Die Rolle des EGR/NAB2/p130Cas Signaltransduktions-Netzwerks in der Progression und Therapieresistenzentwicklung von Tumoren – Identifikation von Biomarkern und therapeutischen Zielen

Das breast cancer antiestrogen resistance 1 (BCAR1) -Gen kodiert für das Adapterprotein p130 Crk-associated substrate (Cas), welches als Knotenpunkt für Signalprotein-Komplexe dient, die Signaltransduktionswege kontrollieren, welche in Tumorzellen häufig dereguliert sind. Unter anderem interagiert p130Cas mit den Kinasen der Src-Familie, der focal adhesion kinase (FAK) und phosphoinositide 3-kinase (PI3K) und ist dadurch an der Kontrolle der Aktivität der rat sarcoma (RAS) /extracellular signal regulated kinase (ERK), PI3K und c-Jun N-terminal kinases (JNK) Signalwege beteiligt. Eine starke Überexpression oder Überaktivierung von p130Cas, die mit Tumorprogression und Therapieresistenz einherging, wurde zunächst in Mammakarzinomen und in den letzten Jahren auch in anderen Tumorentitäten beschrieben. Im Rahmen des Habilitationsprojektes wurden die Mechanismen der Überexpression von p130Cas und der damit assoziierten Therapie-Resistenz-Entstehung in verschiedenen Krebsentitäten untersucht. Außerdem wurden Ansätze zur zielgerichteten p130Cas-Inhibition auf ihre Wirksamkeit geprüft. Es konnte ein Regulations-Netzwerk aus den Transkriptionsfaktoren der early growth response (EGR) -Familie, des EGR-Ko-Regulators NGFI-A binding protein 2 (NAB2) und p130Cas identifiziert werden. Vor allem die starke NAB2-Expression konnte als eine der Ursachen der konstitutiven p130Cas-Expression in Anti-Östrogen-resistenten Zellen/Patientenproben ausgemacht werden. Des Weiteren konnte die Bedeutung des EGR/NAB2/p130Cas-Systems bei der Antwort auf Differenzierungstherapie in Kombination mit NF-B-Inhibition in einem Modell der Akuten Promyelozyten Leukämie (APL) gezeigt werden. Hier scheint p130Cas als Überlebensfaktor zu wirken. In kolorektalen Karzinomen (KRK) wurde ein signifikant erhöhtes Sterberisiko bei BCAR1-exprimierenden rechtsseitigen, Stadium I/II, MSS oder BRAF-mutierten KRK gefunden. Außerdem konnte durch Herunterregulierung der BCAR1/p130Cas-Expression ein verbessertes Ansprechen auf FOLFIRI (Folinsäure/5-FU/Irinotecan) erreicht werden. Diese Ergebnisse verdeutlichen die Rolle von p130Cas und NAB2 als therapeutische Ziele bei Hormonrezeptor-positivien (HR+) Mammakarzinomen und APL, sowie von p130Cas als Prognosefaktor und Behandlungsziel bei bestimmten Subtypen von KRK. Ein weiterer Aspekt der Habilitationsarbeit war die Identifikation von neuen Biomarkern und therapeutischen Zielen für Fernmetastasen bei prämenopausalen Patientinnen mit frühem Hormon-Rezeptor-positivem (HR+) /HER2-negativem Brustkrebs. Durch RNA-Expressionsprofile und Korrelationen mit klinischen Parametern konnten vier Signaturen (risk of recurrence (ROR), progesteron receptor, claudin-low und mammary stemness) mit dem Metastasen-freien Überleben assoziiert und LDL receptor related protein 2 (LRP2), integrin binding Sialoprotein (IBSP) und signal peptide, CUB domain, and EGF-like domain-containing 2 (SCUBE2) als unabhängige prognostische Faktoren identifiziert werden. Zusammenfassend konnte die Rolle von p130Cas als therapeutisches Ziel, als Resistenz-vermittelnder Faktor und als prognostischer Biomarker in verschiedenen Malignitäten bekräftigt werden. Des Weiteren konnten prognostische Gensignaturen und Einzelgenmarker für Fernmetastasen bei prämenopausalem frühen HR+/HER2- Brustkrebs identifiziert werden, die in der zukünftigen klinischen Praxis Anwendung finden könnten

