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Based on new trial data regarding immune checkpoint inhibitors (ICIs), the detection of high-grade microsatellite instability (MSI-H) or underlying deficient mismatch repair protein (dMMR) is now becoming increasingly important for predicting treatment response. For the first time, a PD‑1 ICI (pembrolizumab) has been approved by the European Medicines Agency (EMA) for first-line treatment of advanced (stage IV) dMMR/MSI‑H colorectal cancer (CRC). Further indications, such as dMMR/MSI‑H endometrial carcinoma (EC), have already succeeded (Dostarlimab, 2nd line treatment) and others are expected to follow before the end of 2021. The question of optimal testing in routine diagnostics should therefore be re-evaluated. Based on a consideration of the strengths and weaknesses of the widely available methods (immunohistochemistry and PCR), a test algorithm is proposed that allows quality assured, reliable, and cost-effective dMMR/MSI‑H testing. For CRC and EC, testing is therefore already possible at the primary diagnosis stage, in line with international recommendations (NICE, NCCN). The clinician is therefore enabled from the outset to consider not only the predictive but also the prognostic and predispositional implications of such a test when counseling patients and formulating treatment recommendations. As a basis for quality assurance, participation in interlaboratory comparisons and continuous documentation of results (e.g., QuIP Monitor) are strongly recommended., (© 2021. Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Gene fusions involving the three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, or NTRK3 were identified as oncogenic drivers in many cancer types. Two small molecule inhibitors have been tested in clinical trials recently and require the detection of a NTRK fusion gene prior to therapeutic application. Fluorescence in situ hybridization (FISH) and targeted next-generation sequencing (tNGS) assays are commonly used for diagnostic profiling of gene fusions. In the presented study we applied an external quality assessment (EQA) scheme in order to investigate the suitability of FISH and RNA-/DNA-based tNGS for detection of NTRK fusions in a multinational and multicentric ring trial. In total 27 participants registered for this study. Nine institutions took part in the FISH-based and 18 in the NGS-based round robin test, the latter additionally subdivided into low-input and high-input NGS methods (regarding nucleic acid input). Regardless of the testing method applied, all participants received tumor sections of 10 formalin-fixed and paraffin-embedded (FFPE) tissue blocks for in situ hybridization or RNA/DNA extraction, and the results were submitted via an online questionnaire. For FISH testing, eight of nine (88.8%) participants, and for NGS-based testing 15 of 18 (83.3%) participants accomplished the round robin test successfully. The overall high success rate demonstrates that FISH- and tNGS-based NTRK testing can be well established in a routine diagnostic setting. Complementing this dataset, we provide an updated in silico analysis on the coverage of more than 150 NTRK fusion variants by several commercially available RNA-based tNGS panels., (© 2020 Wiley Periodicals, Inc.)

