Marek Zadrożny

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.

Polymorphism of the homologous recombination repair genes RAD51 and XRCC3 in breast cancer.

The RAD51 protein and its paralog, XRCC3, play an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination. Since DSBs may contribute to the pathogenesis of breast cancer and variability in DNA repair genes may be linked with some cancers, we performed a case-control study (135 cases and 175 controls) to check the association between the genotypes of the Thr241Met polymorphism of the XRCC3 gene and the 135G>C polymorphism of the RAD51 gene and breast cancer occurrence and progression. Genotypes were determined in peripheral blood lymphocytes by RFLP-PCR. We did not find any association between either polymorphism singly and breast cancer occurrence. Both polymorphisms were not related to tumor size, estrogen and progesterone receptors status, cancer type and grade. However, the Thr241Met genotype of the XRCC3 polymorphism slightly increased the risk of local metastasis in breast cancer patients (OR 2.56, 95% CI 1.27-5.17). The combined Thr241Met/135G>C genotype decreased the risk of breast cancer occurrence (OR 0.22, 95% CI 0.08-0.59). Our results suggest that the variability of the DNA homologous recombination repair genes RAD51 and XRCC3 may play a role in breast cancer occurrence and progression, but this role may be underlined by a mutual interaction between these genes.

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.

Free radical scavengers can differentially modulate the genotoxicity of amsacrine in normal and cancer cells.

Amsacrine is an acridine derivative drug applied in haematological malignancies. It targets topoisomerase II enhancing the formation of a cleavable DNA-enzyme complex and leading to DNA fragmentation in dividing cancer cells. Little is known about other modes of the interaction of amsacrine with DNA, by which it could affect also normal cells. Using the alkaline comet assay, we showed that amsacrine at concentrations from the range 0.01 to 10 microM induced DNA damage in normal human lymphocytes, human promyelocytic leukemia HL-60 cells lacking the p53 gene and murine pro-B lymphoid cells BaF3 expressing BCR/ABL oncogene measured as the increase in percentage tail DNA. The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Amifostine at 14 mM decreased the level of DNA damage in normal lymphocytes, had no effect on the HL-60 cells and potentiated the DNA-damaging effect of the drug in BCR/ABL-transformed cells. Vitamin C at 10 and 50 microM diminished the extent of DNA damage in normal lymphocytes, but had no effect in cancer cells. Pre-treatment of the cells with the nitrone spin trap, N-tert-butyl-alpha-phenylnitrone or ebselen, which mimics glutathione peroxidase, reduced the extent of DNA damage evoked by amsacrine in all types of cells. The cells exposed to amsacrine and treated with endonuclease III and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results obtained suggest that free radicals may be involved in the formation of DNA lesions induced by amsacrine. The drug can also methylate DNA bases. Our results indicate that the induction of secondary malignancies should be taken into account as diverse side effects of amsacrine. Amifostine may potentate DNA-damage effect of amsacrine in cancer cells and decrease this effect in normal cells and Vitamin C can be considered as a protective agent against DNA damage in normal cells.

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.

Intraoperative diagnosis of sentinel lymph node metastases in breast cancer treatment with one-step nucleic acid amplification assay (OSNA)

Introduction The aim of the study was to evaluate the clinical usefulness of a one-step nucleic acid amplification assay (OSNA) for intraoperative detection of metastases to sentinel lymph nodes (SLNs) in comparison to examination of frozen sections, and to summarize the results of previous studies. Material and methods We enrolled 98 patients aged 58.13 ±10.74 years treated surgically for breast cancer, and 99 biopsies of SLNs were followed by analysis of 105 SLNs. The central 1 mm slice of SLN was used for examination of frozen sections, whereas 2 outer slices of SLNs were analyzed intraoperatively with OSNA. Detection of isolated tumor cells (ITC), micrometastases or macrometastases with OSNA extended surgery to axillary lymph node dissection. Congruency of results was assessed between OSNA and examination of frozen sections. Results One-step nucleic acid amplification assay detected metastases in 29/105 SLNs in surgery of 27/99 breasts, including ITC in 3/29 SLNs, micrometastases in 12/29 and macrometastases in 14/29. One-step nucleic acid amplification assay detected significantly more metastases to SLNs than examination of frozen sections (p < 0.0001). All 8 inconsistent results were positive in OSNA and negative in examination of frozen sections; ITC were identified in 2/8 SLNs and micrometastases in 6/8 SLNs. Sensitivity for OSNA was calculated as 100%, specificity as 90.47%, and κ was 79.16%. Conclusions One-step nucleic acid amplification assay analysis allows rapid and quantitative detection of mRNA CK19 with high specificity and a low rate of false positives. One-step nucleic acid amplification assay is a reliable tool for intraoperative diagnosis of whole SLNs during surgery of breast cancer. One-step nucleic acid amplification assay minimizes the need for secondary surgery and avoids delays in the adjuvant treatment.

