Prognostic impact of mismatch repair genes germline defects in colorectal cancer patients: are all mutations equal?

Background

Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome, caused by germline mutations in MisMatch Repair (MMR) genes, particularly in MLH1, MSH2 and MSH6. Patients with LS seem to have a more favourable prognosis than those with sporadic CRC, although the prognostic impact of different mutation types is unknown.Aim of our study is to compare survival outcomes of different types of MMR mutations in patients with LS-related CRC.

Methods

302 CRC patients were prospectively selected on the basis of Amsterdam or Revised Bethesda criteria to undergo genetic testing: direct sequencing of DNA and MLPA were used to examine the entire MLH1, MSH2 and MSH6 coding sequence.Patients were classified as mutation-positive or negative according to the genetic testing result.

Results

A deleterious MMR mutation was found in 38/302 patients. Median overall survival (OS) was significantly higher in mutation-positive vs mutation-negative patients (102.6 vs 77.7 months, HR:0.63, 95%CI:0.46–0.89, p = 0.0083). Different types of mutation were significantly related with OS: missense or splicing-site mutations were associated with better OS compared with rearrangement, frameshift or non-sense mutations (132.5 vs 82.5 months, HR:0.46, 95%CI:0.16–0.82, p = 0.0153).

Conclusions

Our study confirms improved OS for LS-patients compared with mutation-negative CRC patients. In addition, not all mutations could be considered equal: the better prognosis in CRC patients with MMR pathogenic missense or splicing site mutation could be due to different functional activity of the encoded MMR protein. This matter should be investigated by use of functional assays in the future.     

Cancer risk in MLH1, MSH2 and MSH6 mutation carriers; different risk profiles may influence clinical management

Background

Lynch syndrome (LS) is associated with a high risk for colorectal cancer (CRC) and extracolonic malignancies, such as endometrial carcinoma (EC). The risk is dependent of the affected mismatch repair gene. The aim of the present study was to calculate the cumulative risk of LS related cancers in proven MLH1, MSH2 and MSH6 mutation carriers.

Methods

The studypopulation consisted out of 67 proven LS families. Clinical information including mutation status and tumour diagnosis was collected. Cumulative risks were calculated and compared using Kaplan Meier survival analysis.

Results

MSH6 mutation carriers, both males and females had the lowest risk for developing CRC at age 70 years, 54% and 30% respectively and the age of onset was delayed by 3-5 years in males. With respect to endometrial carcinoma, female MSH6 mutation carriers had the highest risk at age 70 years (61%) compared to MLH1 (25%) and MSH2 (49%). Also, the age of EC onset was delayed by 5-10 years in comparison with MLH1 and MSH2.

Conclusions

Although the cumulative lifetime risk of LS related cancer is similar, MLH1, MSH2 and MSH6 mutations seem to cause distinguishable cancer risk profiles. Female MSH6 mutation carriers have a lower CRC risk and a higher risk for developing endometrial carcinoma. As a consequence, surveillance colonoscopy starting at age 30 years instead of 20-25 years is more suitable. Also, prophylactic hysterectomy may be more indicated in female MSH6 mutation carriers compared to MLH1 and MSH2 mutation carriers.     

Target gene mutational pattern in Lynch syndrome colorectal carcinomas according to tumour location and germline mutation

Background:

We previously reported that the target genes in sporadic mismatch repair (MMR)-deficient colorectal carcinomas (CRCs) in the distal colon differ from those occurring elsewhere in the colon. This study aimed to compare the target gene mutational pattern in microsatellite instability (MSI) CRC from Lynch syndrome patients stratified by tumour location and germline mutation, as well as with that of sporadic disease.

Methods:

A series of CRC from Lynch syndrome patients was analysed for MSI in genes predicted to be selective MSI targets and known to be involved in several pathways of colorectal carcinogenesis.