The DNA-polymorphism rs849142 is associated with skin toxicity induced by targeted anti-EGFR therapy using cetuximab

Skin toxicity (ST) is a frequent adverse effect (AE) in anti-epidermal growth factor receptor (EGFR)-targeted treatment of metastatic colorectal cancer (mCRC) resulting in decreased quality of life and problems in clinical management. We wanted to identify biomarkers predicting ST in this setting and focused on 70 DNA polymorphisms associated with acne, the (immunoglobulin fragment crystallizable region) Fcγ-receptor pathway, and systemic lupus erythematosus (SLE) applying next-generation-sequencing (NGS). For the analysis patients with mCRC treated with cetuximab were selected from the FIRE-3 study. A training group consisting of the phenotypes low (1) - and high-grade (3) ST (n = 16) and a validation group (n = 55) representing also the intermediate grade (2) were genotyped and investigated in a genotype-phenotype association analysis. The single nucleotide polymorphism (SNP) rs849142 significantly associated with ST in both the training- (p < 0.01) and validation-group (p = 0.04). rs849142 is located in an intron of the juxtaposed with another zinc finger protein 1 (JAZF1) gene. Haplotype analysis demonstrated significant linkage disequilibrium of rs849142 with JAZF1. Thus, rs849142 might be a predictive biomarker for ST in anti-EGFR treated mCRC patients. Its value in the clinical management of AE has to be validated in larger cohorts

Biomarker alterations associated with distinct patterns of metastatic spread in colorectal cancer

Metastatic spread is the most important life-threatening feature of colorectal cancer and is supposed to be mainly driven by alterations in different carcinogenic pathways. The present study compared mutation and expression profiles of distinctive biomarkers in colorectal cancer patients with different clinical metastatic patterns. As for a case-control study, patients were matched according to T category, grading and primary tumour site. Overall, 246 patients with either exclusive lung metastasis (N = 82), exclusive liver metastasis (N = 82) or non-metastatic colorectal cancer (N = 82) were identified. Paraffin-embedded specimens were examined for mutations in the RAS and RAF genes and for the expression of β-catenin and CD133. Clinical endpoints were presence or absence of distant metastasis, formation of metastasis in lungs versus the liver and survival. MAPK pathway mutations in either the KRAS, NRAS or BRAF gene were associated with the development of lung metastasis (63.4%) compared to the control group (47.6%; p = 0.04). MAPK pathway alterations plus high β-catenin expression were associated with metastasis to the lungs but not to the liver (28.0% vs. 13.4%; p = 0.02). High CD133 expression correlated with the development of liver metastasis compared to the control group (30.5% vs. 14.6%; p = 0.02). This data indicates that different patterns of distant spread are associated with specific biomarker alterations and may represent different molecular subtypes of colorectal cancer. However, underlying mechanisms of metastasis formation in different anatomic sites remains unclear. Since knowledge of the anticipated site of distant spread would substantially impact clinical management, further research is needed to identify solid biomarkers for different metastatic patterns

Mixed large cell neuroendocrine carcinoma and squamous cell carcinoma of the colon: detailed molecular characterisation of two cases indicates a distinct colorectal cancer entity

