To determine the prognostic significance of FOXP3(+) lymphocyte (Treg) density in colorectal cancer compared with conventional histopathologic features and with CD8(+) and CD45RO(+) lymphocyte ...densities.
Tissue microarrays and immunohistochemistry were used to assess the densities of CD8(+), CD45RO(+), and FOXP3(+) lymphocytes in tumor tissue and normal colonic mucosa from 967 stage II and stage III colorectal cancers. These were evaluated for associations with histopathologic features and patient survival.
FOXP3(+) Treg density was higher in tumor tissue compared with normal colonic mucosa, whereas CD8(+) and CD45RO(+) cell densities were lower. FOXP3(+) Tregs were not associated with any histopathologic features, with the exception of tumor stage. Multivariate analysis showed that stage, vascular invasion, and FOXP3(+) Treg density in normal and tumor tissue were independent prognostic indicators, but not CD8(+) and CD45RO(+). High FOXP3(+) Treg density in normal mucosa was associated with worse prognosis (hazard ratio HR = 1.51; 95% CI, 1.07 to 2.13; P = .019). In contrast, a high density of FOXP3(+) Tregs in tumor tissue was associated with improved survival (HR = 0.54; 95% CI, 0.38 to 0.77; P = .001).
FOXP3(+) Treg density in normal and tumor tissue had stronger prognostic significance in colorectal cancer compared with CD8(+) and CD45RO(+) lymphocytes. The finding of improved survival associated with a high density of tumor-infiltrating FOXP3(+) Tregs in colorectal cancer contrasts with several other solid cancer types. The inclusion of FOXP3(+) Treg density may help to improve the prognostication of early-stage colorectal cancer.
The aims of the TP53 Colorectal Cancer (CRC) International Collaborative Study were to evaluate the possible associations between specific TP53 mutations and tumor site, and to evaluate the ...prognostic and predictive significance of these mutations in different site, stage, and treatment subgroups.
A total of 3,583 CRC patients from 25 different research groups in 17 countries were recruited to the study. Patients were divided into three groups according to site of the primary tumor. TP53 mutational analyses spanned exons 4 to 8.
TP53 mutations were found in 34% of the proximal colon tumors and in 45% of the distal colon and rectal tumors. They were associated with lymphatic invasion in proximal tumors. In distal colon tumors, deletions causing loss of amino acids were associated with worse survival. In proximal colon tumors, mutations in exon 5 showed a trend toward statistical significance (P < .05) when overall survival was considered. Dukes' C tumors with wild-type TP53 and those with mutated TP53 (proximal tumors) showed significantly better prognosis when treated with adjuvant chemotherapy.
Analysis of TP53 mutations from a large cohort of CRC patients has identified tumor site, type of mutation, and adjuvant treatment as important factors in determining the prognostic significance of this genetic alteration.
Purpose: The major pathologic markers of prognosis in colorectal cancer include vascular invasion by tumor cells, invasion of adjacent
lymph nodes, and perforation of the serosal wall. Recent work ...suggests that a high density of tumor-infiltrating lymphocytes
(TIL) is associated with good outcome independently of these established prognostic markers. The aim of the present study
was to investigate the prognostic significance of TILs and other routinely reported pathologic features in colon cancer, particularly
in relation to the use of adjuvant chemotherapy.
Experimental Design: Pathologic markers, disease-specific survival, and the use of adjuvant chemotherapy were recorded in a retrospective, population-based
series of 1,156 stage III colon cancer patients with a median follow-up time of 52 months.
Results: In patients treated by surgery alone ( n = 851), markers with significant prognostic value included poor histologic grade, T4 stage, N2 nodal status, vascular invasion,
and perforation, but not the presence of TILs. In patients treated with 5-fluorouracil–based chemotherapy ( n = 305), TILs were associated with significantly improved survival hazard ratio (HR), 0.52; 95% confidence interval, 0.30-0.91;
P = 0.02 and perforation with a trend for improved survival (HR, 0.67; 95% confidence interval, 0.27-1.05; P = 0.16). Patients with TILs or perforation seemed to gain more survival benefit from chemotherapy (HR, 0.22 and 0.21, respectively)
than patients without these features (HR, 0.84 and 0.82, respectively).
Conclusion: The apparent survival advantage from 5-fluorouracil associated with TILs and perforation requires confirmation in prospective
studies. Because the presence of TILs reflects an adaptive immune response and perforation is associated with inflammatory
response, these results suggest that there may be interactions between the immune system and chemotherapy leading to improved
survival of colon cancer patients.
The introduction of next generation sequencing (NGS) in the routine diagnostic setting is still in the development phase and has been limited by its complexity. Targeted NGS offers an attractive ...alternative to performing multiple single target assays and is very useful in meeting the increasing clinical demand for testing of multiple genetic aberrations in cancer specimens.