Results:

The most frequently mutated genes belong to the TGF-β superfamily pathway, namely ACVR2A and TGFBR2. A significantly higher frequency of target gene mutations was observed in CRC from patients with germline mutations in MLH1 or MSH2 when compared with MSH6. Mutations in microsatellite sequences (A)7 of BMPR2 and (A)8 of MSH3 were significantly more frequent in the distal CRC. Additionally, we observed differences in MSH3 and TGFBR2 mutational frequency between Lynch syndrome and sporadic MSI CRC regarding tumour location.

Conclusions:

Our results indicate that the pattern of genetic changes differs in CRC depending on tumour location and between Lynch syndrome and sporadic MSI CRC, suggesting that carcinogenesis can occur by different pathways even if driven by generalised MSI.     

Synchronous gynecologic malignancy and preliminary results of Lynch syndrome

Objective

Lynch syndrome is a hereditary cancer syndrome that increases the risks of colorectal and gynecologic malignancies such as endometrial and ovarian cancer. Studies have shown that mutations in mismatch repair genes (MSH2, MSH6, and MLH1) are associated with Lynch syndrome. The aim of our study was to estimate the value of MSH2, MSH6, and MLH1 immunohistochemistry based on family history in a Korean sample.

Methods

Thirty six women with synchronous gynecologic tumors of endometrial and ovarian cancer were identified among patients being treated at our institution. Among them, 32 patients had tumor blocks (total 62 slides) available for analysis. According to a diagnostic algorithm, we performed immunohistochemistry analyses. Staining was scored based on intensity and proportion (negative or 0: intensity undetectable or minimal, proportion <5%; weak or 1+: intensity mild, proportion 5-30%; strong or 2+: intensity moderate to marked, proportion 30-99%).

Results

Among 32 eligible patients, 9 (28%) had a family history of cancer. Six patients (19%) were negative for MLH1; among them, four (4/6) were negative at both sites. Nine patients (28%) were negative for MSH2 or MSH6 at both sites or negative for both MSH2 and MSH6. Among these three patients showed negative staining for both sites. The three patients showing negative staining for MLH1, MSH2, and MSH6 at both sites with family history were considered to be the screening positive groups of Lynch syndrome.

Conclusion

In this study, the frequency of Lynch syndrome associated immunohistochemical staining (MLH1, MSH2, and MSH6) group was estimated as 9% (3/32) among Korean women with synchronous gynecologic tumors.     

Genotyping of BRCA1, BRCA2, p53, CDKN2A, MLH1 and MSH2 genes in a male patient with secondary breast cancer

Background

Some tumour suppressor genes (BRCA2) and mismatch repair genes (MSH2, MLH1) are correlated with an increased risk for male breast cancer.

Case report

Our patient developed secondary breast cancer after the treatment for Hodgkin’s disease in childhood. DNA was isolated from the patients’ blood and screened for mutations, polymorphisms and variants in BRCA1, BRCA2, p53, CDKN2A, MLH1 and MSH2 genes. We found no mutations but common polymorphisms, and three variants in mismatch repair genes.

Conclusions

Nucleotide variants c.2006-6T>C and p.G322D in MSH2 might be correlated with male breast cancer.     

Body mass index in early adulthood and colorectal cancer risk for carriers and non-carriers of germline mutations in DNA mismatch repair genes

Background:

Carriers of germline mutations in DNA mismatch repair (MMR) genes have a high risk of colorectal cancer (CRC), but the modifiers of this risk are not well established. We estimated an association between body mass index (BMI) in early adulthood and subsequent risk of CRC for carriers and, as a comparison, estimated the association for non-carriers.

Methods:

A weighted Cox regression was used to analyse height and weight at 20 years reported by 1324 carriers of MMR gene mutations (500 MLH1, 648 MSH2, 117 MSH6 and 59 PMS2) and 1219 non-carriers from the Colon Cancer Family Registry.

