Grażyna Pasz-Walczak

Angiogenesis Markers Quantification in Breast Cancer and Their Correlation with Clinicopathological Prognostic Variables

Tumoural angiogenesis is essential for the growth and spread of breast cancer cells. Therefore the aim of this study was to assess the diagnostic performance of angiogenesis markers in tumours and there reflecting levels in serum of breast cancer patients. Angiogenin, Ang2, fibroblast growth factor basic, intercellular adhesion molecule (ICAM)-1, keratinocyte growth factor (KGF), platelet-derived growth factor-BB, and VEGF-A were measured using a FASTQuant angiogenic growth factor multiplex protein assay. We observed that breast cancer tumours exhibited high levels of PDGF-BB, bFGF and VEGF, and extremely high levels of TIMP-1 and Ang-2, whereas in serum we found significantly higher levels of Ang-2, PDGF-BB, bFGF, ICAM-1 and VEGF in patients with breast cancer compared to the benign breast diseases patients. Moreover, some of these angiogenesis markers evaluated in tumour and serum of breast cancer patients exhibited association with standard clinical parameters, ER status as well as MVD of tumours. Angiogenesis markers play important roles in tumour growth, invasion and metastasis. Our results suggest that analysis of angiogenesis markers in tumour and serum of breast cancer patients using multiplex protein assay can improve diagnosis and prognosis in this diseases.

ABCB1/MDR1 gene polymorphisms as a prognostic factor in colorectal cancer

ObjectiveTo analyse the single-nucleotide polymorphisms (SNPs): ABCB11236C>T, ABCB12677G>T/A, ABCB13435C>T and haplotypes in the ABCB1/MDR1 gene, which could contribute to genetic risk of colorectal cancer (CRC). Disease association between the ABCB1/MDR1 genotype, allele, haplotype frequencies and histological features, such as TNM classification, localization of primary carcinoma, grade of malignancy, histological type of tumour, lymphoid infiltration and vessel invasion were estimated. In this study, the potential role of SNPs of the ABCB1/MDR1 gene as a prognostic marker for CRC was analysed.Materials and methodsTumour specimens of 95 patients with CRC were studied. Using automated sequencing or PCR-RFLP method, DNA for three common SNPs of ABCB1/MDR1 was extracted and analysed. The results of genotyping and haplotype analysis with histopathological features, grading and clinical staging of neoplasms were correlated.ResultsA statistically significant higher frequency of T1236 allele in T1/T2 (89.7%), M0 groups (81.6%) and I/II clinical staging (82.7%) in comparison with T3/T4 (68.2%), M1 groups (47.4%) and III/IV clinical staging (65.1%) was detected. Furthermore, multivariate analysis according to Coxs proportional hazard model indicated that the T1236 allele is a good, independent prognostic factor and the presence of this allele decreases the risk of death in comparison with a group without this allele (HR = 0.26; p = 0.0424). In addition, a statistically significant higher frequency of C3435 allele and significant differences in the C3435 allele distribution in N1/N2 group (91.7% and 62.5%, respectively) than N0 group (71.2% and 44.9%, respectively) was found. Each of the eight possible haplotypes was noted in M0 or I/II group and only seven in M1 or III/IV group. Haplotype T1236-G2677-C3435 only in less advanced CRC subjects (9.6% in I/II and 9.2% in M0 group) was detected. In addition, significant differences in haplotype distributions between M0 or I/II and M1 or III/IV group were found (p = 0.01 and p = 0.05, respectively).ConclusionsThese results suggest association between T1236 allele and T1236-G2677-C3435 haplotype and less advanced CRC, so these genetic markers may play a role as potentially good prognostic factors. Differences in haplotype distributions and degree of clinical staging may suggest that some other potential SNPs, especially in regulatory region of ABCB1/MDR1 gene, may influence P-glycoprotein function and CRC progression.

