In April 2013 our group published a review on predictive molecular pathology in this journal. Although only 2 years have passed many new facts and stimulating developments have happened in diagnostic ...molecular pathology rendering it worthwhile to present an up-date on this topic. A major technical improvement is certainly given by the introduction of next-generation sequencing (NGS; amplicon, whole exome, whole genome) and its application to formalin-fixed paraffin-embedded (FFPE) tissue in routine diagnostics. Based on this 'revolution' the analyses of numerous genetic alterations in parallel has become a routine approach opening the chance to characterize patients' malignant tumors much more deeply without increasing turn-around time and costs. In the near future this will open new strategies to apply 'off-label' targeted therapies, e.g. for rare tumors, otherwise resistant tumors etc. The clinically relevant genetic aberrations described in this review include mutation analyses of RAS (KRAS and NRAS), BRAF and PI3K in colorectal cancer, KIT or PDGFR alpha as well as BRAF, NRAS and KIT in malignant melanoma. Moreover, we present several recent advances in the molecular characterization of malignant lymphoma. Beside the well-known mutations in NSCLC (EGFR, ALK) a number of chromosomal aberrations (KRAS, ROS1, MET) have become relevant. Only very recently has the clinical need for analysis of BRCA1/2 come up and proven as a true challenge for routine diagnostics because of the genes' special structure and hot-spot-free mutational distribution. The genetic alterations are discussed in connection with their increasingly important role in companion diagnostics to apply targeted drugs as efficient as possible. As another aspect of the increasing number of druggable mutations, we discuss the challenges personalized therapies pose for the design of clinical studies to prove optimal efficacy particularly with respect to combination therapies of multiple targeted drugs and conventional chemotherapy. Such combinations would lead to an extremely high complexity that would hardly be manageable by applying conventional study designs for approval, e.g. by the FDA or EMA. Up-coming challenges such as the application of methylation assays and proteomic analyses on FFPE tissue will also be discussed briefly to open the door towards the ultimate goal of reading a patients' tissue as 'deeply' as possible. Although it is yet to be shown, which levels of biological information are most informative for predictive pathology, an integrated molecular characterization of tumors will likely offer the most comprehensive view for individualized therapy approaches. To optimize cancer treatment we need to understand tumor biology in much more detail on morphological, genetic, proteomic as well as epigenetic grounds. Finally, the complex challenges on the level of drug design, molecular diagnostics, and clinical trials make necessary a close collaboration among academic institutions, regulatory authorities and pharmaceutical companies.
Colon cancer cells frequently carry mutations that activate the β-catenin and mitogen-activated protein kinase (MAPK) signaling cascades. Yet how oncogenic alterations interact to control cellular ...hierarchies during tumor initiation and progression is largely unknown. We found that oncogenic BRAF modulates gene expression associated with cell differentiation in colon cancer cells. We therefore engineered a mouse with an inducible oncogenic BRAF transgene, and analyzed BRAF effects on cellular hierarchies in the intestinal epithelium in vivo and in primary organotypic culture. We demonstrate that transgenic expression of oncogenic BRAF in the mouse strongly activated MAPK signal transduction, resulted in the rapid development of generalized serrated dysplasia, but unexpectedly also induced depletion of the intestinal stem cell (ISC) pool. Histological and gene expression analyses indicate that ISCs collectively converted to short-lived progenitor cells after BRAF activation. As Wnt/β-catenin signals encourage ISC identity, we asked whether β-catenin activity could counteract oncogenic BRAF. Indeed, we found that intestinal organoids could be partially protected from deleterious oncogenic BRAF effects by Wnt3a or by small-molecule inhibition of GSK3β. Similarly, transgenic expression of stabilized β-catenin in addition to oncogenic BRAF partially prevented loss of stem cells in the mouse intestine. We also used BRAF(V637E) knock-in mice to follow changes in the stem cell pool during serrated tumor progression and found ISC marker expression reduced in serrated hyperplasia forming after BRAF activation, but intensified in progressive dysplastic foci characterized by additional mutations that activate the Wnt/β-catenin pathway. Our study suggests that oncogenic alterations activating the MAPK and Wnt/β-catenin pathways must be consecutively and coordinately selected to assure stem cell maintenance during colon cancer initiation and progression. Notably, loss of stem cell identity upon induction of BRAF/MAPK activity may represent a novel fail-safe mechanism protecting intestinal tissue from oncogene activation.
The increasing importance of targeting drugs in the treatment of several tumor entities (breast, colon, lung, malignant melanoma (MM), lymphoma, and so on) and the necessity of a companion diagnostic ...(human epidermal growth factor receptor 2, Kirsten rat sarcoma viral oncogene, epidermal growth factor receptor (EGFR), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), and so on) is leading to new challenges for surgical pathology. As all the biomarkers to be specifically detected are tissue based, a precise and reliable diagnostic is absolutely crucial. To meet this challenge, surgical pathology has adapted a number of molecular methods (semi-quantitative immunohistochemistry, fluorescence in situ hybridization), PCR and its multiple variants, (pyro/Sanger) sequencing, next-generation sequencing, DNA-arrays, methylation analyses, and so on) to be applicable for formalin-fixed paraffin-embedded (FFPE) tissue. To read a patients' tissue as 'deeply' as possible and to obtain information on morphological, genetic, proteomic as well as epigenetic background is the actual task of pathologists and molecular biologists in order to provide the clinicians with information relevant for individualized medicine. The intensified cooperation of clinicians and pathologists will provide the basis of improved clinical drug selection as well as guide development of new cancer gene therapies and molecularly targeted drugs by research units and the pharmaceutical industry. This review will give some information on (1) biomarker detection methods adapted to FFPE tissue, (2) the potency of predictive pathology in tumor detection and treatment and (3) the implications of pathology on the development of new drugs in molecularly targeted and gene therapies.
The transcription factor YBX1 can act as a mediator of signals transmitted via the EGFR-RAS-MAPK axis. YBX1 expression has been associated with tumor progression and prognosis in multiple types of ...cancer. Immunohistochemical studies have revealed dependency between YBX1 expression and individual EGFR family members. We analyzed YBX1 and EGFR family proteins in a colorectal cancer (CRC) cohort and provide functional analyses of YBX1 in the context of EGFR-RAS-MAPK signaling. Immunohistochemistry for YBX1 and EGFR family receptors with two antibodies for YBX1 and EGFR were performed and related to clinicopathological data. We employed Caco2 cells expressing an inducible KRASV12 gene to determine effects on localization and levels of YBX1. Mouse xenografts of Caco2-KRASV12 cells were used to determine YBX1 dynamics in a tissue context. The two different antibodies against YBX1 showed discordant immunohistochemical stainings in cell culture and clinical specimens. Expression of YBX1 and EGFR family members were not correlated in CRC. Analysis of Caco2 xenografts displayed again heterogeneity of YBX1 staining with both antibodies. Our results suggest that YBX1 is controlled via complex regulatory mechanisms involving tumor stroma interaction and signal transduction processes. Our study highlights that YBX1 antibodies have different specificities, advocating their use in a combined manner.
PDF
