Evolutionary trajectories of small cell lung cancer under therapy.

The evolutionary processes that underlie the marked sensitivity of small cell lung cancer (SCLC) to chemotherapy and rapid relapse are unknown1-3. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.

Sarcoidosis involvement of the diaphragm leading to right diaphragmatic elevation: a case report.

A 69-year-old male Caucasian presenting with dyspnea on exertion related to unilateral diaphragmatic dysfunction as caused by sarcoidosis is described. First, right diaphragmatic elevation was unexplained, while the patient presented with a restrictive pattern in lung function testing using bodyplethysmography and with reduced global and diaphragmatic respiratory muscle strength as evidenced by respiratory pressures. Subsequently, surgical diaphragm plication was performed, unfortunately, without any clinical improvement. Microscopic examination of diaphragm sections revealed a lymphocytic myositis with granulomatous pleuritis showing multiple non-caseating epithelioid granulomas. Accordingly, a lymphocytic alveolitis (26% lymphocytes) with an elevated CD4/CD8 T cell ratio of 8.0% and elevated serum parameters (neopterin and sIL-2 receptor) were established. Consequently, the diagnosis of pulmonary sarcoidosis with diaphragm involvement but without extrapulmonary involvement has been established. Therefore, sarcoidosis needs to be considered in any patient presenting with unilateral diaphragmatic dysfunction. The optimal treatment strategy, however, needs to be established in the future.

Comparison of Biocartis IDYLLA ™ cartridge assay with Qiagen GeneReader NGS for detection of targetable mutations in EGFR, KRAS/NRAS, and BRAF genes.

Lung and colorectal cancers (CRC) have two of the highest mortality rates among all cancer types, and their occurrence and the need for personalized diagnostics and subsequent therapy were not influenced by the COVID-19 pandemics. However, due to the disruption of established delivery chains, standard assays for in vitro diagnostics of those cancers were temporarily not available, forcing us to implement alternative testing methods that enabled at least basic therapy decision making. For this reason, we evaluated rapid testing on the Biocartis Idylla™ platform (Biocartis, Mechelen, Belgium) for four important genes commonly mutated in lung and colorectal cancers, namely EGFR, NRAS, KRAS, and BRAF. Clinical specimens from which the mutation status has previously been determined using Next Generation Sequencing (NGS), were retested to determine whether Idylla™ can offer accurate results. To compare the results, the sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) are calculated for each of the mutation types and then combined to determine the values of the Idylla™ system in total, while setting NGS as the gold-standard basis the assays were compared with. Idylla testing thereby displayed acceptable sensitivity and specificity and delivered reliable results for initial therapy decisions.

Oncotype DX Breast Cancer recurrence score resists inter-assay reproducibility with RT2-Profiler Multiplex RT-PCR.

The Oncotype Dx assay is frequently used to test if breast cancer patients can be spared from chemotherapy without negative effects for their future clinical course. However, due to conflicting data on the assay utility, in the recent past its reimbursement situation in Germany was revised; due to continued requests by clinicians for predictive values, our group decided to implement an Oncotype Dx like alternative assay with the objective of obtaining quality and cost optimization. Customized RT2-Profiler assays covering the 21 gene panel of the Oncotype Dx assay were applied to a pilot cohort of breast cancer patients with known Oncotype Dx Recurrence Score (RS). The Ct values obtained with RT2-Profiler-assays were used to calculate the unscaled Recurrence Score (RSu) values and the thereon based RS according to the Oncotype DX assay rules if available. Despite consistent assay performance it was impossible to establish correlations between RT2-Profiler recurrence scores with the respective Oncotype DX values not to mention exact matches. By following the Oncotype DX assay and its interpretation as close as possible we faced several obstructions such as lack of information on RNA amount used, missing units in the single gene expression report, missing references cited in the original study that should explain the determination of the recurrence score formula, and vague information on the normalization of the gene expression impeding the reproduction of Oncotype Dx results in other laboratories. Unfortunately, the Oncotype Dx assay cannot be confirmed by the customized RT2-profiler assay, not least because of the fact that the individual gene measurements are not provided in the medical report, although these are mandatory for the RS calculation. In fact, the "single gene report" only contains unscaled scores of the ER, PR, and Her2 genes without any internationally accepted unit used to describe a transcript quantity. Therefore a direct comparison with the in-house measurement to evaluate its performance is impossible. With regard to our findings and the fact that the Oncotype RS represents a likelihood of the risk of relapse it thus remains impossible to assess the clinical necessity of this assay.