Background: NTRK1, NTRK2 and NTRK3 fusions are present in a plethora of malignancies across different histologies. These fusions represent the most frequent mechanism of oncogenic activation of these receptor tyrosine kinases, and biomarkers for the use of TRK small molecule inhibitors. Given the varying frequency of NTRK1/2/3 fusions, crucial to the administration of NTRK inhibitors is the development of optimal approaches for the detection of human cancers harbouring activating NTRK1/2/3 fusion genes., Materials and Methods: Experts from several Institutions were recruited by the European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group (TR and PM WG) to review the available methods for the detection of NTRK gene fusions, their potential applications, and strategies for the implementation of a rational approach for the detection of NTRK1/2/3 fusion genes in human malignancies. A consensus on the most reasonable strategy to adopt when screening for NTRK fusions in oncologic patients was sought, and further reviewed and approved by the ESMO TR and PM WG and the ESMO leadership., Results: The main techniques employed for NTRK fusion gene detection include immunohistochemistry, fluorescence in situ hybridization (FISH), RT-PCR, and both RNA-based and DNA-based next generation sequencing (NGS). Each technique has advantages and limitations, and the choice of assays for screening and final diagnosis should also take into account the resources and clinical context., Conclusion: In tumours where NTRK fusions are highly recurrent, FISH, RT-PCR or RNA-based sequencing panels can be used as confirmatory techniques, whereas in the scenario of testing an unselected population where NTRK1/2/3 fusions are uncommon, either front-line sequencing (preferentially RNA-sequencing) or screening by immunohistochemistry followed by sequencing of positive cases should be pursued., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Characterization of tumors utilizing next-generation sequencing methods, including assessment of the number of somatic mutations (tumor mutational burden [TMB]), is currently at the forefront of the field of personalized medicine. Recent clinical studies have associated high TMB with improved patient response rates and survival benefit from immune checkpoint inhibitors; hence, TMB is emerging as a biomarker of response for these immunotherapy agents. However, variability in current methods for TMB estimation and reporting is evident, demonstrating a need for standardization and harmonization of TMB assessment methodology across assays and centers. Two uniquely placed organizations, Friends of Cancer Research (Friends) and the Quality Assurance Initiative Pathology (QuIP), have collaborated to coordinate efforts for international multistakeholder initiatives to address this need. Friends and QuIP, who have partnered with several academic centers, pharmaceutical organizations, and diagnostic companies, have adopted complementary, multidisciplinary approaches toward the goal of proposing evidence-based recommendations for achieving consistent TMB estimation and reporting in clinical samples across assays and centers. Many factors influence TMB assessment, including preanalytical factors, choice of assay, and methods of reporting. Preliminary analyses highlight the importance of targeted gene panel size and composition, and bioinformatic parameters for reliable TMB estimation. Herein, Friends and QuIP propose recommendations toward consistent TMB estimation and reporting methods in clinical samples across assays and centers. These recommendations should be followed to minimize variability in TMB estimation and reporting, which will ensure reliable and reproducible identification of patients who are likely to benefit from immune checkpoint inhibitors., (© 2019 The Authors. Genes, Chromosomes & Cancer published by Wiley Periodicals, Inc.)

The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series. Detailed information regarding pre-characterized mutations was not disclosed to the partners. Commercially available and custom-designed cancer gene panels were used for library preparation and subsequent sequencing on several devices of two NGS different platforms. For every case, centrally extracted DNA and FFPE tissue sections for local processing were delivered to each partner site to be sequenced with the commercial gene panel and local bioinformatics. For cancer-specific panel-based sequencing, only centrally extracted DNA was analyzed at seven sequencing sites. Subsequently, local data were compiled and bioinformatics was performed centrally. We were able to demonstrate that all pre-characterized mutations were re-identified correctly, irrespective of NGS platform or gene panel used. However, locally processed FFPE tissue sections disclosed that the DNA extraction method can affect the detection of mutations with a trend in favor of magnetic bead-based DNA extraction methods. In conclusion, targeted NGS is a very robust method for simultaneous detection of various mutations in FFPE tissue specimens if certain pre-analytical conditions are carefully considered.

Inhibition of the PD-L1 (CD274) - PD-1 axis has emerged as a powerful cancer therapy that prevents evasion of tumor cells from the immune system. While immunohistochemical detection of PD-L1 was introduced as a predictive biomarker with variable power, much less is known about copy number alterations (CNA) affecting PD-L1 and their associations with expression levels, mutational load, and survival. To gain insight, we employed The Cancer Genome Atlas (TCGA) datasets to comprehensively analyze 22 major cancer types for PD-L1 CNAs. We observed a diverse landscape of PD-L1 CNAs, which affected focal regions, chromosome 9p or the entire chromosome 9. Deletions of PD-L1 were more frequent than gains (31% vs. 12%) with deletions being most prevalent in melanoma and non-small cell lung cancer. Copy number gains most frequently occurred in ovarian cancer, head and neck cancer, bladder cancer, cervical and endocervical cancer, sarcomas, and colorectal cancers. Fine-mapping of the genetic architecture revealed specific recurrently amplified and deleted core regions across cancers with putative biological and clinical consequences. PD-L1 CNAs correlated significantly with PD-L1 mRNA expression changes in many cancer types, and tumors with PD-L1 gains harbored significantly higher mutational load compared to non-amplified cases (median: 78 non-synonymous mutations vs. 40, P = 7.1e-69). Moreover, we observed that, in general, both PD-L1 amplifications and deletions were associated with dismal prognosis. In conclusion, PD-L1 CNAs, in particular PD-L1 copy number gains, represent frequent genetic alterations across many cancers, which influence PD-L1 expression levels, are associated with higher mutational loads, and may be exploitable as predictive biomarker for immunotherapy regimens. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

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.)

