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Background High tumor mutation burden is shown to be associated with a poor clinical outcome. As the tumor‐derived fraction of circulating cell‐free DNA (cfDNA) is shown to reflect the genetic spectrum of the tumor, we examined whether the mutation burden of cfDNA could be used to predict the clinical outcomes of early‐stage breast cancer (BC) patients. Methods We selected a set of 79 Finnish early‐stage BC cases with a good prognosis based on traditional prognostic parameters but some of which still developed relapsed disease during follow‐up. cfDNA was isolated from the serum collected at the time of diagnosis, sequenced, and compared to matched primary tumors, clinical parameters, and survival data. Results High cfDNA mutation burden was associated with the poor relapse‐free survival (RFS) (P = .016, HR = 2.23, 95% Cl 1.16‐4.27) when patients were divided into high and low mutation burden according to the median number of somatic variants. A high discordance was observed between the matched tumor and cfDNA samples, thus highlighting the challenges related to the liquid biopsy of early‐stage cancer cases. Despite the low number of detected tumor‐specific variants, the presence of tumor‐specific somatic variants in the cfDNA was associated with the poor RFS (P = .009, HR = 2.31, 95% Cl 1.23‐4.31). Conclusions Our results confirm previously observed challenges about the accuracy of liquid biopsy‐based genotyping of early‐stage cancers and support the parallel sequencing of tumor and cfDNA while also demonstrating how the presence of tumor‐specific somatic variants and the high mutation burden in the cfDNA are both associated with the poor RFS, thus indicating the prognostic potential of liquid biopsy in the context of early‐stage cancers., High mutation burden and the presence of tumor‐specific mutations in circulating cell‐free DNA in early‐stage breast cancer patients were observed to be associated with a poor relapse‐free survival. Our results suggest that the liquid biopsy of early‐stage cancers might provide potential benefits when making primary prognosis despite the challenges that are associated with the liquid biopsy of early‐stage cancers.

Breast cancer risk factors and clinical outcomes vary by tumour marker expression. However, individual studies often lack the power required to assess these relationships, and large-scale analyses are limited by the need for high throughput, standardized scoring methods. To address these limitations, we assessed whether automated image analysis of immunohistochemically stained tissue microarrays can permit rapid, standardized scoring of tumour markers from multiple studies. Tissue microarray sections prepared in nine studies containing 20 263 cores from 8267 breast cancers stained for two nuclear (oestrogen receptor, progesterone receptor), two membranous (human epidermal growth factor receptor 2 and epidermal growth factor receptor) and one cytoplasmic (cytokeratin 5/6) marker were scanned as digital images. Automated algorithms were used to score markers in tumour cells using the Ariol system. We compared automated scores against visual reads, and their associations with breast cancer survival. Approximately 65-70% of tissue microarray cores were satisfactory for scoring. Among satisfactory cores, agreement between dichotomous automated and visual scores was highest for oestrogen receptor (Kappa = 0.76), followed by human epidermal growth factor receptor 2 (Kappa = 0.69) and progesterone receptor (Kappa = 0.67). Automated quantitative scores for these markers were associated with hazard ratios for breast cancer mortality in a dose-response manner. Considering visual scores of epidermal growth factor receptor or cytokeratin 5/6 as the reference, automated scoring achieved excellent negative predictive value (96-98%), but yielded many false positives (positive predictive value = 30-32%). For all markers, we observed substantial heterogeneity in automated scoring performance across tissue microarrays. Automated analysis is a potentially useful tool for large-scale, quantitative scoring of immunohistochemically stained tissue microarrays available in consortia. However, continued optimization, rigorous marker-specific quality control measures and standardization of tissue microarray designs, staining and scoring protocols is needed to enhance results.

