Vitamin D receptor gene polymorphisms in relation to the risk of colorectal cancer in the Polish population.

The protective effect of vitamin D against several cancers including colorectal cancer is modulated by the vitamin D receptor (VDR) and its ligand, the active form of vitamin D. VDR response has been found to play a role in various genes encoding proteins involved in crucial cellular pathways. Single nucleotide polymorphisms (SNPs) of the VDR gene that modulate its activity are located in the promoter region, exons 2-9, and their vicinity and also in the 3'UTR region. Some of them have been previously studied in relation to cancer susceptibility and prognosis. The aim of our study was to investigate four polymorphisms, BsmI, ApaI, TaqI, and FokI, of the VDR gene in Polish patients with sporadic colorectal cancer and to evaluate their association with susceptibility to cancer. We found a significant association between the BsmI genotype and cancer (individuals with the bb genotype are more susceptible to cancer compared to those with other genotypes, p = 0.025, Fisher's exact test for 2 × 2 table). Also, the TT genotype at TaqI and the AA genotype at ApaI are correlated with a higher risk of cancer (p = 0.00071 and p = 1.0 × 10(-5), respectively). We found relatively strong linkage disequilibrium between the TaqI and ApaI loci (T with A and t with a, respectively). Both of these loci are associated with cancer. We do not observe any such association for the FokI polymorphism. In conclusion, a small modification in VDR expression may play a role in such a multipathway process as tumorigenesis.

Protein tyrosine phosphatase receptor-like genes are frequently hypermethylated in sporadic colorectal cancer.

The activity of phosphatases could be influenced by genetic, as well as epigenetic alterations. In our study, we have investigated the methylation status of four PTPRs: PTPRM, PTPRT, PTPRR and PTPRZ1, which were pre-selected using microarray techniques as being alternatively methylated in sporadic colorectal cancer (CRC). The analyses were carried out on 131 surgical specimens obtained from sporadic CRC patients. The methylation status of the four genes was examined using methyl specific PCR (MSP). The analysis of promoter methylation using an Illumina 27K microarray revealed four protein tyrosine phosphatases PTPRM, PTPRT, PTPRR and PTPRZ1 as being hypermethylated with ß-value ≥0.2 and P≤0.05. Subsequent analysis using MSP confirmed these observations-the frequency of promoter methylation was significantly higher in tumor cells compared with matched normal tissue for each of the analyzed genes. There was no association observed between the methylation status of PTPRs and either CIMP, K-ras (codon 12) and BRAF (exon 15, V600E) mutations or tumor localization (proximal/distal). The results of our study show a statistically significant difference between promoter methylation in cancerous and healthy tissue. This result supports the hypothesis that the PTPR family has an important role in the etiology of CRC.

Prognostic value of the Fas/Fas ligand system in breast cancer.

Fas and its ligand (FasL) are known to play a crucial role in the genetically controlled mechanism of cell death, and their deregulation in cancer cells is involved in the immune escape of the tumor. The aim of this review is to analyze the current knowledge on the prognostic value of Fas/FasL in breast cancer patients. Both the results of other authors and our own experiences indicate that the lack of Fas ligand, and particularly Fas, is related to a significantly worse prognosis. It probably results from the resistance of Fas-deficient breast tumors to the mechanisms of apoptosis. On the other hand, some results suggest that the Fas/FasL-dependent mechanisms of tumor spread may be different for various target tissues. The expression of the Fas/Fas-ligand system has potential prognostic application in view of current knowledge, and consequently should be considered as an additional prognostic factor in breast cancer patients.

Assessment of three epigenotypes in colorectal cancer by combined bisulfite restriction analysis.