Results:

During 122 304 person-years of observation, we observed diagnoses of CRC for 659 carriers (50%) and 36 non-carriers (3%). For carriers, the risk of CRC increased by 30% for each 5 kg m^–2 increment in BMI in early adulthood (hazard ratio, HR: 1.30; 95% confidence interval, CI: 1.08–1.58; P=0.01), and increased by 64% for non-carriers (HR: 1.64; 95% CI: 1.02–2.64; P=0.04) after adjusting for sex, country, cigarette smoking and alcohol drinking (and the MMR gene that was mutated in carriers). The difference in HRs for carriers and non-carriers was not statistically significant (P=0.50). For MLH1 and PMS2 (MutLα heterodimer) mutation carriers combined, the corresponding increase was 36% (HR: 1.36; 95% CI: 1.05–1.76; P=0.02). For MSH2 and MSH6 (MutSα heterodimer) mutation carriers combined, the HR was 1.26 (95% CI: 0.96–1.65; P=0.09). There was no significant difference between the HRs for MutLα and MutSα heterodimer carriers (P=0.56).

Conclusion:

Body mass index in early adulthood is positively associated with risk of CRC for MMR gene mutation carriers and non-carriers.     

DNA mismatch repair gene polymorphisms affect survival in pancreatic cancer

Purpose.

DNA mismatch repair (MMR) maintains genomic stability and mediates cellular response to DNA damage. We aim to demonstrate whether MMR genetic variants affect overall survival (OS) in pancreatic cancer.

Materials and Methods.

Using the Sequenom method in genomic DNA, we retrospectively genotyped 102 single-nucleotide polymorphisms (SNPs) of 13 MMR genes from 706 patients with pancreatic adenocarcinoma seen at The University of Texas MD Anderson Cancer Center. Association between genotype and OS was evaluated using multivariable Cox proportional hazard regression models.

Results.

At a false discovery rate of 1% (p ≤ .0015), 15 SNPs of EXO1, MLH1, MSH2, MSH3, MSH6, PMS2, PMS2L3, TP73, and TREX1 in patients with localized disease (n = 333) and 6 SNPs of MSH3, MSH6, and TP73 in patients with locally advanced or metastatic disease (n = 373) were significantly associated with OS. In multivariable Cox proportional hazard regression models, SNPs of EXO1, MSH2, MSH3, PMS2L3, and TP73 in patients with localized disease, MSH2, MSH3, MSH6, and TP73 in patients with locally advanced or metastatic disease, and EXO1, MGMT, MSH2, MSH3, MSH6, PMS2L3, and TP73 in all patients remained significant predictors for OS (p ≤ .0015) after adjusting for all clinical predictors and all SNPs with p ≤ .0015 in single-locus analysis. Sixteen haplotypes of EXO1, MLH1, MSH2, MSH3, MSH6, PMS2, PMS2L3, RECQL, TP73, and TREX1 significantly correlated with OS in all patients (p ≤ .001).

Conclusion.

MMR gene variants may have potential value as prognostic markers for OS in pancreatic cancer patients.     

Proficiency of DNA repair genes and microsatellite instability in operated colorectal cancer patients with clinical suspicion of lynch syndrome

Background

Lynch syndrome (LS) diagnosis is underestimated, and most of the patients remain undetected after colorectal resections. The study aims to assess the frequency of LS in patients undergoing surgical treatment for colorectal cancer (CRC).

Methods

A total of 458 CRC patients were operated from January 2005 to December 2008. Positive CRC family history (FH) was present in 118 (25.8%) patients. Histologic sections were reviewed for microsatellite instability (MSI) criteria (Bethesda guidelines), immunohistochemical (IHC) analysis for MLH1, MSH2, MSH6, PMS2 proteins, through the avidin-biotin-peroxidase complex, MSI (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) and BRAF somatic mutation.

Results

Of the 118 patients with FH, 61 (51.69%) met at least one of the revised Bethesda criteria. IHC was abnormal in 8 (13.1%) and MSI in 12 patients (20%). BRAF was negative in all cases. MSI histopathological included: intratumoral lymphocytes (47.5%), expansive tumors (29.5%) mucinous component (27.8%) and Crohn’s like reaction in (14.7%). There was an association between the revised Bethesda criteria with: sex, mucinous histology and Crohn’s like reaction; MSI and IHC with PMS2 and MLH1. Revised Bethesda criteria 4 had 10.6 increased chances to display positive MSI. We have proposed a score to contribute as a practical tool in the diagnosis of LS.

