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Purpose: Gene expression–based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features. Experimental Design: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting. Results: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations. Conclusions: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications. See related commentary by McMullen et al., p. 5271

Arsenic is recognized as a potent carcinogen at high concentrations, but the relationship between environmental arsenic and breast cancer risk has not well been studied. Most research has focused on the effect of arsenic in populations with high endemic exposure, and not in populations with arsenic levels within normal limits. We sought to determine if blood arsenic levels predict the risk of breast and other cancers risk among women in northern Poland. The cohort consisted of 1,702 healthy women, aged 40 and above, identified between 2010 and 2017. Blood arsenic level was determined by inductively coupled plasma mass spectrometry. After an average of 4.5 years of follow‐up (range 0.7–7.3 years), there were 110 incident cases of cancer diagnosed in the cohort, including 68 cases of breast cancer. Women in the highest quartile of arsenic had a highly significant 13‐fold increased risk of developing breast cancer, compared to women in the lowest quartile (hazard ratio [HR] = 13.2; 95% confidence interval [CI] 4.02–43.0). Results were similar for arsenic and all incident cancers (HR quartile 4 vs. quartile 1 = 13.3; 95% CI 4.78–37.0). If confirmed, our study suggests that the blood arsenic level may be a useful predictive marker of cancer risk in women., What's new? Arsenic has long been recognized as a potent carcinogen at high concentrations. But can it affect cancer risk at “normal,” environmental concentrations? In this Polish study, the authors found that women whose blood levels of arsenic were in the highest quartile had a 13‐fold increased risk of developing breast cancer, compared to women in the lowest quartile. If confirmed in further studies, these results suggests that blood arsenic level may be a useful predictive marker of cancer risk in women.

Background Epidemiologic studies have demonstrated a relationship between selenium status and cancer risk among those with low selenium levels. It is of interest to prospectively evaluate the relationship between selenium and cancer among women who reside in a region with ubiquitously low selenium levels. Methods We performed a nested case-control study of baseline serum selenium levels and cancer risk using data and biological samples from 19,573 females that were participants in a biobanking initiative between 2010 and 2014 in Szczecin Poland. Cases included women with any incident cancer (n = 97) and controls (n = 184) were women with no cancer at baseline or follow-up. Serum selenium was quantified using mass spectroscopy. Results The odds ratio associated being below the cutoff of 70.0 μg/L compared to a level above 70.0 μg/L was 2.29 (95% CI 1.26–4.19; P = 0.007). The risks for women in the two middle categories were similar and suggests that the normal range be between 70 μg/L and 90 μg/L. There was evidence for an increased risk of cancer among women in the highest category of selenium levels (i.e., > 90 μg/L), but this association did not achieve statistical significance (OR = 1.63; 95%CI 0.63–4.19; P = 0.31). Conclusions Results from this study suggest that suggest that the optimum serum level of selenium in women living in Poland should be between 70 μg/L and 90 μg/L.

Objective: To evaluate myeloid differentiation primary response gene 88 (MyD88) and Toll-like receptor 4 (TLR4) expression in relation to clinical features of epithelial ovarian cancer, histologic subtypes, and overall survival. Patients and Methods: We conducted centralized immunohistochemical staining, semi-quantitative scoring, and survival analysis in 5263 patients participating in the Ovarian Tumor Tissue Analysis consortium. Patients were diagnosed between January 1, 1978, and December 31, 2014, including 2865 high-grade serous ovarian carcinomas (HGSOCs), with more than 12,000 person-years of follow-up time. Tissue microarrays were stained for MyD88 and TLR4, and staining intensity was classified using a 2-tiered system for each marker (weak vs strong). Results: Expression of MyD88 and TLR4 was similar in all histotypes except clear cell ovarian cancer, which showed reduced expression compared with other histotypes (P

Primary ovarian mucinous tumors can be difficult to distinguish from metastatic gastrointestinal neoplasms by histology alone. The expected immunoprofile of a suspected metastatic lower gastrointestinal tumor is CK7-/CK20+/CDX2+/PAX8-. This study assesses the addition of a novel marker SATB2, to improve the diagnostic algorithm. A test cohort included 155 ovarian mucinous tumors (105 carcinomas and 50 borderline tumors) and 230 primary lower gastrointestinal neoplasms (123 colorectal adenocarcinomas and 107 appendiceal neoplasms). All cases were assessed for SATB2, PAX8 CK7, CK20, and CDX2 expression on tissue microarrays. Expression was scored in a 3-tier system as absent, focal (1-50% of tumor cells) and diffuse ( >50% of tumor cells) and then categorized into either absent/present or nondiffuse/diffuse. SATB2 and PAX8 expression was further evaluated in ovarian tumors from an international cohort of 2876 patients (expansion cohort, including 159 mucinous carcinomas and 46 borderline mucinous tumors). The highest accuracy of an individual marker in distinguishing lower gastrointestinal from ovarian mucinous tumors was CK7 (91.7%, nondiffuse/diffuse cut-off) followed by SATB2 (88.8%, present/absent cut-off). The most effective combination was CK7 and SATB2 with accuracy of 95.3% using the 3-tier interpretation, absent/focal/diffuse. This combination outperformed the standard clinical set of CK7, CK20 and CDX2 (87.5%). Re-evaluation of outlier cases confirmed ovarian origin for all but one case. The accuracy of SATB2 was confirmed in the expansion cohort (91.5%). SATB2 expression was also detected in 15% of ovarian endometrioid carcinoma but less than 5% of other ovarian histotypes. A simple two marker combination of CK7 and SATB2 can distinguish lower gastrointestinal from ovarian primary mucinous tumors with greater than 95% accuracy. PAX8 and CDX2 have value as second-line markers. The utility of CK20 in this setting is low and this warrants replacement of this marker with SATB2 in clinical practice.

