Ulrich Nitsche

Colorectal cancer intrinsic subtypes predict chemotherapy benefit, deficient mismatch repair and epithelial-to-mesenchymal transition.

In most colorectal cancer (CRC) patients, outcome cannot be predicted because tumors with similar clinicopathological features can have differences in disease progression and treatment response. Therefore, a better understanding of the CRC biology is required to identify those patients who will benefit from chemotherapy and to find a more tailored therapy plan for other patients. Based on unsupervised classification of whole genome data from 188 stages I–IV CRC patients, a molecular classification was developed that consist of at least three major intrinsic subtypes (A‐, B‐ and C‐type). The subtypes were validated in 543 stages II and III patients and were associated with prognosis and benefit from chemotherapy. The heterogeneity of the intrinsic subtypes is largely based on three biological hallmarks of the tumor: epithelial‐to‐mesenchymal transition, deficiency in mismatch repair genes that result in high mutation frequency associated with microsatellite instability and cellular proliferation. A‐type tumors, observed in 22% of the patients, have the best prognosis, have frequent BRAF mutations and a deficient DNA mismatch repair system. C‐type patients (16%) have the worst outcome, a mesenchymal gene expression phenotype and show no benefit from adjuvant chemotherapy treatment. Both A‐type and B‐type tumors have a more proliferative and epithelial phenotype and B‐types benefit from adjuvant chemotherapy. B‐type tumors (62%) show a low overall mutation frequency consistent with the absence of DNA mismatch repair deficiency. Classification based on molecular subtypes made it possible to expand and improve CRC classification beyond standard molecular and immunohistochemical assessment and might help in the future to guide treatment in CRC patients.

Tumor Classification of Six Common Cancer Types Based on Proteomic Profiling by MALDI Imaging

In clinical diagnostics, it is of outmost importance to correctly identify the source of a metastatic tumor, especially if no apparent primary tumor is present. Tissue-based proteomics might allow correct tumor classification. As a result, we performed MALDI imaging to generate proteomic signatures for different tumors. These signatures were used to classify common cancer types. At first, a cohort comprised of tissue samples from six adenocarcinoma entities located at different organ sites (esophagus, breast, colon, liver, stomach, thyroid gland, n = 171) was classified using two algorithms for a training and test set. For the test set, Support Vector Machine and Random Forest yielded overall accuracies of 82.74 and 81.18%, respectively. Then, colon cancer liver metastasis samples (n = 19) were introduced into the classification. The liver metastasis samples could be discriminated with high accuracy from primary tumors of colon cancer and hepatocellular carcinoma. Additionally, colon cancer liver metastasis samples could be successfully classified by using colon cancer primary tumor samples for the training of the classifier. These findings demonstrate that MALDI imaging-derived proteomic classifiers can discriminate between different tumor types at different organ sites and in the same site.

Mucinous and Signet-Ring Cell Colorectal Cancers Differ from Classical Adenocarcinomas in Tumor Biology and Prognosis

Objectives:To define the prognostic value of different histological subtypes of colorectal cancer. Background:Most colorectal cancers are classical adenocarcinomas (AC). Less frequent subtypes include mucinous adenocarcinomas (MAC) and signet-ring cell carcinomas (SC). In contrast to established prognostic factors such as TNM and grading, the histological subtype has no therapeutical consequences so far, although it may reflect different biological behavior. Methods:Between 1982 and 2012, a total of 3479 consecutive patients underwent surgery for primary colorectal cancer (AC, MAC, or SC). Clinical, histopathological, and survival data were analyzed. Results:Of all 3479 patients, histological subtype was AC in 3074 cases (88%), MAC in 375 cases (11%), and SC in 30 cases (0.9%). MAC (51%, P < 0.001) and SC (50%, P = 0.029) occurred more frequently in right-sided tumors than AC (28%). Compared with AC, tumor stages and histological grading were higher in MAC and SC (P < 0.001 for each). Rates of angioinvasion were lower in MAC than in AC (5% vs 9%, P = 0.011). Rates of lymphatic invasion were higher in SC than in AC (67% vs 25%, P < 0.001). Five-year cause-specific survival was 67 ± 1% for AC, 61 ± 3% for MAC, and 21 ± 8% for SC (P < 0.001 for difference between the groups). In multivariable analysis, survival did not differ significantly between AC and MAC after correction for tumor stage. However, SC remained an independent prognostic factor associated with worse survival (hazard ratio = 2.5, 95% confidence interval = 1.6–3.8, P < 0.001). Conclusions:MAC and SC are histological subtypes of colorectal cancer with different characteristics than classical AC. Both are diagnosed in more advanced tumor stages, but the dismal prognosis of SC seems to be caused by its intrinsic tumor biology.