Recent investigations have demonstrated the clear heterogeneity of sporadic colorectal cancer (CRC) with regard to CpG island methylation. Two unsupervised cluster analyses revealed that CRCs form three distinct DNA methylation subsets, which are referred to as the high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME, respectively). A recent study by Yagi et al. found a fairly sensitive and specific identification of HME, IME, and LME using two marker panels analyzed by MALDI-TOF mass spectrometry (MassARRAY). However, the expensive equipment required for this method substantially increases the cost and complexity of the assay. In this article, we demonstrate the assessment of HME, IME, and LME in a group of 233 sporadic CRCs using seven markers proposed by Yagi et al. The DNA methylation of each marker was quantified using combined bisulfite restriction analysis (COBRA) and analyzed along with various genetic factors associated with CRC [the BRAF and KRAS mutations, MLH1 methylation and microsatellite instability (MSI)]. The baseline methylation of each marker was generated from pooled DNA isolated from 50 normal colon tissues. We demonstrate that the correlation of HME, IME, and LME epigenotyped by COBRA using different molecular classifiers is similar to that achieved by MassARRAY. Therefore, epigenotyping CRCs using COBRA is a simple, specific, and cost-effective method that has the potential to be widely used in CRC research.

Assessment of chromosomal imbalances in CIMP-high and CIMP-low/CIMP-0 colorectal cancers.

Data presented in a number of recent studies have revealed a negative correlation between CpG island methylator phenotype (CIMP) and chromosomal instability (CIN) measured by a loss of heterozygosity (LOH) of selected loci, suggesting that CIN and CIMP represent two independent mechanisms in sporadic colorectal cancer (CRC) carcinogenesis. However, CIN is a heterogeneous phenomenon, which may be studied not only by employing LOH analysis but also by observing chromosomal imbalances (gains and deletions). The current study aimed to investigate the relationship between CIMP and chromosomal gains and deletions (assessed by comparative genomic hybridization) in a group of 20 CIMP-high and 79 CIMP-low/CIMP-0 CRCs. Our results revealed that the mean numbers of gains and of total chromosomal imbalances were significantly greater (p = 0.004 and p = 0.007, respectively) in the CIMP-low/CIMP-0 group compared to the CIMP-high group, while no significant difference was observed between the mean numbers of losses (p = 0.056). The analysis of copy number changes of 41 cancer-related genes by multiplex ligation-dependent probe amplification showed that CRK gene was exclusively deleted in CIMP-low/CIMP-0 tumors (p = 0.02). Given that chromosomal losses play an important role in tumor suppressor inactivation and chromosomal gains, in the activation of proto-oncogenes, we hypothesize that tumor suppressor inactivation plays similar roles in both CIMP-high and CIMP-low/CIMP-0 CRCs, while the predominance of chromosomal gains in CIMP-low/CIMP-0 tumors may suggest that the activation of proto-oncogenes is the underlying mechanism of CIMP-low/CIMP-0 CRC progression.

Preoperative treatment does not improve the therapeutic results of abdominosacral amputation of the rectum.

BACKGROUND: This study reviewed the impact of preoperative chemoradiotherapy/short-term radiotherapy on abdominosacral amputations of the rectum (ASAR) for the treatment of low-rectum cancers in terms of postoperative morbidity, local recurrence rates, and survival. METHODS: A total of 198 patients with stage II and III tumors located within 6 cm of the anorectal junction underwent ASAR between 1998 and 2008 and were selected for further analysis. Patients were compared according to the following groups: those who had surgery only (Group A) and those who had preoperative chemoradiotherapy/short-term radiotherapy (Group B). RESULTS: There were 44 and 154 patients in Groups A and B, respectively, including 135 males. The median age of the subjects was 63 years (range = 35-88). The median follow-up period was 81 months (range = 23-138). Neither the local recurrence rates (6.8% in Group A vs. 4.6% in Group B, p = 0.544) nor the 5-year relative survival rates (72.4% in Group A vs. 69.3% in Group B, p = 0.127) differed significantly between the groups. CONCLUSION: Preoperative therapy in low-rectum cancer does not improve the therapeutic results of ASAR.

Watch-and-wait strategy in rectal cancer: Is there a tumour size limit? Results from two pooled prospective studies.