Importance Cytotoxic CD8+tumor-infiltrating lymphocytes (TILs) participate in immune control of epithelial ovarian cancer; however, little is known about prognostic patterns of CD8+TILs by histotype and in relation to other clinical factors. Objective To define the prognostic role of CD8+TILs in epithelial ovarian cancer. Design, Setting, and Participants This was a multicenter observational, prospective survival cohort study of the Ovarian Tumor Tissue Analysis Consortium. More than 5500 patients, including 3196 with high-grade serous ovarian carcinomas (HGSOCs), were followed prospectively for over 24 650 person-years. Exposures Following immunohistochemical analysis, CD8+TILs were identified within the epithelial components of tumor islets. Patients were grouped based on the estimated number of CD8+TILs per high-powered field: negative (none), low (1-2), moderate (3-19), and high (≥20). CD8+TILs in a subset of patients were also assessed in a quantitative, uncategorized manner, and the functional form of associations with survival was assessed using penalized B-splines. Main Outcomes and Measures Overall survival time. Results The final sample included 5577 women; mean age at diagnosis was 58.4 years (median, 58.2 years). Among the 5 major invasive histotypes, HGSOCs showed the most infiltration. CD8+TILs in HGSOCs were significantly associated with longer overall survival; median survival was 2.8 years for patients with no CD8+TILs and 3.0 years, 3.8 years, and 5.1 years for patients with low, moderate, or high levels of CD8+TILs, respectively (Pvalue for trend = 4.2 × 10−16). A survival benefit was also observed among women with endometrioid and mucinous carcinomas, but not for those with the other histotypes. Among HGSOCs, CD8+TILs were favorable regardless of extent of residual disease following cytoreduction, known standard treatment, and germlineBRCA1pathogenic mutation, but were not prognostic forBRCA2mutation carriers. Evaluation of uncategorized CD8+TIL counts showed a near-log-linear functional form. Conclusions and Relevance This study demonstrates the histotype-specific nature of immune infiltration and provides definitive evidence for a dose-response relationship between CD8+TILs and HGSOC survival. That the extent of infiltration is prognostic, not merely its presence or absence, suggests that understanding factors that drive infiltration will be the key to unraveling outcome heterogeneity in this cancer.

Purpose To estimate 10-year overall survival (OS) rates for patients with early-onset breast cancer, with and without a BRCA1 mutation, and to identify prognostic factors among those with BRCA1-positive breast cancer. Patients and Methods A total of 3,345 women with stage I to III breast cancer, age ≤ 50 years, were tested for three founder mutations in BRCA1. Information on tumor characteristics and treatments received was retrieved from medical records. Dates of death were obtained from the vital statistics registry. Survival curves for the mutation-positive and -negative subcohorts were compared. Predictors of OS were determined using the Cox proportional hazards model. Results Of the 3,345 patients enrolled onto the study, 233 (7.0%) carried a BRCA1 mutation. The 10-year survival rate for mutation carriers was 80.9% (95% CI, 75.4% to 86.4%); for noncarriers, it was 82.2% (95% CI, 80.5% to 83.7%). The adjusted hazard ratio (HR) associated with carrying a BRCA1 mutation was 1.81 (95% CI, 1.26 to 2.61). Among BRCA1 carriers with a small (< 2 cm) node-negative tumor, the 10-year survival rate was 89.9%. Among BRCA1 mutation carriers, positive lymph node status was a strong predictor of mortality (adjusted HR, 4.1; 95% CI, 1.8 to 8.9). Oophorectomy was associated with improved survival in BRCA1 carriers (adjusted HR, 0.30; 95% CI, 0.12 to 0.75). Conclusion The 10-year survival rate among women with breast cancer and a BRCA1 mutation is similar to that of patients without a BRCA1 mutation. Among women with a BRCA1 mutation, survival was much improved after oophorectomy.

Background Zinc is a micronutrient which is essential for human health, playing a role as a cofactor of enzymes such as dehydrogenases, peptidases and component of zinc finger domains. In organism zinc is involved in metabolic pathways, immune processes, maintaining ion balance between other elements. Recently, it has been reported that zinc may play role in chemoprevention, and its level may be associated with occurrence of cancers.

Mutations in the BRCA1 gene increase susceptibility to both breast and ovarian cancer. In some countries, including several in Eastern Europe, founder mutations in the BRCA1 gene are responsible for a significant proportion of breast cancer cases. To estimate the hereditary proportion of breast cancer in Belarus, we sought the presence of any of three founder mutations in BRCA1 (4153delA, 5382insC and C61G) in 500 unselected cases of breast cancer. These mutations have previously been identified in breast/ovarian cancer families from Belarus and from other Slavic countries, including Poland and Russia. One of the three founder mutations in BRCA1 was present in 38 of 500 unselected cases of breast cancer (7.6%). A mutation was found in 12.6% of women diagnosed before age 50 and 5.6% of women diagnosed after age 50. A mutation was identified in 2 of 251 newborn controls (0.8%). The hereditary proportion of breast cancers in Belarus is among the highest of any countries studied to date.

