 0.168-2.623, p = 0.558, and N-class, RR 3.940, 95% CI 0.933-16.631, p = 0.062). Cytoplasmic claudin 3 expression was also associated with strong nuclear Sip1 expression (p = 0.000053), TNBC phenotype (p = 0.012) and within them, non-basal-like phenotype (p = 0.026). Cytoplasmic claudin 7 was associated with dismal RFS (RR 6.328, 95% CI 1.401-28.593, p = 0.016, vs. T-class, RR 0.692, 95% CI 0.242-1.982, p = 0.493, and N-class, RR 2.981, 95% CI 1.1016-8.749, p = 0.047). Low cytoplasmic expression of claudins 3, 4 and 7 together also predicted poor RFS (RR 6.070, 95% CI 1.347-27.363, p = 0.019, vs. T-class, RR 0.677, 95% CI 0.237-1.934, p = 0.467, and N-class, RR 3.167, 95% CI 1.079-9.290, p = 0.036). CONCLUSIONS: Immunohistochemical expression levels of cytoplasmic claudins 3 and 7 appear to be novel prognostic factors in TNBC.

Rare missense mutations in RECQL and POLG associate with inherited predisposition to breast cancer.

Several known breast cancer susceptibility genes with moderate-to-high risk alleles encode proteins involved in DNA damage response (DDR). As these explain less than half of the hereditary breast cancer cases, additional predisposing alleles are likely to be discovered. Many of the previous studies utilizing massive parallel sequencing have focused on the protein-truncating variants, and the role of rare missense mutations has remained poorly addressed. To identify novel susceptibility factors, we have systematically analyzed the data from our parallel sequencing of 796 DDR genes in 189 Northern Finnish hereditary breast cancer patients for rare missense variants, predicted as deleterious. Thirty-five variants were studied here for the disease association using Finnish breast cancer case (n = 492-2,035) and control (n = 277-1,539) cohorts. As a result, two missense variants in genes involved in DNA replication, RECQL p.I156M and POLG p.L392V, the former involving genomic and the latter mitochondrial DNA replication, showed significant association with risk of breast cancer. Rare RECQL p.I156M allele was observed in breast cancer cases only (6/1,946, 0.3%, p = 0.043), whereas POLG p.L392V was two times more frequent in breast cancer cases (53/2,238, 2.4%) compared to controls (18/1,539, 1.2%, OR = 2.1, 95% CI 1.2-3.5, p = 0.010). Based on the current genetic data, both RECQL p.I156M and POLG p.L392V represent novel breast cancer predisposing alleles.

Long-term cardiovascular morbidity and mortality in patients treated for differentiated thyroid cancer.

OBJECTIVES: Thyroid hormone suppression therapy has been widely used in the treatment of thyroid cancer, but concerns have been raised about the cardiovascular risks of this treatment. The objective of this study was to evaluate long-term cardiovascular morbidity and mortality in patients treated for differentiated thyroid cancer (DTC) and to assess the effect of TSH suppression and radioiodine (RAI) treatment on the cardiovascular outcome. DESIGN: Retrospective cohort study. PATIENTS AND MEASUREMENTS: Patients (n = 901) treated for DTC between 1981 and 2002 at 2 Finnish University hospitals were compared with a randomly chosen reference group (n = 4485) matched for age, gender and the place of residence. Kaplan-Meier and Cox regression analyses were used to estimate the risk of morbidity or death due to different cardiovascular diseases (CVD) after the diagnosis of DTC. RESULTS: Morbidity due to any CVD (hazard ratio [HR] 1.16, 95% confidence interval [CI] 1.05-1.28) and due to all arrhythmias (HR 1.25, CI 1.06-1.48) and atrial fibrillation (AF) (HR 1.29, CI 1.06-1.57) was more frequent in the DTC patients than in the controls. The increased cardiovascular morbidity was confined to patients with a mean TSH level below 0.1 mU/L (HR 1.27, CI 1.03-1.58) and to those treated with RAI (HR 1.18, CI 1.05-1.31). Cardiovascular mortality, however, was lower among the patients than the controls (HR 0.73, CI 0.58-0.92), due to a lower mortality from coronary artery disease. CONCLUSIONS: Differentiated thyroid cancer patients have an increased CVD morbidity, which is mostly accountable to AF and to TSH suppression below 0.1 mU/L.