We present two rare cases of mixed large cell neuroendocrine carcinoma and squamous cell carcinoma of the colon. A literature search revealed only three published cases with similar histology but none of these reports provided profound molecular and mutational analyses. Our two cases exhibited a distinct, colon-like immunophenotype with strong nuclear CDX2 and beta-catenin expression in more than 90% of the tumour cells of both components. We analysed the two carcinomas regarding microsatellite stability, RAS, BRAF and PD-L1 status. In addition, next-generation panel sequencing with Ion AmpliSe (TM) psi Cancer Hotspot Panel v2 was performed. This approach revealed mutations in FBXW7, CTNNB1 and PIK3CA in the first case and FBXW7 and RB1 mutations in the second case. We looked for similar mutational patterns in three publicly available colorectal adenocarcinoma data sets, as well as in collections of colorectal mixed neuroendocrine-non-neuroendocrine neoplasms (MiNENs) and colorectal neuroendocrine carcinomas. This approach indicated that the FBXW7 point mutation, without being accompanied by classical adenoma-carcinoma sequence mutations, such as APC, KRAS and TP53, likely occurs at a relatively high frequency in mixed neuroendocrine and squamous cell carcinoma and therefore may be characteristic for this rare tumour type. FBXW7 codifies the substrate recognition element of an ubiquitin ligase, and inactivating FBXW7 mutations lead to an exceptional accumulation of its target beta-catenin which results in overactivation of the Wnt-signalling pathway. In line with previously described hypotheses of de-differentiation of colon cells by enhanced Wnt-signalling, our data indicate a crucial role for mutant FBXW7 in the unusual morphological switch that determines these rare neoplasms. Therefore, mixed large cell neuroendocrine and a squamous cell carcinoma can be considered as a distinct carcinoma entity in the colon, defined by morphology, immunophenotype and distinct molecular genetic alteration(s)

Patients with colorectal cancer and brain metastasis: The relevance of extracranial metastatic patterns predicting time intervals to first occurrence of intracranial metastasis and survival

To investigate the predictive impact of extracranial metastatic patterns on course of disease and survival in patients with colorectal cancer (CRC) and brain metastasis (BM). A total of 228 patients (134 male [59%], 94 female [41%]) with histologically proven CRC and BM were classified into different groups according to extracranial metastatic patterns. Time intervals to metastatic events and survival times from initial CRC diagnosis, extracranial and intracranial metastasis were analyzed. Extracranial organs mostly affected were liver (102 of 228 [44.7%]) and lung (96 of 228 [42.1%]). Liver and lung metastasis were detected in 31 patients (13.6%). Calculated over the entire course of disease, patients with lung metastasis showed longer OS than patients with liver metastasis or patients without lung metastasis (43.9 vs. 34.6 [p=0.002] vs. 35.0 months [p=0.002]). From the date of initial CRC diagnosis, lung metastasis occurred later in CRC history than liver metastasis (24.3 vs. 7.5 months). Once lung metastasis was diagnosed BM occurred faster than in patients with liver metastasis (15.8 vs. 26.0 months; Δ 10.2 months). Accordingly, OS from the diagnosis of liver metastasis was longer than from lung metastasis (27.1 vs. 19.6 months [p=0.08]). Once BM was present patients with lung metastasis lived longer than patients with liver metastasis (3.8 vs. 1.1 months [p=0.028]). Shortest survival times in all survival categories analyzed revealed patients with concurrent liver and lung metastasis. Patients with CRC and BM form a heterogenous cohort where EM to liver or lung predict survival

An integrated molecular risk score early in life for subsequent childhood asthma risk.

BACKGROUND Numerous children present with early wheeze symptoms, yet solely a subgroup develops childhood asthma. Early identification of children at risk is key for clinical monitoring, timely patient-tailored treatment, and preventing chronic, severe sequelae. For early prediction of childhood asthma, we aimed to define an integrated risk score combining established risk factors with genome-wide molecular markers at birth, complemented by subsequent clinical symptoms/diagnoses (wheezing, atopic dermatitis, food allergy). METHODS Three longitudinal birth cohorts (PAULINA/PAULCHEN, n = 190 + 93 = 283, PASTURE, n = 1133) were used to predict childhood asthma (age 5-11) including epidemiological characteristics and molecular markers: genotype, DNA methylation and mRNA expression (RNASeq/NanoString). Apparent (ap) and optimism-corrected (oc) performance (AUC/R2) was assessed leveraging evidence from independent studies (Naïve-Bayes approach) combined with high-dimensional logistic regression models (LASSO). RESULTS Asthma prediction with epidemiological characteristics at birth (maternal asthma, sex, farm environment) yielded an ocAUC = 0.65. Inclusion of molecular markers as predictors resulted in an improvement in apparent prediction performance, however, for optimism-corrected performance only a moderate increase was observed (upto ocAUC = 0.68). The greatest discriminate power was reached by adding the first symptoms/diagnosis (up to ocAUC = 0.76; increase of 0.08, p = .002). Longitudinal analysis of selected mRNA expression in PASTURE (cord blood, 1, 4.5, 6 years) showed that expression at age six had the strongest association with asthma and correlation of genes getting larger over time (r = .59, p < .001, 4.5-6 years). CONCLUSION Applying epidemiological predictors alone showed moderate predictive abilities. Molecular markers from birth modestly improved prediction. Allergic symptoms/diagnoses enhanced the power of prediction, which is important for clinical practice and for the design of future studies with molecular markers