Tissue-based proteomics reveals FXYD3, S100A11 and GSTM3 as novel markers for regional lymph node metastasis in colon cancer

Regional lymph node metastasis negatively affects prognosis in colon cancer patients. The molecular processes leading to regional lymph node metastasis are only partially understood and proteomic markers for metastasis are still scarce. Therefore, a tissue‐based proteomic approach was undertaken for identifying proteins associated with regional lymph node metastasis. Two complementary tissue‐based proteomic methods have been employed. MALDI imaging was used for identifying small proteins (≤25 kDa) in situ and label‐free quantitative proteomics was used for identifying larger proteins. A tissue cohort comprising primary colon tumours without metastasis (UICC II, pN0, n = 21) and with lymph node metastasis (UICC III, pN2, n = 33) was analysed. Subsequent validation of identified proteins was done by immunohistochemical staining on an independent tissue cohort consisting of primary colon tumour specimens (n = 168). MALDI imaging yielded ten discriminating m/z species, and label‐free quantitative proteomics 28 proteins. Two MALDI imaging‐derived candidate proteins (FXYD3 and S100A11) and one from the label‐free quantitative proteomics (GSTM3) were validated on the independent tissue cohort. All three markers correlated significantly with regional lymph node metastasis: FXYD3 (p = 0.0110), S100A11 (p = 0.0071), and GSTM3 (p = 0.0173). FXYD3 and S100A11 were more highly expressed in UICC II patient tumour tissues. GSTM3 was more highly expressed in UICC III patient tumour tissues. By our tissue‐based proteomic approach, we could identify a large panel of proteins which are associated with regional lymph node metastasis and which have not been described so far. Here we show that novel markers for regional lymph metastasis can be identified by MALDI imaging or label‐free quantitative proteomics and subsequently validated on an independent tissue cohort. Copyright

Integrative marker analysis allows risk assessment for metastasis in stage II colon cancer.

Objectives:Individualized risk assessment in patients with UICC stage II colon cancer based on a panel of molecular genetic alterations. Background:Risk assessment in patients with colon cancer and localized disease (UICC stage II) is not sufficiently reliable. Development of metachronous metastasis is assumed to be governed largely by individual tumor genetics. Methods:Fresh frozen tissue from 232 patients (T3-4, N0, M0) with complete tumor resection and a median follow-up of 97 months was analyzed for microsatellite stability, KRAS exon 2, and BRAF exon 15 mutations. Gene expression of the WNT-pathway surrogate marker osteopontin and the metastasis-associated genes SASH1 and MACC1 was determined for 179 patients. The results were correlated with metachronous distant metastasis risk (n = 22 patients). Results:Mutations of KRAS were detected in 30% patients, mutations of BRAF in 15% patients, and microsatellite instability in 26% patients. Risk of recurrence was associated with KRAS mutation (P = 0.033), microsatellite stable tumors (P = 0.015), decreased expression of SASH1 (P = 0.049), and increased expression of MACC1 (P < 0.001). MACC1 was the only independent parameter for recurrence prediction (hazard ratio: 6.2; 95% confidence interval: 2.4–16; P < 0.001). Integrative 2-step cluster analysis allocated patients into 4 groups, according to their tumor genetics. KRAS mutation, BRAF wild type, microsatellite stability, and high MACC1 expression defined the group with the highest risk of recurrence (16%, 7 of 43), whereas BRAF wild type, microsatellite instability, and low MACC1 expression defined the group with the lowest risk (4%, 1 of 26). Conclusions:MACC1 expression predicts development of metastases, outperforming microsatellite stability status, as well as KRAS/BRAF mutation status.