BACKGROUND: Frequency and predictive factors for a clinical complete response (cCR) in unselected patients are unclear. MATERIAL AND METHODS: Two prospective observational studies were designed and pooled to explore predictive factors for cCR. Both studies evaluated the watch-and-wait strategy in consecutive patients; the first single-institutional study in elderly with a small tumour, the second multi-institutional study in all the patients receiving standard of care preoperative radiotherapy. RESULTS: Four hundred and ninety patients were analysed. Short-course radiotherapy alone, or with consolidation chemotherapy or chemoradiation was given to 40.6%, 40.2% and 19.2% of the patients, respectively. The median interval from the radiation start to the first tumour response assessment was 10.2 weeks for short-course radiation and 13.2 weeks for chemoradiation. Seventy-three patients had cCR and 71 underwent w&w with the median follow-up of 24 months. The regrowth rate was 26.8%. cCR rate was 39.0% for low-risk cancer (cT1-2N0), 16.8% for intermediate-risk (cT3 with unthreatened mesorectal fascia [MRF-] or cT2N+) and 5.4% for high-risk (cT4 or MRF+). In the multivariable analysis, tumour volume (or tumour length and circumferential extent) and cN status were significant predictors for cCR. In circular cancers or with a length ≥7 cm (n = 184), cCR rate was only 2.7%, sustained cCR 1.6% and the sensitivity of cCR diagnosis 23.1%. None of 27 patients with a tumour larger than 120 cm3 achieved cCR. CONCLUSIONS: Considering watch-and-wait strategy is questionable in patients with circular tumours or with tumour length ≥7 cm.

Evaluation of MDA-MB-468 Cell Culture Media Analysis in Predicting Triple-Negative Breast Cancer Patient Sera Metabolic Profiles.

Triple-negative breast cancer (TNBC) is characterized by limited survival, poor prognosis, and high recurrence. Understanding the metabolic adaptations of TNBC could help reveal improved treatment regiments. Here we performed a comprehensive 1H NMR metabolic characterization of the MDA-MB-468 cell line, a commonly used model of TNBC, followed by an analysis of serum samples obtained from TNBC patients and healthy controls. MDA-MB-468 cells were cultured, and changes in the metabolic composition of the medium were monitored for 72 h. Based on time courses, metabolites were categorized as being consumed, being produced, or showing a mixed behavior. When comparing TNBC and control samples (HC), and by using multivariate and univariate analyses, we identified nine metabolites with differing profiles). The serum of TNBC patients was characterized by higher levels of glucose, glutamine, citrate, and acetoacetate and by lower levels of lactate, alanine, tyrosine, glutamate, and acetone. A comparative analysis between MDA-MB-468 cell culture media and TNBC patients' serum identified a potential systemic response to the carcinogenesis-associated processes, highlighting that MDA-MB-468 cells footprint does not reflect metabolic changes observed in studied TNBC serum fingerprint.

Guidelines of the Association of Polish Surgeons and the Polish Society of Surgical Oncology on the accreditation of healthcare centers providing cytoreductive surgery and HIPEC for primary and secondary peritoneal cancers.

Surgical interventions in patients with peritoneal metastases combined with hyperthermic intraperitoneal chemotherapy (HIPEC) and systemic treatment are becoming more common and, when applied to selected patient groups, they reach 5-year survival rates of 32-52%. Good clinical outcomes require experienced and well-equipped healthcare centers, experienced surgical team and adequate patient qualification process. As a result of the discussion on the need for evaluation of quality of care and treatment outcomes and at the request of the Peritoneal Cancer Section of the Polish Society of Surgical Oncology, accreditation standards have been developed and the Accreditation Committee has been established for healthcare centers providing cytoreductive surgery and HIPEC for the management of primary and secondary peritoneal cancers.

Abdominosacral amputation of the rectum for low rectal cancers: ten years of experience.

