Lymph node metastases displaying lower Ki-67 immunostaining activity than the primary breast cancer

The aim of the study was to verify by Ki-67 immunostaining if any difference exists in the cell proliferating fraction between primary breast tumors (PTs) and matching positive axillary lymph nodes (ALNs). PATIENTS AND METHODS: Immunohistochemistry with the monoclonal antibody against Ki-67 was performed in 160 node-positive breast carcinomas and in their respective lymph node metastases. RESULTS: An increase of Ki-67 immunoreactive cells in ALN compared with that of PTs was observed in 84% of cases (ALN: mean 17%, PTs: mean 8%; p < 0.001), whereas 16% of the cases showed Ki-67 value two to six times lower in the ALNs than in the corresponding PTs (ALN: mean 3.2%, PTs mean 12.5%; p < 0.005). The decrease of Ki-67 positive cells in the ALN was independent from the histotype and the histological grade of the tumor. CONCLUSION: A different cell proliferation fraction between PTs and matching positive ALNs was demonstrated and underlined that the existence of a group of patients with decreased number of Ki-67 immunoreactive cells in lymph node metastases compared with that of the primary tumors could be taken into account in the choice of therapeutic strategy

Different expression of thymidylate synthase in primary tumour and metastatic nodes in breast cancer patients.

BACKGROUND: To date an accurate evaluation of predictive markers in breast cancer is mainly conducted at the primary site, although the main goal of the adjuvant therapy is the control of micrometastases. Adjuvant therapy drugs need a high proliferative cell rate to be effective. The proliferating activity can be evaluated by the Ki-67 marker and even by thymidylate synthase (TS), a cell cycle enzyme present in proliferating cells. In this study the TS levels in primary tumours were compared to those of their metastases. PATIENTS AND METHODS: The TS expression and Ki-67 were evaluated by means of immunohistochemistry in 80 primary breast tumours (PTs) and in their matched axillary metastatic lymph-nodes (ALNs). RESULTS: In 16% of patients, malignant cells of involved nodes showed a lower TS expression than the PTs. In the same group, we also found a lower number of Ki-67 immunoreactive cells in lymph node metastases when compared with primary tumours. CONCLUSION: The group of patients with lower TS and Ki-67 expression in lymph node metastatic cells may be less sensitive to 5-fluorouracil and high dose methotrexate requiring them to be treated with other drug combinations

Thymidylate synthase gene promoter polymorphisms are associated with TSmRNA expressions but not with microsatellite instability in colorectal cancer

Abstract BACKGROUND: Microsatellite instability (MSI) is a biological characteristic of most tumours, being involved in 85% of hereditary non-polyposis colorectal cancer (HNPCC). It also occurs in 10-15% of sporadic colorectal cancers (CRC). HNPCC appears to be caused by germline mutations in mismatch repair (MMR) genes, which are responsible for repairing single base-pair mismatches. MSI is also associated with a better response of CRC to adjuvant chemotherapy with fluoropyrimidines. We investigated any relationship between the MSI status and the TSmRNA expression, the polymorphisms of 5-Fluorouracil (5-FU cellular target, the enzyme thymidylate synthase (TS) and TS expression evaluated by means of immunohistochemistry. MATERIALS AND METHODS: A series of 80 colorectal cancers was evaluated for MSI and polymorphisms in the 3'UTR and the 5'UTR of the TS gene by a PCR assay. TSmRNA was quantified by real-time PCR and the TS expression by immunohistochemical assay. RESULTS: There was no significant association between the polymorphisms in the TS gene and the MSI or between the TSmRNA expression and the MSI status. CRC with a 3R/3R or 2R/3R genotype showed a significantly higher TSmRNA expression than those with the 2R/2R genotype (p = 0.001 and p = 0.028, respectively). Another significant association was found between the TSmRNA expression and the TS immunohistochemical determination (p = < 0.05). No association was found between the polymorphism of the 3'UTR and the TSmRNA expression. CONCLUSION: Our data show that there is no association between MSI status and the polymorphisms in the 3' and 5' UTRs and the TS expression. Tumour samples displaying the 3R/3R or 2R/3R genotype of TS have higher TSmRNA levels than the 2R/2R genotype. Polymorphic variant of the 3'UTR does not influence the TSmRNA level. We found a relationship between the TSmRNA expression, evaluated by real-time PCR, and with the TS level determined by immunohistochemical assay. Thus, genotyping of the 5'UTR and quantification of the TSmRNA expression in human CRC could be considered as predictors for response to SFU-based chemotherapy. The evaluation of the TS expression by means of immunohistochemistry assay remains a safe and reliable assay in CRC

Expression of a specific Thymidylate synthase polimorfic allele in metastatic colorectal patients is regulated by Myeloid Zinc Finger 1.