Conclusions

The frequence of LS in resected CRC patients was 2.6%. The criterion 4 Revised Bethesda was associated more strongly with the presence of MSI.     

Expression of young HERV-H loci in the course of colorectal carcinoma and correlation with molecular subtypes

Background

Expression of the human endogenous retrovirus (HERV)-H family has been associated with colorectal carcinomas (CRC), yet no individual HERV-H locus expression has been thoroughly correlated with clinical data.Here, we characterized HERV-H reactivations in clinical CRC samples by integrating expression profiles, molecular patterns and clinical data. Expression of relevant HERV-H sequences was analyzed by qRT-PCR on two well-defined clinical cohorts (n = 139 pairs of tumor and adjacent normal colon tissue) including samples from adenomas (n = 21) and liver metastases (n = 16). Correlations with clinical and molecular data were assessed.

Results

CRC specific HERV-H sequences were validated and found expressed throughout CRC disease progression. Correlations between HERV-H expression and lymph node invasion of tumor cells (p = 0.0006) as well as microsatellite instable tumors (p < 0.0001) were established. No association with regard to age, tumor localization, grading or common mutations became apparent. Interestingly, CRC expressed elements belonged to specific young HERV-H subfamilies and their 5′ LTR often presented active histone marks.

Conclusion

These results suggest a functional role of HERV-H sequences in colorectal carcinogenesis. The pronounced connection with microsatellite instability warrants a more detailed investigation. Thus, HERV-H sequences in addition to tumor specific mutations may represent clinically relevant, truly CRC specific markers for diagnostic, prognostic and therapeutic purposes.     

Clinicopathological significance of indoleamine 2,3-dioxygenase 1 expression in colorectal cancer

Background:

Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolising enzyme that induces immune tolerance by modulating T-cell responses. Carcinomas may create an immunosuppressive state via IDO1 expression. Here we examined a possible contribution of IDO1 on this phenomenon and investigated whether IDO1 has prognostic value in colorectal cancer (CRC).

Methods:

IDO1 expression was investigated by quantitative PCR and western blotting in three colon cancer cell lines, in basal state and after interferon (IFN)-γ stimulation. Semi-quantitative immunohistochemistry was used to evaluate IDO1 expression in 265 pT1-4N0-2Mx-staged CRCs. Results were related to clinical variables and correlated with amounts of CD3⁺ and CD8⁺ T lymphocytes, which were quantitatively evaluated using image analysis.

Results:

In vitro expression of IDO1 depended on IFN-γ stimulation. Higher IDO1 expression at the tumour invasion front was an independent adverse prognostic factor in pT1-4N1Mx-staged CRC. It was associated with overall survival (P=0.001) and with metachronous metastases (P=0.018). IDO1 expression was not associated with the presence of CD3⁺ or CD8⁺ T lymphocytes.

Conclusion:

Higher IDO1 expression at the tumour invasion front is involved in CRC progression and correlates with impaired clinical outcome, suggesting that IDO1 is an independent prognostic indicator for CRC.     

Factors that contribute to faecal cyclooxygenase-2 mRNA expression in subjects with colorectal cancer

Background:

We previously reported that a faecal cyclooxygenase-2 (COX-2) mRNA assay was useful for identifying colorectal cancer (CRC). This study sought to investigate the factors that contribute to faecal COX-2 mRNA expression in subjects with CRC.

Methods:

The study cohort comprised 78 patients with CRC and 36 control subjects. The expressions of COX-2, β-2-microglobulin (B2M), carcinoembryonic antigen (CEA), E-cadherin (E-cad), and CD45 mRNA in faeces and COX-2 mRNA expression in tissue were determined by quantitative real-time RT–PCR.

Results:

The level of faecal expression of COX-2 mRNA in CRC was significantly higher than that in controls. A significant correlation was found between faecal COX-2 mRNA expression and faecal B2M, CEA, E-cad, or CD45 mRNAs, markers of exfoliated total cells, colonocytes, and leukocytes, respectively. A significant correlation was found between the expression of COX-2 mRNA in faeces and tumour surface area, COX-2 mRNA expression in primary tumour. There was no difference in faecal COX-2 mRNA expression between proximal CRC and distal CRC.

