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Review Article

Free Access

Molecular Pathology: A Requirement for Precision Medicine in Cancer

Dietel M.

Author affiliations

Institute of Pathology, Charité, University Medicine Berlin, Berlin, Germany

Corresponding Author

Prof. Dr. Manfred Dietel

Institute of Pathology

Charité, University Medicine Berlin

Charitéplatz 1, 10117 Berlin, Germany

manfred.dietel@charite.de

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Oncol Res Treat 2016;39:804-810

Abstract

The increasing importance of targeting drugs and check-point inhibitors in the treatment of several tumor entities (breast, colon, lung, malignant melanoma, lymphoma, etc.) and the necessity of a companion diagnostic (HER2, (pan)RAS, EGFR, ALK, BRAF, ROS1, MET, PD-L1, etc.) is leading to new challenges for surgical pathology. Since almost 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 (amplicon, whole exome, whole genome), DNA arrays, methylation analyses, etc.) to be applicable for formalin-fixed paraffin-embedded tissue. Reading a patient's tissue as ‘deeply' as possible and obtaining information on the morphological, genetic, proteomic and epigenetic background are the tasks of pathologists and molecular biologists and provide the clinicians with information relevant for precision medicine. Intensified cooperation between clinicians and pathologists will provide the basis of improved clinical drug selection and guide development of new cancer gene therapies and molecularly targeted drugs by research units and the pharmaceutical industry.

© 2016 S. Karger GmbH, Freiburg


Introduction

Targeted drugs and check-point inhibitors, in particular therapeutic antibodies, kinase inhibitors and poly(ADP-ribose) polymerase (PARP) inhibitors, are transforming current treatment strategies in oncology. Prior to application almost all of them require a so-called pre-therapeutic companion or complementary diagnostic to identify molecular alterations serving as targets. More precisely, a well-defined biomarker, often a characteristic genetic alteration or particular protein (over-)expression, that indicates the efficacy of the respective drug has to be identified in the tumor tissue of individual patients. These analyses are mostly done by predictive molecular pathology applying conventional or high-throughput techniques. This is the basis of personalized or precision medicine.

This new and extremely exciting field of medicine creates a number of challenges, which can be managed only by new ways of thinking and new organizational structures. An example is the scope of the interdisciplinary tumor board. At present, this comprises oncologists, radiologists, pathologists, surgeons and organ specialists, but needs to be extended to account for the clinically relevant molecular profiling information by including specially trained pathologists. It is pivotal to further integrate ‘molecular knowledge' into diagnostic pathology training.

Due to the complexity of new combination treatment, new designs of clinical studies have to be defined and approved by the regulatory authorities. Despite the increasing importance of molecular information for tumor classifications for the foreseeable future, the classical morphology-based WHO classification will remain in place, but will certainly be supplemented by genetic, proteomic, metabolomic and other data so that finally the combination of all the provided data will lead to an optimal strategy for patient care.

Methodological Considerations

Since formalin fixation and paraffin embedding (FFPE) is and will be the most widely used method for tissue fixation in the diagnostic setting, the high-throughput technologies have been adapted to the quality of DNA and RNA in FFPE tissue. This opens the door to extended molecular analyses for almost all tissues, including cytological preparations and even blood (liquid biopsy). It has to be emphasized that carefully controlled pre-analytical steps, standard operating procedures and bio-mathematic knowledge are of absolute importance. In addition, the laboratories working in clinical diagnostics should constantly perform external quality controls.

Next Generation Sequencing and Liquid Biopsy

Next generation sequencing (NGS) has recently been adapted to diagnostic requirements, and a growing number of diagnostic laboratories and companies offer a variety of NGS-based services. However, different NGS-based approaches such as amplicon-based NGS, whole genome (WGS) or whole exome NGS (WES) exist and are, as yet, suited for different applications. With respect to tumor diagnostics, amplicon-based NGS is predominantly applied in molecular pathology. This is due to the fact that: (1) the vast majority of sequence information generated by WGS and WES cannot be translated into clinical intervention, (2) WGS and WES are not ready for FFPE-based NGS, (3) bioinformatics for WGS and WES data requires both germ line and tumor data, (4) the sequencing coverage (coverage describes the average number of NGS reads that align to known reference bases) appears to be too low, especially in cases of low tumor cell content, (5) the turn-around times are too long in a diagnostic setting, and (6) the costs (consumables, data analysis, bioinformatics and data handling) are much too high.

