Poor prognosis of KRAS or BRAF mutant colorectal liver metastasis without microsatellite instability

Background

The discovery of practical biomarkers is important to realize personalized medicine for patients with malignant neoplasias, including colorectal cancer (CRC).

Purpose

The aim of this study was to determine reliable prognostic biomarkers by the analysis of patients with resectable colorectal liver metastases (CRLM).

Methods

Genomic DNA was obtained from the CRLM tissues of a cohort of 126 patients with CRLM with curative hepatic resection. The KRAS/BRAF mutation spectrum and microsatellite instability (MSI) status were successfully analyzed in 100 of the 126 CRLM tissues and these findings were examined in relation to the patients’ clinical outcomes.

Results

The cohort of 100 CRLM patients consisted of 46 with synchronous and 54 with metachronous liver metastasis. Overall survival and disease-free survival at 5 years were 57.4 and 24.9 %, respectively. MSI analysis revealed that none of the 100 CRLM specimens showed any evidence of MSI. By KRAS/BRAF mutation analysis, the analyzed CRLM patients were divided into 3 groups; KRAS-mutant (KRAS-Mt; n = 27), BRAF-mutant (BRAF-Mt; n = 3), and wild-types of both genes (Wild-type; n = 70). In the survival analysis, both KRAS-Mt and BRAF-Mt patients showed significantly poorer prognoses compared with Wild-type patients. Furthermore, although the population with the BRAF mutation was small, this mutation had a significant negative impact on disease-free survival.

Conclusions

In this study, all tumors in the cohort of CRLM patients were non-MSI tumors, suggesting MSI cancer in primary CRC would rarely reveal metastatic potential. KRAS and BRAF mutations are suggested to be poor prognostic factors in CRLM. Genetic information has an essential role as a prognostic marker and could contribute to the decisions on treatment strategy for CRLM.     

Molecular pathways in colorectal cancer

Colorectal cancer (CRC) is the second most common newly diagnosed cancer and accounts for the second highest number of cancer related deaths in Australia, the third worldwide and of increasing importance in Asia. It arises through cumulative effects of inherited genetic predispositions and environmental factors. Genomic instability is an integral part in the transformation of normal colonic or rectal mucosa into carcinoma. Three molecular pathways have been identified: these are the chromosomal instability (CIN), the microsatellite instability (MSI), and the CpG Island Methylator Phenotype (CIMP) pathways. These pathways are not mutually exclusive, with some tumors exhibiting features of multiple pathways. Germline mutations are responsible for hereditary CRC syndromes (accounting for less than 5% of all CRC) while a stepwise accumulation of genetic and epigenetic alterations results in sporadic CRC. This review aims to discuss the genetic basis of hereditary CRC and the different pathways involved in the process of colorectal carcinogenesis.     

Chromosomal and microsatellite instability in sporadic gastric cancer

BACKGROUND: Gastric cancer can progress through two pathways of genomic instability: chromosomal (CIN) and microsatellite instability (MSI). It is hypothesized that these two pathways are not always independent and that some tumors show overlap between these two mechanisms. METHODS: A total of 98 sporadic gastric cancers were classified based on their MSI status, using microsatellite assay with BAT26. Evidence for CIN was investigated by identifying loss of heterozygosity (LOH) events on chromosome arms, 5q, 10p, 17p, 17q, and 18q, which are regions harboring tumor suppressor genes that are significant in gastric cancer development. RESULTS: Twelve tumors (12%) showed high-frequency MSI (MSI-H). Overall, 43 of the tumors (44%) had at least one LOH event, with most frequent chromosomal losses observed on 10p and 18q (30%, respectively), followed by 5q (21%), 17p (14%), and 17q (12%). Interestingly, overlap was observed between CIN and MSI pathways. Of 43 cancers with LOH events, four (9%) were also MSI-H. It was also found that 48% of cancers without MSI-H had no LOH events identified, comprising a subgroup of tumors that were not representative of either of these two pathways of genomic instability. CONCLUSION: These results suggest that molecular mechanisms of genomic instability are not necessarily independent and may not be fully defined by either the MSI or CIN pathways in sporadic gastric cancers.     