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.

The majority of patients with solid malignancies die from metastatic burden. However, our current understanding of the mechanisms and resulting patterns of dissemination is limited. Here, we analyzed patterns of metastatic progression across 16 major cancer types in a cohort of 1008 patients with metastatic cancer autopsied between 2000 and 2013 to assess cancer specific progression patterns of disease and related risk predictions. The frequency and location of metastases were evaluated in and across 1) 16 major cancers, 2) smoking- and non-smoking-related cancers and 3) adeno- and squamous cell carcinoma. Associations between primary and secondary sites were analyzed by the fractional and the relative risk methods. We detected significantly different cancer specific patterns of metastatic progression with specific relative risk profiles for secondary site involvement. Histology and smoking etiology influenced these patterns. Backward analysis showed that metastatic patterns help to predict unknown primary sites. Solid malignancies maintain a unique and recurrent organ tropism to specific secondary sites which does not appear to be strongly influenced by advances in cancer medicine as shown by comparison with previous data sets. The delineated landscape of metastatic progression patterns is a comprehensive data resource to both clinical and basic scientists which aids fostering new hypotheses for cancer research and cancer therapies.

Cancer medicine relies on the paradigm that cancer is an organ- and tissue-specific disease, which is the basis for classifying tumors. With the extensive genomic information now available on tumors it is possible to conduct analyses to reveal common genetic features across cancer types and to explore whether the established anatomy-based tumor classification is actually reflected on the genetic level, which might provide important guides to new therapeutic directions. Here, we have conducted an extensive analysis of the genetic similarity of tumors from 14 major cancer entities using somatic mutation data from 4,796 cases available through The Cancer Genome Atlas (TCGA) based on all available genes as well as different cancer-related gene sets. Our analysis provides a systematic account of the genetic similarity network for major cancer types and shows that in about 43% of the cases on average, tumors of a particular anatomic site are genetically more similar to tumors from different organs and tissues (trans-similarity) than to tumors of the same origin (self-similarity). The observed similarities exist not only for carcinomas from different sites but are also present among neoplasms from different tissue origin, such as melanoma, acute myeloid leukemia, and glioblastoma. The current WHO cancer classification is therefore reflected on the genetic level by only about 57% of the tumors. These results provide a rationale to reconsider organ- and tissue-specificity in cancer and contribute to the discussion about whether personalized therapies targeting specific genetic alterations may be transferred to cancers from other anatomic sites with similar genetic properties., (© 2014 UICC.)

Molecular testing is becoming an important part of the diagnosis of any patient with cancer. The challenge to laboratories is to meet this need, using reliable methods and processes to ensure that patients receive a timely and accurate report on which their treatment will be based. The aim of this paper is to provide minimum requirements for the management of molecular pathology laboratories. This general guidance should be augmented by the specific guidance available for different tumour types and tests. Preanalytical considerations are important, and careful consideration of the way in which specimens are obtained and reach the laboratory is necessary. Sample receipt and handling follow standard operating procedures, but some alterations may be necessary if molecular testing is to be performed, for instance to control tissue fixation. DNA and RNA extraction can be standardised and should be checked for quality and quantity of output on a regular basis. The choice of analytical method(s) depends on clinical requirements, desired turnaround time, and expertise available. Internal quality control, regular internal audit of the whole testing process, laboratory accreditation, and continual participation in external quality assessment schemes are prerequisites for delivery of a reliable service. A molecular pathology report should accurately convey the information the clinician needs to treat the patient with sufficient information to allow for correct interpretation of the result. Molecular pathology is developing rapidly, and further detailed evidence-based recommendations are required for many of the topics covered here., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Aims: Atherosclerosis and cancer share common risk factors and involve similar molecular pathomechanisms. Most clinical and epidemiological studies show a positive correlation between atherosclerosis and smoking-related cancers and heterogeneous results for non-smoking-related cancers. However, up-to-date large-scale autopsy studies including a detailed analysis of cancer types are lacking. Therefore, we sought to investigate the relation between major cancer types and the grade of atherosclerosis in a recent well-powered autopsy cohort., Methods: In 2101 patients, both autopsy data and clinical data including demographics, disease groups, tumour type, cause of death and grade of atherosclerosis were reviewed and statistically analysed., Results: We found cancer in general is associated with less atherosclerosis (OR 0.60, p<0.0001). In particular, haematological neoplasm and sarcomas were associated with much less atherosclerosis (OR=0.45, p<0.0001 and OR=0.43, p=0.087), while carcinomas were associated with moderately less atherosclerosis (OR=0.72, p=0.002). Furthermore, non-smoking-related cancers were associated with much less atherosclerosis (OR=0.41, p<0.0001), while possibly smoking-related cancer and smoking-related cancer showed no significant association. In a comprehensive analysis of 21 cancer types, biliary tract cancer, lymphomas/lymphoid leukaemias and kidney cancer were associated with much less atherosclerosis (OR=0.19, p<0.0001; OR=0.41, p<0.0001; and OR=0.48, p=0.029). In an exploratory analysis of treatment strategies, we found that tumours with a recommendation of oxazaphosphorines and pyrimidine antagonist treatment were significantly associated with less atherosclerosis (OR=0.33, p=0.0068 and OR=0.58, p=0.012)., Conclusions: In conclusion, the study showed an inverse association between cancer and atherosclerosis postmortem that depends on the cancer type and suggests a possible impact of chemotherapy regimens., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

