Agnieszka Kołacińska

Polymorphisms of the promoter regions of matrix metalloproteinases genes MMP-1 and MMP-9 in breast cancer

SummaryPurposeMatrix metalloproteinases play a crucial role in the cancer invasion and metastasis, angiogenesis and tumorigenicity. A single guanine insertion – the 1G/2G polymorphism in the promoter of the matrix metalloproteinase 1 (MMP-1) gene creates a binding site for the transcription factor AP-1 and thus may affect the transcription level of MMP-1. The C→T substitution at the polymorphic site of the MMP-9 gene promoter results in a higher transcription activity of the T-allelic promoter trough the loss of binding site for a repressor protein. The aim of this work was to investigate the influence of 1G/2G and C→T polymorphisms on the MMP-1 and MMP-9 level and therefore on the occurrence and progression of breast cancer.Experimental designWe investigated the distribution of genotypes and frequency of alleles of the 1G/2G and C→T polymorphisms for 270 patients with breast cancer and 300 healthy women served as control. The genotypes were determined by RFLP-PCR. Additionally, we estimated the level of MMP-1 and MMP-9 antigens in tumor samples and normal breast tissue using ELISA.ResultsThe levels of MMP-1 in tumor samples of node positive patients ware significantly higher than in samples of node negative patients (p<0.05). Increased level of MMP-9 correlates with Bloom-Richardson grading III (p<0.05), increased tumor size (p<0.05) and absence of estrogen and progesterone receptors (p<0.01). Additionally, both MMP-1 and MMP-9 levels were higher in tumor than in the normal breast tissue. We showed the higher risk of metastasis development in lymph node for the 2G/2G genotype (OR=2.14; CI 95% 1.24;3.69) and the 2G allele carriers (OR=1.68; CI 95% 1.19;2.39). We found correlation between the T allele (OR=2.61; CI 95% 1.33;4.87), 2G (OR=2.58; CI 95% 1.35;4.91) and malignance.ConclusionThe results suggest that MMP-1 is responsible for the local invasion and MMP-9 is associated with the malignance and the growth of the tumor. We suggest that the 2G allele of the 1G/2G MMP-1 gene polymorphism may be associated with the lymph node metastasis in patients with breast cancer and therefore it can be considered as a progression marker in this disease.

Polymorphisms of the BRCA2 and RAD51 genes in breast cancer.

SummaryWe performed a case-control study (150 cases and 150 controls) to test the association between three polymorphisms in BRCA2 and RAD51 genes and breast cancer risk. Genotypes were determined in DNA from blood cells by PCR–RFLP. Cancer occurrence was strongly associated with the BRCA2 Met/1915Thr homozygous polymorphic variants, whereas heterozygous variant was associated with significant reduction in breast cancer risk. Gene-gene interaction between the BRCA2-Met1915Thr Thr/Thr and BRCA2-Met784Val Met/Met homozygous variants increased the risk. Therefore, the Met1915Thr polymorphism in the BRCA2 gene may be considered as an independent marker of breast cancer.

Polymorphisms of the DNA Mismatch Repair Gene HMSH2 in Breast Cancer Occurence and Progression

SummaryThe response of the cell to DNA damage and its ability to maintain genomic stability by DNA repair are crucial in preventing cancer initiation and progression. Therefore, polymorphism of DNA repair genes may affect the process of carcinogenesis. The importance of genetic variability of the components of mismatch repair (MMR) genes is well documented in colorectal cancer, but little is known about its role in breast cancer. hMSH2 is one of the crucial proteins of MMR. We performed a case-control study to test the association between two polymorphisms in the hMSH2 gene: an A → G transition at 127 position producing an Asn → Ser substitution at codon 127 (the Asn127Ser polymorphism) and a G → A transition at 1032 position resulting in a Gly → Asp change at codon 322 (the Gly322Asp polymorphism) and breast cancer risk and cancer progression. Genotypes were determined in DNA from peripheral blood lymphocytes of 150 breast cancer patients and 150 age-matched women (controls) by restriction fragment length polymorphism and allele-specific PCR. We did not observe any correlation between studied polymorphisms and breast cancer progression evaluated by node-metastasis, tumor size and Bloom-Richardson grading. A strong association between breast cancer occurrence and the Gly/Gly phenotype of the Gly322Asp polymorphism (odds ratio 8.39; 95% confidence interval 1.44–48.8) was found. Therefore, MMR may play a role in the breast carcinogenesis and the Gly322Asp polymorphism of the hMSH2 gene may be considered as a potential marker in breast cancer.