P21 (WAF1) expression in colorectal cancer: correlation with P53 and cyclin D1 expression, clinicopathological parameters and prognosis.

P21 (WAF1), P53 and cyclin D1 belong to the cell cycle-regulating family of proteins, and the loss of activity of proteins P53 and P21 (WAF1) seems to be one of the most important regulatory mechanisms of carcinogenesis in colorectal cancer. The purpose of this study was to assess the relationship between P21 (WAF1), P53 and cyclin D1 immunoreactivity, and to evaluate the prognostic significance of their expression. Tissue sections from 122 paraffin-embedded colorectal carcinomas were immunostained with monoclonal antibodies. Positivity for P21 (WAF1) was found in 48 cases (39%), positivity for P53 in 96 cases (70%) and positivity for cyclin D1 in all the cases (100%). Statistical analyses revealed a statistically significant inverse correlation between P53 and P21 (WAF1)-immunopositivity and between P21 (WAF1)-immunopositivity and the degree of cyclin D1-immunopositivity, as well as an inverse correlation between P21 (WAF1) expression and clinical stage. In univariate analysis, down-regulation of P21 (WAFI) expression was associated with poor prognosis, but multivariate analysis did not confirm its independent prognostic significance. In Coxs analysis only regional lymph node invasion and hepatic metastases were proven as independent prognostic parameters. Our investigation results suggest that in colorectal cancer, the induction of P21 (WAF1) may occur mostly in a P53-dependent pathway. P21 (WAF1), as the main cyclin-dependent kinase (CDK)-inhibitor, may also inhibit the activity of cyclins such as cyclin D1.

WWOX expression in colorectal cancer—a real-time quantitative RT-PCR study

The WWOX gene is a tumour suppressor gene affected in various types of malignancies. Numerous studies showed either loss or reduction of the WWOX expression in variety of tumours, including breast, ovary, liver, stomach and pancreas. Recent study demonstrated that breast cancer patients exhibiting higher WWOX expression showed significantly longer disease-free survival in contrast to the group with lower relative WWOX level. This work was undertaken to show whether similar phenomena take place in colon tumours and cell lines. To assess the correlation of WWOX gene expression with prognosis and cancer recurrence in 99 colorectal cancer patients, we performed qRT-PCR analysis. We also performed analysis of WWOX promoter methylation status using MethylScreen method and analysis of loss of heterozygosity (LOH) status at two WWOX-related loci, previously shown to be frequently deleted in various types of tumours. A significantly better disease-free survival was observed among patients with tumours exhibiting high level of WWOX (hazard ratio = 0.39; p = 0.0452; Mantel–Cox log-rank test), but in multivariate analysis it was not an independent prognostic factor. We also found that although in colorectal cancer WWOX expression varies among patients and correlates with DFS, the exact mode of decrease in this type of tumour was not found. We failed to find the evidence of LOH in WWOX region, or hypermethylation in promoter regions of this gene. Although we provide the evidence for tumour-suppressive role of WWOX gene expression in colon, we were unable to identify the molecular mechanism responsible for this.

HMGI(Y) gene expression in colorectal cancer: comparison with some histological typing, grading, and clinical staging.

We investigated HMGI(Y) gene expression in 81 pairs of frozen samples obtained from colorectal carcinomas and adjacent normal colorectal mucosas and in four samples from colorectal mucosa from patients without neoplastic diseases. In this group, HMGI(Y)-positive/-negative expression was compared with some histological features, grading, and clinical staging of neoplasms investigated to assess its potential role as a prognostic marker for colorectal cancer. Expression of HMGI(Y) gene was found in 51 of 81 cases of colorectal cancers, while, in normal mucosa, expression of this gene was not observed. HMGI(Y) gene expression was associated with more advanced tumors (T3, T4) and metastases to lymph nodes (N1, N2). The most interesting finding was that expression of this gene correlated with distant metastases. HMGI(Y) gene expression was detected in all cases classified as M1 (n = 19, p = 0.0008). We did not find any association between age, gender, tumor localization, histological type and this gene expression.

cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

BackgroundRecently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers.MethodsTo this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry.ResultsWe found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations.ConclusionIn terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis.