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.

In February 2011, a group of pathologists from different departments in Europe met in Zurich, Switzerland, to discuss opportunities and challenges for pathology in the era of personalized medicine. The major topics of the meeting were assessment of the role of pathology in personalized medicine, its future profile among other biomedical disciplines with an interest in personalized medicine as well as the evolution of companion diagnostics. The relevance of novel technologies for genome analysis in clinical practice was discussed. The participants recognize that there should be more initiatives taken by the pathology community in companion diagnostics and in the emerging field of next-generation sequencing and whole genome analysis. The common view of the participants was that the pathology community has to be mobilized for stronger engagement in the future of personalized medicine. Pathologists should be aware of the challenges and the analytical opportunities of the new technologies. Challenges of clinical trial design as well as insurance and reimbursement questions were addressed. The pathology community has the responsibility to lead medical colleagues into embracing this new area of genomic medicine. Without this effort, the discipline of pathology risks losing its key position in molecular tissue diagnostics.

The success of targeted therapy depends on reliable biomarkers that indicate the presence and accessibility of the target molecules with which the therapeutic drug will interact. In principle, this is independent of whether the drug is a small molecule, such as kinase inhibitors, or a therapeutic antibody, such as trastuzumab or cetuximab. The functional system of target-drug interaction is described as 'predictive medicine', one of the great hopes in the treatment of cancer. The major challenges are the precise prediction of the patient's individual prognosis, of the probability of metastases, and of individual therapeutic response. The questions, challenges, and possible answers related to this complex issue as well as the prerequisites for the development of more efficient and less toxic cancer therapy are discussed., (Copyright (c) 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Due to continuous technical developments and new insights into the high complexity of many diseases, in particular the pathogenesis of cancer, molecular pathology is a rapidly growing field gaining centre stage in the clinical management of tumours as well as in the pharmaceutical development of new anti-cancer drugs. Activated signalling components are the targets for classical therapeutic agents and newly developed inhibitors. The application of the compounds in clinical trials has revealed promising results; however, the current diagnostic procedures available for determining which patients will primarily benefit from rational tumour therapy are insufficient. To read a patients' tissue as "deeply" as possible, gaining information on the morphology and on genetic, proteomic and epigenetic alterations will . be the new task of surgical pathologists experienced in molecular diagnostics, in order to provide the clinicians with information relevant for an individualized medicine. Among the different high-throughput technologies, DNA microarrays are now the first array approaches close to entering routine diagnostics. Technically advanced and well-established microarray platforms can now be evaluated by distinct bioinformatic tools capable of both identifying novel genes associated with disease development and also clusters of genes predicting the clinical outcome of an individual tumour. DNA microarrays have been efficiently used for the classification of tumour subtypes, the prediction of metastatic potential and drug response. In the current review we will focus in particular on the new possibilities of predicting the efficacy of anti-neoplastic drugs as a diagnostic tool of pathologists seeking an efficient individualized therapy.