To this end, we carried out a blinded validation study on 113 tumours in a diagnostic laboratory and compared mutation results from targeted NGS with those from Sanger sequencing, pyrosequencing, competitive allele specific TaqMan polymerase chain reaction (CAST PCR) and Cobas assays. DNA was extracted from formalin fixed, paraffin embedded (FFPE) tissue samples that included core biopsies, resections and cytology samples from three common and one rare cancer types non-small cell lung cancer (NSCLC), colorectal cancer (CRC), malignant melanoma (MM) and gastrointestinal stromal tumour (GIST). Libraries were prepared using the TruSight Tumour 26 gene panel and NGS was carried out on the MiSeq instrument.
Results from NGS were concordant with the mutational status determined by other platforms in 107 of the 113 cases tested (94.7%). The sequencing quality for NGS failed in four of the six false negative cases, while a further two samples gave false negative results because the c-KIT mutations were located outside the range of the NGS panel. One NSCLC sample contained an EGFR mutation previously detected by the Cobas assay. Reanalysis of the NGS data for this sample using a cut-off allele frequency of 1% revealed the mutation had an allele frequency of 2%, which was below the recommended software-determined threshold of 3%. NGS detected 113 additional mutations that were not previously known from analysis by the conventional methods. Twenty-six of these have known clinical importance, 37 have potential clinical significance, while 50 were novel mutations with unknown clinical significance. NGS detected variants using inputs of 10–20 ng of FFPE extracted DNA and from specimens with a tumour cell content less than 50%, for which when possible we recommend microdissection.
We conclude that results from targeted NGS are highly concordant with those from other mutation testing platforms. Targeted NGS is suitable for a range of sample types received in the diagnostic pathology laboratory, including those with limited material or with low tumour cell content (TCC). This work has allowed us to determine the quality parameter settings required in order to obtain robust mutation data by NGS.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The purpose of this investigation is to determine if Epstein Barr virus (EBV), high risk human papillomavirus (HPV), and mouse mammary tumour viruses (MMTV) co-exist in some breast cancers.
All the ...specimens were from women residing in Australia. For investigations based on standard PCR, we used fresh frozen DNA extracts from 50 unselected invasive breast cancers. For normal breast specimens, we used DNA extracts from epithelial cells from milk donated by 40 lactating women. For investigations based on in situ PCR we used 27 unselected archival formalin fixed breast cancer specimens and 18 unselected archival formalin fixed normal breast specimens from women who had breast reduction surgery. Thirteen of these fixed breast cancer specimens were ductal carcinoma in situ (dcis) and 14 were predominantly invasive ductal carcinomas (idc).
EBV sequences were identified in 68%, high risk HPV sequences in 50%, and MMTV sequences in 78% of DNA extracted from 50 invasive breast cancer specimens. These same viruses were identified in selected normal and breast cancer specimens by in situ PCR. Sequences from more than one viral type were identified in 72% of the same breast cancer specimens. Normal controls showed these viruses were also present in epithelial cells in human milk - EBV (35%), HPV, 20%) and MMTV (32%) of 40 milk samples from normal lactating women, with multiple viruses being identified in 13% of the same milk samples.
We conclude that (i) EBV, HPV and MMTV gene sequences are present and co-exist in many human breast cancers, (ii) the presence of these viruses in breast cancer is associated with young age of diagnosis and possibly an increased grade of breast cancer.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The cancer stem cell hypothesis may explain why conventional chemotherapies are unable to fully eradicate cancers. In this study, we examined both the prognostic and predictive significance of ...putative cancer stem cell markers in colorectal cancer. In this study, immunohistochemistry for three candidate cancer stem cell markers (CD133, Oct-4 and Sox-2) and for six other postulated prognostic markers (CK7, CK20, Cox-2, Ki-67, p27 and p53) were performed using tissue microarrays containing 501 primary colorectal cancer cases. Receiver-operating characteristic analysis was used to determine cut-off scores for positive protein expression. Multivariate analysis revealed that positive expression for CD133 and Oct-4 was associated with significantly worse survival in patients treated by surgery alone (P=0.023 and P<0.001, respectively) and in patients treated with 5-fluorouracil-based chemotherapy (P=0.001 and P=0.021, respectively). Stage III patients with negative CD133 expression showed an apparent survival benefit from 5-fluorouracil treatment (P=0.002), but not those with positive CD133 expression. Positive expression of CD133 was also associated with poorer clinical response to chemotherapy in stage IV patients (P=0.006). In summary, the putative cancer stem cell markers CD133 and Oct-4 showed strong prognostic significance in colorectal cancer. Our results show for the first time that CD133+ colorectal tumors are more resistant to 5-fluorouracil-based chemotherapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Hypermethylation of CpG island loci within gene promoter regions is a frequent event in colorectal cancer that is often associated