HNPCC-associated small bowel cancer: clinical and molecular characteristics

BACKGROUND & AIMS: The risk for small bowel cancer (SBC) is significantly increased in hereditary nonpolyposis colorectal cancer (HNPCC). HNPCC-associated SBCs are poorly characterized. METHODS: Thirty-two SBCs were characterized according to clinical, pathologic, and germline mutation data. Histomorphologic characteristics, microsatellite instability (MSI) testing, mismatch repair (MMR) protein expression, and frameshift mutations of 7 coding mononucleotide repeats were investigated in 17 SBCs. RESULTS: Median age at diagnosis was 39 years. Fifty percent of SBCs were located in the duodenum. The Amsterdam criteria were fulfilled in 50% of patients; 45% of patients had no personal history of previous malignancies. Two patients had a positive family history for SBC. Pathogenic germline mutations were identified in 81%; high MSI was detected in 95% and loss of MMR protein expression in 89% of cases. TGFBR2 , BAX , MSH3 , MSH6 , ACVR2 , AIM2 , and SEC63 frameshift mutations were detected in 69%, 59%, 59%, 35%, 82%, 56%, and 56%, respectively. An expansive growth pattern of the tumor border and an intense intratumoral lymphocytic infiltrate were present in 75%, respectively. CONCLUSIONS: HNPCC-associated SBC often manifests at a young age and may be the first disease manifestation. Endoscopy may detect 50% of tumors. Considering recent data on gastric cancer, we propose endoscopic screening of mutation carriers starting at 30 years of age because clinical criteria cannot define a high-risk group. In addition, our study shows that histopathologic criteria, MSI, and MMR immunohistochemistry are often similar to these features in HNPCC.     

Microsatellite instability—a useful diagnostic tool to select patients at high risk for hereditary non-polyposis colorectal cancer: a study in different groups of patients with colorectal cancer

BACKGROUND: Clinical diagnosis of hereditary non-polyposis colorectal cancer (HNPCC) is based on a typical family history. As molecular genetic testing is predominantly restricted to these families, gene carriers not meeting the clinical criteria may be missed. AIMS: To examine the value of microsatellite instability (MSI) as a tool to increase the likelihood for uncovering a mismatch repair germline mutation in patients with colorectal cancer and to identify a genotype-phenotype relation in families with verified mutations. METHODS: Systematic search for germline mutations (hMSH2 and hMLH1 genes) was performed in 96 patients: 57 fulfilled the Amsterdam criteria (group 1) and 12 the looser HNPCC criteria (group 2). Seventeen patients showed familial clustering of cancers (group 3) and 10 patients under 50 years had sporadic cancer (group 4), the latter of whom all exhibited MSI+ tumours. RESULTS: A similar proportion of germline mutations was found in patients who fulfilled the clinical criteria of HNPCC and had MSI+ tumours (groups 1 and 2; 15/39) compared with patients who did not meet these clinical criteria but who had MSI+ tumours (groups 3 and 4; 8/27 patients). Affected relatives of patients with hMLH1 mutations showed a significantly higher frequency of colorectal cancer but a lower frequency of endometrium cancer than those with hMSH2 mutations. CONCLUSIONS: MSI in tumour tissue is a useful criterion for selecting patients who should be tested for germline mutations in the mismatch repair genes hMSH2 and hMLH1 irrespective of their family history. Among carriers of hMSH2 mutations the tumour spectrum was broader than among carriers of hMLH1 mutations.     