By contrast, amplicon-based NGS strategies can overcome most of these disadvantages. Since the first reports showing that it is feasible to use small amounts of FFPE-derived DNA for NGS-based analyses [2,] several protocols have been established that adapt amplicon length as well as DNA extraction methods to the specific requirements of FFPE tissue and biopsy samples.

Perspectives on Proteomics: The Next Level of Molecular Tumor Profiling?

Pan-cancer mutational profiling efforts by the consortia TCGA (the cancer genome atlas) or ICGC (international cancer genome consortium) and many other groups have demonstrated a high complexity of the mutational landscapes in most cancers. The average squamous cell lung cancer, for instance, has been shown to harbor over 800 genetic aberrations including exon mutations, copy number variations and rearrangements. Even though only a minority of the observed mutations are believed to belong to the group of driver mutations that are causally linked with cancer pathology, the functional and, therefore, clinical implications of many less frequent mutations for individual patients are largely unknown. Even if a (rare) mutation is known to affect protein function, in principle, modulations of such effects through other mutations or epigenetic as well as posttranscriptional and posttranslational regulation may differ substantially among tumors. A solution would be to complement genomic with proteomic profiling of tumor tissue to help relating mutational profiles to protein changes as more direct indicators of tumor cell function. For a comprehensive functional characterization, the value of the protein-based approaches lies in providing a more global (simultaneous measurements of several thousand proteins) and unbiased view of the functional implications of mutational profiles and will likely play an important role in future molecular tumor profiling.

Molecular Diagnostics of Tumors (Molecular Classification)

The demands of clinicians on tissue-based diagnostic analyses of solid and hematological tumors are becoming more and more challenging. This is particularly true if therapeutical options need companion diagnostics. While pathology reports on tumor type, WHO code, pTNM status, dignity (i.e. benign, malignant, in situ carcinoma or borderline lesion), histogenesis and prognosis remain the skeleton of tumor classification, the predictive relevance is limited. This means that conventional histopathology often has to be refined by molecular data for selecting the optimal treatment. The most relevant examples are given below.

Breast Cancer

In breast cancer, the classical morphology-based classification has been largely replaced by the molecular classification [3]. Based on gene expression profiling and immunohistochemistry (IHC), at least 5 different subtypes can be distinguished: luminal A, luminal B/HER2 negative, luminal B/HER2 positive, HER2 positive/non-luminal, and triple negative. The molecular subtypes were introduced as the main classification system for clinical decisions in breast cancer at the St. Gallen conference 2011; this classification was slightly modified at the St Gallen 2013 meeting [4].

Breast cancer subtypes can be determined by gene expression analysis. However, in clinical practice, the standard approach is the immunohistochemical investigation of estrogen receptor, progesterone receptor and HER2. The differential diagnosis of luminal A versus luminal B tumors can be a challenge in some situations. Ki67 has been suggested as a marker for luminal A versus B tumors; however, the cut-points for Ki67 are still under debate and international efforts for further standardization are still ongoing.

Over the past few years, several diagnostic assays have been developed to define a low-risk group of luminal A tumors that have an excellent prognosis with endocrine therapy alone. The OncotypeDX [5] assay, the Endopredict [6] assay and the PAM50/Prosigna [7] test are based on mRNA analysis of gene expression in FFPE tissue and can be used to define low-risk tumors that might not need a chemotherapy treatment. Some tests (Endopredict [8] and Prosigna) have also been used to identify which patients would benefit from extended endocrine therapy of more than 5 years.

In addition to mRNA-based gene expression profiling, mutation-based classification is under evaluation in breast cancer. The most commonly mutated genes are p53 and PIK3CA. Tumors with PIK3CA mutations have recently been shown to have a reduced response to neoadjuvant anti-HER2 therapy; further validation studies are ongoing [9].

