Cancer of unknown primary (CUP) through the lens of precision oncology: a single institution perspective.

PURPOSE: For patients with cancer of unknown primary (CUP), treatment options are limited. Precision oncology, the interplay of comprehensive genomic profiling (CGP) and targeted therapies, aims to offer additional treatment options to patients with advanced and hard-to-treat cancers. We aimed to highlight the use of a molecular tumor board (MTB) in the therapeutic management of CUP patients. METHODS: In this single-center observational study, CUP patients, presented to the MTB of the Comprehensive Cancer Center Munich LMU, a tertiary care center, were analyzed retrospectively. Descriptive statistics were applied to describe relevant findings. RESULTS: Between June 2016 and February 2022, 61 patients with unfavorable CUP were presented to the MTB, detected clinically relevant variants in 74% (45/61) of patients, of which 64% (29/45) led to therapeutic recommendation. In four out of 29 patients (14%), the treatment recommendations were implemented, unfortunately without resulting in clinical benefit. Reasons for not following the therapeutic recommendation were mainly caused by the physicians' choice of another therapy (9/25, 36%), especially in the context of worsening of general condition, lost to follow-up (7/25, 28%) and death (6/25, 24%). CONCLUSION: CGP and subsequent presentation to a molecular tumor board led to a high rate of therapeutic recommendations in patients with CUP. Recommendations were only implemented at a low rate; however, late GCP diagnostic and, respectively, MTB referral were found more frequent for the patients with implemented treatment. This contrast underscores the need for early implementation of CGP into the management of CUP patients.

Treatment outcome of atypical EGFR mutations in the German National Network Genomic Medicine Lung Cancer (nNGM).

BACKGROUND: Atypical EGFR mutations occur in 10%-30% of non-small-cell lung cancer (NSCLC) patients with EGFR mutations and their sensitivity to classical epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI) is highly heterogeneous. Patients harboring one group of uncommon, recurrent EGFR mutations (G719X, S768I, L861Q) respond to EGFR-TKI. Exon 20 insertions are mostly insensitive to EGFR-TKI but display sensitivity to exon 20 inhibitors. Clinical outcome data of patients with very rare point and compound mutations upon systemic treatments are still sparse to date. PATIENTS AND METHODS: In this retrospective, multicenter study of the national Network Genomic Medicine (nNGM) in Germany, 856 NSCLC cases with atypical EGFR mutations including co-occurring mutations were reported from 12 centers. Clinical follow-up data after treatment with different EGFR-TKIs, chemotherapy and immune checkpoint inhibitors were available from 260 patients. Response to treatment was analyzed in three major groups: (i) uncommon mutations (G719X, S7681, L861Q and combinations), (ii) exon 20 insertions and (iii) very rare EGFR mutations (very rare single point mutations, compound mutations, exon 18 deletions, exon 19 insertions). RESULTS: Our study comprises the largest thus far reported real-world cohort of very rare EGFR single point and compound mutations treated with different systemic treatments. We validated higher efficacy of EGFR-TKI in comparison to chemotherapy in group 1 (uncommon), while most exon 20 insertions (group 2) were not EGFR-TKI responsive. In addition, we found TKI sensitivity of very rare point mutations (group 3) and of complex EGFR mutations containing exon 19 deletions or L858R mutations independent of the combination partner. Notably, treatment responses in group 3 (very rare) were highly heterogeneous. Co-occurring TP53 mutations exerted a non-significant trend for a detrimental effect on outcome in EGFR-TKI-treated patients in groups 2 and 3 but not in group 1. CONCLUSIONS: Based on our findings, we propose a novel nNGM classification of atypical EGFR mutations.

LIN28 promotes tumorigenesis in colorectal cancer but is not associated with metastatic spread.

BACKGROUND: Tumor stem cells play a role in metastatic spread in colorectal cancer (CRC). The oncogene LIN28A/B, a prognostic marker in CRC, is involved in tumorigenesis and maintains stem cell function. Therefore, it was the aim of the present study to clarify whether LIN28A/B is involved in metastatic spread in CRC. METHODS: Expression of LIN28A/B was analyzed in patients with colon adenocarcinoma in a matched case-control study comparing patients with corresponding liver metastases (n = 42) and patients without hepatic spread within five years (n = 42) by applying immunohistochemistry. Further, LIN28A/B expression was correlated with stem cell associated markers (SOX2, CD133). RESULTS: LIN28A and B expression significantly correlated with SOX2 expression (p = .02, and p = .04 respectively) but not with CD133 expression. This correlation between LIN28 A/B and SOX2 was not reflected in differences in hepatic spread. In this respect, there was no significant association between LIN28A/B expression and liver metastases. CONCLUSION: LIN28A/B might be involved in tumor initiation and progression in CRC but is not associated with hepatic spread.