CHEK2 c.1100delC mutation is associated with an increased risk for male breast cancer in Finnish patient population.

BACKGROUND: Several susceptibility genes have been established for female breast cancer, of which mutations in BRCA1 and especially in BRCA2 are also known risk factors for male breast cancer (MBC). The role of other breast cancer genes in MBC is less well understood. METHODS: In this study, we have genotyped 68 MBC patients for the known breast or ovarian cancer associated mutations in the Finnish population in CHEK2, PALB2, RAD51C, RAD51D, and FANCM genes. RESULTS: CHEK2 c.1100delC mutation was found in 4 patients (5.9%), which is significantly more frequent than in the control population (OR: 4.47, 95% CI 1.51-13.18, p = 0.019). Four CHEK2 I157T variants were also detected, but the frequency did not significantly differ from population controls (p = 0.781). No RAD51C, RAD51D, PALB2, or FANCM mutations were found. CONCLUSIONS: These data suggest that the CHEK2 c.1100delC mutation is associated with an increased risk for MBC in the Finnish population.

Case-control analysis of truncating mutations in DNA damage response genes connects TEX15 and FANCD2 with hereditary breast cancer susceptibility.

Several known breast cancer susceptibility genes encode proteins involved in DNA damage response (DDR) and are characterized by rare loss-of-function mutations. However, these explain less than half of the familial cases. To identify novel susceptibility factors, 39 rare truncating mutations, identified in 189 Northern Finnish hereditary breast cancer patients in parallel sequencing of 796 DDR genes, were studied for disease association. Mutation screening was performed for Northern Finnish breast cancer cases (n = 578-1565) and controls (n = 337-1228). Mutations showing potential cancer association were analyzed in additional Finnish cohorts. c.7253dupT in TEX15, encoding a DDR factor important in meiosis, associated with hereditary breast cancer (p = 0.018) and likely represents a Northern Finnish founder mutation. A deleterious c.2715 + 1G > A mutation in the Fanconi anemia gene, FANCD2, was over two times more common in the combined Finnish hereditary cohort compared to controls. A deletion (c.640_644del5) in RNF168, causative for recessive RIDDLE syndrome, had high prevalence in majority of the analyzed cohorts, but did not associate with breast cancer. In conclusion, truncating variants in TEX15 and FANCD2 are potential breast cancer risk factors, warranting further investigations in other populations. Furthermore, high frequency of RNF168 c.640_644del5 indicates the need for its testing in Finnish patients with RIDDLE syndrome symptoms.

Adjuvant Capecitabine in Combination With Docetaxel, Epirubicin, and Cyclophosphamide for Early Breast Cancer: The Randomized Clinical FinXX Trial.