Germline mutations in the DNA mismatch repair genes MSH2 and MLH1 account for a significant proportion of hereditary non-polyposis colorectal cancer (HNPCC) families. One approach by which development of an efficient DNA-testing procedure can be implemented is to describe the nature and frequency of common mutations in particular ethnic groups. Two hundred and twenty-six patients from families matching the Amsterdam II diagnostic criteria or suspected HNPCC criteria were screened for MSH2 and MLH1 germline mutations. Fifty different pathogenic mutations were found, 25 in MSH2 and 25 in MLH1. Twenty-four of these had not previously been described in other populations. Among our 78 families with MSH2 or MLH1 mutations, 54 (69.2%) were affected by recurrent mutations including 38 found at least twice in our own series. Two of the most frequent alterations were a substitution of A to T at the splice donor site of intron 5 of MSH2 and a missense change (A681T) of MLH1 found in 10 and eight families, respectively. Among large deletions detected by the multiplex ligation-dependent probe amplification assay, exon 9 deletions in the MSH2 gene were found in two families. Our results indicate that a screening protocol specific for the Polish population that is limited to the detection of all reported mutations will result in the identification of the majority of changes present in MLH1 and MSH2 genes in Polish HNPCC kindreds.

In Belarus and other Slavic countries, founder mutations in the BRCA1 gene are responsible for a significant proportion of breast cancer cases, but the data on contribution of these mutations to ovarian cancers are limited. To estimate the proportion of ovarian cancers in Belarus, which are dependent on BRCA1 Slavic founder mutations, we sought the presence of three most frequent mutations (BRCA1: 5382insC, C61G and, 4153delA) in 158 consecutive unselected cases of ovarian cancer. One of the three founder mutations was present in 25 of 158 unselected cases of ovarian cancer (15.8 %). We recommend that all cases of ovarian cancer in Belarus be offered genetic testing for these founder mutations. Furthermore, genetic testing of the Belarusian population will provide the opportunity to prevent a significant proportion of ovarian cancer.

Magnesium plays a key role in many essential cellular processes. It is recognized that magnesium deficiency may lead to many disorders like cardiovascular disease, diabetes mellitus, hypertension, myocardial infarction or even cancer. Connection between magnesium concentration in body fluids and cancer occurrence still becomes unclear. Magnesium ions are crucial cofactors for enzymes involved DNA repairing. Thereby magnesium deficiency may result in genomic instability and consequently cause cancer. The aim of our study was to investigate if serum magnesium concentration may be useful biomarker for early detection of prostate and laryngeal cancer and also if serum Mg concentration may be marker of selection for CT of the lung, laryngeal examination, PSA levels or prostate biopsy. Independently we have investigated a prospective study where our aim was to investigate if the serum Mg concentration is a marker of increased breast cancer risk. We performed retrospective investigation in laryngeal cancer patients (n = 123) and prostate cancer groups (n = 166). Each group was compared with healthy controls group, which consists of equal to cases amount of controls. Controls and cases were matched by age, gender, negative supplementation status, negative status of hormonal contraceptives and smoking status. In the prospective study (sera collected before cancer diagnosis) Mg concentration was determined in BRCA (-) women and compared to controls. Mg serum levels ( 20800 µg/l) are associated with 2-fold higher occurrence of laryngeal cancers. Magnesium serum level is not a useful marker for selection of the patients for prostate cancer screening. Finally, magnesium concentration may be a marker for breast cancer risk in BRCA (-) patients but further investigation is needed.

Iron plays an important role in many metabolic processes, is included in the delivery of oxygen to cells and redox processes. Deficiency or excess of iron can lead to multiple organ failures and in extreme cases to death. The latest studies show that the iron can significantly influence the risk of cancer development and progression. Scientists reported association between high levels of iron in the serum and the risk of colon, liver, stomach and breast cancers. The low iron level was detected in patients with bladder and lung cancers. There are also studies in which authors showed lower iron level in patients with colorectal cancer and breast or they did not find any correlation between iron levels and the risk of breast cancer.

Recently, the 1100delC variant of cell cycle checkpoint kinase 2 (CHEK2) has been reported to confer a colorectal cancer risk in hereditary non-polyposis-colorectal cancer (HNPCC) and HNPCC-related families in the Netherlands. To investigate whether CHEK2 mutations confer increased cancer risk in HNPCC and HNPCC-related families in Poland, we genotyped 463 probands from HNPCC and HNPCC-related families, and 5,496 controls for 4 CHEK2 alleles (1100delC, IVS2+1G>A, del5395, I157T). All 463 probands were screened for mutations in the HNPCC-related genes MSH2, MLH1 and MSH6. A positive association was observed for HNPCC-related cancer and the I157T missense CHEK2 mutation (OR = 1.7; p = 0.007), but not for the truncating alleles (OR = 1.0; p = 1.0). The association with the I157T was seen both for the 117 cases who fulfill Amsterdam criteria (OR = 1.9; p = 0.1) and for the 346 cases who do not fulfill the criteria (OR = 1.6; p = 0.03). One hundred forty-five of the 463 families had a mutation in MSH2, MLH1 or MSH6 (MMR-positive families). A positive association between the CHEK2 I157T mutation and HNPCC-related cancer was observed only for MMR-negative cases (OR = 2.1; p = 0.0004), but not for MMR-positive cases (OR = 0.8; p = 0.9). The association with I157T was particularly strong for MMR-negative cases with familial colorectal cancer (2 or more first-degree relatives affected) (OR = 2.5; p < 0.0001). We conclude that the I157T variant of CHEK2 increases the risk of colorectal cancer among MMR-negative, HNPCC/HNPCC-related families in Poland.