Conclusion:

COX-2 mRNA expression in faeces seems to originate from tumour lesion and to be affected by factors such as the number of exfoliated cells, exfoliation of inflammatory cells, COX-2 mRNA expression in tumour, and tumour size.     

DNA mismatch repair-related protein loss as a prognostic factor in endometrial cancers

Objective

Recent investigations have revealed DNA mismatch repair (MMR) gene mutations are closely related with carcinogenesis of endometrial cancer; however the impact of MMR protein expression on prognosis is not determined. Correlations between MMR-related protein expression and clinicopathological factors of endometrial cancers are analyzed in the present study.

Methods

A total of 191 endometrial cancer tissues treated between 1990 and 2007 in our hospital were enrolled. Immunoreactions for MSH2, MLH1, MSH6, and PMS2 on tissue microarray specimens and clinicopathological features were analyzed retrospectively.

Results

Seventy-six cases (40%) had at least one immunohistochemical alteration in MMR proteins (MMR-deficient group). There were statistically significant differences of histology, International Federation of Gynecology and Obstetrics (FIGO) stage, and histological grade between MMR-deficient group and the other cases (MMR-retained group). Response rate of first-line chemotherapy in evaluable cases was slightly higher in MMR-deficient cases (67% vs. 44%, p=0.34). MMR-deficient cases had significantly better progression-free and overall survival (OS) compared with MMR-retained cases. Multivariate analysis revealed MMR status was an independent prognostic factor for OS in endometrial cancers.

Conclusion

MMR-related proteins expression was identified as an independent prognostic factor for OS, suggesting that MMR was a key biomarker for further investigations of endometrial cancers.     

Detection of DNA mismatch repair proteins in fresh human blood lymphocytes - towards a novel method for hereditary non-polyposis colorectal cancer (Lynch syndrome) screening

Background

A broad population-based assay to detect individuals with Lynch Syndrome (LS) before they develop cancer would save lives and healthcare dollars via cancer prevention. LS is caused by a germline mutation in a DNA mismatch repair (MMR) gene, especially protein truncation-causing mutations involving MSH2 or MLH1. We showed that immortalized lymphocytes from LS patients have reduced levels of full-length MLH1 or MSH2 proteins. Thus, it may be feasible to identify LS patients in a broad population-based assay by detecting reduced levels of MMR proteins in lymphocytes.

Methods

Accordingly, we determined whether MSH2 and MLH1 proteins can also be detected in fresh lymphocytes. A quantitative western blot assay was developed using two commercially available monoclonal antibodies that we showed are specific for detecting full-length MLH1 or MSH2. To directly determine the ratio of the levels of these MMR proteins, we used both antibodies in a multiplex-type western blot.

Results

MLH1 and MSH2 levels were often not detectable in fresh lymphocytes, but were readily detectable if fresh lymphocytes were first stimulated with PHA. In fresh lymphocytes from normal controls, the MMR ratio was ~1.0. In fresh lymphocytes from patients (N > 50) at elevated risk for LS, there was a bimodal distribution of MMR ratios (range: 0.3-1.0).

Conclusions

Finding that MMR protein levels can be measured in fresh lymphocytes, and given that cells with heterozygote MMR mutations have reduced levels of full-length MMR proteins, suggests that our immunoassay could be advanced to a quantitative test for screening populations at high risk for LS.     

Early-onset breast cancer in a Lebanese family with Lynch syndrome due to MSH2 gene mutation

Background

There are still controversies about the integration of breast cancer as a part of the disease spectrum in Lynch syndrome.

Methods

A regular follow-up of a Lebanese pedigree with Lynch syndrome due to a point mutation of MSH2 gene at the splice donor site of intron 3 started in 1996.