Ovarian Cancer

Sequence analysis of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 to reveal their genomic status is the first routinely performed molecular test for ovarian cancer. According to data provided during the TCGA project, high-grade serous carcinoma, the most frequent histological subtype of ovarian cancer (70%), shows germline mutations within BRCA1/2 in ca. 20% of primary tumors; an additional 6% show somatic mutations. BRCA mutations lead to a homologous recombination deficiency (HRD), which is characterized by a reduced ability to repair DNA double-strand breaks [10]. Although the inability to perform DNA repair adequately is the origin of an increased cancer risk, it is also the reason for the high sensitivity of BRCA-mutated tumors to agents that induce DNA single-strand breaks and thereby produce a synthetic lethality. In particular, BRCA-mutated ovarian cancer is highly sensitive to platinum-based chemotherapy, which seems to be the reason for the comparably favorable prognosis of this molecular tumor subtype [11]. PARP inhibitors are the most recently approved targeted therapeutics for ovarian cancer. In January 2015, the European Medicines Agency (EMA) approved the PARP inhibitor olaparib (Astra Zeneca) for maintenance therapy of recurrent platinum-sensitive high-grade serous ovarian, tubal or peritoneal carcinoma revealing germline or somatic BRCA mutations.

Colorectal Cancer

Antibody-mediated blockade of the epidermal growth factor receptor (EGFR) is a therapeutic option in the treatment of advanced colorectal cancer (CRC). However, in several clinical trials it was shown that only those patients with cancers bearing no mutation in the RAS gene benefit from EGFR-targeting antibodies such as cetuximab and panitumumab [12], [13]. Mutations of the gene result in a constitutional activation of the EGFR pathway and, thus, might completely abolish the effects of an upstream inhibition of EGFR. Therefore, exclusion of (K/N)RAS hot spot mutations in exon 2-4 have become mandatory for the application of EGFR-targeting antibodies in the therapy of advanced colon cancer.

Other genetic changes such as BRAF and PI3K mutations have also been shown to be associated with a lacking EGFR antibody response [14,] but the data generated in a different study [15] are too controversial to include these markers in routine predictive molecular testing and therapy decision making to date. It is foreseeable, however, that in the near future the number of markers predicting therapy response in CRC will rise with novel data generated from larger CRC cohorts and with the introduction of novel therapy strategies, which may include antibody targeting of BRAF.

Another application of molecular pathology in CRC refers to a principal genetic mechanism causing the disease. In approximately 10-15% of CRCs microsatellite instability (MSI), a consequence of DNA mismatch repair deficiency, is found. High MSI (MSI-H) is a hallmark alteration of hereditary nonpolyposis CRC/Lynch syndrome-associated tumors, but is also found in sporadic colon cancers [16]. MSI-H has a significant impact on tumor biology. This is reflected by a more favorable prognosis for this molecular CRC subtype and a lacking response of MSI-H CRCs to 5΄-fluorouracil monotherapy [17].

To date, knowledge of the MSI status is required to address 2 routine applications/requirements. First, following the revised Bethesda guidelines [18], colon cancer is tested for MSI to evaluate the possibility of Lynch syndrome. In combination with immunohistochemical analysis of the mismatch repair proteins (MLH1, MSH2, PMS2, and MSH6) and EPCAM [19], the molecularly determined MSI status guides further genetic counselling. Second, the MSI status is determined according to conventional histological grading in poorly differentiated cancers as well as in mucinous carcinoma and signet-ring cell carcinoma. Only in the absence of MSI-H is poor histological/high-grade differentiation (G3-4) considered a prognostic marker, while cancers with MSI-H are considered low grade, irrespective of the conventional histological appearance.

Non-Small Cell Lung Cancer

To date the detection of EGFR and anaplastic lymphoma kinase (ALK) alterations is the standard of molecular testing in non-small cell lung cancer (NSCLC) (∼15% EGFR, chromosome 7; ∼3% ALK, chromosome 2) [20]. Both mainly occur in advanced-stage adenocarcinomas (ADC). Although their frequency in squamous cell carcinoma (SCC) is very low, under certain conditions (e.g. young patients or non-smokers) testing should also be considered. KRAS and EGFR testing is performed almost always by NGS, and ALK testing mainly by fluorescence in situ hybridization (FISH) [21]. Recent studies demonstrated that a ‘carefully validated' ALK IHC is eligible for multicenter routine testing [22], at least as a screening tool and in samples that may be unsuitable for the FISH approach, e.g. very small amount of tumor cells, high amount of osseous tissue.