Apoptosis-, proliferation, immune function-, and drug resistance- related genes in ER positive, HER2 positive and triple negative breast cancer.

The aim of our study was to examine an association between gene expression assessed using a 23-gene microarray and receptor status of breast cancer samples categorized as ER positive, HER2 positive and triple negative subtypes. The ER positive cohort was subsequently divided into Luminal A, Luminal B HER2 negative and Luminal B HER2 positive subtypes. Core- needle biopsies were collected from 78 female patients with inoperable locally advanced breast cancer or resectable tumors suitable for downstaging, before any treatment. Expressions of 23 genes were determined by means of TagMan Low Density Arrays. Analysis of variance was used to select genes with discriminatory potential between receptor subtypes. We introduced a correction for false discovery rates (presented as q values) due to testing multiple hypothesis. Pairwise post-hoc comparisons of receptor subtypes were performed using Tukey s HSD test. Five genes out of a 23-gene microarray differed significantly in relation to breast cancer receptor-based subtypes. Among these five genes, we identified: BCL2 (p=0.0002, q=0.0009), MKI67 (p=0.0037, q=0.0064), IGF1R (p=0.0040, q=0.0064), FOXC1 (p=0.0113, q=0.0135) and IRF1 (p=0.0435, q=0.0416) as ones showing ER positive, HER2 positive and triple negative -subtype specific expression profiles. When incorporating Luminal A, Luminal B HER2 negative, Luminal B HER2 positive subtypes into analysis, four genes: BCL2 (p=0.0006, q=0.0034), MKI67 (p=0.0078, q=0.0198), FOXC1 (p=0.0102, q=0.0198) and IGF1R (p=0.0174, q=0.0254) were selected. Elevated levels of IGF1R and BCL2 were significantly linked with Luminal A subtype. Triple negative breast cancer subtype was associated with higher expression of IRF1, FOXC1 and MKI67. In HER2 positive cohort lower expression of all five analyzed genes was noted.

Association of microRNA-93, 190, 200b and Receptor Status in Core Biopsies from Stage III Breast Cancer Patients

Oncologists now favor more personalized treatment strategies in breast cancer patients. Gene expression analysis has been widely used, but less is known about epigenetic factors, for example, microRNAs (miRNAs). The aim of this study was to determine the relationship between selected miRNAs and receptor status in core biopsies sampled before preoperative chemotherapy in stage III locally advanced breast cancer (LABC) patients. In 37 LABC core biopsies, three miRNAs per sample were analyzed: hsa-miR-93-5p, hsa-miR-190a, and hsa-miR-200b-3p, and hsa-miR-103a-3p as an endogenous control (TaqMan(®) RT-PCR; Applied Biosystems). Receptor status was determined by a dedicated pathologist. The Mann-Whitney U, Shapiro-Wilk, and Levenes tests were used to compare related samples. Levels of miRNA-93 differed significantly in core biopsies of LABC patients with different expressions of ER (estrogen receptor) and PR (progesterone receptor). Higher levels of miRNA-93 were found in ER-negative (p=0.0027) and PR-negative patients (p=0.0185). Levels of miRNA-190 and 200b did not differ significantly in core biopsies of LABC patients who expressed ER and PR differently (p=0.7727, p=0.9434, p=0.6213, and p=0.1717). Levels of miRNA-93, 190, and 200b were not significantly different in core biopsies of LABC patients with different HER2 (human epidermal growth factor 2) expressions (p=0.8013, p=0.2609, and p=0.3222). The assessment of core biopsy miRNA profiles and receptor-based subtypes may identify new signaling pathways for improved breast cancer classification.

