A Phase II Study of Telisotuzumab Vedotin in Patients With c–MET-positive Stage IV or Recurrent Squamous Cell Lung Cancer (LUNG-MAP Sub-study S1400K,NCT03574753)

IntroductionLung-MAP S1400K was designed to evaluate the response to telisotuzumab vedotin, an antibody-drug conjugate targeting c-MET, in patients with c-MET–positive squamous cell carcinoma (SCC).Patients and MethodsPatients with previously treated SCC with c-MET–positive tumors (H score ≥ 150, Ventana SP44 assay) were enrolled into 2 cohorts: Cohort 1 (immune checkpoint inhibitor-naive) and Cohort 2 (immune checkpoint inhibitor refractory). Telisotuzumab vedotin 2.7 mg/kg was administered intravenously every 3 weeks until disease progression or unacceptable toxicity. Response assessments were performed every 6 weeks. The primary endpoint was response by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Secondary endpoints included progression-free survival, overall survival, response within cohort, duration of response, and toxicities. Interim analysis was planned after 20 evaluable patients, with ≥ 3 responses needed to continue enrollment.ResultsForty-nine patients (14% of screened patients) were assigned to S1400K, 28 patients enrolled (15 in Cohort 1 and 13 in Cohort 2), and 23 were eligible. S1400K closed on December 21, 2018 owing to lack of efficacy. Two responses (response rate of 9%; 95% confidence interval, 0%-20%) were reported in cohort 1 (1 complete and 1 unconfirmed partial response), whereas 10 patients had stable disease, with a disease control rate of 52%. The median overall and progression-free survival was 5.6 and 2.4 months, respectively. There were 3 grade 5 events (2 pneumonitis, in Cohort 2, and 1 bronchopulmonary hemorrhage, in Cohort 1).ConclusionTelisotuzumab vedotin failed to meet the pre-specified response needed to justify continuing enrollment to S1400K. Pneumonitis was an unanticipated toxicity observed in patients with SCC.     

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung,Melanoma, and Gastrointestinal Malignancies