Further testing should be considered for ROS1 (chromosome 6, translocation, ∼0.5-2%, mainly ADC). Diagnosis is performed using FISH [23]. IHC and PCR are possible; however, so far these have not been standardized. In addition to several other ROS1 inhibitors, the drug crizotinib has recently been approved. Interestingly, crizotinib (an ALK/ROS1/cMET inhibitor) was originally designed as an MET inhibitor. Thus, testing for MET (chromosome 7, amplification/overexpression ∼10%) seems to be the next step [24]. MET alterations are more frequent in ADC than in SCC, and diagnosis is made by FISH (different scoring systems!) and IHC (be aware of discordant IHC/FISH results!) [24]. However, a clinical phase III trial (MET inhibitor) was stopped recently and its role will need further evaluation [25]. Nevertheless, MET plays an important role concerning EGFR therapy resistance [26].

Today EGFR, ALK and ROS1 are in the focus, and new drugs under investigation may target more than one alteration (see table 1 and page 441 in [27]). Further examples are on the way (representing encouraging studies) [28]: RET (chromosome 10; inversion/translocation; 1-2%, FISH); HER2 (chromosome 17; exon 20 insertion, deletion, 1-2%, PCR, but not protein expression as known in breast and gastric cancer); and BRAF (chromosome 7, activating point mutation V600E, fusion protein, 1-3%, PCR).

So far the data for SCC are not as encouraging as for ADC. The main 2 targets to test for are FGFR1 (chromosome 8, 20%, amplification, only 1% in ADC, detection by FISH) and DDR2 (chromosome 1, point mutation, 2%, PCR).

The implementation of NGS in the daily routine might help us identify different mutations in a single tumor (not only concerning the above-mentioned targets) in the context of tumor heterogeneity. This will not only broaden the spectrum of targeted therapy, but will also help to gain a better understanding of the questions of drug resistance, e.g. the role of T790M mutations. To detect and monitor the T790M mutation during the development of EGFR inhibitor resistance, the newly established liquid biopsies technique (mutation detection from blood) will facilitate the diagnostic procedure and can provide the basis for alternative target therapy, e.g. using osimertinib, a third generation tyrosine kinase inhibitor.

Malignant Melanoma

The incidence of malignant melanoma (mm) has increased rapidly in the past few years, partly due to changes in diagnostic criteria and improvement of screening methods, but also due to behavioral changes such as increased exposure to ultraviolet radiation [29]. A first breakthrough in the therapy of mm was achieved with the approval of several targeted therapies for patients with BRAF-mutated metastatic melanoma, e.g. vemurafenib and later dabrafenib [30]. About 50% of metastatic melanoma harbor a mutation in the oncogene BRAF, with V600E being the most common (75% of V600 mutations), followed by V600K (20%) and V600R.

The first results of the BRIM3 study revealed a prolonged overall survival of 13.9 months (vemurafenib) compared to 9.6 months (dacarbicine) in patients carrying BRAF mutations [31]. With these targeted therapies, 1 of the biggest challenges in the therapy of patients with BRAF-mutated tumors is still the primary and secondary resistance that most patients develop over time. The success of the therapy is often restricted by the activation of the MAPK signaling pathway in BRAF wild type cells, and the resulting activation of proliferation followed by a fast relapse [32].

Besides BRAF mutations, mm harbor several other oncogenic alterations, such as mutations in the GTPase NRAS and the tyrosine kinase KIT. An activating mutation in NRAS can be found in about 20% of all mm, which is mainly associated with a worse prognosis [33]. Since NRAS cannot be targeted directly, testing for NRAS alterations in mm initially became of interest as a potential predictive factor for downstream applications, e.g. for MEK inhibition.

KIT mutations in mm are rare. About 15% of mm harbor KIT mutations [34], mainly in exon 11 and exon 13. KIT inhibitors such as sunitinib, dasatinib and imatinib, although primarily not developed for the treatment of mm, turned out to be interesting therapy options for patients with KIT-positive melanoma.

Due to the limited long-term effect of BRAF inhibitors, efforts are being made to combine different strategies to improve therapy outcome. A promising approach is the complete inhibition of the MAPK pathway by combining BRAF and MEK inhibitors. The first results of a phase I/II study for the combination of dabrafenib and trametinib showed an increase of median progression-free survival from 5.8 months with dabrafenib alone to 9.4 months with the dabrafenib plus trametinib, and the combination showed even less toxicity compared to the treatment with 1 of the agents alone [35]. Furthermore, the use of ERK inhibitors is being tested for patients with MAPK pathway-dependent resistance to RAF or MEK inhibitors [36].