BACKGROUND: Abdominoperineal resection for rectal cancer is related to the high frequency of local recurrences, risk of inadvertent bowel perforation, and disease-positive tumor margin. An alternative technique to this procedure, however, is the abdominosacral amputation of the rectum (ASAR). The aim of this study was to report on the technique and share our experience of ASAR on the cohort of consecutively operated patients. METHODS: In its anterior stage, ASAR follows the rules of total mesorectal excision. In its posterior part, the patient is positioned in a prone jackknife position and the coccyx and the last sacral vertebra (if necessary) are removed, enabling a sharp and directly visualized resection of the tumor and other structures critical to local recurrence. Between 1998 and 2007, a total of 210 low-rectal cancer patients were so treated at our clinic. RESULTS: Bowel perforation occurred in 9 patients, the circumferential resection margin was positive in 16 patients, and 38 patients had local wound complications. Seven (4.4%) of 158 patients with 2-year follow-up developed local recurrence, whereas 5-year observed and relative survivals were 68.3% and 73.2%, respectively. CONCLUSIONS: ASAR has a low risk of bowel perforation, circumferential resection margin involvement, and local wound complications. The local recurrence rate is lower and survival better than with conventional abdominoperineal resection.

Infralevator lymphatic drainage of low-rectal cancers: preliminary results.

BACKGROUND: Some low-rectal cancers may spread into or recur in the inguinal lymph nodes despite optimal resection of the primary tumor. Hence, we hypothesized that lymphatic drainage of low-rectal malignancies may be inhomogeneous and that an extramesorectal route may be involved in at least some cases. The idea of our preliminary study was to analyze the potential lymphatic drainages in low-rectal cancer patients. METHODS: The first stage of the experiment included two consecutive low-rectal adenocarcinoma patients (free from inguinal lymph node metastases), in whom the lymphatics of the primary tumor were traced with Patentbalu dye. During the second stage the records of 206 consecutive low-rectal cancer patients were analyzed for presence of inguinal lymph node metastases. RESULTS: An evaluation of specimens from two rectal cancer patients revealed extramesorectal lymphatic drainage of the primary tumor besides the mesorectal route. An analysis of 206 patients revealed six cases of inguinal node metastases. Median age of patients was 55 years. They were all diagnosed with rectal adenocarcinoma, T3 or T4 tumors with G2 or G3 grade. CONCLUSION: The demonstration of an alternative route of lymphatic drainage suggests that more radical surgical procedures are necessary for successful treatment of low-rectal cancers.

Constitutive and dynamic phosphorylation and acetylation sites on NUCKS, a hypermodified nuclear protein, studied by quantitative proteomics.

Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS) is a 27 kDa chromosomal protein of unknown function. Its amino acid composition as well as the structure of its DNA binding domain resembles that of high mobility group A (HMGA) proteins, chromosomal proteins known as modulators of chromatin conformation and regulators of transcription. Conformation and function of the HMGA proteins are regulated by phosphorylation and acetylation. So far 19 phosphorylation sites had been reported in NUCKS. In this study, we have identified all known and six additional phosphorylation sites, and also mapped multiple sites of acetylation, methylation and formylation. We measured cell cycle dependent changes of phosphorylation and acetylation of NUCKS in HeLa cells through stable isotope labeling by amino acids in cell culture (SILAC), using the dephosphorylated protein for normalization. We identified sites that were highly phosphorylated or dephosphorylated in mitotically arrested cells as well as sites that were constitutively phosphorylated. The extent of acetylation is reduced in mitotically and G1 arrested cells. Analysis of human cancer specimens revealed that in tissues the extent of acetylation, formylation and methylation is higher than in cultured cells. In breast cancer samples, seven acetylation, three methylation, and three formylation sites were mapped in NUCKS. Of the 243 amino acids, at least 36 can be modified with a total of 57 posttranslational modifications. Thus, NUCKS appears to have the highest ratio of modified to unmodified residues of any protein so far described.

The Influence of Tumor Microenvironment on ATG4D Gene Expression in Colorectal Cancer Patients.