Association of the Human Bocavirus With Tonsil Squamous Cell Carcinomas.

Background: The human bocavirus (HBoV) is known to persist latently in the infected host cells and seems to replicate its DNA via the DNA damage response system, which is frequently defect in tumors and correlates with microsatellite instability (MSI). Because HBoV is able to persist in the infected tissues, induces pro-fibrotic and pro- cancerogenic cytokines in vivo and in vitro, and is detected in colorectal and lung tumors, the virus may be involved in cancerogenesis at least as a cofactor. Recently it was shown that the adenotonsillar tissue is an important site of HBoV1 persistence and replication. Considering the background that approximately 60% of oropharyngeal cancers were thought to be attributable to a HPV infection, a co-participation of HBoV in terms of a chronic virus infection might play a role in the cancerogenesis of tonsil tumors. Methods: Formalin-fixed, paraffin-embedded tonsil tumor samples were screened for HBoV and HPV DNA. Positive tissue sections were afterward subjected to fluorescence in situ hybridization (FISH) analysis to identify HBoV and HPV infected cells. By use of an in vitro cell culture model with primary tonsil fibroblasts, keratinocytes, and lymphocytes infected by HBoV we tried to find the target cells of virus replication. MSI testing was based on a previously published protocol using a de-multiplexed PCR followed by fluorescent detection of PCR products in a capillary sequencing device. Results: In total 62 of 103 (60, 19%) of the tonsil squamous cell carcinomas tested positive for HBoV DNA and 66 of 103 (66%) samples were identified as HPV positive. The FISH analysis revealed both double infection of HPV and HBoV in the same cells as well as single infections of both viruses within the tumor tissue. Twenty-two of 62 HBoV positive tumors tested HPV negative, 40 of 62 tissue sections were HBoV and HPV positive. We analyzed 21 out of the 62 HBoV positive tumors for MSI. Of those four tonsils displayed MSI in at least 1 of 10 microsatellite markers. Conclusion: Our findings support the hypothesis that human bocavirus infections as a cofactor may have an impact on tumor development in tonsils, although it still remains possible that HBoV solely displays a tumor tropism.

NGS-dataset of putative driver mutations associated with benign peritoneal strumosis.

A rare case of benign peritoneal strumosis was screened for driver mutations in genes relevant to currently approved cancer therapies. Therefore, three formalin fixed paraffin embedded issue sections were screened with the GeneReader Actionable Insights NGS panel (Qiagen, Hilden, Germany) for the occurrence of driver mutations. Several mutations were identified in drug-targetable genes, such as ALK, EGFR, and BRAF. The majority of identified mutations were single nucleotide variant, but also a insertion/deletion mutation was identified. The presented dataset is the first NGS dataset available from a patient with benign peritoneal strumosis.

Microsatellite instability testing in colorectal cancer using the QiaXcel advanced platform.

BACKGROUND: Microsatellite instability (MSI) is a major predictive and diagnostic marker in several cancers including colorectal carcinomas. Diagnostic testing for microsatellites is generally performed using capillary sequencers, which requires expensive high-end equipment including expensive chemistry using fluorescent dyes labelling the PCR products of interest. In this study we have modified such a diagnostic protocol and established the microsatellite testing on the QiaXcel Advanced platform. METHODS: MSI testing was based on a previously established protocol describing a multiplex PCR followed by fluorescent detection of PCR products in a capillary sequencing device. Ten microsatellites were included in the new protocol: BAT25, BAT26, BAT40, D2s123, D10s197, D13s153, D17s250, D18s58, D5s346, and MycI. In this protocol the PCR was demultiplexed and established on the QiaXcel Advanced system (Qiagen, Hilden, Germany). RESULTS: Making use of a series of FFPE control samples with known MSI status including those with and without MSI a protocol for MSI testing was successfully established on the QiaXcel Advanced platform. CONCLUSIONS: MSI testing for human colorectal cancers using the QiaXcel Advanced system could serve as an economic acceptable tool for rapid diagnostics in laboratories that do not have access to a capillary sequencing unit.