with transcriptional silencing and has been referred to as CIMP+. ...DNA hypomethylation can occur in concert with CIMP+, although these two phenomena appear not to be related in colorectal cancer. The authors investigated here whether the methylation level of LINE‐1 repeats, a surrogate marker for genomic methylation, was associated with the level of CpG island methylation in colorectal cancers and in matching normal colonic mucosa from 178 patients. The MethyLight assay was used to quantitate the methylation of CpG islands within the MLH1, P16(INK4A), TIMP3, DAPK, APC, ER and MYOD genes. A real‐time, methylation‐specific polymerase chain reaction assay was also used to quantitate the methylation of LINE‐1 repeats. In colorectal cancer, no associations were seen between methylation levels in LINE‐1 repeats and CpG island loci, including a new CpG island panel that was recently proposed for CIMP+. In normal colonic mucosa, however, the methylation level of LINE‐1 repeats was inversely correlated with CpG‐island methylation of the MLH1, P16, TIMP3, APC, ER and MYOD genes. The methylation level of LINE‐1 repeats in normal colonic mucosa also showed significant associations with common polymorphisms in the methylene tetrahydrofolate reductase and methylene tetrahydrofolate dehydrogenase genes involved in methyl group metabolism. Further investigation of genomic and CpG island methylation in normal colonic mucosa and the possible influences of environmental and genetic factors may provide new insights into the development of CIMP+ colorectal cancer. (Cancer Sci 2007; 98: 1454–1460)
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Inhibition of the phosphatidylinositol‐3‐kinase (PI3K) signaling pathway is a cancer treatment strategy that has entered into clinical trials. We performed a meta‐analysis on the frequency of ...prominent genetic (PIK3CA mutation, PIK3CA amplification and PTEN deletion) and protein expression (high PI3K, PTEN loss and high pAkt) aberrations in the PI3K pathway in gastric cancer (GC) and colorectal cancer (CRC). We also performed laboratory analysis to investigate the co‐occurrence of these aberrations. The meta‐analysis indicated that East Asian and Caucasian GC patients differ significantly for the frequencies of PIK3CA Exon 9 and 20 mutations (7% vs. 15%, respectively), PTEN deletion (21% vs. 4%) and PTEN loss (47% vs. 78%), while CRC patients differed for PTEN loss (57% vs. 26%). High study heterogeneity (I2 > 80) was observed for all aberrations except PIK3CA mutations. Laboratory analysis of tumors from East Asian patients revealed significant differences between GC (n = 79) and CRC (n = 116) for the frequencies of PIK3CA amplification (46% vs. 4%) and PTEN loss (54% vs. 78%). The incidence of GC cases with 0, 1, 2 and 3 concurrent aberrations was 14%, 52%, 27% and 8%, respectively, while for CRC it was 10%, 60%, 25% and 4%, respectively. Our study consolidates knowledge on the frequency, co‐occurrence and clinical relevance of PI3K pathway aberrations in GC and CRC. Up to 86% of GC and 90% of CRC have at least one aberration in the PI3K pathway, and there are significant differences in the frequencies of these aberrations according to cancer type and ethnicity.
What's new?
Aberrations in the PI3K‐Akt pathway have been observed in many types of cancer cells, and inhibiting this pathway is a promising therapeutic strategy. However, it's important to have a more complete molecular picture in order to identify patient subgroups that are most likely to respond to therapy. In this study, the authors conducted both a meta‐analysis and single‐laboratory analysis to determine the frequency and co‐occurrence of PI3K pathway aberrations, in gastric cancer (GC) and colorectal cancer (CRC). The study also found differences in the frequency of aberrations according to ethnicity and cancer type.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Background & Aims: The microsatellite instability (MSI) phenotype is a characteristic of the hereditary nonpolyposis colorectal cancer syndrome as well as approximately 15% of sporadic colon and ...gastric tumors. It is a valuable diagnostic marker for the identification of hereditary nonpolyposis colorectal cancer cases and may be a molecular predictive marker for the identification of colon cancer patients who benefit from chemotherapy. To evaluate MSI, a reference panel was proposed at an international consensus meeting, comprised of 2 mononucleotide (BAT-25, BAT-26) and 3 dinucleotide repeats. Analysis of BAT-26 is sufficient for detecting the MSI phenotype in most, but not all, cases. Additional results with dinucleotide markers can sometimes lead to incorrect classification of MSI tumors. Methods: We describe here a single fluorescent multiplex system comprising 5 quasimonomorphic mononucleotide repeats for the detection of MSI tumors. Results: None of 184 germline DNA samples, including 56 from African subjects, was found to contain allelic size variations in more than 2 of these markers. In contrast, all MSI tumors showed allelic size variations in 3 or more of the microsatellites. Using this assay, we confirmed (or reclassified in 6 cases) the MSI status of 124 colon and 50 gastric primary tumors and 16 colon cell lines. Conclusions: We propose that using a pentaplex polymerase chain reaction system allows accurate evaluation of tumor MSI status of DNA with 100% sensitivity and specificity without the need to match normal DNA. This assay is simpler to use than those involving dinucleotides and is more specific than using BAT-26 alone.
GASTROENTEROLOGY 2002;123:1804-1811
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