Novel methods for in vitro modeling of pancreatic cancer reveal important aspects for successful primary cell culture.

BACKGROUND: Pancreatic cancer remains a fatal disease. Experimental systems are needed for personalized treatment strategies, drug testing and to further understand tumor biology. Cell cultures can serve as an excellent preclinical platform, but their generation remains challenging. METHODS: Tumor cells from surgically removed pancreatic ductal adenocarcinoma (PDAC) specimens were cultured under novel protocols. Cellular growth and composition were analyzed and culture conditions were continuously optimized. Characterization of cell cultures and primary tumors was performed via hematoxylin and eosin (HE) and immunofluorescence (IF) staining. RESULTS: Protocols for two- and three-dimensional PDAC primary cell cultures could successfully be established. Primary cell culture depended on dissociation techniques, growth factor supplementation and extracellular matrix components containing Matrigel being crucial for the transformation to three-dimensional PDAC organoids. The generated cultures showed to be highly resemblant to established PDAC primary cell cultures. HE and IF staining for cell culture and corresponding primary tumor characterization could successfully be performed. CONCLUSIONS: The work presented herein shows novel and effective methods to successfully establish primary PDAC cell cultures in a distinct time frame. Factors contributing to cell growth and differentiation could be identified with important implications for further primary cell culture protocols. The established protocols might serve as novel tools in personalized tumor therapy.

[IgG4-Related Orbitopathy as an Important Differential Diagnosis of Advanced Silent Sinus Syndrome. German version]. / IgG4-assoziierte Orbitopathie als wichtige Differenzialdiagnose eines fortgeschrittenen Silent-Sinus-Syndroms.

BACKGROUND: Immunoglobulin (Ig)G4-related disease is classified as an immune-mediated disease. The etiology of this condition has not been explained to date. Manifestations of the disease are diverse, and simultaneous involvement of multiple organs is not unusual. CASE REPORT: We report the case of a patient referred to us after multiple unsuccessful paranasal sinus operations who presented with enophthalmos and a resultant migratory keratitis with a suspected diagnosis of silent sinus syndrome. Preservation of the orbit was no longer feasible. After five years without a definitive diagnosis, we ascertained that this was a case of IgG4-related disease. DISCUSSION: IgG4-related disease represents an important element in the differential diagnosis of chronic advanced diseases of the orbit and paranasal sinuses. The diagnosis should be considered in the case of unclear disease presentations. Typical histological findings include a storiform pattern of fibrosis, vasculopathy, and tissue infiltration by IgG4 plasma cells.

IgG4-related orbitopathy as an important differential diagnosis of advanced silent sinus syndrome.

BACKGROUND: Immunoglobulin (Ig)G4-related disease is classified as an immune-mediated disease. The etiology of this condition has not been explained to date. Manifestations of the disease are diverse, and simultaneous involvement of multiple organs is not unusual. CASE REPORT: We report the case of a patient referred to us after multiple unsuccessful paranasal sinus operations who presented with enophthalmos and a resultant migratory keratitis with a suspected diagnosis of silent sinus syndrome. Preservation of the orbit was no longer feasible. After five years without a definitive diagnosis, we ascertained that this was a case of IgG4-related disease. DISCUSSION: IgG4-related disease represents an important element in the differential diagnosis of chronic advanced diseases of the orbit and paranasal sinuses. The diagnosis should be considered in the case of unclear disease presentations. Typical histological findings include a storiform pattern of fibrosis, vasculopathy, and tissue infiltration by IgG4 plasma cells.

[Detection and interpretation of somatic variants in molecular pathology]. / Detektion und Interpretation somatischer Varianten in der Molekularpathologie.

BACKGROUND: Due to the increasing amount of data and sources of information, data evaluation is a crucial step in parallel sequencing. OBJECTIVES: Illustration of pitfalls in evaluating the variant list of parallel sequencing and recommendations regarding software tools and databases. METHODS: Description of filtering steps used, demonstration of criteria and recommendations for annotation by examples from everyday work, comparative analysis of databases with somatic variants, description of the installation of an individualized database. RESULTS: Variant filtering is a multistep process using information from different databases. The plausibility of variant calling should be verified using the Integrative Genomics Viewer and variants should be described according to the Human Genome Variation Society (HGVS) recommendations. Different databases, which all show advantages and disadvantages, are available for variant interpretation. An individualized database can be built up with the open-source tool cBioPortal. CONCLUSIONS: Different tools and databases might be used for the analysis of parallel sequencing data. The application depends on, amongst other things, the local situation and has to be extensively validated.