Cancer characteristics in Swedish families fulfilling criteria for hereditary nonpolyposis colorectal cancer

BACKGROUND & AIMS: The present study quantified the prevalence of families that fulfill the Amsterdam or Bethesda criteria for hereditary nonpolyposis colorectal cancer (HNPCC) in the whole Swedish population and investigated the extent to which tumors in the classified families are HNPCC-related. METHODS: The families of the Swedish Family-Cancer Database with at least 4 generations (N = 566,877) were classified according to the Amsterdam and the Bethesda criteria. Survival methods were used to assess the risk of cancer in the classified families, the prognosis of cancer patients, and the risk of subsequent malignancies after colorectal adenomas and after colorectal/endometrial adenocarcinomas. RESULTS: The Bethesda criteria identified 0.9% of all Swedish families and 11.2% of patients with colorectal cancer. Families that fulfilled the Bethesda criteria showed increased risks of cancer in the colorectum, endometrium, small bowel, ovary, stomach, bile ducts, renal pelvis, and ureter; members of Bethesda criteria families were at decreased risks of lung and cervical cancers. The prognosis of cancer in the ureter, renal pelvis, stomach, ovary, and colorectum, but not in the endometrium, was better in Bethesda criteria than in nonclassified families. CONCLUSIONS: Most malignancies in the classified families reflect typical features of HNPCC (association with subsequent malignancies, accelerated adenoma-carcinoma sequence, and better survival). The data presented in this study should help to define surveillance strategies for members of families that fulfill the criteria for HNPCC testing.     

Management of hepatic metastasis from colorectal cancers: an update

Approximately 50%–60% of patients with colorectal cancers will develop liver lesions in their life span. Despite the potential of surgical resection to provide long-term survival in this subset of patients, only 15%–20% are found to be resectable. The introduction of new neoadjuvant chemotherapeutic agents and the expanding criteria of resection have enhanced the overall 5-year survival from 30% to 60% in the past decade. The use of technical innovations such as staged resection; portal vein embolization, and repeat resection have allowed higher resection rates in patients with bilobar disease. Extrahepatic primary and liver-exclusive recurrent disease no longer represent an absolute contraindication to resection. The role of regional therapy using hepatic arterial infusion is being redefined for liver-exclusive unresectable disease. Adjuvant chemotherapy in combination with regional therapies is being looked at from fresh perspectives. Ablative approaches have gained a firm role both as an adjunct to surgical resection and in the management of patients who are not surgical candidates. Overall, the management of hepatic metastasis from colorectal cancers requires a multimodal approach.     

DNA microsatellite instability and mismatch repair protein loss in adenomas presenting in hereditary non-polyposis colorectal cancer

BACKGROUND AND AIM: Hereditary non-polyposis colorectal cancer (HNPCC), as its name implies, is associated with few adenomas, and the early evolution of colorectal neoplasia is poorly understood. In this study our aim was to clarify the genetic profiles of benign polyps in subjects with HNPCC using a combined molecular and immunohistochemical approach. METHODS: Thirty adenomas and 17 hyperplastic polyps were obtained from 24 affected HNPCC subjects. DNA was extracted from paraffin embedded tissue by microdissection and analysed for the presence of microsatellite instability (MSI) and mutations in five genes known to be targets in mismatch repair deficiency (TGFbetaRII, IGF2R, BAX, hMSH3, and hMSH6). Serial sections were stained by immunohistochemistry for hMLH1 and hMSH2. RESULTS: Twenty four (80%) of 30 adenomas showed MSI. Of MSI positive adenomas, 66.7% showed MSI at more than 40% of markers (high level of MSI (MSI-H)). Two of 17 hyperplastic polyps revealed MSI at one marker (low level of MSI (MSI-L)). A significant association was found between MSI-H and high grade dysplasia in adenomas (p=0.004). Eight of nine adenomas with mutations of coding sequences revealed high grade dysplasia and all nine were MSI-H. Four of the nine ranged in size from 2 to 5 mm. The presence of the hMSH6 mutation was significantly correlated with high levels of MSI (80% of markers) (p<0.02). Twenty four adenomas gave evaluable results with immunohistochemistry. One of six (17%) microsatellite stable, six of seven (86%) MSI-L, and 11 of 11 (100%) MSI-H adenomas showed loss of either hMLH1 or hMSH2. CONCLUSIONS: Most adenomas in subjects with a definite diagnosis of HNPCC show MSI (80%). The finding of MSI-L is usually associated with loss of expression of hMLH1 or hMSH2, unlike the situation in MSI-L sporadic colorectal cancer. The transition from MSI-L to MSI-H correlated with the finding of high grade dysplasia and mutation of coding sequences and may be driven by mutation of secondary mutators such as hMSH3 and hMSH6. Advanced genetic changes may be present in adenomas of minute size.     