Thymidylate Synthase (TS) is the target enzyme for fluoropyrimidine anticancer drugs. Its expression is regulated by the number of functional upstream stimulatory factor (USF) E box consensus elements present on its 5’ untranslated region. To date are known different polymorphisms, the first one consisting of 2 or 3 repeat of a 28 bp sequence, a further single nucleotide polymorphism (SNP) consisting in a G>C substitution within the second repeat of 3R (3RG>3RC) and recently it has been identified an additional SNP a G>C substitution at the 12th nucleotide in the first repeat of the 2R allele (2RG>2RC). These polymorphisms can influence TS expression, in particular 3R/3R genotype and the presence of 3RG alleles are associated to an increased transcriptional activity and to higher TS levels. The sequence of promoter region of colorectal cancer (CRC) samples was subjected to an in silico analysis (http://www.cbrc.jp/research/db/TFSEARCH.html) to search for all potential transcription factors binding this region. We found that Myeloid zinc finger 1(MZF-1) binds the analyzed consensus. By the literature it is known that this factor induces invasion and in vivo metastasis in CRC, so we investigated a possible correlation between TS and MZF-1 expression in the same pathological samples. Materials and Methods: we analyzed the distribution of these polimorphisms in a group of 68 healthy Caucasian subjects, in the normal tissue, in primary tumour and in liver metastasis of 13 CRC patients. Tandem repeat length and the presence of SNP was determined by direct sequencing of genomic DNA. TS and MZF 1 expression were analyzed by immunohistochemistry. Results: In healthy population the allele frequency was respectively 2RG(35%) 3RG (44%) 3RC (21%), in colorectal patients while both primary that normal and metastatic samples showed the same genotype: 2RG/3RG. TS and MZF-1 expression were related and gradually increased from normal tissue (negative) to the primary tumour (focally positive) in the metastases (overexpressing). Conclusions: These unexpected results lead to the hypothesis of a genetic selection towards a more aggressive disease and enough suggest that regardless of genotype other factors are involved in regulation of TS expression as MZF 1, therefore the only genetic marker is not a valid predictor of eventual fluoropyrimidine response

Relationship between Thymidylate synthase and p53 and response versus taxane adjuvant chemotherapy for breast carcinoma

Many drugs can be used for adjuvant therapy of breast cancer, including anthracyclines, cyclophosphamide, 5-fluorouracil (5-fU) and, recently, taxanes (TXT) have shown promising results. 5-FU blocks thymidylate synthase (TS) which cross-links p53 mRNA, inhibiting its synthesis. TS overexpression is one of the main mechanisms involved in 5-FU drug resistance. Enough p53 mutations can confer resistance to chemotherapy using anthracyclines and 5-FU, while are associated with improved responses to TXT. The aim of this study was to examine the TS and p53 levels in tumor samples and to compare the efficacy of FEC (5-FU, epirubicin, cyclophosphamide) and TXT chemotherapy in a group of patients with differing TS and p53 status. We examined 84 breast tumor samples using immunohistochemistry. TS and p53 levels were inversely related, and TS and p53 positivity was significantly associated with the failure of FEC treatment and with a good response to TXT therapy (p <0.001). This confirms the predictive role of these two markers, which should be considered when choosing the appropriate adjuvant therapy for breast cancer

Differing expression of metalloprotease and of adhesion molecules in signet-ring cell and intestinal colorectal carcinoma.

Abstract. Background: Pure signet-ring cell colorectal carcinoma (SRCC) is an infrequent and highly malignant histological variant of colorectal cancer (CRC), while it is present as a histological component in colorectal carcinomas more frequently. Materials and Methods: The aim of this work was to widen the knowledge of the biological factors involved in the pathogenesis and aggressiveness of SRCC by the identification and evaluation of possible molecular abnormalities. By means of immunohistochemistry the expression of the proteolytic degradation enzyme matrix metalloprotease (MMP)-1, that is a collagenase specifically degrading collagens I, II, III and of the adhesion proteins Ecadherin, β-catenin and fibronectin which are usually involved in the carcinogenesis of conventional colorectal tumours was investigated. Results: SRCCs showed a significantly greater MMP-1 expression compared to the ordinary intestinal colorectal cancer (ICRC) and a significantly reduced E-cadherin, β-catenin and fibronectin expression. Conclusion: The biological aggressiveness and strong metastatic behaviour of SRCC could be due to high MMP-1 and low expression of the adhesion molecules

Thymidylate Synthase polymorphism and microsatellite instability: association in colorectal cancer

5-Fluorouracil (5FU) is the main drug used for the treatment of colorectal cancer (CRC) and Thymidilate Synthase (TS) is its target enzyme. TS gene has regulatory tandemly repeated sequences in its 5'' and 3''untraslated region (5''-3'' UTR). CRC often shows a kind of genomic instability called Microsatellite Instability (MSI) that is associated with TS levels and survival. Our data show that the genotype 2R/2R (homozygosity for 2 tandem repeat sequences in the 5''UTR) is more frequently associated with MSI+ and lower TS levels. More over we did not find any significant association between the 2R/3R (heterozygosity for 2 and 3 tandem repeat sequences in the 5''UTR) and 3R/3R (homozygosity for 3 tandem repeat sequences in the 5'' UTR) genotypes with the MSI+ and MSI-, while these genotypes were associated with a higher TS expression. As a consequence we can hypothesise that patients bearing CRC with the MSI+, the 2R/2R genotype and with low TS levels could have a better prognosis and they could not be drug resistant