Working under the assumption that up- or downregulation of genes implicated in chemoresistance may be the result of altered function of regulatory transcription factors (TF), over-represented TF-binding sites of gene lists previously associated with doxorubicin resistance were the target of our search. First, a data warehouse was set up containing 52 genes which were present in at least two gene lists; of those, proximal promoter sequences (1 kb upstream and 0.05 kb downstream of the transcriptional start sites) could be retrieved from genomic databases for 45 genes using the EZ-Retrieve. The TOUCAN tool MotifScanner, which searches the TRANSFAC database, was used to detect TF-binding sites (TFBSs) in our set of sequences. The statistics tool of the Java program TOUCAN was applied to the data with the appropriate expected frequencies file to compare the measured prevalence to a background model. The most significantly over-represented TFBS was that of E47 (p=0.00024, prevalence: 0.2 vs. background: 8.19E-6). In summary, based on the results of our analysis it is hypothesized that the E47 transcription factor may contribute to doxorubicin resistance.

Hormone replacement therapy (HRT) has gained widespread and in some areas indiscriminate use. In reference to recent epidemiological studies which showed unexpected and controversial associations of HRT use with malignant tumours, here we review the current understanding of the dynamics of tumour growth. The pathomorphological characteristics and sex hormone sensitivity of cancers of the breast, endometrium, ovary and colon are discussed. The development of cancer from the first malignant tumour cell to clinical diagnosis takes many years. Hormones can influence tumour growth, but it is questionable whether hormones induce malignant tumours de novo. It is much more likely that hormones 'merely' promote the growth of already existing tumour cells. The long developmental process of tumours is in apparent contradiction to results of some epidemiological studies that describe an increased cancer risk, implying primary initiation, in HRT users within observation periods of 1-6 years. The mechanisms of initiation versus promotion of hormone-sensitive cancers, particularly breast cancer, are only partly understood. The conventional methods of epidemiological studies cannot detect potential risk factors without bias if they do not include a pathomorphological component on growth characteristics. The results of previous studies should be interpreted with great caution with regard to tumour biology.

Background: The morphologic diagnosis of tumor specimens with precise tumor typing, staging, and grading remains the basis of almost all cancer treatments. Thus, in each tumor case, a histologic diagnosis of the highest quality should be the physician's priority. In approximately 10-20% of tumor cases, diagnostic uncertainty remains to some degree, requiring a second opinion in determining the biologic behavior, the histogenesis, the grade of dedifferentiation, or any other parameter. Facilitating the communication between pathologists and the exchange of cases, telepathology gains more and more importance. To benefit from this technical development, the International Union Against Cancer (UICC) has decided to establish a Telepathology Consultation Center (UICC-TPCC) for interested pathologists around the world., Methods: The communication and exchange of histologic images works via the Internet. To ensure constant documentation, a case-based data base and image archive is used. Special TPCC software handles all requests to the TPCC and controls the interaction among requesting pathologists, TPCC, and UICC experts (transferring, reading, answering, logging, storing, etc.). All necessary data for controlling the telepathology service are stored in a customized SQL data base. The necessary equipment for the requesting pathologist is a personal computer; a digital or television camera/frame grabber, which is attached to a microscope; and access to the Internet. The requesting party contacts the TPCC's World Wide Web server and uploads the images and the clinical data of their case. To ensure uninterrupted functioning, the hardware will be part of a high-level communication center, which is connected via ATM (asynchronous transfer mode, 155 megabits per second) to the Internet., Results: The UICC has decided to establish the TPCC at the Institute of Pathology at the Charité, Humboldt-University, Berlin, Germany. The TPCC will not make the diagnoses itself but will involve an affiliated specialized expert pathologist. He or she will be on the panel of UICC experts who will constitute the "diagnostic backbone" of the TPCC. The center will function as follows: If a pathologist anywhere on the globe is confronted with the diagnosis of a difficult tumor case, he takes digitized histologic images (5-40 in number) and sends them along with sufficient clinical data to the server of the UICC-TPCC, asking for a second opinion. The center checks the case and transfers it to one of the UICC experts. This expert makes his or her diagnostic suggestion, which is then transferred back to the requesting pathologist via the UICC-TPCC., Conclusions: The UICC-TPCC will be able to provide rapid and inexpensive diagnostic aid to pathologists all over the world, offering the possibility of a second opinion in accordance with the UICC-TNM and World Health Organization (WHO) standards. During the first and second year, the UICC-TPCC will be financed by sponsors. Telepathology makes the distribution of new developments of diagnostic standards, e.g., of the TNM system, WHO terminology, new tumor classifications, and updated information on actual technologies, globally accessible in a direct and rapid way. It also enables a high quality of education and teaching., (Copyright 2000 American Cancer Society.)