The newly discovered approach of treatment with targeted therapies combined with novel immunotherapies, e.g. with the anti-CTLA4 antibody ipilimumab or anti-PD-1 and anti-PD-L1 antibodies showed great promise for a prolonged therapeutic efficacy [37].

Cancer of Unknown Primary

In about 5% of tumors the anatomical site of origin cannot be determined despite extensive examinations [38]. These cancers are termed ‘cancer of unknown primary' (CUP). Histologically, the vast majority of CUPs present as carcinoma, mainly ADC, followed by low or undifferentiated carcinoma, SCC and neuro-endocrine carcinoma. The remaining ca. 10% of cases comprise melanoma, sarcoma, germ cell tumors and very rarely lymphoma [38]. Typically, at the time of diagnosis the patients present with metastases in multiple organs.

To determine the tissue of origin in CUP, clinical and pathological data as well as results from advanced imaging technologies are integrated, and in most instances are sufficient. In cases in which this procedure is not successful, molecular assays can be of help to provide the patient with a site-specific therapy. At present 2 multi-marker profiling assays for the determination of the tissue of origin in CUP are commercially available. The CancerType ID assay (Biotheranostics, CA, USA) analyses the expression levels of 87 candidate and 5 housekeeping genes by qPCR. The gene expression signature is then compared against a proprietary database consisting of 30 tumor types (54 subtypes). The Cancer Origin Test (Rosetta Genomics) profiles the expression of 64 micro RNAs by microarray and enables the identifications of 42 tumor types ([39]). The accuracy of both assays has been evaluated in retrospective studies in which the primary tumor was subsequently found and demonstrated good concordance [40].

Mesenchymal Tumors

Within the heterogeneous group of human soft tissue tumors, particularly the gastrointestinal stromal tumors (GISTs; the most common mesenchymal tumors in the gastrointestinal tract) became more interesting regarding the possibility of targeted therapy with the tyrosine kinase inhibitor imatinib.

In 1998, the discovery of the KIT receptor tyrosine kinase expression in almost all of these tumors, as well as the KIT mutations, led to a modern point of view [41]. Approximately 85% of all GISTs exhibit the more frequent KIT mutation or the much more uncommon platelet-derived growth factor receptor (PDGFR) alpha mutation. Several different domains of KIT exist in which oncogenic mutations occur; the location is relevant for sensitivity to targeted inhibitors and in some cases for prognosis. Most of the KIT mutations were found on the juxta-membrane domain of exon 11, and most of the PDGFR alpha mutations were D842V substitutions of the PDGFR alpha tyrosine kinase 2 domain detected on exon 18 [42].

A small subset (< 1% of total GIST cases) features BRAF V600E mutations. Approximately 10% of all adult GISTs and most GISTs in children show none of these mutations. In 2001, the first case report of successfully targeted therapy of metastatic GIST with the tyrosine kinase inhibitor imatinib led to a change of therapy [43]. Now, this therapy is recommended for first-line treatment of patients with unresectable and/or metastatic GIST as well as for adjuvant therapy in high-risk patients. Tumors with a KIT mutation in exon 11 are most sensitive to imatinib, and GISTs with a PDGFR alpha mutation in exon 18 (D842V) seem to be resistant to this therapy [44]. GISTs with a KIT mutation in exon 9 need a higher dose of imatinib (> 400 mg/day) to gain a longer progression-free survival.

Immune Oncology

For decades it was not clear how tumor cells are able to escape immune surveillance even though they express neo-antigens at their cell membrane and, thus, should be recognized by the lymphocytes. After intensive research in this field it became clear that the tumor cells express certain molecules at their surface, e.g. CTLA4 und PD1/PD-L1 and many more (called checkpoint receptors), which make them ‘invisible' to the immune system and by this inhibit the execution of effector functions of T lymphocytes.