Autologous fat transfer to the subcutaneous tissue in the context of breast reconstructive procedures

Autologous fat transfer (AFT) is an appropriate technique for aesthetic rejuvenation of the face, aesthetic enhancement of hands, correction of the facial appearance in various disorders and constitutes a surgical alternative of treatment of numerous breast deformities ranging from distorting posttraumatic scars, post-eczema lesions, post-burn deformities to partial or total breast reconstruction. Our work is aimed to familiarize dermatologists with the technique of harvesting and implanting the aspirate of adipose cells in patients consulted for deformities of the breast. In addition, the review summarizes the most common applications of AFT in the breast reconstructive procedures. In summary, AFT is an oncologically safe, relatively complication-free, minimally invasive surgical technique, which can be used to correct a wide range of deformities, which are commonly seen by dermatologists, in the area of the face, trunk and extremities. The procedure can correct a wide range of breast deformities, from contour or single quadrant deformities up to the state after mastectomy.

Data on Single Nucleotide Polymorphism of DNA Repair Genes and Breast Cancer Risk from Poland

Single nucleotide polymorphisms (SNPs) may modify the risk of cancer. They may be then regarded as potential markers of carcinogenesis. The aim of this study was to analyze the frequency of genotypes and alleles of SNPs in DNA repair genes and to investigate the influence this genetic variation exerts on breast cancer in Polish females. The test group comprised 600 females with breast cancer and 600 healthy controls. Genomic DNA was isolated and the SNPs in DNA repair genes were determined by High-Resolution Melter (HRM) technique. Following polymorphisms were analysed: Arg399Gln (rs25487) of the XRCC1, Gly322Asp (rs4987188) of the hMSH2, Lys751Gln (rs13181) of the XPD, Arg188His (rs3218536) of the XRCC2, P871L (rs799917) of the BRCA1 and N372H (rs144848) of the BRCA2 gene. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for each genotype and allele. Statistically significant correlations were identified between 4 single nucleotide polymorphisms and the breast cancer risk: rs25487 rs4987188 rs13181 and rs799917. The alleles XRCC1-Gln (OR 5.11; 95% CI 5.68–11.64, p < .0001), hMSH2-Asp (OR 4.66; 95% CI 3.90–5.56, p < .0001), XPD-Gln (OR 2.65; 95% CI 2.24–3.14, p < .0001) and BRCA1-L (OR 1.45; 95% CI 1.24–1.71, p < .0001) genes were strongly correlated with this malignancy. No correlation was found between the studied SNPs and tumor grading nor the lymph node status. Further research on larger groups is warranted to determine the influence of above-mentioned genetic variants on breast cancer risk.

Autologous Breast Reconstruction with the Denervated, Extended Latissimus Dorsi Musculocutaneous Flap

Breast reconstruction with latissimus dorsi (LD) musculocutaneous flap allows to recreate a symmetrical breast mound with the contralateral side. Procedure of extended LD flap avoids need for implant also in moderately-sized breasts. The complication rate and donor site morbidity are low and patient satisfaction with this procedure is high. The twitching transposed LD muscle causes significant discomfort for the patients and thus diminishes the satisfaction after the surgery. The muscular contraction deformities can be treated noninvasively with botulin toxin injections or surgically by excising at least 1 cm segment of the most proximal aspect the thoracodorsal nerve. Autologous breast reconstruction with the pedicled, extended, denervated LD flap is a well-established surgical technique with high rate of long-term good aesthetic outcomes with the natural tissue consistency. The routine division of the thoracodorsal nerve will reduce the complication of troublesome breast movements.

SURGICAL METHODS OF THE INFRAMAMMARY FOLD CORRECTION IN BREAST RECONSTRUCTION

Mastectomy for breast cancer requires radical resection of the breast parenchyma, often including the tissue located within the inframammary fold (IMF). Reconstruction of the breast should include correction of IMF in order to achieve its natural-looking, slightly ptotic appearance. In case of two-stage surgery, the procedure is usually done when replacing the expander with prosthesis. Compliation of the IMF corrective surgical methods applicable in breast reconstruction with silicone implants (expander and/or prosthesis). IMF corrective operations are divided according to the scope of the surgery into methods using only stitches (interrupted sutures or continuous suture anchored to the chest wall) or the patient’s own tissues (pedicled adipo-fascial flaps from chest wall or capsular flaps). There are several modern techniques of surgical IMF correction after mastectomy, which allow to obtain naturally ptotic, reconstructed breast. An optimal outcome of the IMF correction requires individually selected method from the surgical armamentarium in relation to the severity of the asymmetry, the quality of the surrounding tissues and the size of the prosthesis. Multidisciplinary planning of the overall surgical treatment by oncological and plastic surgeons for the breast cancer patients is essential to optimize the radicality of the resection and the favourable aesthetic result of reconstruction.