We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated ≥500× coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at ≥5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n = 27), deletions (n = 5), insertions (n = 3), and multinucleotide variants (n = 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines.In modern oncologic practice, patients with advanced-stage non-small cell lung cancer (NSCLC),^{1, 2} melanoma,^{3, 4} and colorectal adenocarcinoma^{5, 6} are often treated with targeted therapies as standard of care or after enrollment in clinical trials. Molecular mutation analysis is the preferred testing modality to guide therapeutic decision making and/or eligibility for biological studies. Therefore, laboratory-developed mutation assays require robust workflows that produce high-quality sequence information from routine clinical specimens, namely formalin-fixed, paraffin-embedded (FFPE) samples. As molecular testing transitions from an ancillary tool to a seminal requirement for optimal oncologic patient management, multiplex sequencing assays with clearly defined content and bioinformatics workflows are essential for accurate and consistent results, reporting, and patient management.Published guidelines endorse which genes to test in a particular tumor type and provide timeframes for receipt of actionable results, but they also grant individual laboratories autonomy to perform mutation testing using any suitable validated method.² Historically at our institution, single-gene mutation analysis for clinically relevant genes was performed either in-house or at a Clinical Laboratory improvement Amendment–certified reference laboratory. Depending on the result, reflex testing was performed for additional genes per mutation frequency or designated algorithms. Unfortunately, this approach introduced considerable turn-around time delays and unnecessary cost, particularly when send-out testing was required. Therefore, we sought testing modalities that could analyze multiple clinically relevant mutations simultaneously, accurately, and expeditiously.Next-generation sequencing (NGS) technologies have revolutionized genomic medicine by allowing high-throughput, parallel sequencing of the human genome.⁷ Currently, however, a large proportion of clinical NGS endeavors are supported by larger academic institutions with shared access to established genomic and bioinformatics research infrastructures, and routine clinical implementation of NGS is complicated by mitigating factors, such as clinical performance, laboratory expertise, lengthy turn-around times, and cost.⁸ Thus, we investigated affordable methods to detect clinically relevant somatic mutations in NSCLC, melanoma, and gastrointestinal (GI) malignancies that generated high-quality sequencing data from FFPE samples, and offered manageable turn-around times. Targeted amplicon-based library preparation methods combined with parallel sequencing offered a practical solution, and recent studies have demonstrated the utility of this approach.^{9, 10}Reversible terminal dideoxynucleotide sequencing chemistry by Illumina (San Diego, CA) consistently generates accurate and reproducible sequencing data.^{11, 12} To use this chemistry for clinical testing, we purchased the bench-top NGS sequencer, the Illumina MiSeq, and paired it with the MiSeq-compatible Illumina TruSight tumor (TST) 26-target amplicon-based library preparation kit. TST targets 26 genes and 174 amplicons selected from College of American Pathologists/National Comprehensive Cancer Network guidelines, relevant publications, and late-phase pharmaceutical clinical trials (Supplemental Table S1). TST offered several advantages over other commercially available mutation testing kits, such as bidirectional targeting of the positive and negative DNA strands, full-exon coverage as opposed to hotspot analysis, and robust vendor-supplied bioinformatics techniques optimized for somatic variant detection. More important, TST library preparation is optimized for FFPE samples, multiple safeguards exist to detect FFPE variant artifacts, and deep sequencing of TST libraries consistently yields high depths of coverage of targeted regions.Somatic mutation testing for many of the TST genes has clinical utility in a wide variety of solid tumors. For example, testing for CTNNB1 exon 3 is performed clinically for diagnostic and prognostic purposes in pediatric desmoid tumors, select PIK3CA hotspot mutations are positive prognostic factors for breast carcinoma, and multiple exons in PDGFRA and KIT are routinely tested in GI stromal tumors to predict response to targeted therapies. More important for our intended validation purposes, all of the clinically relevant genes and regions mutated in NSCLC, melanoma, and colonic adenocarcinoma that were tested in our routine clinical practice were represented. In addition, we could easily incorporate the TST NGS into a 5 business day workflow model, and a cost-analysis demonstrated a reasonable cost per test.Last, TST NGS data are processed from raw sequence (FASTQ) to called variants with on-board MiSeq Reporter software version 2.3, and variant annotations can be performed with Illumina''s VariantStudio software version 2.1 software using standard desktop and laptop computers. The ease of library preparation, sequencing, and data analysis with tools provided by a single vendor best fit our clinical priorities and the resources available at our academic molecular pathology laboratory.Herein, we present our results from the clinical validation of TST NGS using 80 sequenced samples that were selected from 100 FFPE patient samples (40 NSCLCs, 30 melanomas, and 30 GI malignancies). During our validation, we achieved high depths of coverage for multiple clinically relevant variants when multiplexing 8 to 12 samples on a single MiSeq flow cell. TST NGS consistently demonstrated sensitivities comparable to reference assays, showed 100% concordance with known variants, detected novel variants in many samples, and uncovered variants missed by less-sensitive testing modalities. The TST variant-calling pipeline was robust and showed high concordance when compared with an alternative analysis pipeline, and we used an in-house custom Java program to assess laboratory-defined quality control (QC) metrics and streamline clinical reporting (developed by G.H.S., Emory University, http://github.com/ghsmith/coverageQc). More important, although the results detailed herein represent the experience of a single institution, the data and validation strategies shown herein are broadly applicable to most clinical molecular laboratories interested in offering NGS for NSCLCs, melanomas, and GI malignancies as well as many other solid tumors.     

Bidirectional Glenn shunt for right ventricular endomyocardial fibrosis

A 25-year-old man in New York Heart Association functional class IV with right ventricular endomyocardial fibrosis received a palliative bidirectional Glenn shunt. Despite a stormy postoperative convalescence the bidirectional Glenn shunt provided good long-term palliation.     

Participation of CD45, NKR-P1A and ANK61 antigen in rat hepatic NK cell (pit cell)mediated target cell cytotoxicity

AIM Several triggering receptors have been described to be involved in natural killer (NK) cellmediated target cytotoxicity. In these studies, NK cells derived from blood or spleen were used. Pit cells are liver-specific NK cells that possess a higher level of natural cytotoxicity and a different morphology when compared to blood NK cells. The aim of this study was to characterize the role of the NK-triggering molecules NKR-P1A, ANK61 antigen, and CD45 in pit cell-mediated killing of target cells. METHODS 51 Cr-release and DNA fragmentation were used to quantify target cell lysis and apoptosis, respectively. RESULTS Flow cytometric analysis showed that pit cells expressed CD45, NKR-P1A, and ANK61 antigen. Treatment of pit cells with monoclonal antibody ( mAb ) to CD45 ( ANK74 ) not only inhibited CC531s or YAC-1 target lysis but also apoptosis induced by pit cells. The mAbs to NKRP1A (3.2.3) and ANK61 antigen (ANK61) had no effect on pit cell-mediated CC531s or YAC-1 target cytolysis or apoptosis, while they did increase the Fcγ receptor positive (FcγR+) P815 cytolysis and apoptosis. This enhanced cytotoxicity could he inhibited by 3,4-dichloroisocoumarin, an inhibitor of granzymes. CONCLUSION These results indicate that CD45 participates in pit cell-mediated CC531s and YAC-1 target cytolysis and apoptosis. NKR-P1A and ANK61 antigen on pit cells function as activation structures against FcγR+ P815 cells, which was mediated by the perforin/granzyme pathway.     