[Standardized determination of tumor-infiltrating lymphocytes in breast cancer : A prognostic marker for histological diagnosis]. / Standardisierte Bestimmung tumorinfiltrierender Lymphozyten beim Mammakarzinom : Ein prognostischer Marker für die histologische Diagnostik.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) have been identified as prognostic parameter in breast cancer. OBJECTIVES: The aim of this review article is to provide an overview on the clinical and analytical validation of TILs in breast cancer. MATERIALS AND METHODS: Summary of international guidelines of the TIL working group as well as clinical and translational studies. RESULTS, CONCLUSIONS: Breast carcinomas with a high TIL level have an improved response to neoadjuvant chemotherapy. Triple-negative and HER2-positive carcinomas with increased TIL levels have improved survival. TILs are a new prognostic biomarker for routine histopathological diagnosis.

Scoring of tumor-infiltrating lymphocytes: From visual estimation to machine learning.

The extent of tumor-infiltrating lymphocytes (TILs), along with immunomodulatory ligands, tumor-mutational burden and other biomarkers, has been demonstrated to be a marker of response to immune-checkpoint therapy in several cancers. Pathologists have therefore started to devise standardized visual approaches to quantify TILs for therapy prediction. However, despite successful standardization efforts visual TIL estimation is slow, with limited precision and lacks the ability to evaluate more complex properties such as TIL distribution patterns. Therefore, computational image analysis approaches are needed to provide standardized and efficient TIL quantification. Here, we discuss different automated TIL scoring approaches ranging from classical image segmentation, where cell boundaries are identified and the resulting objects classified according to shape properties, to machine learning-based approaches that directly classify cells without segmentation but rely on large amounts of training data. In contrast to conventional machine learning (ML) approaches that are often criticized for their "black-box" characteristics, we also discuss explainable machine learning. Such approaches render ML results interpretable and explain the computational decision-making process through high-resolution heatmaps that highlight TILs and cancer cells and therefore allow for quantification and plausibility checks in biomedical research and diagnostics.

A 2015 update on predictive molecular pathology and its role in targeted cancer therapy: a review focussing on clinical relevance.

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 evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014.

BACKGROUND: The morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is gaining momentum as evidence strengthens for the clinical relevance of this immunological biomarker. Accumulating evidence suggests that the extent of lymphocytic infiltration in tumor tissue can be assessed as a major parameter by evaluation of hematoxylin and eosin (H&E)-stained tumor sections. TILs have been shown to provide prognostic and potentially predictive value, particularly in triple-negative and human epidermal growth factor receptor 2-overexpressing BC. DESIGN: A standardized methodology for evaluating TILs is now needed as a prerequisite for integrating this parameter in standard histopathological practice, in a research setting as well as in clinical trials. This article reviews current data on the clinical validity and utility of TILs in BC in an effort to foster better knowledge and insight in this rapidly evolving field, and to develop a standardized methodology for visual assessment on H&E sections, acknowledging the future potential of molecular/multiplexed approaches. CONCLUSIONS: The methodology provided is sufficiently detailed to offer a uniformly applied, pragmatic starting point and improve consistency and reproducibility in the measurement of TILs for future studies.

[Mutational tumor profiles beyond organ and tissue specificity: implications for diagnostics and clinical study design]. / Mutationsprofile von Tumoren jenseits von Organ- und Gewebespezifität : Implikationen für Diagnostik und klinisches Studiendesign.

The diagnostics and therapy of malignant tumors are based on the paradigm that cancer is an organ and tissue-specific disease. Comprehensive tumor mutation profiling data that has recently become available from next generation sequencing projects has made it possible to analyze whether the established anatomical tumor classification is reflected on the genetic level. Here, we review the results of a study on 4796 tumors of 14 major cancer types from the cancer genome atlas (TCGA) database, that on average 43% of tumors of a particular type are genetically more similar to tumors of a different anatomical origin and that the genetic tumor type corresponds to the anatomical type in only 57% of the cases. Furthermore, we discuss the implications of the complex mutation profiles and similarity patterns across cancers for diagnostics and clinical study design and explain why the comprehensive genomic data should be complemented by functional proteomic analyses.