We studied whether or not single nucleotide polymorphisms (SNPs), which have been shown to modify the risk of breast cancer in women with a BRCA1 mutation, are associated with cancer risk in unselected (non-hereditary) breast cancer patients. We genotyped seven SNPs in six distinct genes ( PHB , RAD51 , ITGB3 , TGFB1 , VEGF , MTHFR ) in 1100 unselected Polish breast cancer patients and 1100 controls. The frequencies of genotypes were similar in cases and controls. In a subgroup analysis, we found a positive association between the homozygous genotype PHB 1630C/T and medullary breast cancer (odds ratio (OR) = 4.0, 95% confidence interval (CI) 1.1–14.0). PHB 1630C/T was also associated with tumours negative for oestrogen receptor (OR = 2.2, 95% CI 1.13–4.4) or progesterone receptor (OR = 2.8, 95% CI 1.4–5.8). Our results show that, in general, the single nucleotide polymorphisms which modify the risk of hereditary breast cancer in Poland do not modify the risk of sporadic breast cancer. The PHB 1630 C/T single nucleotide polymorphism was associated with breast cancers with clinical features typical for BRCA1 -positive tumours and is deserving of further study.

Among women with a BRCA1 mutation, the lifetime risks of breast and ovarian cancer are elevated. Several measures for reducing cancer risk in carriers of BRCA1 mutations have been proposed, including prophylactic surgery and tamoxifen chemoprevention. It is not yet known to what extent women with mutations have adopted these various preventive measures. We surveyed 414 Polish women with a BRCA1 mutation who had received counseling about various preventive strategies. Each woman completed a survey for a minimum of 18 months after receiving her genetic result. A high proportion of women reported having had an oophorectomy to reduce breast and ovarian cancer risk (49.1%). In contrast, only 11% had taken tamoxifen and only 5% had undergone a preventive mastectomy. Most of the women (81%) had a screening mammogram during the follow-up period. Oral contraceptives and breastfeeding are believed to protect against ovarian cancer, but only 9% of women had taken the birth control pill for 3 years or more and 27% had breastfed for 1 year or more. In summary, approximately one-half of Polish women with a BRCA1 mutation had taken an active step to reduce their risk of breast cancer within 18 months of receiving a positive result. A greater effort should be made to promote breastfeeding and use of oral contraceptive as risk-reducing measures.

Purpose: To investigate the contribution of CHEK2 mutations to early-onset breast cancer in Poland and to establish the characteristic features of these cancers. Experimental Design: We studied 3,228 women diagnosed with breast cancer under the age of 51 years and 5,496 population controls. CHEK2 mutations were detected by RFLP-PCR or allele-specific oligonucleotide-PCR assays. Clinical and pathologic features of CHEK2-positive cases and CHEK2-negative cases were compared. Results: A truncating CHEK2 mutation (1100delC or IVS2+1G>A) was seen in 47 of 3,228 cases and in 34 of 5,496 controls (odds ratio, 2.4; P = 0.0001). The CHEK2 I157T missense mutation was present in 207 of 3,228 cases, compared with 264 of 5,496 controls (odds ratio, 1.4; P = 0.002). Breast cancers in women with a CHEK2 mutation were more commonly of lobular histology (21.5% versus 15.8%; P = 0.05), of size >2 cm (54.8% versus 43.5%; P = 0.01), or of multicentric origin (28.7% versus 19.5%; P = 0.01) than were cancers from women without a CHEK2 mutation. Bilateral cancers were equally common in both subgroups. Conclusion: Three founder alleles in CHEK2 contribute to early-onset breast cancer in Poland. Breast tumors which arise in carriers of CHEK2 mutations seem to be similar to those of breast cancers in the population at large.

Purpose: To investigate the contribution of CDKN2A A148T common variant to early-onset breast cancer in Poland, and to establish the characteristic features of these cancers. Experimental design: We studied 3,069 women diagnosed with breast cancer under the age of 51 and 3,493 population controls. CDKN2A variant were detected using RFLP-PCR and confirmed by genomic sequencing. Clinical and pathological features of CDKN2A-positive cases and CDKN2A-negative cases were compared. Results: A148T variant was identified in 157 of 3,069 women with breast cancer (5.1%). Overall, the odds ratio for early-onset breast cancer, given a CDKN2A variant was 1.4 (95% CI 1.075–1.725; P = 0.012). Breast tumors in women with the CDKN2A variant were more commonly intraductal cancers (DCIS) with micro-invasion (14.8% vs. 8.5%; P = 0.035). Carriers and non-carriers were similar with respect to tumor size, laterality, multicentricity, nodal status, family history and estrogen-receptor status. Conclusion: The CDKN2A A148T variant seems to contribute to early-onset breast cancer in Poland. Breast tumors which arise in carriers of A148T variant appear to be similar to those of breast cancers in the population at large.

The population frequencies of the CDKN2A common variants remain undetermined. In Poland, there is a common variant of the CDKN2A: an alanine to threonine substitution (A148T), which has been detected in other populations. We have recently showed that it is significantly overrepresented among Polish melanoma patients when compared to general population. Herein, we ascertained the prevalence of the A148T variant in 3,583 unselected cancer cases and 3,000 random control subjects from the same Polish population. We evaluated eleven different malignancies, representing the majority of all common cancer sites. Positive association with A148T variant was observed for lung cancer (OR, 2.0; p = 0.0052). A similar trend, although nonsignificant after the Bonferroni correction, was observed for colorectal cancer (OR, 1.5; p = 0.5499). These results suggest that A148T variant may be associated with a multi-organ cancer risk in the Polish population.

Etiologic factors affecting bladder tumor have been well confirmed and it is widely recognized that carcinogenic substances in urine may play an important role in a pathogenesis. Carcinoma developing in a defunctionalized bladder, although uncommon, does occur. We report a case of a transitional cell carcinoma (TCC) found in a remaining bladder of a male patient and a review of the most relevant literature.