Results

A 26-year-old pregnant woman, mutation carrier, developed an aggressive breast cancer, refractory to standard chemotherapy regimens. The microsatellite analysis of the tumor showed an unstable pattern for markers BAT25 and BAT26. The immunohistochemical staining was negative for MSH2 and MSH6 and normal for MLH1 and PMS6 enzymes.

Conclusion

The segregation of the mutation with the disease phenotype and these results suggest that MSH2 inactivation may be involved in the accelerated breast carcinogenesis and might be considered in the cancer screening program.     

Design and validation of an oligonucleotide microarray for the detection of genomic rearrangements associated with common hereditary cancer syndromes

Background

Conventional Sanger sequencing reliably detects the majority of genetic mutations associated with hereditary cancers, such as single-base changes and small insertions or deletions. However, detection of genomic rearrangements, such as large deletions and duplications, requires special technologies. Microarray analysis has been successfully used to detect large rearrangements (LRs) in genetic disorders.

Methods

We designed and validated a high-density oligonucleotide microarray for the detection of gene-level genomic rearrangements associated with hereditary breast and ovarian cancer (HBOC), Lynch, and polyposis syndromes. The microarray consisted of probes corresponding to the exons and flanking introns of BRCA1 and BRCA2 (≈1,700) and Lynch syndrome/polyposis genes MLH1, MSH2, MSH6, APC, MUTYH, and EPCAM (≈2,200). We validated the microarray with 990 samples previously tested for LR status in BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, MUTYH, or EPCAM. Microarray results were 100% concordant with previous results in the validation studies. Subsequently, clinical microarray analysis was performed on samples from patients with a high likelihood of HBOC mutations (13,124), Lynch syndrome mutations (18,498), and polyposis syndrome mutations (2,739) to determine the proportion of LRs.

Results

Our results demonstrate that LRs constitute a substantial proportion of genetic mutations found in patients referred for hereditary cancer genetic testing.

Conclusion

The use of microarray comparative genomic hybridization (CGH) for the detection of LRs is well-suited as an adjunct technology for both single syndrome (by Sanger sequencing analysis) and extended gene panel testing by next generation sequencing analysis. Genetic testing strategies using microarray analysis will help identify additional patients carrying LRs, who are predisposed to various hereditary cancers.     

Digital pattern recognition-based image analysis quantifies immune infiltrates in distinct tissue regions of colorectal cancer and identifies a metastatic phenotype

Background:

Several studies in colorectal cancer (CRC) indicate a relationship between tumour immune infiltrates and clinical outcome. We tested the utility of a digital pattern recognition-based image analysis (DPRIA) system to segregate tissue regions and facilitate automated quantification of immune infiltrates in CRC.

Methods:

Primary CRC with matched hepatic metastatic (n=7), primary CRC alone (n=18) and primary CRC with matched normal (n=40) tissue were analysed immunohistochemically. Genie pattern recognition software was used to segregate distinct tissue regions in combination with image analysis algorithms to quantify immune cells.

Results:

Immune infiltrates were observed predominately at the invasive margin. Quantitative image analysis revealed a significant increase in the prevalence of Foxp3 (P<0.0001), CD8 (P<0.0001), CD68 (<0.0001) and CD31 (<0.0001) positive cells in the stroma of primary and metastatic CRC, compared with tumour cell mass. A direct comparison between non-metastatic primary CRC (MET−) and primary CRC that resulted in metastasis (MET+) showed an immunosuppressive phenotype, with elevated Foxp3 (P<0.05) and reduced numbers of CD8 (P<0.05) cells in the stroma of MET+ compared with MET− samples.

Conclusion:

By combining immunohistochemistry with DPRIA, we demonstrate a potential metastatic phenotype in CRC. Our study accelerates wider acceptance and use of automated systems as an adjunct to traditional histopathological techniques.     

Cytosine-based nucleoside analogs are selectively lethal to DNA mismatch repair-deficient tumour cells by enhancing levels of intracellular oxidative stress

Background:

DNA mismatch repair deficiency is present in a significant proportion of a number of solid tumours and is associated with distinct clinical behaviour.