Despite great progress in research on the subject, the involvement of autophagy in colorectal cancer (CRC) pathogenesis (initiation, progression, metastasis) remains obscure and controversial. Autophagy is a catabolic process, fundamental to cell viability and connected with degradation/recycling of proteins and organelles. In this study, we aimed at investigating the relative expression level of mRNA via Real-Time PCR of 16 chosen genes belonging to Atg8 mammalian orthologs and their conjugation system, comprising GABARAP, GABARAPL1, GABARAPL2, MAP1LC3A, MAP1LC3B, MAP1LC3C, ATG3, ATG7, ATG10, ATG4A, ATG4B, ATG4C, ATG4D, and three genes encoding proteins building the multimeric ATG16L1 complex, namely ATG5, ATG12, and ATG16L1, in 73 colorectal tumors and paired adjacent normal colon mucosa. Our study demonstrated the relative downregulation of all examined genes in CRC tissues in comparison to adjacent noncancerous mucosa, with the highest rate of expression in both tumor and non-tumor tissues observed for GAPARBPL2 and the lowest for MAP1LC3C. Moreover, in patients with advanced-stage tumors and high values of regional lymph nodes, statistically significant downregulation of ATG4D expression in adjacent normal cells was observed. Our study confirms the role of autophagy genes as cancer suppressors in colorectal carcinogenesis. Furthermore, in regard to the ATG4D gene, we observed the influence of tumor microenvironments on gene expression in adjacent colon mucosa.

Fas/Fas-ligand expressions in primary breast cancer are significant predictors of its skeletal spread.

BACKGROUND: Bones belong to the most frequent localizations of breast cancer metastases. Several studies on female breast malignancies have indicated that Fas/Fas-ligand status may have a significant impact on survival. Hence, the aim of our study was to determine if these molecules might serve as the predictors of skeletal dissemination in radically-treated breast cancer patients. PATIENTS AND METHODS: Tumor samples from 147 radically-treated breast cancer patients were studied immunohistochemically for Fas/Fas-ligand expression. RESULTS: Both Fas and Fas-ligand expression in the primary tumor were considerably less frequent among breast cancer patients with bone metastases compared to women without skeletal spread. Moreover, negative staining for Fas or the lack of Fas-ligand expression proved to be significant predictors for the survival free from bone metastases under univariate analysis. CONCLUSION: Our results suggest that the probability of bone metastases may be assessed on the basis of Fas/Fas-ligand expression in primary breast cancer. Consequently, their determination seems crucial for further prognosis and determination of adjuvant treatment.

[The results of breast conserving treatment in breast cancer]. / Wyniki leczenia oszczedzajacego chorych na raka sutka.

UNLABELLED: The aim of the work was to assess the results of breast conserving treatment (BCT) in early breast cancer in patients treated in Lower Silesian Oncology Centre. MATERIAL AND METHODS: Analysis of data from 167 women treated between 1997 and June 2003 with BCT and adjuvant therapy was performed. RESULTS: Only in 14 patients (8.3%) failure of treatment was observed. In 7 cases (4.8%) there was local recurrence, all of them underwent mastectomy m. Patey and they are all alive without disease symptoms. In 6 women (3.6%) dissemination of cancer was observed. Death of 5 patients (3%) between 21 and 68 months from operation took place. In Cox-Mantel test only dependency between N status and dissemination (p < 0.05) was noted. Better results of treatment were connected with estrogen receptor (ER) overexpression. Our results do not differ from data published by leading oncology centers around the world.

Peritoneal metastases of colorectal origin - cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC). The financial aspect.

The incidence of peritoneal carcinomatosis of colorectal cancer amounts to 5%-15% for synchronous metastases and as much as 40% in cases of local recurrence. Best results are obtained for cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC). This treatment offers much better outcomes, leading to 5-year survival rates of as much as 30%-50%. The procedures require significant experience in abdominal surgery, are time-consuming (mean duration of the procedure ranging from 6 to 8 hours) and are burdened by complications that are due not only to the procedure itself but also to the intraperitoneal administration of the cytostatic drug at elevated temperature (41.5 °C). After the procedure, patients are required to be admitted to intensive care units due to potential complications associated with the extent and duration of the procedure as well as chemotherapy administered in hyperthermia. Postoperative management of these patients requires appropriate experience of the entire medical and nursing team. Cytoreductive surgeries combined with HIPEC as highly specialized medical procedures should be assessed for their potential long-term benefits and their costs should be appropriately calculated with consideration to realistic reimbursement rates. Realistic valuation and reimbursement covering the overall average cost of the procedure is recommended by the National Consultant in Surgical Oncology as well as the ESMO consensus guidelines.