Consequently, the drug-based blockade of this escape mechanism could make the tumor cells recognizable again for lymphocytes, in particular T regulatory lymphocytes, which results in tumor cell death. The drugs to achieve this are therapeutic monoclonal antibodies directed against, for example, PD1 or PD-L1. This therapeutical approach, termed cancer immunotherapy, has shown to be very effective in a number of tumor entities, including mm, cancer of the lung, bladder, stomach, some sarcomas, and Hodgkin's disease [45].

Due to the fact that in most tumor types the membranous expression of PD1/PD-L1 is to some degree correlated with the efficacy of the therapeutic drugs, e.g. nivolumab, pembrolizumab, durvalumab, and atezolizumab, a biomarker-based companion or complementary diagnostic appears to be indispensable to select those patients who would benefit from the treatment from those who would not, i.e. whose tumor does not express PD-L1.

The method of choice for defining the PD-L1 expression of tumor cells, and in some instances also of immune cells, is IHC. Although there is an ongoing debate on the optimal antibody and detection system and the criteria of evaluation (tumor cells and/or immune cells, only membrane vs. cytoplasmic staining, cutoffs, etc.), the standardization is a worldwide and on-going project. The German Society of Pathology and its quality assurance initiative QuIP has done a first harmonization study [46] and will perform ring trials in the next few months to achieve a reliable diagnostic around the country.

Conclusion and Perspective

It has to be stated that the effect of the development of targeted therapy has been dramatic, the number of new drugs in clinical studies is enormous and the resulting challenges for predictive pathology/companion diagnostic are indeed substantial. Since currently the vast majority of the assays are tissue based, the responsibility for accurate performance lies in the hands of pathologists. The scientific societies have to be alert to cover this chance. Education of doctors and technicians, quality control of technical procedures and the intellectual interpretation of the results are crucial to provide reliable results. Clearly this will play an increasing role in the future structure of tissue-based diagnostics.

The multilayer analyses of malignant tumors produce an increasing volume of data, creating a high complexity of information for more or less each tumor. In future, this situation may become even more challenging with the new attempts to subclassify tumors by gene expression-based classification systems, or classification systems based on immune scores [47,48].

To manage this situation, interdisciplinarity becomes an important prerequisite in cancer treatment (fig. 1). This is true in daily work and in scientific projects. A consequence of this situation is the necessity to build up comprehensive cancer centers that can provide the broad spectrum of knowledge and experience.

Fig. 1

Organizational diagram of the cooperation between the Institute of Pathology and the Comprehensive Cancer Center at the Charité.

http://www.karger.com/WebMaterial/ShowPic/527450

Disclosure Statement

The author has nothing to declare.


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  43. Joensuu H, Roberts PJ, Sarlomo-Rikala M, et al.: Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med 2001;344:1052-1056.
  44. Corless CL, Schroeder A, Griffith D, et al.: PDGFRA mutations in gastrointestinal stromal tumors: Frequency, spectrum and in vitro sensitivity to imatinib. J Clin Oncol 2005;23:5357-5364.
  45. Melero I, Berman DM, Aznar MA, et al.: Evolving synergistic combinations of targeted immunotherapies to combat cancer. Nat Rev Cancer 2015;15:457-472.
  46. Scheel AH, Dietel M, Heukamp LC, et al.: Harmonized PD-L1 immunohistochemistry for pulmonary squamous-cell and adenocarcinomas. Mod Pathol 2016;29:1165-1172.
  47. Guinney J, Dienstmann R, Wang X, et al.: The consensus molecular subtypes of colorectal cancer. Nat Med 2015;21:1350-1356.
  48. Galon J, Mlecnik B, Bindea G, et al.: Towards the introduction of the ‘immunoscore' in the classification of malignant tumours. J Pathol 2014;232:199-209.

Author Contacts

Prof. Dr. Manfred Dietel

Institute of Pathology

Charité, University Medicine Berlin

Charitéplatz 1, 10117 Berlin, Germany

manfred.dietel@charite.de


Article / Publication Details

First-Page Preview
Abstract of Review Article

Received: September 30, 2016
Accepted: November 07, 2016
Published online: November 25, 2016
Issue release date: December 2016

Number of Print Pages: 7
Number of Figures: 1
Number of Tables: 0

ISSN: 2296-5270 (Print)
eISSN: 2296-5262 (Online)

For additional information: https://www.karger.com/ORT


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  43. Joensuu H, Roberts PJ, Sarlomo-Rikala M, et al.: Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med 2001;344:1052-1056.
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