The most sensitive technique for the detection of germline mutations is exon by exon sequencing of the gene under investigation using genomic DNA as a template for analysis. This approach, however, has cost and sensitivity limitations that can, at least in part, be overcome by RNA-based analysis. Germline mutations of MLH1 and MSH2 are the most frequent cause of the inherited susceptibility to colorectal and other epithelial cancers known as hereditary non-polyposis colorectal cancer (HNPCC). We compared the analysis of the MLH1 and MSH2 genes using mRNA and genomic DNA as starting material from 21 HNPCC patients. All samples were investigated by RT-PCR, sequencing of cDNA and simultaneous sequencing of genomic DNA. The cDNA was generated using specific primers complementary to the ends of MLH1 and MSH2 genes, respectively. Mutations in MLH1 and MSH2 were detected in 11 out of 21 unrelated patients. In 10 out of 11 cases, mutations were detected independently of the type of primers used for reverse transcription (RT). One novel missense mutation (K751R) in MLH1 was detected using this method. One nonsense mutation (E205X) in MSH2 was only detectable when RT was performed using MSH2 gene-specific primers. Shorter PCR products indicative of alternatively spliced transcripts were not observed when MLH1 or MSH2 specific cDNA RT primers were employed to generate template, except in one case where exon skipping was observed for exons 9 and 10. In this report we demonstrate that primers specific for RT of MLH1 and MSH2 are crucial for increasing the sensitivity of cDNA analysis. DNA sequencing using RNA as a basis for template construction may be a valuable and economical alternative to genomic DNA sequencing. Hum Mutat 17:52–60, 2001. © 2001 Wiley-Liss, Inc.

The aim of the study is identification of correlations between the serum concentrations of iron and the risk of breast cancer among female BRCA1 mutations carriers and unselected breast cancer patients. The first group (99 triplet) selected for the trial were Polish women, positive for at least one of three founder mutations in BRCA1 gene dominating in Poland (5382insC, C61G, 4153delA). Serum was collected at the time of breast cancer diagnosis. Persons with detected tumor were considered as cases and the others were considered as controls. One case and two controls were paired regarding many criteria (e.g. age, family cancer history, cigarettes smoking, adnexectomy status ) to achieve the maximum of similarity between them. The subjects of the second group (28 triplet) selected for the trial were Polish women, positive for at least one of three founder mutations in BRCA1 gene. Serum was collected 1-2 years before breast cancer diagnosis. One case and two controls were matched for year of birth, past history of cancer, adnexectomy status and cigarettes smoking to achieve the maximum of similarity between them. The subjects of the third group were unselected cancer for the trial were Polish women. Serum was collected during breast cancer diagnosis before treatment. One case and one control were matched for year of birth (+/- 3 years), number and location of cancer among Io relatives, smoking-the number of pack years (+/- 10%), adnexectomy status, CHEK2 mutation. The proportion of cases and control in the first quartile was taken as a reference to calculate the odds ratio, confidence interval and p-value of the multivariate conditional logistic regression. The iron was quantitatively measured by ICP-MS (Inductively Coupled Plasma Mass Spectrometry), (model Elan DRC-e 6100 th, PerkinElmer). This study shows that concentration levels of iron in blood serum are a strong factors associated with an additionally increased risk of breast cancer among BRCA1 mutation carriers and unselected breast cancer patients. For iron concentration in BRCA1 carriers (serum collected at the time of breast cancer diagnosis) all quartiles above the first one had a decreased risk of breast cancer. The results are shown in Table ​Table11. Table 1 Association between Fe serum concentration and risk of breast cancer in BRCA 1 carriers (serum collected at the time of breast cancer diagnosis). For iron concentration in BRCA1 carriers (serum collected 1-2 years before breast cancer diagnosis) the last quartile had a decreased risk of breast cancer. The results are shown in Table ​Table22. Table 2 Association between Fe serum concentration and risk of breast cancer in BRCA 1 carriers (serum collected 1-2 years before breast cancer diagnosis). Individuals classified in the III and IV quartile had lower risk of breast cancer however results were statistically insignificant. The strongest result and lowest cancer risk was for Fe > 1350 μg/l (OR= 0,289, p= 0,0426) For iron concentration in unselected breast cancer patients (serum was collected during breast cancer diagnosis before treatment) all quartiles were statistically insignificant. The results are shown in Table ​Table33. Table 3 Association between Fe serum concentration and risk of breast cancer Individuals classified in the III quartile had lower risk of breast cancer however results were statistically insignificant. The strongest result and lowest cancer risk was for Fe > 1000 μg/l (OR= 0,6698, p= 0,25554). For iron concentration in unselected breast cancer patients (serum was collected during breast cancer diagnosis before treatment) before 55 years old, third quartile had a decreased risk of breast cancer. The results are shown in Table ​Table44. Table 4 Association between Fe serum concentration and risk of cancer in group of patients < 55 years old. Individuals classified in the III quartile had lower risk of breast cancer (OR=0,248, p=0,019); The strongest result and lowest cancer risk was for Fe concentration higher than 987 μg/l (III quartile) ; max 1285 μg/l (III quartile)