Pre-clinical validation of a next generation sequencing testing panel.

OBJECTIVE: Next Generation Sequencing (NGS) has become a useful tool for gene mutation testing which is required for targeted therapies. The aim of this study was to validate the GeneRead QIAact Actionable Insights Tumor Panel (Qiagen) on the GeneReader System in a diagnostic laboratory setting. METHODS: The GeneRead QIAact Actionable Insights Tumor Panel allows the analysis of 773 variant positions in 12 genes (ALK, BRAF, EGFR, ERBB2, ERBB3, ESR1, KIT, KRAS, NRAS, PDGFRA, PIK3CA and RAF1). For the validation of the panel we used a commercial available multiplex reference standard carrying 11 mutations in defined positions, samples from interlaboratory tests, and FFPE tumor samples from patients which were tested previously for mutations in KRAS, NRAS, BRAF, EGFR, KIT, and/or PDGFRA with pyrosequencing. RESULTS: Among the 122 tested samples, 121 samples (99.2%) were successfully sequenced. The sensitivity and specificity for detecting variants was 100% and results proved to be reproducible and precise. 119 (98.3%) results were concordant to the expected results. The differences between NGS and pyrosequencing observed in two samples were due to a wrong analysis by the pyrosequencing software which did not cover the present mutations. CONCLUSION: Overall, the GeneRead QIAact Actionable Insights Tumor Panel was specific and sensitive for mutation analysis for targeted therapies and can be incorporated into laboratory diagnostics' daily practice.

Cost Saving Opportunities in NSCLC Therapy by Optimized Diagnostics.

With an incidence of 68 new cases per 100,000 people per year, an estimated total number of up to 350,000 new non-small-cell lung cancer (NSCLC) cases are diagnosed each year in the European Union. Up to 10% of NSCLC patients are eligible for therapy with novel ALK (anaplastic lymphoma kinase) inhibitors, as they have been diagnosed with a mutation in the gene coding for ALK. The ALK inhibitor therapy costs add up to approx. 9,000 € per patient per month, with treatment durations of up to one year. Recent studies have shown that up to 10% of ALK cases are misdiagnosed by nearly 40% of pathologic investigations. The current state-of-the-art ALK diagnostic procedure comprises a Fluorescent in situ Hybridization (FISH) assay accompanied by ALK inhibitor therapy (Crizotinib). The therapy success ranges between a full therapy failure and the complete remission of the tumor (i.e., healing), but the biomedical and systemic reasons for this range remain unknown so far. It appears that the variety of different ALK mutations and variants contributes to the discrepancy in therapy results. Although the major known fusion partner for ALK in NSCLC is the Echinoderm microtubule-associated protein-like 4 (EML4), of which a minimum of 15 variants have been described, an additional 20 further ALK fusion variants with other genes are known, of which three have already been found in NSCLC. We hypothesize that the wide variety of known (and unknown) ALK mutations is associated with a variable therapy success, thus rendering current companion diagnostic procedures (FISH) and therapy (Crizotinib) only partly applicable in ALK-related NSCLC treatment. In cell culture, differing sensitivity to Crizotinib has been shown for some fusion variants, but it is as yet unknown which of them are really biologically active in cancer patients, and how the respective variants affect the response to Crizotinib treatment. Moreover, it has been demonstrated that translocated ALK genes can also be observed in healthy tissues and are not compulsorily associated with tumors. Therefore, it is important to keep in mind that even for the known variants of ALK fusion genes, the biological function is not known for all variants, and that no information is available on the homogeneity of ALK fusion variants within a single tumor. These facts, in concert with data for ALK mutation prevalence and therapy outcomes of a German cohort of NSCLC patients, support the hypothesis that, by using novel companion diagnostic tools in combination with therapy outcome predictions, massive cost savings could be possible in European Health Care systems without a loss of patient care.

Pneumocystis jirovecii-induced chronic interstitial lung disease in Waldenström's macroglobulinemia.