The first description of sentinel node biopsy in a patient with amelanotic melanoma of the glans penis

We present the first description of sentinel node biopsy in a patient with amelanotic melanoma of the glans penis. The patient underwent partial amputation of the penis due to tumor of the glans. Pathologic examination of the postoperative specimen revealed the presence of a very rare malignancy--amelanotic melanoma. Sentinel node biopsy, with the use of the combined radiotracer/blue dye technique, was performed. Preoperative lymphoscintigraphy was performed the day before surgery. During surgery, blue dye mapping and intraoperative detection of gamma radiation were used. Two sentinel nodes were identified in the left inguinal region and one sentinel node in the right inguinal region. All sentinel nodes were an intense violet color; in each case, the level of radiation in the sentinel node was almost 20 times higher than the level of radiation in the node bed. Routine hematoxylin and eosin staining and immunohistochemistry (HMB-45) revealed the presence of micrometastasis in one of the sentinel nodes harvested from the left inguinal region. Consequently, left inguinal, iliac and obturatory lymphadenectomies were performed. The final pathologic examination revealed the presence of one metastasis (diameter, 2 mm) in one of the resected non-sentinel nodes. No relapse has been observed during 18 months of follow-up.

Prediction of survival for patients with advanced colorectal cancer using 1H High‐resolution magic angle spinning nuclear MR spectroscopy

To evaluate whether the metabolic profiles of colorectal cancer specimens can be used for prediction of survival.

Isolated metastasis to the foot as an extremely rare presenting feature of primary endometrial cancer.