IMPORTANCE: Capecitabine is not considered a standard agent in the adjuvant treatment of early breast cancer. The results of this study suggest that addition of adjuvant capecitabine to a regimen that contains docetaxel, epirubicin, and cyclophosphamide improves survival outcomes of patients with triple-negative breast cancer (TNBC). OBJECTIVE: To investigate the effect of capecitabine on long-term survival outcomes of patients with early breast cancer, particularly in subgroups defined by cancer estrogen receptor (ER) and progesterone receptor (PR) content, and HER2 content (human epidermal growth factor receptor 2). DESIGN, SETTING, AND PARTICIPANTS: This is an exploratory analysis of the multicenter FinXX randomized clinical trial that accrued 1500 women in Finland and Sweden between January 27, 2004, and May 29, 2007. About half received 3 cycles of docetaxel followed by 3 cycles of cyclophosphamide, epirubicin, and fluorouracil (T+CEF), while the other half received 3 cycles of docetaxel plus capecitabine followed by 3 cycles of cyclophosphamide, epirubicin, and capecitabine (TX+CEX). Data analysis took place between January 27, 2004, and December 31, 2015. MAIN OUTCOMES AND MEASURES: Recurrence-free survival (RFS). RESULTS: Following random allocation, 747 women received T+CEF, and 753 women received TX+CEX. Five patients were excluded from the intention-to-treat population (3 had overt distant metastases at the time of randomization; 2 withdrew consent). The median age of the remaining 1495 patients was 53 years at the time of study entry; 157 (11%) had axillary node-negative disease; 1142 (76%) had ER-positive cancer; and 282 (19%) had HER2-positive cancer. The median follow-up time after random allocation was 10.3 years. There was no significant difference in RFS or overall survival between the groups (hazard ratio [HR], 0.88; 95% CI, 0.71-1.08; P = .23; and HR, 0.84, 95% CI, 0.66-1.07; P = .15; respectively). Breast cancer-specific survival tended to favor the capecitabine group (HR, 0.79; 95% CI, 0.60-1.04; P = .10). When RFS and survival of the patients were compared within the subgroups defined by cancer steroid hormone receptor status (ER and/or PR positive vs ER and PR negative) and HER2 status (positive vs negative), TX+CEX was more effective than T+CEF in the subset of patients with TNBC (HR, 0.53; 95% CI, 0.31-0.92; P = .02; and HR, 0.55, 95% CI, 0.31-0.96; P = .03; respectively). CONCLUSIONS AND RELEVANCE: Capecitabine administration with docetaxel, epirubicin, and cyclophosphamide did not prolong RFS or survival compared with a regimen that contained only standard agents. Patients with TNBC had favorable survival outcomes when treated with the capecitabine-containing regimen in an exploratory subgroup analysis. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00114816.

Primary neuroendocrine breast carcinomas are associated with poor local control despite favourable biological profile: a retrospective clinical study.

BACKGROUND: Breast carcinomas with neuroendocrine features (NEBC) are a very rare entity of mammary neoplasms, WHO classification of which has recently been revised. There are very limited data available about the clinical behaviour and treatment options of NEBC. METHODS: We collected retrospectively patients with NEBC from Oulu and Helsinki University Hospitals in 2007-2015. There were 43 NEBC cases during the period. RESULTS: The incidence of NEBC from all breast cancers varied from 0.1% in Helsinki to 1.3% in Oulu. The mean tumor size was 2.2 cm and 23 patients (55.8%) had no lymph node metastases when diagnosed. In total, 4 patients (9.3%) had distant metastases at the time of diagnosis. High estrogen receptor (ER) expression was observed in 41 (97.7%) patients. When non-metastatic NEBC were compared to a prospective set of ductal carcinomas (n = 506), NEBC were more often HER2 negative (p = 0.046), ER positive (p = 0.0062) and the NEBC patients were older (p < 0.0005) than patients with ductal carcinomas. Plasma chromogranin A correlated only to higher age at diagnosis (p = 0.0028). Relapse-free survival (p = 0.0013), disease-free survival (p = 0.024) and overall survival (p = 0.0028) favoured ductal carcinomas compared to NEBC, while no difference was observed in distant disease-free survival or in breast cancer-specific survival. CONCLUSIONS: There is remarkable variation in the incidence of NEBC in Finland, which is likely to be explained by differences in the use of neuroendocrine marker immunostainings. Poor local control and worse overall survival may be linked to the more aggressive biology of the disease, despite its association with apparently indolent prognostic factors.

Toll-like receptor 9 expression is associated with breast cancer sensitivity to the growth inhibitory effects of bisphosphonates in vitro and in vivo.

Bisphosphonates are standard treatments for bone metastases. When given in the adjuvant setting, they reduce breast cancer mortality and recurrence in bone but only among post-menopausal patients. Optimal drug use would require biomarker-based patient selection. Such biomarkers are not yet in clinical use. Based on the similarities in inflammatory responses to bisphosphonates and Toll-like receptor (TLR) agonists, we hypothesized that TLR9 expression may affect bisphosphonate responses in cells. We compared bisphosphonate effects in breast cancer cell lines with low or high TLR9 expression. We discovered that cells with decreased TLR9 expression are significantly more sensitive to the growth-inhibitory effects of bisphosphonates in vitro and in vivo. Furthermore, cancer growth-promoting effects seen with some bisphosphonates in some control shRNA cells were not detected in TLR9 shRNA cells. These differences were not associated with inhibition of Rap1A prenylation or p38 phosphorylation, which are known markers for bisphosphonate activity. However, TLR9 shRNA cells exhibited increased sensitivity to ApppI, a metabolite that accumulates in cells after bisphosphonate treatment. We conclude that decreased TLR9-expression sensitizes breast cancer cells to the growth inhibitory effects of bisphosphonates. Our results suggest that TLR9 should be studied as a potential biomarker for adjuvant bisphosphonate sensitivity among breast cancer patients.

Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility.

Breast cancer is strongly influenced by hereditary risk factors, a majority of which still remain unknown. Here, we performed a targeted next-generation sequencing of 796 genes implicated in DNA repair in 189 Finnish breast cancer cases with indication of hereditary disease susceptibility and focused the analysis on protein truncating mutations. A recurrent heterozygous mutation (c.904_916del, p.Arg304ValfsTer3) was identified in early DNA damage response gene, MCPH1, significantly associating with breast cancer susceptibility both in familial (5/145, 3.4%, P = 0.003, OR 8.3) and unselected cases (16/1150, 1.4%, P = 0.016, OR 3.3). A total of 21 mutation positive families were identified, of which one-third exhibited also brain tumors and/or sarcomas (P = 0.0007). Mutation carriers exhibited significant increase in genomic instability assessed by cytogenetic analysis for spontaneous chromosomal rearrangements in peripheral blood lymphocytes (P = 0.0007), suggesting an effect for MCPH1 haploinsufficiency on cancer susceptibility. Furthermore, 40% of the mutation carrier tumors exhibited loss of the wild-type allele. These findings collectively provide strong evidence for MCHP1 being a novel breast cancer susceptibility gene, which warrants further investigations in other populations.

Telomeric G-quadruplex-forming DNA fragments induce TLR9-mediated and LL-37-regulated invasion in breast cancer cells in vitro.

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously showed that synthetic and self-derived DNA fragments induce TLR9-mediated breast cancer cell invasion in vitro. We investigated here the invasive effects of two nuclease-resistant DNA fragments, a 9-mer hairpin, and a G-quadruplex DNA based on the human telomere sequence, both having native phosphodiester backbone. Cellular uptake of DNAs was investigated with immunofluorescence, invasion was studied with Matrigel-assays, and mRNA and protein expression were studied with qPCR and Western blotting and protease activity with zymograms. TLR9 expression was suppressed through siRNA. Although both DNAs induced TLR9-mediated changes in pro-invasive mRNA expression, only the telomeric G-quadruplex DNA significantly increased cellular invasion. This was inhibited with GM6001 and aprotinin, suggesting MMP- and serine protease mediation. Furthermore, complexing with LL-37, a cathelicidin-peptide present in breast cancers, increased 9-mer hairpin and G-quadruplex DNA uptake into the cancer cells. However, DNA/LL-37 complexes decreased invasion, as compared with DNA-treatment alone. Invasion studies were conducted also with DNA fragments isolated from neoadjuvant chemotherapy-treated breast tumors. Also such DNA induced breast cancer cell invasion in vitro. As with the synthetic DNAs, this invasive effect was reduced by complexing the neoadjuvant tumor-derived DNAs with LL-37. We conclude that 9-mer hairpin and G-quadruplex DNA fragments are nuclease-resistant DNA structures that can act as invasion-inducing TLR9 ligands. Their cellular uptake and the invasive effects are regulated via LL-37. Although such structures may be present in chemotherapy-treated tumors, the clinical significance of this finding requires further studying.

Current follow-up of a breast cancer patient.

The number of patients with breast cancer is on the rise, which leads to an increasing number of patients in need of follow-up and support for recovery from treatments. The disease will recur in one out of five patients. With the exception of imaging of the breasts carried out at intervals of 1 to 2 years, frequent appointments or imaging have not been shown to be beneficial for the prognosis. Follow-up care of breast cancer is well suited to be carried out by the nursing staff. The status of mobile applications developed for patient monitoring as part of the surveillance is becoming more clear.