Comparative genomic hybridization (CGH) is a new method of screening a tumor for genetic changes. The alterations are classified as DNA gains and losses and reveal a characteristic pattern that includes mutations at the chromosomal and subchromosomal levels. Although DNA from fresh-frozen tissue is recommended as the starting material, archival specimens can also be analyzed. Various examples are presented that illustrate the changes that occur in different tumor entities, and preneoplastic lesions, the differentiation of primary tumors from metastases, and the investigation of tumor cell lines that are resistant to cytostatic drugs. These examples emphasize that CGH can extend the possibilities for genetic diagnosis in tumor pathology.

Comparative genomic hybridization (CGH) is a molecular cytogenetic method for the detection of chromosomal imbalances between a tumor and a normal genome. In order to produce quantitative and reproducible results, we developed an image analysis program that allows the detection and mapping of the genetic alterations. The result is represented as a CGH sum karyogram in which the genetic changes are documented as color coded-chromosomes. The aim of our investigation is to correlate the genotype with the phenotype on the basis of CGH sum karyograms and thus to achieve a genetic characterization that will supplement the morphological tumor description.

Multidrug resistance (MDR), especially that associated with overexpression of MDR1 and its product, P-glycoprotein (Pgp), is thought to play a role in the outcome of therapy for some human tumors; however, a consensus conclusion has been difficult to reach, owing to the variable results published by different laboratories. Many factors appear to influence the detection of Pgp in clinical specimens, including its low and heterogeneous expression; conflicting definitions of detection end points; differences in methods of sample preparation, fixation, and analysis; use of immunological reagents with variable Pgp specificity and avidity and with different recognition epitopes; use of secondary reagents and chromogens; and differences in clinical end points. Also, mechanisms other than Pgp overexpression may contribute to clinical MDR. The combined effect of these factors is clearly important, especially among tumors with low expression of Pgp. Thus, a workshop was organized in Memphis, Tennessee, to promote the standardization of approaches to MDR1 and Pgp detection in clinical specimens. The 15 North American and European institutions that agreed to participate conducted three preworkshop trials with well-characterized MDR myeloma and carcinoma cell lines that expressed increasing amounts of Pgp. The intent was to establish standard materials and methods for a fourth trial, assays of Pgp and MDR1 in clinical specimens. The general conclusions emerging from these efforts led to a number of recommendations for future studies: (a) although detection of Pgp and MDR1 is at present likely to be more reliable in leukemias and lymphomas than in solid tumors, accurate measurement of low levels of Pgp expression under most conditions remains an elusive goal; (b) tissue-specific controls, antibody controls, and standardized MDR cell lines are essential for calibrating any detection method and for subsequent analyses of clinical samples; (c) use of two or more vendor-standardized anti-Pgp antibody reagents that recognize different epitopes improves the reliability of immunological detection of Pgp; (d) sample fixation and antigen preservation must be carefully controlled; (e) multiparameter analysis is useful in clinical assays of MDR1/Pgp expression; (f) immunostaining data are best reported as staining intensity and the percentage of positive cells; and (g) arbitrary minimal cutoff points for analysis compromise the reliability of conclusions. The recommendations made by workshop participants should enhance the quality of research on the role of Pgp in clinical MDR development and provide a paradigm for investigations of other drug resistance-associated proteins.