Tumour infiltrating lymphocytes and apoptosis are independent features in colorectal cancer stratified according to microsatellite instability status

BACKGROUND: The presence of high level DNA microsatellite instability (MSI-H) in colorectal cancer is associated with an improved prognosis, as is the presence of tumour infiltrating lymphocytes (TILs). It is not clear if TILs contribute directly to the survival advantage associated with MSI-H cancers through activation of an antitumour immune response. AIMS: To correlate TIL and apoptosis rates in colorectal cancer stratified by MSI status. METHODS: The distribution of TILs was characterised and quantified in a selected series of 102 sporadic colorectal cancers classified according to levels of MSI as 32 MSI-H, 30 MSI-low (MSI-L), and 40 microsatellite stable (MSS). Archival blocks were immunostained using the T cell markers CD3 and CD8, and the B cell marker CD20. Apoptosis of malignant epithelial cells was quantified by immunohistochemistry with the M30 CytoDEATH antibody. RESULTS: Positive staining with anti-CD3 and negative staining with anti-CD20 identified virtually all TILs as T cells. The majority of CD3+ TILs (>75%) also stained with anti-CD8. TILs were most abundant in MSI-H colorectal cancers in which 23/32 (72%) scored as TIL positive. Only 5/40 (12.5%) MSS tumours and 9/30 (30%) MSI-L cancers were TIL positive (p<0.0001). MSI-H cancers showed a twofold higher rate of apoptosis (mean (SD) 3.52 (0.34)%) than the MSS cancers (1.53 (0.23)%) while the MSI-L subgroup had an intermediate level (2.52 (0.35)%) (p<0.0001). Overall, there was a small (r=0.347) but significant linear correlation between CD3+ and M30+ random apoptosis counts (p<0.001). However, TILs and apoptosis showed little colocalisation. CONCLUSIONS: While TILs might be expected to explain the increased apoptotic rate and improved prognosis of MSI-H cancers, it is likely that TILs and apoptosis are independent characteristics of MSI-H cancers.     

Early-Age-at-Onset Colorectal Cancer and Microsatellite Instability as Markers of Hereditary Nonpolyposis Colorectal Cancer