Endometrial cancer is the most common invasive cancer of the female genital tract, with an increasing incidence rate. Abnormal uterine bleeding is the presenting symptom in 75-90% of cases [1, 2]. The majority of patients with endometrial cancer are diagnosed with no evidence of extra-uterine spread (70-80% stage I), which gives patients better prognosis [1, 3]. In more advanced disease the sites commonly affected outside the uterus are pelvic and para-aortic lymph nodes and the ovaries [4]. Similarly to uterine sarcomas, distant metastases in advanced or recurrent endometrial cancer most commonly involve the lungs, liver, central nervous system and skin [4–6]. Metastases to bones have been described in 2-15% of patients with metastatic disease, and the most common site of osseous metastases are vertebrae, with pelvic bones, ribs and sternum [3, 7]. Isolated metastases to bone extremities are extremely rare, and thought to result from the haematological spread of cancer cells [5]. The review of the Medline database by searching the items endometrial cancer and metastases to extremities showed twenty such cases, with only nine cases when metastatic tumour of the foot was the first manifestation of endometrial cancer (Table I) [3, 5, 7–13]. Table I Isolated metastasis to the bones of the extremities as the first manifestation of endometrial cancer – review of the literature A 74-year-old female suffered from pain and swelling of the right foot from October 2006. In the X-ray picture and bone scintigraphy, suspected lesion of the right calcaneus, talus and metatarsal bones was detected (Figure 1). Afterwards it was histologically verified in the biopsy as metastatic cancer (Figure 2). The patient had no vaginal bleeding or other gynaecological symptoms, but subsequent computed tomography scans showed an enlarged uterus. Uterine curettage confirmed the diagnosis of endometrial cancer. The patient was treated with total abdominal hysterectomy, bilateral salpingo-oophorectomy without pelvic lymph node dissection in December 2006. The histological diagnosis was a moderate differentiated (G2) endometrioid endometrial cancer invading the outer half of the myometrium (> 1/2) with invasion of cervical stroma. The fallopian tubes and the ovaries showed no signs of metastases. No evidence of macroscopic abdominopelvic metastases was found at surgery. Her disease was classified as clinical stage IVB according to FIGO 2009 staging, but in the TNM classification it was pT2 Nx M2. Figure 1 Radiograph of the right foot demonstrating metastases of the endometrial cancer Figure 2 Metastatic endometrial cancer cells in the fine needle aspiration of the tumour of the foot (H + E, 200× magnification) In January 2007 the patient was admitted to the department of palliative radiotherapy. She received irradiation by Co 60 until a total dose of 20 Gy to the tumour of the right foot, with complete resolution of symptoms. The patient was discharged from hospital in March 2007. During the observation from surgery in December 2006 until June 2010 no other metastases were detected. The patient remains alive and asymptomatic 43 months after the diagnosis of metastatic endometrial cancer to the foot. Our report presented in the previous section has three main peculiar features: 1) it demonstrates endometrial cancer that presented with osseous metastasis, which is a rare occurrence, 2) the metastatic tumour was located in the foot, which is extremely rare, 3) the bone lesion was a single bone metastasis, which is unusual in such cases. Additionally, the case in which isolated metastatic tumour of the extremity was the first manifestation of endometrial cancer is the tenth such case reported in the literature (Table I), and the second one located in the calcaneus and involving talus and metatarsal bones [8]. Despite the rarity of metastases to the upper and lower extremities, there is a need to have a high index of suspicion for metastasis in patients with a history of endometrial cancer who present with swelling or bony tenderness. The initial diagnosis can be challenging, as the symptoms of pain and swelling are often attributed to other more common benign conditions such as soft tissue inflammation, trauma, arthritis, and osteomyelitis [7]. It is important to consider bone metastasis as a possible diagnosis also in patients without history of cancer, but with osseous pain not responding to conservative treatment [5, 9–13]. Appropriate imaging may include plain X-ray picture and radionuclide bone scans [3]. Technetium diphosphonate bone scans can be positive up to 18 months before a lesion is detectable on plain X-ray [14]. Therefore, a biopsy should be performed in patients with suspected lesions, and who demonstrate evidence of bony destruction [4]. The treatment strategy in patients with confirmed isolated metastatic lesion in the bone extremity still remains a topic of controversy, because of the few descriptions available in the literature and the different bone sites involved. For these reasons, a common suitable treatment regimen cannot be established and treatment should be tailor suited to each patient. The treatment of irradiation with or without surgery, hormone therapy and chemotherapy is reported as effective in most cases and may be curative [4, 5, 7, 9, 11, 12]. The main goal of treatment should be to eliminate or palliate pain and prolong survival.

TRAIL protein expression in breast cancer cells correlates with nuclear grade

Introduction TRAIL protein may serve as an escape mechanism for cancer cells from the immune response. The aim of the study was to assess whether the presence of TRAIL protein correlates with unfavourable prognostic factors in breast carcinoma. Material and methods The study group was composed of breast cancer patients treated surgically in the Department of Surgical Oncology, Medical University of Lodz, Poland, from January to December 2003. Inclusion criteria for the study were fulfilled by 117 women. The immunohistochemical study of TRAIL protein expression was performed in 118 breast carcinomas diagnosed in the study group. TRAIL protein expression was correlated with other variables: tumour size, lymph node status, grade, histological type of carcinoma, oestrogen and progesterone receptor status, HER2 expression, presence of lymphovascular invasion and age of the patient. Results Expression of TRAIL protein was present in 73% of breast carcinomas. The percentage of TRAIL-expressing breast carcinoma cells correlated with the nuclear grade (τ = 0.26, p < 0.05; Tau Kendall test). The intensity of TRAIL expression (intensity of staining) in breast carcinoma cells correlated with the nuclear grade (τ = 0.15, p < 0.05; Tau Kendall test). TRAIL expression in breast carcinoma did not correlate with other studied variables. Conclusions Our analysis revealed that expression of TRAIL protein in breast carcinoma cells correlates with nuclear grade of carcinoma.