Mutations in the CHEK2 gene confer an approximately two-fold increase in the risk of cancer in many organs including the breast, colon, kidney, prostate and thyroid. In addition, we have recently identified a positive association of similar magnitude between a CHEK2 mutation and bladder cancer, endometrial cancer and low-grade ovarian cancer (unpublished data). Surveillance is available for most CHEK2-associated cancers. CHEK2 is not a high-risk gene, but it is associated with multi-cancer susceptibility, and founder alleles in CHEK2 are common in Poland (5.5% of the general population); therefore CHEK2 is responsible for development of more cases of cancer in Poland than founder mutations known to date in high-risk genes (including those in BRCA1, MSH2 and MLH1). In 2005 in cooperation with the Center of Medical Diagnosis and Hematology (BIOS) and the journal Wprost we performed population screening of CHEK2 mutations in Warsaw. This study was started by publication of an article describing the association of the CHEK2 gene and multi-site cancer predisposition. Readers of Wprost journal were invited for genetic consultation and CHEK2 gene testing. Patients who responded to the invitation were interviewed in the BIOS centre. During the interview the goals of the study were explained, informed consent was obtained, a first session of genetic counselling was given and a blood sample was obtained for DNA analysis. A detailed family history of cancer was taken from all patients (first and second-degree relatives included). During a period of 6 months, 3,826 individuals participated in this study. We identified: 192 carriers of a CHEK2 mutation, 240 families with hereditary susceptibility to breast and/or ovarian cancer, 62 families with familial colorectal cancer (including HNPCC). In addition, BRAC1 testing was performed in all individuals with a positive family history of breast or ovarian cancer. Twenty-one BRCA1 mutation carriers were identified. CHEK2 and BRCA1 mutation status was investigated using two independent blood samples. Then, all patients were invited for a second session and final genetic counselling was given. Appropriate surveillance was recommended for patients with familial cancer aggregations, as well as for BRAC1 and CHEK2 mutation carriers. We believe that this action broadened the knowledge about the role of genetic counselling and DNA testing in cancer prevention in Poland. It has also shown that many people in our population need such counselling and surveillance. We believe that this action will help reduce cancer morbidity and mortality in Poland.

A single founder allele of the CHEK2 gene has been associated with predisposition to breast and prostate cancer in North America and Europe. The CHEK2 protein participates in the DNA damage response in many cell types and is therefore a good candidate for a multisite cancer susceptibility gene. Three founder alleles are present in Poland. Two of these result in a truncated CHEK2 protein, and the other is a missense substitution of an isoleucine for a threonine. We ascertained the prevalence of each of these alleles in 4,008 cancer cases and 4,000 controls, all from Poland. The majority of the common cancer sites were represented. Positive associations with protein-truncating alleles were seen for cancers of the thyroid (odds ratio [OR] 4.9; P=.0006), breast (OR 2.2; P=.02), and prostate (OR 2.2; P=.04). The missense variant I157T was associated with an increased risk of breast cancer (OR 1.4; P=.02), colon cancer (OR 2.0; P=.001), kidney cancer (OR 2.1; P=.0006), prostate cancer (OR 1.7; P=.002), and thyroid cancer (OR 1.9; P=.04). The range of cancers associated with mutations of the CHEK2 gene may be much greater than previously thought.

Background: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is similar to 4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC. Patients and methods: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies. Results: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years. Conclusion: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

The study was conducted to determine if serum copper level could be a useful marker for selection for control examinations and if serum copper level is a risk factor in developing cancer. Copper was quantitatively measured in diluted serum samples by inductively coupled plasma mass spectrometry (ICP-MS) using mass spectrometer (Elan DRC-e, PerkinElmer) in standard mode. In our study, there were two independent groups of patients examined. In the first, retrospective model, there were patients diagnosed with prostate cancer (n = 166) and laryngeal cancer (n = 123) matched with healthy controls. This study showed that serum copper level above 1250 µg/l may be a useful marker for laryngeal examination, but is not a useful marker for prostate cancer early detection. In the second, prospective model, there were patents diagnosed with breast cancer (n = 42) matched with unaffected controls. Serum from breast cancer patients was collected 3 - 41 months before cancer diagnosis. This part of study showed that there is a tendency that breast cancer risk is about two times lower when copper serum level is in range between 1035 - 1311 µg/l. Further investigations are needed.