Manifold mechanisms of resistance can be expressed by malignancies. Profound information on this aspect is a prerequisite for comprehensive individual chemotherapy. Based on both morphological and functional findings, the diagnosis of P-Glycoprotein (P-Gp) mediated Multidrug Resistance (MDR) can be verified. In order to describe minimum criteria for conclusive diagnosis, morphological and biochemical findings (Immunocytochemistry, RT-PCR, ultrastructural and Laser-Scan microscopy) and functional data (cytostatic drug transport, proliferation) were correlated in tumor cell lines of the MDR phenotype as opposed to cells with atypical resistance. Frequently, single features of MDR are found in atypical, P-Gp negative resistance. Accumulation deficits for mitoxantrone based on vesicular drug transport were found in P-Gp negative gastric carcinoma cell line EPG85-257RNOV. Nocodazole blocked microtubule formation which is essential for vesicle transfer from perinuclear regions to the periphery of the cytosol. Cytochalasin blocked exocytosis of drug containing vesicles. MDR modulators were ineffective. Alternatively, P-Gp mediated drug extrusion and exocytosis of drug containing vesicles may constitute complementary mechanisms of resistance. In gastric carcinoma cells EPG85-257DAU, MDR modulators do increase cytosolic daunorubicin load, but drug binding to nuclear target sites is still inhibited due to drug containment within vesicles. To complicate matters, MDR modulators may be functional even in MDR negative cells, as shown in a panel of melanoma cell lines. Data show that conclusive diagnosis of P-Gp-mediated MDR should be based on more than one experimental approach including immunocytochemistry, a sensitive assay such as RT-PCR and--whenever feasible--a functional test.

DNA is a very stable molecule and fixation as well as routine histological processing does not destroy its molecular structure. Hence the possibility exists to use this material for molecular genetic analyses. The polymerase chain reaction (PCR) opens the chance to amplify DNA to huge amounts from pathological paraffin and plastic blocks and to use this DNA for further examinations, such as detection of mutations or translocations in malignant tumors, e.g. t(14;18) in follicular lymphomas, bcr/abl in chronic myeloic leukemia, t(11;22) in Ewing sarcomas or of the ras-gene in colon cancer, overexpression of tumor related mRNA, e.g. mdr1-mRNA of the multidrug associated P-Glycoprotein, or detection of foreign DNA from viruses or bacteriae, as well as analysis of hereditary diseases. PCR in its various forms (conventional PCR, PCR with direct sequencing, reverse transcriptase (RT)-PCR, nested primer PCR, quantitative RT-PCR, inverse PCR, degenerated primer (DOP) PCR, in situ PCR, in situ RT-PCR etc.) has proven to supplement routine diagnostic work in many occasions, however, it has to be emphasized that up to now there exists no example for a complete replacement of histological or immunhistological examination by PCR. As consequence, histology remains the first and most important step towards a relevant diagnosis supplemented e.g. by PCR and other techniques of molecular biology.

Humoral hypercalcemia of malignancy may reflect the synthesis and secretion of biologically active parathyroid hormone-related protein (PTHrP) by a given tumor. In the present study we investigated 25 human non-endocrine carcinomas which were clinically associated with hypercalcemia (Ca > 11 mg%). By applying PTHrP-specific immunocytochemistry, PTHrP could be detected in all tumors. The intra-tumorous distribution was heterogeneous with strong positivity in relatively few cells or weak positivity in the majority of cells. Surprisingly, in the PTHrP producing cells none of the marker proteins typical of endocrine cells (neuron-specific enolase, Leu-7 antigen, chromogranin, synaptophysin and endocrine granule constituent) was found. On the other hand, PTHrP producing cells of endocrine origin, such as medullary cancer, or normal and adenomatous parathyroid glands, all produce these endocrine markers. Thus for the first time, the existence of peptide hormone producing tumor cells is reported without expression of endocrine markers. This indicates a special mechanism of PTHrP secretion.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dependent growth and differentiation of 6 tumor cell lines has been determined by the use of the monolayer proliferation assay. Cell lines of 4 gastro-intestinal carcinomas, 1 malignant schwannoma, and 1 malignant histiocytoma have been established and characterized. Cells were incubated for 4, 7, and 11 days in the presence of 0.8 or 8 nM 1,25(OH)2D3 and for control without addition of the hormone. Proliferation rates of 1,25(OH)2D3 treated cells were compared with cell growth in the untreated controls. Five out of 6 cell lines showed a 1,25(OH)2D3 dependent growth pattern. With 8 nM 1,25(OH)2D3 they were all inhibited. With 0.8 nM, 3 of them were inhibited at any time of the test period, whereas 1 was stimulated at day 4 and inhibited at days 7 and 11. One cell line was stimulated at days 4, 7, and 11 when incubated with 0.8 nM 1,25(OH)2D3. No striking morphological changes could be observed in the presence of 1,25(OH)2D3. We conclude that 1,25(OH)2D3 dependent cells in vitro are not necessarily growth-inhibited by this compound. Thus, 1,25(OH)2D3 is not an exclusively proliferation inhibiting agent.