Daple is a novel non-receptor GEF required for trimeric G protein activation in Wnt signaling

Wnt signaling is essential for tissue homeostasis and its dysregulation causes cancer. Wnt ligands trigger signaling by activating Frizzled receptors (FZDRs), which belong to the G-protein coupled receptor superfamily. However, the mechanisms of G protein activation in Wnt signaling remain controversial. In this study, we demonstrate that FZDRs activate G proteins and trigger non-canonical Wnt signaling via the Dishevelled-binding protein, Daple. Daple contains a Gα-binding and activating (GBA) motif, which activates Gαi proteins and an adjacent domain that directly binds FZDRs, thereby linking Wnt stimulation to G protein activation. This triggers non-canonical Wnt responses, that is, suppresses the β-catenin/TCF/LEF pathway and tumorigenesis, but enhances PI3K-Akt and Rac1 signals and tumor cell invasiveness. In colorectal cancers, Daple is suppressed during adenoma-to-carcinoma transformation and expressed later in metastasized tumor cells. Thus, Daple activates Gαi and enhances non-canonical Wnt signaling by FZDRs, and its dysregulation can impact both tumor initiation and progression to metastasis. DOI: http://dx.doi.org/10.7554/eLife.07091.001

A robust genomic signature for the detection of colorectal cancer patients with microsatellite instability phenotype and high mutation frequency

Microsatellite instability (MSI) occurs in 10–20% of colorectal tumours and is associated with good prognosis. Here we describe the development and validation of a genomic signature that identifies colorectal cancer patients with MSI caused by DNA mismatch repair deficiency with high accuracy. Microsatellite status for 276 stage II and III colorectal tumours has been determined. Full‐genome expression data was used to identify genes that correlate with MSI status. A subset of these samples (n = 73) had sequencing data for 615 genes available. An MSI gene signature of 64 genes was developed and validated in two independent validation sets: the first consisting of frozen samples from 132 stage II patients; and the second consisting of FFPE samples from the PETACC‐3 trial (n = 625). The 64‐gene MSI signature identified MSI patients in the first validation set with a sensitivity of 90.3% and an overall accuracy of 84.8%, with an AUC of 0.942 (95% CI, 0.888–0.975). In the second validation, the signature also showed excellent performance, with a sensitivity 94.3% and an overall accuracy of 90.6%, with an AUC of 0.965 (95% CI, 0.943–0.988). Besides correct identification of MSI patients, the gene signature identified a group of MSI‐like patients that were MSS by standard assessment but MSI by signature assessment. The MSI‐signature could be linked to a deficient MMR phenotype, as both MSI and MSI‐like patients showed a high mutation frequency (8.2% and 6.4% of 615 genes assayed, respectively) as compared to patients classified as MSS (1.6% mutation frequency). The MSI signature showed prognostic power in stage II patients (n = 215) with a hazard ratio of 0.252 (p = 0.0145). Patients with an MSI‐like phenotype had also an improved survival when compared to MSS patients. The MSI signature was translated to a diagnostic microarray and technically and clinically validated in FFPE and frozen samples. Copyright

High HSP27 and HSP70 expression levels are independent adverse prognostic factors in primary resected colon cancer