Tumor infiltrating CD8+ T lymphocyte count is independent of tumor TLR9 status in treatment naïve triple negative breast cancer and renal cell carcinoma.

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor of the innate immune system that is widely expressed in cancers. We demonstrated that low tumor TLR9 expression predicts poor disease-specific survival in triple negative breast cancer (TNBC) and renal cell carcinoma (RCC). We hypothesized that this is because TLR9 expression affects tumor immunophenotype. To begin to test this, we compared the number of tumor infiltrating CD8+ T lymphocytes with TLR9 expression in treatment naïve breast cancer (n = 197) and RCC (n = 94) cohorts with known TLR9 expression status. CD8+ T lymphocyte counts were assayed with image analysis after immunohistochemistry (IHC). Tumor TLR9 expression was not correlated with CD8+ T cell counts in breast cancer or RCC. CD8+ T cell counts were significantly associated with tumor proliferation index in TNBC, but not in non-TNBC. CD8+ T cell counts were also significantly associated with tumor grade in non-TNBC, but not in TNBC. In RCC, CD8+ T cell counts were significantly associated with tumor stage. CD8+ T cell counts were significantly associated with prognosis in TNBC and RCC, but the presence of CD8+ T cells in these tumors had opposite effects on disease-specific survival: High CD8+ counts were associated with better prognosis in TNBC and worse prognosis in RCC. Among TNBC patients, those with low tumor TLR9 and low CD8+ T cell counts had the poorest prognosis (log-rank p = 0.0002 vs. high tumor TLR9 and high CD8+ T cell count). In conclusion, pre-treatment tumor TLR9 status is not associated with tumor infiltrating CD8+ T lymphocytes in TNBC or RCC. The combination of TLR9 and CD8+ TIL count might be a novel composite prognostic marker in TNBC.

KEAP1 Genetic Polymorphisms Associate with Breast Cancer Risk and Survival Outcomes.

PURPOSE: Defective oxidative stress response may increase cancer susceptibility. In tumors, these rescue mechanisms may cause chemo- and radioresistance impacting patient outcome. We previously showed that genetic variation in the nuclear factor erythroid 2-related factor 2 (NFE2L2) is associated with breast cancer risk and prognosis. Here we further studied this pathway by investigating Kelch-like ECH-associated protein 1 (KEAP1). EXPERIMENTAL DESIGN: Five tagging SNPs in the KEAP1 gene were genotyped in 996 breast cancer cases and 880 controls from two Finnish case-control sets. KEAP1 protein expression was studied in 373 invasive breast cancer tumors. RESULTS: rs34197572 genotype TT was associated with increased risk of breast cancer in the KBCP samples [P = 1.8×10(-4); OR, 7.314; confidence interval (CI), 2.185-24.478]. rs11085735 allele A was associated with lower KEAP1 protein expression (P = 0.040; OR,= 3.545) and high nuclear NRF2 expression (P = 0.009; OR, 2.445) and worse survival in all invasive cases (P = 0.023; HR, 1.634). When including treatment data, rs11085735 was associated with recurrence-free survival (RFS; P = 0.020; HR, 1.545) and breast cancer-specific survival (P = 0.016; HR, 1.683) and rs34197572 with overall survival (P = 0.045; HR, 1.304). rs11085735 associated with RFS also among tamoxifen-treated cases (P = 0.003; HR, 3.517). Among radiotherapy-treated cases, overall survival was associated with rs34197572 (P = 0.018; HR, 1.486) and rs8113472 (P = 0.025; HR, 1.455). RFS was associated with rs9676881 (P = 0.024; HR, 1.452) and rs1048290 (P = 0.020; HR, 1.468) among all invasive cases and among estrogen receptor (ER)-positive tamoxifen-treated cases (P = 0.018; HR, 2.407 and P = 0.015; HR, 2.476, respectively). CONCLUSIONS: The present findings suggest that the investigated SNPs have effects related to oxidative stress induced by cancer treatment, supporting involvement of the NRF2/KEAP1 pathway in breast cancer susceptibility and patient outcome.