Infections with Pneumocystis jirovecii can result in asymptomatic colonization or induce life threatening clinical symptoms. However, there appears to be a 'gray area' between colonization and severe pneumonia that remains underestimated so far. We describe a case with chronic interstitial lung disease and chronic cough that was attributed to P. jirovecii. The patient's history of chronic cough, although very likely being fostered by the underlying Waldenström's macroglobulinemia and interstitial lung disease, was most likely caused by P. jirovecii infection. This gives raise to the hypothesis that P. jirovecii infections do not necessarily induce life threatening pneumonia. Consequently, serial testing is required in eligible patients with positive PCR results in order to discriminate between colonization, 'gray zone' infection, and beginning pneumonia.

Fatal HBoV-1 infection in adult female cystic fibrosis patient.

A clinical case of fatal HBoV infection in an adult cystic-fibrosis patient awaiting lung transplantation is reported. The case is important as the genetic background of the underlying disease is congruent with the background of the sole permissive permanent cell culture CuFi-8 which originates also from a CF patient donor.

Presurgical mapping of basal cell carcinoma or squamous cell carcinoma by confocal laser endomicroscopy compared to traditional micrographic surgery: a single-centre prospective feasibility study.

BACKGROUND: At present, no ideal diagnostic tools exist in the market to excise cancer tissue with the required safety margins and to achieve optimal aesthetic results using tissue-conserving techniques. OBJECTIVES: In this prospective study, confocal laser endomicroscopy (CLE) and the traditional gold standard of magnifying glasses (MG) were compared regarding the boundaries of in vivo basal cell carcinoma and squamous cell carcinoma. MATERIALS & METHODS: Tumour diameters defined by both methods were measured and compared with those determined by histopathological examination. Nineteen patients were included in the study. RESULTS: The CLE technique was found to be superior to excisional margins based on MG only. Re-excision was required in 68% of the cases following excision based on MG evaluation, but only in 27% of the cases for whom excision margins were based on CLE. CONCLUSION: Our results are promising regarding the distinction between tumour and healthy surrounding tissue, and indicate that presurgical mapping of basal cell carcinoma and squamous cell carcinoma is possible. The tool itself should be developed further with special attention to early detection of skin cancer.

PD-L1 expression in non-small cell lung cancer: Correlations with genetic alterations.

Inhibition of the PD-1/PD-L1 pathway may induce anticancer immune responses in non-small cell lung cancer (NSCLC). Two PD-L1 immunohistochemistry (IHC) assays have been approved as companion diagnostic tests for therapeutic anti-PD-1 antibodies. However, many aspects of PD-L1 prevalence and association with genetically defined subtypes have not been addressed systematically. Here, we analyzed PD-L1 expression in 436 genetically annotated NSCLC specimens enriched for early stages using PD-L1 antibody 5H1. Expression of PD-L1 was detected in the tumor cells (TC) (34% of cases) and in associated immune cells (IC) (49%) across all stages of NSCLC, either alone or in combination. PD-L1 IHC-positive TC, but not IC showed significantly higher PD-L1 RNA expression levels. Expression in TC was associated with TP53, KRAS and STK11 mutational status in adenocarcinomas (AD) and with NFE2L2 mutations in squamous cell carcinomas (SQ). No correlations with histological subtype, clinical characteristics and overall survival were found. The presence of PD-L1-positive IC was significantly associated with patients' smoking status in AD. The findings are in agreement with the emerging concept that tumors with high mutational burden are more likely to benefit from immunotherapy, since TP53 and KRAS mutations are linked to smoking, increased numbers of somatic mutations and expression of neoantigens. Current clinical studies focus on stage IIIB and IV NSCLC; however, PD-L1 expression occurs in earlier stages and might be a predictive biomarker in clinical trials testing (neo-) adjuvant strategies.

Lung Infection by Human Bocavirus Induces the Release of Profibrotic Mediator Cytokines In Vivo and In Vitro.

Human Bocavirus subtype 1 (HBoV1) is associated with respiratory diseases and may contribute to chronic lung diseases by persisting in the infected host. Here the question was addressed if HBoV infections could contribute to fibrogenesis processes as suggested by previously published clinical observations. Cytokine profiles induced by HBoV infection in CuFi-8 air-liquid interphase cell cultures and in bronchoalveolar lavage fluid (BALF) of 20 HBoV-positive and 12 HBoV-negative patients were analysed by semi-quantitative Western spot blot analyses. Although lots of cytokines were regulated independently of HBoV status, several cytokines associated with lung fibrosis and tumour development, e.g., EGF, VEGF, TARC (CCL17), TNF-α, TNF-ß, TIMP-1, were clearly upregulated in the HBoV-positive cohort. These findings suggest that the development of lung fibrosis might be triggered by HBoV induced cytokine expression.