p130Cas Is Correlated with EREG Expression and a Prognostic Factor Depending on Colorectal Cancer Stage and Localization Reducing FOLFIRI Efficacy

p130 Crk-associated substrate (p130Cas) is associated with poor prognosis and treatment resistance in breast and lung cancers. To elucidate p130Cas functional and clinical role in colorectal cancer (CRC) progression/therapy resistance, we performed cell culture experiments and bioinformatic/statistical analyses of clinical data sets. p130Cas expression was associated with poor survival in the cancer genome atlas (TCGA) data set. Knockdown/reconstitution experiments showed that p130Cas drives migration but, unexpectedly, inhibits proliferation in CRC cells. TCGA data analyses identified the growth factor epiregulin (EREG) as inversely correlated with p130Cas. p130Cas knockdown and simultaneous EREG treatment further enhanced proliferation. RNA interference and EREG treatment experiments suggested that p130Cas/EREG limit each other’s expression/activity. Inverse p130Cas/EREG Spearman correlations were prominent in right-sided and earlier stage CRC. p130Cas was inducible by 5-fluorouracil (5-FU) and FOLFIRI (folinic acid, 5-FU, irinotecan), and p130Cas and EREG were upregulated in distant metastases (GSE121418). Positive p130Cas/EREG correlations were observed in metastases, preferentially in post-treatment samples (especially pulmonary metastases). p130Cas knockdown sensitized CRC cells to FOLFIRI independent of EREG treatment. RNA sequencing and gene ontology analyses revealed that p130Cas is involved in cytochrome P450 drug metabolism and epithelial-mesenchymal transition. p130Cas expression was associated with poor survival in right-sided, stage I/II, MSS (microsatellite stable), or BRAF-mutated CRC. In summary, p130Cas represents a prognostic factor and potential therapeutic target in CRC

The association of immunosurveillance and distant metastases in colorectal cancer

BACKGROUND Colorectal cancer (CRC) is the third most common malignancy worldwide, but the key driver to distant metastases is still unknown. This study aimed to elucidate the link between immunosurveillance and organotropism of metastases in CRC by evaluating different gene signatures and pathways. MATERIAL AND METHODS CRC patients undergoing surgery at the Department of General, Visceral and Transplantation Surgery at the Ludwig-Maximilian University Hospital Munich (Munich, Germany) were screened and categorized into M0 (no distant metastases), HEP (liver metastases) and PER (peritoneal carcinomatosis) after a 5-year follow-up. Six patients of each group were randomly selected to conduct a NanoString analysis, which includes 770 genes. Subsequently, all genes were further analyzed by gene set enrichment analysis (GSEA) based on seven main cancer-associated databases. RESULTS Comparing HEP vs. M0, the gene set associated with the Toll-like receptor (TLR) cascade defined by the Reactome database was significantly overrepresented in HEP. HSP90B1, MAPKAPK3, PPP2CB, PPP2R1A were identified as the core enrichment genes. The immunologic signature pathway GSE6875_TCONV_VS_FOXP3_KO_TREG_DN with FOXP3 as downstream target was significantly overexpressed in M0. RB1, TMEM 100, CFP, ZKSCAN5, DDX50 were the core enrichment genes. Comparing PER vs. M0 no significantly differentially expressed gene signatures were identified. CONCLUSION Chronic inflammation might enhance local tumor growth. This is the first study identifying immune related gene sets differentially expressed between patients with either liver or peritoneal metastases. The present findings suggest that the formation of liver metastases might be associated with TLR-associated pathways. In M0, a high expression of FOXP3 + tumor infiltrating lymphocytes (TILs) seemed to prevent at least in part metastases. Thus, these correlative findings lay the cornerstone to further studies elucidating the underlying mechanisms of organotropism of metastases