Immune checkpoints in aggressive breast cancer subtypes.

Immune checkpoints are molecules referred to inhibitory pathways in the immune system that play a pivotal role in prevention of autoimmunity and oncogenesis. The aim of the study was to evaluate expression levels of selected immune checkpoints- PD-1 (programmed cell death protein 1), and PD-L1 (programmed cell death 1 ligand 1) in breast cancer patients, suitable for breast conservation and sentinel node biopsy and determine their associations with clinicopathological factors.Expression of the genes coding for PD-1 and PD-L1 was analyzed in formalin-fixed paraffin-embedded specimens using real-time PCR. mRNA expression levels were determined using beta actin (ACTB) as an endogenous control. There was a trend towards significance between higher PD-1 and PD-L1 levels in triple negative breast cancers (p=0.1). Higher PD-L1 expression was also found in aggressive breast cancer subtypes e.g. triple negative and HER2 (human epidermal growth factor receptor 2) -positive as compared with subtypes with better prognosis such as luminal A and luminal BHER2-negative (p=0.05). There was a trend towards significance in higher PD-1 levels in triple negative and HER-2 positive breast cancers (p=0.1). A statistically significant difference was found between PD-L1 expression and tumor grade (p=0.01). Elevated PD-L1 levels were noted in G3 tumors. Immunogenicity appears to be gaining importance in triple negative and HER2-positive molecular subtypes of breast cancer, and the results in this study provide a basis for further investigation into the role of immune checkpoints in breast cancer.

Pathological complete response in younger and older breast cancer patients

Introduction Pathologic complete response (pCR) after neoadjuvant systemic treatment for inoperable locally advanced breast cancer is defined as complete microscopic disappearance of invasive cancer in both the breast and axilla in the postoperative specimen. The aim of the study was to characterize the groups of younger (≤ 40 years old) and older (≥ 70 years old) breast cancer patients who achieved a pCR. Material and methods One hundred thirty-eight consecutive patients aged between 30 and 78 years with locally advanced breast cancer, operated on after neoadjuvant systemic treatment between November 2007 and June 2010, were analyzed. In this group 9 women (6.5%) were 40 years of age or younger, and 12 patients (8.7%) were 70 years of age or older. Results In the younger group, pCR was achieved in 1 patient with triple negative, invasive ductal breast cancer, G3, BRCA 1 mutation, treated with cisplatin. A near pCR was achieved in 2 other patients, with triple negative, invasive ductal breast cancer, G3, treated with AT. The pCR in the breast was found in a HER2 positive patient. In older patients, pCR was achieved in 2 patients with triple negative, invasive ductal breast cancer, G3, treated with AT or FEC. Pathologic complete response in the axilla was achieved in 1 patient with triple negative, ductal carcinoma. The pCR rates were significantly higher in triple negative breast cancer in both groups (p = 0.047 and p = 0.018, respectively). Conclusions Pathologic complete response was significantly associated with receptor- based subtypes in both young and old women.

Interleukin-6 and the IL-6 (-174) C/G polymorphism in breast pathologies and in HIV-infected patients.

Introduction Breast cancer and acquired immunodeficiency syndrome (AIDS) are key issues for modern medicine. The aim of the current study was to present how cytokines, in the example of IL-6 and its polymorphism, can affect these two conditions. Material and methods Thirty-one women with benign breast tumours, 42 breast cancer patients and 40 HIV-infected females were enrolled in the study. Serum IL-6 levels were determined by ELISA. The IL-6 polymorphism was genotyped by PCR-RFLP. Results Serum IL-6 in patients with benign breast tumours was significantly lower than in females with breast cancer (p = 0.017) and HIV-infected women (p = 0.032). We did not find statistically significant differences in serum IL-6 level between females with breast cancer and HIV-infected women (p = 0.749). Comparing the distribution of genotypes and frequency of the IL-6 (–174) C/G polymorphism between the three study groups – breast cancer patients, patients with benign breast tumours, and HIV-infected patients – we did not find any statistically significant differences. Conclusions IL-6 can play an important role in pathogenesis of breast cancer and HIV infection and its level is higher than in the control group irrespective of distribution of genotypes and frequency of the IL-6 (–174) C/G polymorphism.