Epidemiology of driver mutations in lung cancer in a German tertiary hospital in patients with testing indication.

BACKGROUND: KRAS, BRAF, EGFR and ALK-mutation testing is a prerequisite for non-small-cell lung cancer treatments, but there remains limited epidemiological information about such mutations in German cohorts. MATERIALS & METHODS: Between February 2010 and June 2015, a total of 1080 tumor samples from 1019 non-small-cell lung cancer patients were analyzed for KRAS, BRAF, EGFR and ALK-mutations by Therascreen-pyrosequencing and FISH. RESULTS: Mutation patterns differed dependent on the histological subtype and sex. Mainly, adenocarcinomas were mutated and formed the major histological group. Double mutations were observed also not explicitly screened for. In our German cohort, female adenocarcinoma patients had statistically significantly higher rates EGFR mutations than male patients. DISCUSSION: The different mutation patterns dependent on histological phenotypes warrant further epidemiological studies while suggesting different mechanisms of cancerogenesis.

Spontaneous regression of non-small cell lung cancer after biopsy of a mediastinal lymph node metastasis: a case report.

INTRODUCTION: Spontaneous regression of cancer is defined as a complete or partial, temporary or permanent disappearance of tumor in the absence of specific therapy. With only a few cases reported, spontaneous regression is extremely rare in primary lung cancer. Regarding spontaneous regression in lung cancer, recent investigations revealed the role of immunological mechanisms, thus indicating potential treatment options by specific immunotherapy in the future. CASE PRESENTATION: A 76-year-old Caucasian man with progressive dyspnea presented to our hospital. A computed tomography scan revealed a tumor mass in the upper lobe of his right lung and enlarged mediastinal lymph nodes. A biopsy of a paratracheal lymph node by mediastinoscopy disclosed metastatic lung cancer. By immunohistochemical findings the tumor was classified as large cell carcinoma. Diagnosed with clinical stage IIIA non-small cell lung cancer, a neoadjuvant therapy concept was indicated. However, before starting chemoradiation, a computed tomography scan showed a regression of both the tumor mass in the upper lobe of his right lung and the mediastinal lymphadenopathy. As a repeated computed tomography scan showed further regression, we agreed with our patient to perform routine follow-up instead of starting therapy. To date, no relapse has been reported. CONCLUSIONS: Given the circumstances that regression started after the biopsy and involved both the tumor in the upper lobe of his right lung and the mediastinal lymph node metastases, an immune response is a reasonable explanation for the observed spontaneous regression in this case.

Low frequency of EGFR mutations in pleural mesothelioma patients, Cologne, Germany.

EGFR mutations were previously found in patients suffering from peritoneal mesothelioma but have not yet been described in pleural mesothelioma. The aim of the present study was the identification of EGFR mutations in patients suffering from pleural mesothelioma. Pleural mesothelioma tissue from 31 patients was used to analyze possible mutations in the EGFR gene comprising the exons 18-21 with the codons 719, 768, 790, 858+861, 731+734, 785, 797+801, 831, and 868 with pyrosequencing. The results indicate that 31 pleural mesothelioma patients show a wild-type EGFR gene when analyzing the codons D19, 768, 790, 858+861, 731+734, 785, 797+801, 831, and 868, whereas 2 patients have a mutation in the EGFR gene in codon 719. Sanger sequencing of the EGFR codon 785 was used for the determination of a polymorphism in the sequencing of tumor-free patients and pleural mesothelioma patients with a distribution of a wild-type homozygous sequence with guanine, a wild-type heterozygous sequence having guanine and adenine, a wild-type homozygous sequence with adenine, and a wild-type heterozygous sequence with adenine and guanine. Next, the identification of less EGFR mutations in the EGFR gene of the pleural mesothelioma an up to this time unknown polymorphism in the EGFR gene was identified which could be wrongly interpreted as a mutation.