Mixed large cell neuroendocrine carcinoma and squamous cell carcinoma of the colon: detailed molecular characterisation of two cases indicates a distinct colorectal cancer entity

We present two rare cases of mixed large cell neuroendocrine carcinoma and squamous cell carcinoma of the colon. A literature search revealed only three published cases with similar histology but none of these reports provided profound molecular and mutational analyses. Our two cases exhibited a distinct, colon-like immunophenotype with strong nuclear CDX2 and beta-catenin expression in more than 90% of the tumour cells of both components. We analysed the two carcinomas regarding microsatellite stability, RAS, BRAF and PD-L1 status. In addition, next-generation panel sequencing with Ion AmpliSe (TM) psi Cancer Hotspot Panel v2 was performed. This approach revealed mutations in FBXW7, CTNNB1 and PIK3CA in the first case and FBXW7 and RB1 mutations in the second case. We looked for similar mutational patterns in three publicly available colorectal adenocarcinoma data sets, as well as in collections of colorectal mixed neuroendocrine-non-neuroendocrine neoplasms (MiNENs) and colorectal neuroendocrine carcinomas. This approach indicated that the FBXW7 point mutation, without being accompanied by classical adenoma-carcinoma sequence mutations, such as APC, KRAS and TP53, likely occurs at a relatively high frequency in mixed neuroendocrine and squamous cell carcinoma and therefore may be characteristic for this rare tumour type. FBXW7 codifies the substrate recognition element of an ubiquitin ligase, and inactivating FBXW7 mutations lead to an exceptional accumulation of its target beta-catenin which results in overactivation of the Wnt-signalling pathway. In line with previously described hypotheses of de-differentiation of colon cells by enhanced Wnt-signalling, our data indicate a crucial role for mutant FBXW7 in the unusual morphological switch that determines these rare neoplasms. Therefore, mixed large cell neuroendocrine and a squamous cell carcinoma can be considered as a distinct carcinoma entity in the colon, defined by morphology, immunophenotype and distinct molecular genetic alteration(s)

Molecular Prognostic Factors for Distant Metastases in Premenopausal Patients with HR+/HER2− Early Breast Cancer

Molecular factors that drive metastasis in premenopausal patients with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2−), early breast cancer (EBC) are largely unknown. To identify markers/signatures contributing to metastasis, we analyzed molecular changes in tumors from premenopausal patients who developed metastasis (M1) and who did not (M0). Ninety-seven premenopausal patients with HR+/HER2− EBC were included (M1, n = 48, median distant metastasis-free survival (DMFS): 54 (7–184) months; M0, n = 49, median follow-up: 149 (121–191) months). Gene expression profiling on tumor RNA (Breast Cancer 360TM panel, Nanostring) was performed, followed by comprehensive bioinformatic and statistical analyses. Significantly enhanced ROR (risk of recurrence) scores and reduced signature scores of PGR (progesterone receptor), claudin-low, and mammary stemness were determined in M1. These differences were significantly associated with shorter DMFS in univariate survival analyses. Gene set enrichment analysis showed an enriched mTORC1 pathway in M1. Moreover, a metastasis signature of 19 differentially expressed genes (DEGs) that were DMFS-related was defined. Multivariate analysis including the four signatures, 19 DEGs, pN, and pT status, identified LRP2, IBSP, and SCUBE2 as independent prognostic factors. We identified prognostic gene signatures and single-gene markers for distant metastasis in premenopausal HR+/HER2− EBC potentially applicable in future clinical practice