BackgroundThe expression of Heat Shock Proteins (HSPs) is increased in various cancers and has been shown to correlate with biological tumor behaviour. This study aimed to investigate the impact of HSP70, HSP60 and HSP27 expression in colon cancer.Material and methodsHSP expression was determined by immunohistochemistry on a tissue microarray with 355 primary resected colon carcinomas of all stages. Expression patterns were correlated with pathologic features (UICC pTNM category, tumor grading) and survival.ResultsExpression of HSP27, HSP60 and HSP70 ranged from negative to high. There was no correlation between HSP27, HSP60 and HSP70 expression among each other and with UICC pT category, presence of lymph node or distant metastases or tumor grading. High HSP70 expression was associated with worse overall survival (p < 0.001) and was an independent prognostic factor (p = 0.004) in multivariate analysis including the pathological parameters mentioned above. For patients without lymph node or distant metastases (UICC stages I/II) and with complete tumor excision, HSP70 expression was the only independent prognostic factor for survival (p = 0.001) and superior to UICC pT category. In left sided UICC stage I/II carcinomas, high HSP27 expression also had adverse prognostic impact and was an independent prognostic factor (p = 0.016) besides HSP70 (p = 0.002).ConclusionHigh HSP70 and HSP27 expression is associated with worse clinical outcome in colon cancer. Determination of tumoral HSP70 and HSP27 may be used as additional biomarker for risk stratification especially for UICC stage I/II patients.

Right Sided Colon Cancer as a Distinct Histopathological Subtype with Reduced Prognosis

Background/Aims: Recent data suggest that tumors of the right and left colon should be distinguished as they differ in clinical and molecular characteristics. Methods: A total of 1,319 patients who underwent surgical resection for colon cancer (CC) were investigated. Tumors between the ileocecal valve and the hepatic flexure were classified as right CC (RCC), tumors between the splenic flexure and the rectum as left CC (LCC). Results: RCC revealed a higher cause-specific mortality risk (hazard ratio 1.36, 95% CI 1.10-1.68, p = 0.005) and lower 5-year cause-specific (RCC 64.9%, 95% CI 60.4-69.4, LCC 70.7%, 95% CI 67.2-74.2, p = 0.032) and disease-free (RCC 56.0%, 95% CI 51.5-60.5, LCC 59.9%, 95% CI 56.2-63.6, p = 0.025) survival rates. RCCs were more often microsatellite instable (RCC 37.2%, LCC 13.0%, p < 0.001) and more often showed KRAS (RCC 42.5%, LCC 18.9%, p = 0.001) and BRAF mutations (RCC 26.6%, LCC 3.2%, p < 0.001). Conclusion: RCC and LCC differ significantly regarding clinical, histopathological and molecular genetic features and can be considered as distinct entities. The reduced prognosis of RCC may be caused by higher rates of microsatellite instability, KRAS and BRAF mutations.

The E3 ligase RNF43 inhibits Wnt signaling downstream of mutated β-catenin by sequestering TCF4 to the nuclear membrane

Cancer-associated mutations in RNF43 prevent its inhibitory actions on the Wnt–β-catenin pathway in the nucleus. RNF43 halts Wnt at the nucleus Wnt signaling is critical to development and is often reactivated in cancer. The E3 ubiquitin ligase RNF43 inhibits Wnt–β-catenin signaling. Rather than promoting the degradation of cell surface Wnt receptors, Loregger et al. found that RNF43 sequestered TCF4, a β-catenin partner and transcription factor, at the nuclear membrane through a mechanism independent of its E3 ligase function. When expressed in frog embryos, RNF43 bearing mutations like those found in human gastrointestinal tumors increased Wnt signaling. Coexpression of wild-type RNF43 suppressed Wnt signaling even in cells with a constitutively active mutant of β-catenin, indicating tumor-suppressive activity of RNF43 functioning downstream of β-catenin. Given its fundamental role in development and cancer, the Wnt–β-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of the Frizzled family. We detected endogenous RNF43 in the nucleus of human intestinal crypt and colon cancer cells. We found that RNF43 physically interacted with T cell factor 4 (TCF4) in cells and tethered TCF4 to the nuclear membrane, thus silencing TCF4 transcriptional activity even in the presence of constitutively active mutants of β-catenin. This inhibitory mechanism was disrupted by the expression of RNF43 bearing mutations found in human gastrointestinal tumors, and transactivation of the Wnt pathway was observed in various cells and in Xenopus embryos when the RING domain of RNF43 was mutated. Our findings indicate that RNF43 inhibits the Wnt pathway downstream of oncogenic mutations that activate the pathway. Mimicking or enhancing this inhibitory activity of RNF43 may be useful to treat cancers arising from aberrant activation of the Wnt pathway.