PURPOSE: Early-age-at-onset colorectal cancer and microsatellite instability are characteristic features of hereditary nonpolyposis colorectal cancer. Our aim was therefore to investigate whether these features might be useful markers in screening for hereditary nonpolyposis colorectal cancer and mismatch repair gene mutations. METHODS: From 1,132 consecutive patients who underwent surgery for colorectal cancer at our department between 1980 and 1999, we selected all patients 40 years of age or younger (study group, n = 59) and a subset of patients 40 years of age or older (control group, n = 60) who were matched for gender and pathologic TNM stage. Patients for whom a complete family cancer history or microsatellite status was unavailable were excluded from the study. Family cancer histories, retrieved from archival charts, were reassessed. Microsatellite status was investigated with the five microsatellites from the Bethesda recommended panel (BAT-26, BAT-25, D2S123, D5S346, and D17S250). On the basis of the number of altered microsatellites ( 2, 1, or 0), tumors were considered as having high or low instability or microsatellite stability, respectively. Mutation analysis for MLH1 and MSH2 genes was performed only in cases of high instability. DNA was investigated for mutations by single-strand conformational polymorphism and sequencing analysis. RESULTS: Data from 95 patients (study group: n = 37, 18 males, mean age 35 years; control group: n = 58, 29 males, mean age 62 years) were available for analysis. Four patients (study group, n = 3; control group, n = 1) fulfilled the Amsterdam II criteria for hereditary nonpolyposis colorectal cancer. Of the 37 study group tumors, 12 (32.4 percent) showed high-frequency microsatellite instability, and 25 had microsatellite stability, whereas among the 58 control group tumors, 4 (7 percent) showed high-frequency microsatellite instability, and 54 had microsatellite stability (P < 0.002). Mismatch repair gene mutation analysis was performed in 12 cases (study group, n = 7; control group, n = 5). We found four mutations (MSH2 119delG, MLH1 ex9 684insT, MSH2 Gln239Stop, and MLH1 del0.8 Kb) in the study group patients and none in the control group. Of four hereditary nonpolyposis colorectal cancer patients who underwent mismatch repair gene mutation analysis, one had a mutation. CONCLUSIONS: Early-age-at-onset colorectal cancer is significantly correlated with high-frequency microsatellite instability tumor status and is a useful criterion to identify hereditary nonpolyposis colorectal cancer patients. Moreover, when used in association with high-frequency microsatellite instability status, it is effective in selecting patients for mismatch repair gene mutation analysis.     

Colorectal hepatic metastasis: Evolving therapies

The approach for colorectal hepatic metastasis has advanced tremendously over the past decade. Multidrug chemotherapy regimens have been successfully introduced with improved outcomes. Concurrently, adjunct multimodal therapies have improved survival rates, and increased the number of patients eligible for curative liver resection. Herein, we described major advancements of surgical and oncologic management of such lesions, thereby discussing modern chemotherapeutic regimens, adjunct therapies and surgical aspects of liver resection.     

KRAS,NRAS, BRAF signatures,and MMR status in colorectal cancer patients in North China

We assessed the clinicopathological features and prognostic values of KRAS, NRAS, BRAF, and DNA mismatch repair status in colorectal cancer (CRC) to provide real-world data in developing countries. We enrolled 369 CRC patients and analyzed the correlation between RAS/BRAF mutation, mismatch repair status with clinicopathological features, and their prognostic roles. The mutation frequencies of KRAS, NRAS, and BRAF were 41.7%, 1.6%, and 3.8%, respectively. KRAS mutations and deficient mismatch repair (dMMR) status were associated with right-sided tumors, aggressive biological behaviors, and poor differentiation. BRAF (V600E) mutations are associated with well-differentiated and lymphovascular invasion. The dMMR status predominated in young and middle-aged patients and tumor node metastasis stage II patients. dMMR status predicted longer overall survival in all CRC patients. KRAS mutations indicated inferior overall survival in patients with CRC stage IV. Our study showed that KRAS mutations and dMMR status could be applied to CRC patients with different clinicopathological features.     

Prospective results of surveillance colonoscopy in dominant familial colorectal cancer with and without Lynch syndrome

BACKGROUND & AIMS: Lynch syndrome is an autosomal dominant predisposition to colorectal cancer caused by mutations in DNA mismatch repair genes; colorectal cancer risk is high. Few studies have addressed colorectal cancer risk in individuals from dominant families without mismatch repair deficiency. We sought to establish whether these individuals are also at increased risk by examining the incidence of advanced neoplasia during surveillance. METHODS: In this prospective cohort study, BAT26 testing of tumors was carried out at 2 tertiary centers on 125 individuals from 97 families (with a dominant colorectal cancer history) to classify families as Lynch syndrome (microsatellite unstable) or non-Lynch syndrome (microsatellite stable). Colonoscopy results in 288 at-risk family members were compared. RESULTS: Twenty-nine families were classified as Lynch syndrome and 68 as non-Lynch syndrome. Seven hundred seventy-six colonoscopies were undertaken. High-risk adenomas occurred in 7 of 91 (7.7%) Lynch syndrome individuals and 15 of 197 (7.6%) non-Lynch syndrome individuals, adjusted relative risk 1.15 (95% CI: 0.6-2.3). Cancer was observed only in Lynch syndrome individuals (4/91; 4.4%), Fisher exact test, P = .010. Multiple adenomas were only seen in non-Lynch syndrome individuals (13/197; 6.6%), Fisher exact text, P = .06. CONCLUSIONS: Individuals with an autosomal dominant family history of colorectal cancer with and without evidence of Lynch syndrome are at equal risk of high-risk adenomas during surveillance, but colorectal cancer was only seen in Lynch syndrome. Therefore non-Lynch syndrome individuals do require colonoscopic surveillance, but the interval could be lengthened because risk of (interval) cancer is low. Lynch syndrome individuals require short surveillance intervals as is the recommended practice.     