Matriptase-2 (TMPRSS6) has been identified as a breast cancer risk factor. Here, we examined relationships between TMPRSS6 genetic variations and breast cancer risk and survival, and determined the gene and protein expressions in breast tumors and assessed their clinical importance. Thirteen TMPRSS6 polymorphisms were genotyped in 462 invasive breast cancer cases and 458 controls. Gene expression was analyzed from 83 tumors and protein expression from 370 tumors. We then assessed the statistical significance of associations among genotypes, clinicopathological characteristics and survival. The TMPRSS6 variant rs2543519 was associated with breast cancer risk (p = 0.032). Multivariate analysis showed that four variants had effects on survival—rs2543519 (p = 0.017), rs2235324 (p = 0.038), rs14213212 (p = 0.044) and rs733655 (p = 0.021)—which were used to create a group variable that was associated with poorer prognosis correlating with more alleles related to reduced survival (p = 0.006; risk ratio, 2.375; 95% confidence interval, 1.287–4.382). Low gene expression was related to triple-negative breast cancer (p = 0.0001), and lower protein expression was detected in undifferentiated (p = 0.019), large (p = 0.014) and ductal or lobular tumors (p = 0.036). These results confirm the association of TMRRSS6 variants with breast cancer risk and survival. Matriptase-2 levels decrease with tumor progression, and lower gene expression is seen in poor-prognosis-related triple-negative breast cancers. Our study is the first to show that matriptase-2 gene variants are related to breast cancer prognosis, supporting matriptase-2 involvement in tumor development.

We investigated the expression and prognostic significance of matrix metalloproteinase (MMP) -7, its relation to β-catenin expression and clinicopathological factors in epithelial ovarian cancer. The expression of MMP-7 was analyzed immunohistochemically in a series of 284 primary epithelial ovarian cancers, their 36 metastases and 8 normal ovaries. In cancers with endometrioid histology, a high percentage area of MMP-7 expression and an intense MMP-7 signal was significantly associated with nuclear positivity of β-catenin in cancer cells (p = 0.003, χ2 = 8.853 and p = 0.030, χ2 = 4.713, respectively). In all tumors and nonendometrioid subgroup, a low percentage area of MMP-7 positive tumor cells was significantly correlated with a high histological grade of the tumor (p = 0.003 and 0.005, respectively), in all tumors also with advanced stage of the tumor (p = 0.002) and large primary residual tumor (p = 0.005). A 10-year disease-related survival (DRS) was significantly better when the percentage area of MMP-7 expression in cancer cells was high, when compared to low (p = 0.0008). A high percentage area of intense MMP-7 signal in cancer cells predicted a significantly more favorable DRS and recurrence-free survival (RFS) (p = 0.0003 and 0.0052, respectively). In multivariate analysis, a high percentage area of intense MMP-7 signal in tumor cells was an independent prognostic factor, predicting favorable DRS and RFS. The present study showed that intense MMP-7 signal in tumor cells is an independent prognostic factor predicting better survival in epithelial ovarian cancer. © 2006 Wiley-Liss, Inc.

High hydrostatic pressure (HP) has recently been shown to increase cellular heat shock protein 70 (Hsp70) level in a specific way that does not involve transcriptional activation of the gene, but rather the stabilisation of the mRNA for Hsp70. In this study, we investigated whether there are other observable changes caused by HP stress, and compared them with those induced by certain other forms of stressors. A chondrocytic cell line T/C28a4 was exposed to 30 MPa continuous HP, heat shock at 43 degrees C, and increased cytosolic calcium concentration by the addition of sarco-endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin (25 nM) or calcium ionophore A23187 (1 microM) in the cultures. The protein synthesis was studied by in vitro metabolic labelling followed by one- and two-dimensional polyacrylamide gel electrophoresis, and mass spectrometry was utilized to confirm the identity of the protein spots on two-dimensional gels. Continuous 30 MPa HP increased remarkably the relative labelling of Hsp70. Labelling of Hsp90 was also increased by 15-20%, although no clear change was evident at the protein level in Western blots. Elevated intracellular Ca(2+) concentration induced by thapsigargin and calcium ionophore A23187 increased mainly the synthesis of glucose-regulated protein 78 (Grp78/BiP), whereas Hsp70 and Hsp90 were decreased by the treatment. Heat shock was the strongest inducer of Hsp70 and Hsp90. This study further confirmed the induction of Hsp70 in chondrocytic cells exposed to high HP, but it also showed that calcium-mediated responses are unlikely to cause the stress response observed in the hydrostatically pressurized cells.