Recurrent CYP2C19 deletion allele is associated with triple-negative breast cancer.

BACKGROUND: Using a genome-wide approach, we have previously observed an increase in the frequency of rare copy number variants (CNVs) in familial and early-onset breast cancer cases when compared to controls. Moreover, the biological networks of the CNV disrupted genes differed between the two groups. Here, six of the previously observed CNVs were selected for further investigation. Four of these were singletons and disturbed the following genes: DCLRE1C, CASP3, DAB2IP and ITGA9, encoding proteins that are part of the TP53 and ß-estradiol centered network. The two others were recurrent alleles and disrupted CDH19 and CYP2C19 genes. Of these, CDH19 encodes a cadherin functioning as a cell-cell adhesion receptor and CYP2C19 a CYP450 enzyme with a major function in estrogen catabolism. METHODS: The exact breakpoints of the six previously observed CNV deletion alleles were defined by using qPCR, nested PCR and sequencing. The prevalence of these CNVs was investigated in 842 Northern Finnish breast cancer cases, unselected for family history of cancer and age at disease onset, as well as in 497 healthy female controls by using multiplex PCR. Also the association of the relatively common CDH19 and CYP2C19 deletion alleles with different clinical parameters was studied. RESULTS: No significant differences in the carrier frequencies between cases and controls were found for any of the studied CNVs. However, the deletion in CYP2C19 showed a significant association with triple-negative breast cancer (p=0.021). CONCLUSION: Our results indicate that inherited changes in CYP2C19 gene participating in estrogen catabolism have an influence on the molecular subtype of breast cancer.

Outcome of patients with HER2-positive breast cancer treated with or without adjuvant trastuzumab in the Finland Capecitabine Trial (FinXX).

BACKGROUND: Little information is available about survival outcomes of patients with HER2-positive early breast cancer treated with adjuvant capecitabine-containing chemotherapy with or without trastuzumab. PATIENTS AND METHODS: One thousand and five hundred patients with early breast cancer were entered to the Finland Capecitabine trial (FinXX) between January 2004 and May 2007, and were randomly assigned to receive either three cycles of adjuvant TX (docetaxel, capecitabine) followed by three cycles of CEX (cyclophosphamide, epirubicin, capecitabine; TX-CEX) or three cycles of docetaxel followed by three cycles of CEF (cyclophosphamide, epirubicin, fluorouracil; T-CEF). The primary endpoint was recurrence-free survival (RFS). The study protocol was amended in May 2005 while study accrual was ongoing to allow adjuvant trastuzumab for patients with HER2-positive cancer. Of the 284 patients with HER2-positive cancer accrued to FinXX, 176 (62.0%) received trastuzumab after amending the study protocol, 131 for 12 months and 45 for nine weeks. The median follow-up time was 6.7 years. RESULTS: Patients with HER2-positive cancer who received trastuzumab had better RFS than those who did not (five-year RFS 89.2% vs. 75.9%; HR 0.41, 95% CI 0.23-0.72; p = 0.001). Patients treated with trastuzumab for 12 months or nine weeks had similar RFS. There was no significant interaction between trastuzumab administration and the type of chemotherapy. Four (2.3%) patients treated with trastuzumab had heart failure or left ventricular dysfunction, three of these received capecitabine. CONCLUSION: Adjuvant trastuzumab improves RFS of patients treated with TX-CEX or T-CEF. Few patients had cardiac failure.

The UGT1A6_19_GG genotype is a breast cancer risk factor.

Validation of an association between the UGT1A6_19_T>G (rs6759892) polymorphism and overall breast cancer risk. A pilot study included two population-based case-control studies from Germany (MARIE-GENICA). An independent validation study comprised four independent breast cancer case-control studies from Finland (KBCP, OBCS), Germany (BBCC), and Sweden (SASBAC). The pooled analysis included 7418 cases and 8720 controls from all six studies. Participants were of European descent. Genotyping was done by MALDI-TOF MS and statistical analysis was performed by logistic regression adjusted for age and study. The increased overall breast cancer risk for women with the UGT1A6_19_GG genotype which was observed in the pilot study was confirmed in the set of four independent study collections (OR 1.13, 95% CI 1.05-1.22; p = 0.001). The pooled study showed a similar effect (OR 1.09, 95% CI 1.04-1.14; p = 0.001). The risk effect on the basis of allele frequencies was highly significant, the pooled analysis showed an OR of 1.11 (95% CI 1.06-1.16; p = 5.8 × 10(-6)). We confirmed the association of UGT1A6_19_GG with increased overall breast cancer risk and conclude that our result from a well powered multi-stage study adds a novel candidate to the panel of validated breast cancer susceptibility loci.

