Hans Juergen Grote

Methylation Assay for the Diagnosis of Lung Cancer on Bronchial Aspirates: A Cohort Study

Purpose: Recent studies have detected aberrant promoter methylation of adenomatous polyposis coli promoter 1 A (APC), cyclin-dependent kinase inhibitor-2A (p16INK4a), retinoic acid receptor β2, and RAS association domain family protein 1 (RASSF1A) in bronchial aspirates and suggested their use as biomarkers for lung cancer diagnostics. The purpose of this study was to validate these candidate marker genes in a retrospective cohort study. Experimental Design: Bronchial aspirates collected from a cohort comprising 247 patients with suspected lung cancer were investigated retrospectively regarding aberrant promoter methylation using a quantitative methylation-specific real-time PCR (QMSP). Results: Eighty-nine patients were diagnosed with primary lung cancer, 102 had benign lung disease, and 56 showed miscellaneous other conditions. A panel consisting of APC, p16INK4a, and RASSF1A emerged as useful combination. This panel detected aberrant methylation in bronchial aspirates of 22 of 35 (63%) and 21 of 44 (44%) centrally and peripherally located primary lung cancers, respectively. Bronchial aspirates also showed aberrant methylation in 5 of 7 (71%) patients with a recurrent lung cancer and in 8 of 30 (27%) cases without tumor recurrence. In contrast, only 1 of 102 patients with benign lung disease displayed a (false) positive test result. Rarely, aberrant methylation was found in patients with other malignancies (3 of 16). The QMSP assay correctly confirmed lung cancer in 8 of 12 (67%) cases with an ambiguous cytology. Moreover, it disclosed 9 of 26 (35%) of peripheral tumors lacking simultaneous cytologic or histologic diagnosis of malignancy. Conclusions: Our findings suggest that the QMSP assay could be applied as a reflex test in cases of suspected lung cancer that defy a definite diagnosis by conventional methods. Thus, the assay could be a useful diagnostic adjunct especially regarding peripheral tumors.

Avelumab for patients with previously treated metastatic or recurrent non-small-cell lung cancer (JAVELIN Solid Tumor): dose-expansion cohort of a multicentre, open-label, phase 1b trial

BACKGROUND Avelumab, a human Ig-G1 monoclonal antibody targeting PD-L1 and approved in the USA for the treatment of metastatic Merkel cell carcinoma, has shown antitumour activity and an acceptable safety profile in patients with advanced solid tumours in a dose-escalation phase 1a trial. In this dose-expansion cohort of that trial, we assess avelumab treatment in a cohort of patients with advanced, platinum-treated non-small-cell lung cancer (NSCLC). METHODS In this dose-expansion cohort of a multicentre, open-label, phase 1 study, patients with progressive or platinum-resistant metastatic or recurrent NSCLC were enrolled at 58 cancer treatment centres and academic hospitals in the USA. Eligible patients had confirmed stage IIIB or IV NSCLC with squamous or non-squamous histology, measurable disease by Response Evaluation Criteria In Solid Tumors version 1.1 (RECIST v1.1), tumour biopsy or archival sample for biomarker assessment, and Eastern Cooperative Oncology Group performance status 0 or 1, among other criteria. Patient selection was not based on PD-L1 expression or expression of other biomarkers, including EGFR or KRAS mutation or ALK translocation status. Patients received infusional avelumab monotherapy 10 mg/kg every 2 weeks until disease progression or toxicity. The primary objective was to assess safety and tolerability. This trial is registered with ClinicalTrials.gov, number NCT01772004; enrolment in this cohort is closed and the trial is ongoing. FINDINGS Between Sept 10, 2013, and June 24, 2014, 184 patients were enrolled and initiated treatment with avelumab. Median follow-up duration was 8·8 months (IQR 7·2-11·9). The most common treatment-related adverse events of any grade were fatigue (46 [25%] of 184 patients), infusion-related reaction (38 [21%]), and nausea (23 [13%]). Grade 3 or worse treatment-related adverse events occurred in 23 (13%) of 184 patients; the most common (occurring in more than two patients) were infusion-related reaction (four [2%] patients) and increased lipase level (three [2%]). 16 (9%) of 184 patients had a serious adverse event related to treatment with avelumab, with infusion-related reaction (in four [2%] patients) and dyspnoea (in two [1%]) occurring in more than one patient. Serious adverse events irrespective of cause occurred in 80 (44%) of 184 patients. Those occurring in more than five patients (≥3%) were dyspnoea (ten patients [5%]), pneumonia (nine [5%]), and chronic obstructive pulmonary disease (six [3%]). Immune-related treatment-related events occurred in 22 patients (12%). Of 184 patients, 22 (12% [95% CI 8-18]) achieved a confirmed objective response, including one complete response and 21 partial responses. 70 (38%) had stable disease. Overall, 92 (50%) of 184 patients achieved disease control (they had a confirmed response or stable disease as their best overall response). One patient was initially thought to have died from grade 5 radiation pneumonitis during the study; however, this adverse event was subsequently regraded to grade 3 and the death was attributed to disease progression. INTERPRETATION Avelumab showed an acceptable safety profile and antitumour activity in patients with progressive or treatment-resistant NSCLC, providing a rationale for further studies of avelumab in this disease setting. FUNDING Merck KGaA and Pfizer.

Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors.

Summary The relationship between integrin expression and function in pathologies is often contentious as comparisons between human pathological expression and expression in cell lines is difficult. In addition, the expression of even integrins &agr;v&bgr;6 and &agr;v&bgr;8 in tumor cell lines is not comprehensively documented. Here, we describe rabbit monoclonal antibodies (RabMabs) against the extracellular domains of &agr;v integrins that react with both native integrins and formalin fixed, paraffin embedded (FFPE) human tissues. These RabMabs, against &agr;v&bgr;3 (EM22703), &agr;v&bgr;5 (EM09902), &agr;v&bgr;6 (EM05201), &agr;v&bgr;8 (EM13309), and pan-&agr;v (EM01309), recognize individual integrin chains in Western blots and in flow cytometry. EM22703 detected a ligand-induced binding site (LIBS), reporting an epitope enhanced by the binding of an RGD-peptide to &agr;v&bgr;3. &agr;v&bgr;8 was rarely expressed in human tumor specimens, and weakly expressed in non-small-cell lung carcinoma (NSCLC). However, ovarian carcinoma cell lines expressed &agr;v&bgr;8, as did some melanoma cells, whereas U87MG glioma lacked &agr;v&bgr;8 expression. We observed an unexpected strong expression of &agr;v&bgr;6 in tumor samples of invasive ductal breast adenoma, colorectal carcinoma (CRC), and NSCLC. &agr;v&bgr;3 was strongly expressed in some invasive NSCLC cohorts. Interestingly, PC3 prostate cell and human prostate tumors did not express &agr;v&bgr;3. The RabMabs stained plasma membranes in FFPE-immunohistochemistry (IHC) samples of tumor cell lines from lung, ovary, colon, prostate, squamous cell carcinoma of head and neck (SCCHN), breast, and pancreas carcinomas. The RabMabs are unique tools for probing &agr;v integrin biology, and suggest that especially &agr;v&bgr;6 and &agr;v&bgr;8 biologies still have much to reveal.

9p21 Deletion in the Diagnosis of Malignant Mesothelioma in Serous Effusions Additional to Immunocytochemistry, DNA-ICM, and AgNOR Analysis

The diagnosis of malignant mesothelioma (MM) in serous effusions is difficult but may be achieved by the application of adjuvant methods.

Reproducibility of immunohistochemical scoring for epidermal growth factor receptor expression in non-small cell lung cancer: round robin test.

CONTEXT The addition of cetuximab to first-line chemotherapy substantially prolonged survival in patients with advanced non-small cell lung cancer whose tumors expressed high levels of epidermal growth factor receptor (EGFR; immunohistochemistry score of ≥200 on a scale of 0-300). OBJECTIVE To evaluate the interobserver reproducibility of this EGFR immunohistochemistry scoring system, based on both the tumor cell membrane staining intensity (graded 0-3+) and the percentage of cells staining at each intensity. DESIGN In parts 1 (initial feasibility study) and 2 of this 2-part round robin test, sections of different non-small cell lung cancer tissue microarrays were stained in a central reference laboratory. Following reference evaluation, EGFR expression in 30 selected tumor cores was characterized in serial sections by lung cancer pathology specialists. The reproducibility of scoring by different raters was assessed. Analysis of between-rater agreement was based on the allocation of EGFR immunohistochemistry scores into low- (<200) and high- (≥200) EGFR expression groups. RESULTS After discussion with raters of the issues impacting reproducibility identified in part 1 and following adjustment of processes, part 2 of the round robin test showed a high interobserver agreement in EGFR immunohistochemistry scoring, with an overall concordance rate of 90.9% and a mean κ coefficient of 0.812. Specimens with a reference EGFR immunohistochemistry score of lower than 200 and of 200 or higher showed mean concordance rates of 94.7% and 85.6%, respectively. CONCLUSIONS After appropriate training, assessing EGFR expression by this immunohistochemistry-based method allowed a highly reproducible allocation of non-small cell lung cancers into clinically relevant high- or low-EGFR expression groups.

[Quantitative methylation-specific PCR for the diagnosis of lung cancer].