Hereditary nonpolyposis colorectal cancer in young colorectal cancer patients: high-risk clinic versus population-based registry

BACKGROUND & AIMS: Early onset colorectal cancer (CRC) is an important feature of hereditary nonpolyposis colorectal cancer (HNPCC). We sought to compare rates of genetically defined HNPCC among individuals with early onset CRC drawn from a high-risk clinic and a population-based cancer registry. METHODS: Probands with CRC diagnosed before 36 years of age were enrolled from a high-risk CRC clinic at the University of California, San Francisco (UCSF), and a population-based Kaiser Permanente (KP) Health Plan cancer registry. Probands provided cancer family histories and tumors for microsatellite instability (MSI) testing and MSH2/MLH1 protein immunostaining. Germline MSH2 and MLH1 mutational analysis was performed. RESULTS: Forty-three probands were enrolled from UCSF and 23 from KP. The UCSF and KP probands had similar median age of onset of CRC (30 vs. 31 years) and the percentage with any personal or family history of another HNPCC-related cancer (70% vs. 74%). However, 28 of 40 (70%) of the UCSF tumors were MSI-H compared with 6 of 18 (33%) of KP tumors (P = 0.01), and 13 germline MSH2 or MLH1 mutations were found in the UCSF group compared with 0 in the KP group (P = 0.0001). In a multivariate analysis, institution (P = 0.002) and the total number of colorectal cancers in the family (P = 0.0001) were independent predictors of MSH2 or MLH1 mutation. CONCLUSIONS: Family history of cancer is an important feature of HNPCC, even among individuals with early onset CRC. Caution must be undertaken when extrapolating data regarding HNPCC from high-risk clinic populations to the general population.     

Colorectal cancer screening

Colorectal cancer is one of the most common malignancies in Australia, and screening to detect it an earlier stage is cost‐effective. Furthermore, detection and removal of precursor polyps can reduce incidence. Currently, there are limited data to determine the screening rate in Australia, but it is certainly lower than the 80% screening rate considered desirable. Whether colonoscopy is used as the screening test or to follow up positive results of an initial non‐invasive test, it plays a fundamental role. Despite high sensitivity and specificity, it is expensive and invasive with measurable risk and is not acceptable as an initial test to many participants. It does not provide complete protection, and interval cancers between planned colonoscopies are associated with proximal location, origin in sessile serrated adenomas and operator‐dependent factors. An essential component of colorectal screening is the measurement of colonoscopy quality indicators, such as caecal intubation and adenoma detection rates, which are known to be associated with the rate of interval cancer. The non‐invasive screening test currently recommended in Australia is biennial testing for faecal occult blood between the ages of 50 and 75 using a faecal immunochemical test, with positives evaluated by colonoscopy. This is provided through the National Bowel Cancer Screening Programme, currently for those at the ages of 50, 55, 60 and 65 years, with full implementation of biennial screening by 2020. To improve screening in Australia, the most fruitful approach may be to acknowledge that there is a choice of screening tests and to focus on the goal of improving overall participation rate and being able to measure this.