Familial breast cancer was first recognized in the Roman medical literature of 100 AD [1]. The first documentation of familial clustering of breast cancer in modern times was published by Broca, who reported 10 cases of breast cancer in 4 generations of his wife's family [2]. In the middle of the nineteen nineties it was proven at the molecular level that a substantial number of breast and ovarian cancers has hereditary monogenic aetiology [3,4]. Evaluation of frequency of pedigree-clinical signs characteristic for strong aggregations of breast/ovarian cancers among consecutive cases of cancers of these organs as well as analyses of cancer incidence in monozygotic twins indicate that about 30% of breast and ovarian cancers develop because of a strong genetic predisposition [5]. In other breast/ovarian cancers the significance of genetic factors was underestimated. However, recently it has been possible to show the characteristic constitutional background influencing development of cancer also in patients with sporadic neoplasms. Therefore now scientists think that in almost all patients with cancer a certain genetic background should be detectable, although influencing cancer risk to a various degree. Genetic abnormalities strongly related to cancer are called high risk changes (genes) and abnormalities influencing cancer development to a lower degree are called moderate risk changes (genes). Most frequently strong genetic predisposition to breast/ovarian cancers are related to mutations in the BRCA1 and BRCA2 genes and most often it appears as syndromes of hereditary breast cancer -site-specific (HBC-ss), hereditary breast-ovarian cancer (HBOC) and hereditary ovarian cancer (HOC). In family members of families with HBC-ss syndrome only breast cancers but not ovarian cancers are observed. In HBOC syndrome families both breast and ovarian cancers are diagnosed, and in HOC syndrome only ovarian but not breast cancers are detected. Operational clinical-pedigree criteria which we use in order to diagnose "definitively" or "with high probability" the discussed syndromes are summarized in Table ​Table1.1. In the vast majority of cancer cases related to moderate risk genes family history is negative. HBC-ss, HBOC and HOC syndromes are clinically and molecularly heterogeneous. Mutations in the BRCA1 and BRCA2 genes are the most frequent cause of these syndromes. Table 1 Pedigree-clinical diagnostic criteria of HBC-ss, HBOC and HOC syndromes [6] BRCA1 syndrome In this syndrome women carry a germline mutation in the BRCA1 gene. Carriers of a BRCA1 mutation have approximately 50-80% lifetime risk of breast cancer and 40% risk of ovarian cancer [7]. We estimate that these risks are 66% for breast cancer and 44% for ovarian cancer in the Polish population (Table ​(Table2).2). Both risks appear to be dependent on the type and localization of the mutation [8-10]. Our findings suggest that the risk of breast cancer in women with 5382insC is two times higher than in women with 4153delA. Table 2 Risk of breast and ovarian cancer in BRCA1 mutation carriers in Poland [8] Incomplete penetrance of BRCA1 suggests that other factors, genetic and non-genetic modifiers, are important in carcinogenesis in the mutation carriers. The risk of ovarian cancer is modified by VNTR locus for HRAS 1 and is increased 2-fold in BRCA1 carriers harbouring one or two rare alleles of HRAS 1 [11]. We reported that the 135G>C variant in the RAD51 gene is strongly protective (OR = 0.5) against both ovarian and breast cancer [12,13]. Carriers of a BRCA1 mutation are also at about 10% lifetime risk of fallopian tube and peritoneal cancers [14]. These data about the frequency of ovarian cancer in BRCA1 carriers appear to reflect the combined frequency of ovarian, fallopian tube and peritoneal cancers, because these tumours were diagnosed as ovarian cancers in the past, and because they share similar morphology and cause elevated levels of the marker CA 125. The risk of cancer at other sites may be increased in carriers of a BRCA1 mutation as well, but the evidence is controversial and needs further studies. Breast and ovarian cancer in BRCA1 carriers have particular clinical characteristics. The mean age at onset of breast cancer is about 42-45 years [15,16] and of ovarian cancer is about 54 years [17,18]. 18-32% of breast cancers are bilateral [19,20]. These are rapidly growing tumours: >90% of cases have G3 grade at the time of diagnosis and almost all ovarian cancers in women with a BRCA1 mutation are diagnosed in FIGO stage III°/IV°. Medullary, atypical medullary, ducal and oestrogen receptor negative (ER) breast tumours are common in BRCA1 carriers. BRCA1 mutation-positive tumours constitute about 10-15% of all ER-breast cancers [21-23]. Most carriers of a BRCA1 mutation report a positive family history of breast or ovarian cancer (Figure ​(Figure1).1). However, 45% of BRCA1 carriers report a negative family history, mainly because of paternal inheritance and incomplete penetrance (Figure ​(Figure3)3) [20]. Figure 1 Family with HOC syndrome and diagnosed constitutional 4153delA BRCA1 gene mutation. Figure 3 Patient with ovarian cancer and detected 5382insC BRCA1 mutation from family with negative family history.

Hereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome) is an autosomal dominantly inherited syndrome predisposing to the early development of cancers of the colon, rectum, endometrium, small bowel, and urinary tract and accounts for ∼5% of all colon cancer cases.1 There are at least five genes involved in this cancer predisposition and they include MLH1 ,2 MSH2 ,3 MSH6 ,4 PMS2 , and PMS1 .5 Currently, more than 300 different mutations have been described in these genes which account for approximately 500 HNPCC kindreds from different parts of the world.6 MLH1 and MSH2 genes show abnormalities in more than 90% of HNPCC families with identified germline mutations7 (http://www.nfdht.nl). The majority of reported MLH1 and MSH2 mutations are dispersed throughout the 35 exons of these two genes. However, some changes are recurrent and are described as founder mutations in particular populations.8–11 In order to develop efficient DNA testing, it is important to describe the nature and frequency of mutations that are characteristic of particular ethnic groups. The MSH2 and MLH1 mutation spectrum has not been investigated in the eastern European region and therefore there is no knowledge about any recurrent mutations which may significantly aid in the mutation screening procedures for this region. Here, we describe the results of DNA/RNA based mutation sequencing of both the MSH2 and MLH1 genes in a series of HNPCC families from Poland (89 cases) and the Baltic States (12 cases). ### Patients A total of 101 unrelated patients affected by colorectal cancer or an HNPCC associated cancer (endometrium, small bowel, urinary tract) were from 17 families which fulfilled the Amsterdam II criteria12 and from 84 families matching our modified criteria of suspected HNPCC, one colorectal cancer patient with a first degree relative affected by an HNPCC associated cancer, …

Keywords: CDKN24, CHEK2, NOD2, cancer risk There is continuing interest in identifying low-penetrance genes which are associated with increased susceptibility to common types of cancer. There are several approaches to this problem, including the use of chip-based single nucleotide polymorphism (SNP) arrays to interrogate a large number of genes simultaneously and pre-selecting candidate genes of interest. Candidate genes for cancers of a particular site may be selected because they are known to predispose to malignancies of other organs, or because they are mutated somatically in the cells from the cancer of interest. It is possible that missense variants of genes for which truncating mutations are clearly pathogenic may also be deleterious, but with reduced penetrance. In this situation the association may be overlooked unless large numbers of cancers are studied. In our centre we performed population-based studies of common variants of three genes: a tumour-suppressor gene CDKN2A (OMIM 600160), NOD2 (OMIM 605956) involved in the chronic inflammation process, and CHEK2 (OMIM 604373) participating in the DNA damage response. To determine whether CDKN2A common variant A148T may be associated with an increased risk of malignancies at different sites of origin we genotyped a series of 8,263 unselected cancer cases and compared the frequency of the change observed in this population to 3,000 controls in Poland. To establish the range of cancer types associated with three CHEK2 mutations (VS2+1G → A, 1100delC, and I157T) we genotyped 4,008 unselected cases of cancer and 4,000 controls in Poland. In order to define the range of cancer phenotypes associated with the NOD2 3020insC mutation we examined 2,604 unselected invasive cancers of 12 different types and 1,910 controls from Poland.