Diagnostic challenges of an unusually large schwannoma of the mandible: Report of a case

Schwannomas are slow-growing, benign neoplasms arising from the Schwann cells and are commonly reported as peripheral tumors in the head and neck region. Central intramandibular schwannomas are extremely rare lesions. We report a case of intramandibular schwannoma in a 70?year old male patient. Panoramic radiography revealed a large, multilocular radiolucent lesion with distinct borders involving the right mandibular body and ramus. A complete excision was achieved by removing the tumor followed by reconstruction of the mandible. The clinical, radiological, and histopathological features are discussed within the context of this case.     

Specific and early detection of IgG, IgA and IgM antibodies to Mycobacterium tuberculosis 38kDa antigen in pulmonary tuberculosis

The objective was to apply the purified 38kDa protein antigen of Mycobacterium tuberculosis in ELISA to estimate the IgG, IgA and IgM antibody levels in sera and circulating immune complexes of tuberculosis patients. Sera from smear and culture positive tuberculosis patients were positive for anti 38kDa IgG, IgA and IgM antibodies, with a sensitivity of 61%, 30% and 10%, respectively, and with a specificity of 100% for IgG. The sensitivity of the test improved to a level of 68% for IgG+IgA and of 71.4% for IgG+IgA+IgM without significantly compromising the specificity (IgG of 100%, IgG+IgA of 96%, IgG+IgA+IgM of 90%). Among the smear, culture-negative but X-ray-positive cases, 60% were serum positive for IgG antibody, while in smear-negative but culture-positive cases, 54% were positive for IgG antibody. Measurement of 38kDa antibodies showed a greater than 95% sensitivity in smear and culture-positive, and smear-negative and culture-positive patients, through a combination of assays for serum IgG and circulating immune complex antibodies, while the specificity was 100%.     

Prevention of murine antiphospholipid syndrome by BAFF blockade

OBJECTIVE: This study was undertaken to determine whether BAFF blockade can be used to prevent or treat antiphospholipid syndrome in a mouse model. METHODS: Eight- and 12-week-old (NZW x BXSB)F(1) mice were treated with BAFF-R-Ig or TACI-Ig alone or in addition to a short course of CTLA-4Ig. Mice were monitored for thrombocytopenia and proteinuria. Sera were tested for anticardiolipin antibodies (aCL), BAFF levels, and levels of soluble vascular cell adhesion molecule and E-selectin. Mice were killed at 17, 22, or 32 weeks of age, and kidneys and hearts were subjected to histologic examination. Spleen cells were phenotyped and enzyme-linked immunospot assays for autoantibody-producing B cells were performed. RESULTS: Both BAFF-R-Ig and TACI-Ig prevented disease onset and significantly prolonged survival. Treated mice had significantly smaller spleens than controls, with fewer B cells and fewer activated and memory T cells. BAFF blockade did not prevent the development of aCL, and there was only a modest delay in the development of thrombocytopenia. However, treated mice had significantly less nephritis and myocardial infarcts than did controls. CONCLUSION: Our findings suggest that aCL are generated in the germinal center, which is relatively independent of BAFF. Effector function of antiplatelet antibodies was only modestly affected by BAFF blockade. In contrast, myocardial infarctions were prevented, suggesting that triggering of thromboses requires both autoantibodies and mediators of inflammation. Similarly, renal damage requires both immune complexes and effector cells. The dissociation between autoantibody production and inflammation that may occur with B cell-depleting therapies underscores the role of B cells as effector cells in the autoimmune response.     

A method for simultaneous study of the karyotype, morphology, and immunologic phenotype of mitotic cells in hematologic malignancies

A major problem in the cytogenetic analysis of hematologic neoplasms has been an inability to identify the cell from which the chromosomes were obtained. We describe a procedure that allows simultaneous analysis of karyotype and cell cytology in mitotic cells. The method differs from conventional cytogenetic analysis in that after mild hypotonic treatment, the cells are cytocentrifuged onto glass slides. In mitotic cells, this procedure often results in adequate spread of the chromosomes within the intact cell membrane. The cytoplasmic structure also remains intact, so that cytologic preparations are of good quality. Morphologic and immunologic identification of mitotic cells can be done using routine hematologic stains, such as Giemsa or Sudan black B, and various antisera using immunofluorescence techniques. The chromosomes can be simultaneously analyzed either without banding on slides stained with Giemsa or with Q-banding on slides stained with immunofluorescence techniques. Identification of numerical and structural karyotype aberrations thus is possible in morphologically identified cells.