Growth control of normal cells is regulated by highly complex interactions of environmental signals and cell inherent conditions. Disorder of this system may result in the multi-step event of malignant transformation. This review discusses some aspects of the interaction of growth controlling agents, cell receptors, signal transmission, and cellular response. It focuses on the role of cell receptors that recognize and transduce information of cancer initiating and promoting agents to the cell: e.g. viruses, cancerogens, peptide and steroid hormones, growth factors, local growth promoting agents, and other environmental factors. All these growth modulators may be involved in triggering cell division. The mutual interactions of promotor induced signals, genes, and oncogenes are also discussed.

Highly organized multicellular organisms require a complex system of intercellular communication to coordinate the great variety of physiological functions. Major components of this system are the (i) signalling substances delivering the information and (ii) the corresponding cellular receptors recognizing the information and translating them to special intracellular organelles of the target cell. For the process of signal binding and transmission various types of receptor proteins are known playing a key role in the mutual understanding between cells. The present paper comprehends lectures on functions and deficiencies of the different types of cell receptors given during the Symposion on "Receptors in Pathology" during the 11th European Congress of Pathology. Each section is written under the personal responsibility of the author indicated.

The tumor markers CA 19-9 and CA 125 defined by the monoclonal antibodies 19-9 and OC 125 were investigated with respect to organ specificity and tumor sensitivity. Normal and tumor tissue specimens, and blood samples from 34 patients with pancreatic carcinomas, 40 with ovarian carcinomas and 39 with miscellaneous tumors were examined. CA 19-9 and CA 125 were determined by immunohistochemistry (IH) applied to sections of the tumors and adjacent normal tissues. In parallel, the antigens were measured in the patients' sera by radioimmunoassay (RIA). By means of IH CA 19-9 and CA 125 were detected in normal surface cells from many different organs. Both antigens were also found in tissue sections of various types of tumors and in the sera of the corresponding patients. Thus, organ specificity could not be demonstrated. Sensitivity of CA 19-9 was found to be high for pancreatic carcinomas, i.e., 88% of the tumors expressed the antigen shown by IH and 85% of the sera revealed concentrations above the cut-off value (greater than 37 units/ml). Evidence for CA 125 was high in ovarian carcinomas with a tissue positivity in 83% and elevated (greater than 35 units/ml) serum levels in 70% of patients. Comparing IH and RIA case by case a discrepancy was found in 14% of cases with positive IH and low serum values a vice versa. Reasons for this finding are small tumor mass not producing elevated serum levels, retention of the antigen inside the tumor cells because of defective release mechanisms demonstrable only by IH, or heterogeneity of tumors with only focal antigen expression not present in the tissue sections investigated and thus disclosable only by RIA. The relevance of immunohistochemical detection of the antigens for therapeutic planning is discussed.

Cell culture systems allow the examination of cell populations in a functional state. To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines. Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: Disintegration, culture media supplemented with basal additions, special supplements (growth factors, hormones), and attachment factors. The proliferation rates of the attained cell strains were evaluated by determination of cell doubling times. Procedures for how to obtain a relatively high plating efficiency (approx. 70% in our series of 219 attempts) of primary growth in vitro are described: (1) Mechanical disintegration is superior to enzymatic digestion. If mechanical treatment alone did not produce a sufficient number of viable cells, additional digestion with collagenase/dispase revealed a higher number of proliferating primary cultures than with trypsin. (2) Proliferation of cell cultures from normal and tumorous tissues of epithelial origin was superior in Leibovitz L 15 medium (58 of 87 (67%) cases). Cultures from mesenchymal tissues and tumors were found to have shortest cell doubling times in MEM and RPMI 1640 (16 of 23 (70%) cases). The media were supplemented with the basal additions indicated. (3) In approx. 30% of the cases special supplements like growth factors or hormones increased cell replication, although they were almost always not essential for cell growth. (4) Attachment factors only rarely contributed to the initiation of primary monolayer cultures. The application of various culture conditions does not lead to a protocol optimal for all tissues, for all probes of the same type of tumor, or for all tumor specimens of unique differentiation.