Customized Array Comparative Genomic Hybridization Analysis of 25 Phosphatase-encoding Genes in Colorectal Cancer Tissues.

BACKGROUND/AIM: Molecular mechanisms of alterations in protein tyrosine phosphatases (PTPs) genes in cancer have been previously described and include chromosomal aberrations, gene mutations, and epigenetic silencing. However, little is known about small intragenic gains and losses that may lead to either changes in expression or enzyme activity and even loss of protein function. MATERIALS AND METHODS: The aim of this study was to investigate 25 phosphatase genes using customized array comparative genomic hybridization in 16 sporadic colorectal cancer tissues. RESULTS: The analysis revealed two unique small alterations: of 2 kb in PTPN14 intron 1 and of 1 kb in PTPRJ intron 1. We also found gains and losses of whole PTPs gene sequences covered by large chromosome aberrations. CONCLUSION: In our preliminary studies using high-resolution custom microarray we confirmed that PTPs are frequently subjected to whole-gene rearrangements in colorectal cancer, and we revealed that non-polymorphic intragenic changes are rare.

The BAX gene as a candidate for negative autophagy-related genes regulator on mRNA levels in colorectal cancer.

Autophagy is a catabolic process, which is involved in the maintenance of intracellular homeostasis by degrading redundant molecules and organelles. Autophagy begins with the formation of a double-membrane phagophore, followed by its enclosure, thus leading to the appearance of an autophagosome which fuses with lysosome. This process is highly conserved, precisely orchestrated and regulated by autophagy-related genes. Recently, autophagy has been widely studied in different types of cancers, including colorectal cancer. As it has been revealed, autophagy plays two opposite roles in tumorigenesis, as a tumor suppressor and a tumor enhancer/activator, and therefore is called a double-edge sword. Recently, interaction between autophagy and apoptosis has been found. Therefore, we aimed to study the mRNA levels of genes engaged in autophagy and apoptosis in colorectal cancer tissues. Colorectal cancer and adjacent healthy tissues were obtained from 73 patients diagnosed with primary colorectal cancer. Real-time PCR analysis employing Universal Probe Library was used to assess the expression of the seven following selected genes: BECN1, UVRAG, ULK1, ATG13, Bif-1, BCL2 and BAX. For all but one of the tested genes, a decrease in expression was observed. An increase in expression was observed for BAX. BAX expression decreases consistently from early to more advanced stages. High expression of BAX was strongly associated with negative UVRAG expression. The high expression of the BAX gene seems to be a negative regulator of autophagy in colorectal cancer cells. The relative downregulation of autophagy-related genes was observed in colorectal cancer samples.

High PTPRQ Expression and Its Relationship to Expression of PTPRZ1 and the Presence of KRAS Mutations in Colorectal Cancer Tissues.

BACKGROUND: The risk of sporadic colorectal cancer (CRC) is strongly influenced by Iifestyle, environmental and genetic factors. Protein tyrosine phosphatases belong to a group of enzymes whose role in CRC has not yet been intensively studied. They play an important role in activation/de-activation of many enzymes, influencing cell biology by catalyzing reactions opposing those catalyzed by kinases. Protein tyrosine phosphatase receptor-like type Q (PTPRQ) and protein tyrosine phosphatase receptor-like type Z polypeptide 1 (PTPRZ1) have both been shown to be important in development of many cancer types including CRC. MATERIALS AND METHODS: The expression level of PTPRQ and PTPRZ1 was determined by real-time polymerase chain reaction in 16 CRC tissues obtained from patients diagnosed with adenocarcinoma coli. RESULTS: We revealed a high level of PTPRQ expression (p=0.0080), as well as an association between expression levels of PTPRQ and PTPRZ1 (p<0.0001). Moreover PTPRQ expression was higher in tissues presenting with Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation (p=0.0293). CONCLUSION: We confirmed the contribution of PTPRZ1 and especially PTPRQ in CRC development, supporting the hypothesis that PTPRQ is a candidate oncogene, playing a crucial role in phosphorylation/dephosphorylation signaling pathways.