ZusammenfassungDie rasche, möglichst frühe Diagnose des Lungenkarzinoms stellt eine große Herausforderung dar. Die Sensitivität der konventionellen histo- und zytomorphologischen Verfahren ist diesbezüglich unbefriedigend. Der vorliegende Beitrag beleuchtet Technik, Chancen und Probleme des Nachweises aberranter Promotormethylierungen als Biomarker für die Lungenkarzinomdiagnostik an Materialien der pulmonalen Exfoliativzytologie.AbstractEfficient, preferably early diagnosis of lung cancer represents a major challenge. Under this aspect the sensitivity of conventional histomorphology and cytomorphology procedures is unsatisfactory. This review highlights technical aspects, possibilities and drawbacks of the application of aberrant promoter methylation as a biomarker for lung cancer diagnostics using specimens of pulmonary exfoliative cytology.Efficient, preferably early diagnosis of lung cancer represents a major challenge. Under this aspect the sensitivity of conventional histomorphology and cytomorphology procedures is unsatisfactory. This review highlights technical aspects, possibilities and drawbacks of the application of aberrant promoter methylation as a biomarker for lung cancer diagnostics using specimens of pulmonary exfoliative cytology.

Quantitative methylierungsspezifische PCR zur Lungenkarzinomdiagnostik

ZusammenfassungDie rasche, möglichst frühe Diagnose des Lungenkarzinoms stellt eine große Herausforderung dar. Die Sensitivität der konventionellen histo- und zytomorphologischen Verfahren ist diesbezüglich unbefriedigend. Der vorliegende Beitrag beleuchtet Technik, Chancen und Probleme des Nachweises aberranter Promotormethylierungen als Biomarker für die Lungenkarzinomdiagnostik an Materialien der pulmonalen Exfoliativzytologie.AbstractEfficient, preferably early diagnosis of lung cancer represents a major challenge. Under this aspect the sensitivity of conventional histomorphology and cytomorphology procedures is unsatisfactory. This review highlights technical aspects, possibilities and drawbacks of the application of aberrant promoter methylation as a biomarker for lung cancer diagnostics using specimens of pulmonary exfoliative cytology.Efficient, preferably early diagnosis of lung cancer represents a major challenge. Under this aspect the sensitivity of conventional histomorphology and cytomorphology procedures is unsatisfactory. This review highlights technical aspects, possibilities and drawbacks of the application of aberrant promoter methylation as a biomarker for lung cancer diagnostics using specimens of pulmonary exfoliative cytology.

PD-L1 immunostaining scoring for non-small cell lung cancer based on immunosurveillance parameters

Non-Small Cell Lung Cancer (NSCLC) is the leading cause of cancer death globally, and new immunotherapies developed and under development targeting PD-1/PD-L1 checkpoint inhibition require accurate patient selection to assure good clinical outcome. PD-L1 immunohistochemistry is the current biomarker assay used for patient selection, but still imprecise in predicting therapy response. Exploring this issue, we performed computational tissue analysis of PD-L1 immunostaining in procured NSCLC tissues (n = 50) using the Merck KGaA anti-PD-L1 clone MKP1A07310. Staining patterns and PD-L1 cut-off points were interrogated using relevant cancer immune-surveillance biomarkers. Groups with high PD-L1 expression levels (above 25/50% staining cut-off points) were enriched for a biomarker profile in the tumor-nest and microenvironment indicating escape from host-immunity, as represented by increased numbers of cells positive for CD8 and Granzyme B (immune-effectors), FOXP3 (immune-suppressive), and CD68 (P < 0.05). Manual analysis of PD-L1 staining patterns identified tumors with an immune-induced reactive pattern relevant for immunotherapy that would ordinarily be excluded by the arbitrary 25% staining threshold (P < 0.05). Conversely, some cases with completely or predominantly immune-independent constitutive PD-L1 staining patterns that indicate insensitivity to immunotherapy may have been incorrectly selected using this staining cut-off point criterion. Therefore, we propose differentiation of reactive vs constitutive PD-L1 staining patterns to improve the accuracy of this biomarker assay in selecting NSCLC patients for PD-1/PD-L1 immunotherapy.

Quantitative methylierungsspezifische PCR zur Lungenkarzinomdiagnostik@@@Quantitative methylation-specific PCR for the diagnosis of lung cancer

ZusammenfassungDie rasche, möglichst frühe Diagnose des Lungenkarzinoms stellt eine große Herausforderung dar. Die Sensitivität der konventionellen histo- und zytomorphologischen Verfahren ist diesbezüglich unbefriedigend. Der vorliegende Beitrag beleuchtet Technik, Chancen und Probleme des Nachweises aberranter Promotormethylierungen als Biomarker für die Lungenkarzinomdiagnostik an Materialien der pulmonalen Exfoliativzytologie.AbstractEfficient, preferably early diagnosis of lung cancer represents a major challenge. Under this aspect the sensitivity of conventional histomorphology and cytomorphology procedures is unsatisfactory. This review highlights technical aspects, possibilities and drawbacks of the application of aberrant promoter methylation as a biomarker for lung cancer diagnostics using specimens of pulmonary exfoliative cytology.Efficient, preferably early diagnosis of lung cancer represents a major challenge. Under this aspect the sensitivity of conventional histomorphology and cytomorphology procedures is unsatisfactory. This review highlights technical aspects, possibilities and drawbacks of the application of aberrant promoter methylation as a biomarker for lung cancer diagnostics using specimens of pulmonary exfoliative cytology.