Cancer beyond organ and tissue specificity: next-generation-sequencing gene mutation data reveal complex genetic similarities across major cancers.

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.

Prognostic impact of tumour-infiltrating immune cells on biliary tract cancer.

BACKGROUND: Biliary tract cancers (BTC) are relatively rare malignant tumours with poor prognosis. It is known from other solid neoplasms that antitumour inflammatory response has an impact on tumour behaviour and patient outcome. The aim of this study was to provide a comprehensive characterisation of antitumour inflammatory response in human BTC. METHODS: Tumour-infiltrating T lymphocytes (CD4+, CD8+, and Foxp3+), natural killer cells (perforin+), B lymphocytes (CD20+), macrophages (CD68+) as well as mast cells (CD117+) were assessed by immunohistochemistry in 375 BTC including extrahepatic (ECC; n=157), intrahepatic (ICC; n=149), and gallbladder (GBAC; n=69) adenocarcinomas. Overall and intraepithelial quantity of tumour-infiltrating immune cells was analysed. Data were correlated with clinicopathological variables and patient survival. RESULTS: The most prevalent inflammatory cell type in BTC was the T lymphocyte. Components of the adaptive immune response decreased, whereas innate immune response components increased significantly in the biliary intraepithelial neoplasia - primary carcinoma - metastasis sequence. BTC patients with intraepithelial tumour-infiltrating CD4+, CD8+, and Foxp3+ T lymphocytes showed a significantly longer overall survival. Number of total intraepithelial tumour-infiltrating Foxp3+ regulatory T lymphocytes (HR: 0.492, P=0.002) and CD4+ T lymphocytes (HR: 0.595, P=0.008) were tumour grade- and UICC-stage-independent prognosticators. The subtype-specific evaluation revealed that the tumour-infiltrating lymphocytic infiltrate is a positive outcome predictor in ECC and GBAC but not in ICC. CONCLUSION: Our findings characterise the immune response in cholangiocarcinogenesis and identify inflammatory cell types that influence the outcome of BTC patients. Further, we show that BTC subtypes show relevant differences with respect to density, quality of inflammation, and impact on patient survival.

Viscoelasticity-based magnetic resonance elastography for the assessment of liver fibrosis in hepatitis C patients after liver transplantation.

PURPOSE: Despite advantages in antiviral therapy of hepatitis C (HCV) in recent years, progressing liver fibrosis remains a major problem for patients suffering from hepatitis C after liver transplantation. Therefore, effective non-invasive methods for the assessment of liver fibrosis are needed in order to guide treatment decisions and predict prognosis in these patients. The aim of this study was to prospectively assess the diagnostic accuracy of viscoelasticity-based magnetic resonance (MR) elastography for the assessment of liver fibrosis in HCV patients after liver transplantation. MATERIALS AND METHODS: After IRB approval, a total of 25 patients, who had received a liver graft due to chronic hepatitis C underwent both liver biopsy and MR elastography. Two viscoelastic constants, the shear elasticity µ and the powerlaw exponent α were calculated by fitting the frequency function of the complex shear modulus with the viscoelastic springpot-model. RESULTS: A strong positive correlation between shear elasticity µ and the stage of fibrosis could be found (R = 0.486, p = 0.0136). The area under the receiver operating curve (AUROC) of MR elastography based on µ for diagnosis of severe fibrosis (F ≥ 3) was 0.87 and 0.65 for diagnosis of significant fibrosis (F ≥ 2). The powerlaw exponent α did not correlate with the stage of fibrosis. CONCLUSION: MR elastography represents a promising non-invasive procedure for the assessment of higher grades of fibrosis in HCV patients after liver transplantation. The poor correlation for lower grades of fibrosis suggests unknown mechanical interactions in the transplanted liver.

Understanding diseases by mouse click: the promise and potential of computational approaches in Systems Biology.

Computational modelling approaches can nowadays build large-scale simulations of cellular behaviour based on data describing detailed molecular level interactions, thus performing the space- and time-scale integrations that would be impossible just by intuition. Recent progress in the development of both experimental methods and computational tools has provided the means to generate the necessary quantitative data and has made computational methods accessible even to non-theorists, thereby removing a major hurdle that has in the past made many experimentalists hesitate to invest serious effort in formulating quantitative models. We describe how computational biology differs from classical bioinformatics, how it emerged from mathematical biology and elucidate the role it plays for the integration of traditionally separated areas of biomedical research within the larger framework of Systems Biology.