Expression of Toll-like receptor-9 is associated with poor progression-free survival in prostate cancer.

Toll-like receptor-9 (TLR9) is a member of the innate immune system and recognizes bacterial and vertebrate DNA in cells. In addition to being expressed in cells of the immune system, it is widely expressed in various types of human cancer, including prostate cancer. We have previously demonstrated that synthetic TLR9 ligands induce invasion in TLR9-expressing prostate cancer cells in vitro. However, the role of TLR9 in the pathophysiology of prostate cancer is unclear. The expression of TLR9 in radical prostatectomy samples (n=186) was studied using immunohistochemistry. TLR9 staining scores were compared with tumor stage, Gleason score and prostate-specific antigen (PSA) concentration prior to treatment and progression-free survival. Results revealed that 124 (66.7%) of the tumors were strongly positive, 59 (31.7%) were weakly positive and 3 (1.6%) were negative, for cytoplasmic TLR9 immunostaining in cancer cells. There was no significant association between cytoplasmic TLR9 expression and distributions of pT-class, prostatectomy sample margin status, Gleason score and preoperative PSA value. Prostate cancer-specific progression-free survival was significantly longer for patients whose tumors were graded as negative for cytoplasmic TLR9 expression, as compared with patients whose tumors were strongly immunopositive for cytoplasmic TLR9 (P=0.009). In the Cox regression analysis, high TLR9 expression was an independent marker of poor prognosis in prostate cancer. Expression of TLR9 is associated with poor progression-free survival in prostate cancer patients who were treated by radical prostatectomy with curative intent.

Vimentin, zeb1 and Sip1 are up-regulated in triple-negative and basal-like breast cancers: association with an aggressive tumour phenotype.

In epithelial-to-mesenchymal transition (EMT) epithelial cancer cells achieve mesenchymal features, essentially helping them to metastasize. There is some evidence that EMT could be increased in triple-negative (TNBC) or basal-like breast cancers, although more precise mechanisms considering e.g. EMT-regulating transcription factors are largely unknown. We assessed immunohistochemically vimentin (separately in in situ areas and in invasive cells) as an indicator of EMT, and also EMT-regulating transcription factors zeb1 (separately in stroma and tumour) and Sip1 (in nuclei and cytoplasm) in histological samples of 231 women with local or locally advanced invasive breast cancer. 51.1 % of patients had TNBC and 48.9 % oestrogen and progesterone receptor-positive and HER2 negative breast cancer. Basal-like breast cancers were defined as TNBC that also expressed epidermal growth factor receptor EGFR and/or cytokeratin 5/6. Vimentin expression in invasive cells was higher in TNBCs (p = 9 × 10(-12)) compared to non-TNBC tumours. Vimentin (p = 2 × 10(-6)), nuclear Sip1 (p = 0.035) and zeb1 in stroma (p = 0.013) were overexpressed in basal-like cancers compared to non-basal-like TNBCs. In non-TNBC group findings between studied markers and clinicopathological factors were rare. However, in TNBC cases, vimentin expression in invasive cells associated with poor differentiation (p = 0.00007), zeb1 expression in cancer cells with higher grade (p = 0.002), vascular invasion (p = 0.036) and larger T-class (p = 0.027), whereas stromal zeb1 associated with lymphatic vessel invasion (p = 0.036) and vascular invasion (p = 0.039). High nuclear Sip1 expression was prognostic for poor disease-free survival (p = 0.002) in the whole cohort. The current results emphasize the increased role of EMT in TNBC and especially in basal-like breast cancers. These observations also support the role of studied parameters in tumour progression.