Material and methods The study was performed in five groups. For four of them (cancers of the lung, larynx, colon, prostate) and matched healthy controls serum was collected before treatment but during diagnosis. Whereas for breast cancer group serum was collected 1 2 years before tumor diagnosis. Cases and controls in each study groups were matched for year of birth (+/-3 years), number and location of cancer among Io relatives, smoking the number of pack years (+/10%) and adnexectomy (only for breast cancer group). Se level in serum was determined in all individuals by inductively coupled plasma mass spectrometry (ICP-MS) using Elan DRC-e ICP-Mass Spectrometer, Perkin Elmer. For selenium measurements strong quality control criteria was applied: reference material (SeronoformTM) was measured every four samples, cases and controls samples were tested alternately, correction value: +/5%. After obtaining results from mass spectrometry, individuals in each group were divided into four categories (quartiles). Comparison of number of cases and controls was performed in each quartile. To evaluate the occurrence of cancers, individuals with the lowest selenium level (quartile) was taken as reference category. Results It was observed that detailed analysis of selenium concentrations showed that in groups of lung, laryngeal, colorectal and prostate cancers, and low Se level in serum is associated with an increase of cancers frequency. It was also observed that when Se level is higher the number of cancers decreases. In contrast, selenium level >100 μg/l in serum was associated with increased incidence of breast cancer (OR 5.89; p = 0.0004; CI = 2.19 15.84).

The largest worldwide population screening for cancer family syndromes was initiated in January 2001 in the West Pomeranian Region of Poland with 1.7 m inhabitants. In the first step in the period January 2001 - May 2002 family doctors and nurses collected questionnaires asking about cancer family history among 1st and 2nd degree relatives from 1,258,401 of 1.5 m individuals (87%) who were insured in the West Pomeranian Regional Health Foundation. Up to now about 1.15 m questionnaires have been evaluated by geneticists/oncologists. According to questionnaire data around 2% of families fulfilled criteria to be suspected for cancer family syndrome. Family members (usually 1-2 representatives per family) from suspected families were invited for detailed examination. After pedigree and clinical examination as well as DNA/RNA analyses, high genetic predisposition to neoplasms was diagnosed in 10,525 families. Diagnosis of the following syndromes was established definitively or with high probability: 1. Hereditary breast/ovarian cancer syndrome - 4121 families including 438 families with diagnosed BRCA1/2 mutation. 2. Familial syndromes of colorectal cancer: a) HNPCC - 568 families including 63 families with diagnosed MSH2/MLH1 mutation, b) FAP - 22 families, c) Late onset colorectal cancer aggregations - 459 families. 3. Other hereditary organ specific syndromes: a) hereditary stomach cancer - 1250 families, b) hereditary renal cancer - 565 families, c) hereditary laryngeal cancer - 206 families, d) hereditary prostate cancer - 170 families, e) NFI - 66 families, f) VHL - 36 families, g) Retinoblastoma - 4 families, h) Peutz Jeghers syndrome - 3 families, i) juvenile polyposis - 2 families. 4. Organ-specific familial aggregations: a) familial lung cancer - 242 families, b) familial leukaemia/lymphoma - 77 families, c) familial liver cancer - 68 families, d) familial cervical cancer - 30 families, e) familial pancreatic cancer - 73 families, f) familial melanoma - 44 families, g) familial bladder - 19 families. 5. Unspecified cancer family aggregations - 3319 families. Appropriate management has been introduced in all identified families with high predisposition to malignancies.

Keywords: BRCA1, BRCA2, CHEK2, NOD2, p16, NBS1, breast cancer Background It is a known fact that cancer risk is influenced by both environmental and genetic factors. Parameters such as the kind and site of the tumour and family history depend on the proportion between both and the population determines to a great extent the characteristics of the genetic background. Here we focus on breast cancer in the Polish population. Despite complex population dynamics in the last centuries, the Poles seem to be surprisingly homogeneous in their genetics. A sequence-based screening of BRCA1 positive patients showed just 9 polymorphisms of BRCA1, where 91% of individuals shared just 3 common founder mutations [1]. The result is consistent with other Slavic countries [2-4]. In contrast, a similar screening in neighbouring Germany revealed 77 distinct BRCA1 mutations, 18 of them shared by 68% of BRCA1 positive patients [5]. Highly penetrating mutations, such as those of BRCA1, are mostly detected in conspicuous family aggregations via genetic linkage studies. Therefore, they may be detected with the help of just a few families even in heterogeneous populations. However, the probability of finding a new highly penetrating gene for the Polish population seems rather low; mutations of BRCA1 and BRCA2 already cover ~70% of cases of strong familial aggregations of breast and ovarian cancers [1]. The advantage of genetically homogeneous populations relies instead on the increased power of finding medium- and low-risk markers via large association studies that would otherwise be blurred in more complex populations.