Retrospective analysis of local recurrence rate in breast cancer patients treated at the department of surgical oncology in Łódź between 2009 and 2013.

UNLABELLED The aim of the study was to analyze clinicopathological features in breast cancer patients with local recurrence (LR). MATERIAL AND METHODS A retrospective analysis of database of breast cancer patients operated on in the Department of Surgical Oncology in Łódź from 2 January 2009 to 30 June 2013, identified 1080 women with primary breast cancer and 11 patients with LR. RESULTS LR rate was 0.23% per year. True recurrence (TR) occurred more frequently in patients with luminal B molecular subtype, in HER-2 positive and in triple-negative subgroups. In one patient with luminal -A subtype new primary (triple negative) occurred. TR were noted predominantly in patients with axillary lymph nodes metastases and with luminal B subtype who did not receive adjuvant chemotherapy but were given only endocrine therapy. LR were observed more frequently in patients who did not receive adjuvant radiotherapy or this treatment was delayed. Minimal surgical margins in postoperative specimens measured by pathologist were 4-25 mm, mean 9.5 mm. CONCLUSIONS The LR rate in patients operated on breast cancer in the Department of Surgical Oncology between 2009 and 2013 was low. TR was diagnosed in patients with non- luminal A breast cancer despite wide surgical margins, especially if the patients did not receive optimal adjuvant systemic treatment or radiotherapy was delayed or omitted. Complete cancer excision followed by an immediate implementation of optimal adjuvant treatment seems to be crucial especially in patients with poor tumor biology.

Local Antibiotic Therapy in Rectal Cancer Surgery

UNLABELLED Infectious complications and their consequences are still key issues in rectal cancer surgery. Currently, intravenous antibiotic administration is a recognized method for lowering the rate of these complications. The aim of the study was to assess the efficacy of complementary application of a gentamicin-impregnated sponge in the perineal wound or in the vicinity of intestinal anastomosis after abdominoperineal resection or low anterior resection. MATERIAL AND METHODS 112 patients with primary rectal cancer were enrolled in this study. 42 patients were treated with a gentamicin sponge and drainage (group A) and 70 individuals were treated with drainage alone (group B). In the aforementioned groups a routine short-term regimen of antibiotic prophylaxis was used. We applied gentamicin-impregnated sponges in 27 patients in whom anterior resection was performed and in 15 patients from the abdominoperineal resection group (64% and 36%, respectively). In the control group, 44 anterior resections and 26 abdominoperineal resections were carried out (63% and 37%, respectively). RESULTS We did not observe statistically significant differences in the incidence of suppurative complications (intraabdominal abscess, perineal wound infection): 4 cases (9.52%) in group A and 9 (12.58%) in group B and anastomotic leakage with clinical manifestation after low anterior resection: 1 case (3.7%) in group A and 2 (4.5%) in group B. Postoperative fever of unknown origin was noted more often in group B: 23 patients (32.8%) versus 10 patients (23.8%) in group A and this difference was statistically significant (p<0.05). Hospitalization after surgery was also significantly longer in group B (9-37 days, median 11 days) as compared with group A (8-26 days, median 13 days) (p<0.05). CONCLUSIONS Local antibiotic therapy in rectal cancer surgery lowered the incidence of postoperative fever of unknown origin and permitted shorter hospitalization after surgery. Local gentamicin application in rectal cancer surgery did not change significantly the rate of infectious complications.