Methods:

To identify the therapeutic agents that might show selectivity for mismatch repair-deficient tumour cells, we screened a pair of isogenic MLH1-deficient and MLH1-proficient tumour cell lines with a library of clinically used drugs. To test the generality of hits in the screen, selective agents were retested in cells deficient in the MSH2 mismatch repair gene.

Results:

We identified cytarabine and other related cytosine-based nucleoside analogues as being selectively toxic to MLH1 and MSH2-deficient tumour cells. The selective cytotoxicity we observed was likely caused by increased levels of cellular oxidative stress, as it could be abrogated by antioxidants.

Conclusion:

We propose that cytarabine-based chemotherapy regimens may represent a tumour-selective treatment strategy for mismatch repair-deficient cancers.     

Berberine may rescue Fusobacterium nucleatum-induced colorectal tumorigenesis by modulating the tumor microenvironment

Background

Accumulating evidence links colorectal cancer (CRC) with the intestinal microbiota. However, the disturbance of intestinal microbiota and the role of Fusobacterium nucleatum during the colorectal adenoma-carcinoma sequence have not yet been evaluated.

Methods

454 FLX pyrosequencing was used to evaluate the disturbance of intestinal microbiota during the adenoma-carcinoma sequence pathway of CRC. Intestinal microbiota and mucosa tumor-immune cytokines were detected in mice after introducing 1,2-dimethylhydrazine (DMH), F. nucleatum or Berberine (BBR), using pyrosequencing and Bio-Plex Pro™ cytokine assays, respectively. Protein expressions were detected by western blotting.

Results

The levels of opportunistic pathogens, such as Fusobacterium, Streptococcus and Enterococcus spp. gradually increased during the colorectal adenoma-carcinoma sequence in human fecal and mucosal samples. F. nucleatum treatment significantly altered lumen microbial structures, with increased Tenericutes and Verrucomicrobia (opportunistic pathogens) (P < 0.05 = in wild-type C57BL/6 and mice with DMH treatment). BBR intervention reversed the F. nucleatum-mediated increase in opportunistic pathogens, and the secretion of IL-21/22/31, CD40L and the expression of p-STAT3, p-STAT5 and p-ERK1/2 in mice, compared with mice fed with F. nucleatum alone.

Conclusions

F. nucleatum colonization in the intestine may prompt colorectal tumorigenesis. BBR could rescue F. nucleatum-induced colorectal tumorigenesis by modulating the tumor microenvironment and blocking the activation of tumorigenesis-related pathways.     

D2-40, Podoplanin,and CD31 as a Prognostic Predictor in Invasive Ductal Carcinomas of the Breast

Purpose

Distant metastasis and recurrence are major prognostic factors associated with breast cancer. Both lymphovascular invasion (LVI) and blood vessel invasion (BVI) are important routes for metastasis to regional lymph nodes and for systemic metastasis. Despite the importance of vascular invasion as a prognostic factor, application of vascular invasion as a histopathological criterion is controversial. The aim of this study was to distinguish LVI from BVI in prognosis and recurrence of breast cancer using an endothelial subtype specific immunohistochemical stain (podoplanin, D2-40, and CD31).

Methods

Sections from 80 paraffin-embedded archival specimens of invasive breast cancer were stained for podoplanin, D2-40, or CD31 expression. Immunohistochemical staining results were correlated with clinicopathological features, such as tumor size, status of lymph node metastases, estrogen receptor status, progesterone receptor status, human epidermal growth factor receptor-2 expression, and recurrence. Patients with ductal carcinoma in situ and stage IV breast cancer were excluded.

Results

A significant correlation was found between D2-40 LVI positivity and lymph node metastasis (p=0.022). We found a significant correlation between D2-40 LVI positivity and recurrence of breast cancer (p=0.014). However, no significant correlation was found between BVI and recurrence. A poorer disease free survival was shown for D2-40 positive LVI (p=0.003). In a multivariate analysis, the presence of D2-40 LVI positivity revealed a significant association with decreased disease-free survival.

Conclusion

D2-40 LVI positivity was a more prognostic predictor of breast cancer than BVI.