Faculty Bibliography

PURPOSE: To establish a novel panel of cancer-specific methylated genes for cancer detection and prognostic stratification of early stage non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Identification of differentially methylated regions (DMRs) was performed with bumphunter on "The Cancer Genome Atlas (TCGA)" dataset, and clinical utility was assessed using quantitative methylation-specific PCR assay in multiple sets of primary NSCLC and body fluids that included serum, pleural effusion, and ascites samples. RESULTS: A methylation panel of 6 genes (CDO1, HOXA9, AJAP1, PTGDR, UNCX, and MARCH11) was selected from TCGA dataset. Promoter methylation of the gene panel was detected in 92.2% (83/90) of the training cohort with a specificity of 72.0% (18/25) and in 93.0% (40/43) of an independent cohort of stage IA primary NSCLC. In serum samples from the later 43 stage IA subjects and population-matched 42 control subjects, the gene panel yielded a sensitivity of 72.1% (31/41) and specificity of 71.4% (30/42). Similar diagnostic accuracy was observed in pleural effusion and ascites samples. A prognostic risk category based on the methylation status of CDO1, HOXA9, PTGDR, and AJAP1 refined the risk stratification for outcomes as an independent prognostic factor for an early stage disease. Moreover, the paralogue group for HOXA9, predominantly overexpressed in subjects with HOXA9 methylation, showed poor outcomes. CONCLUSION: Promoter methylation of a panel of 6 genes has potential for use as a biomarker for early cancer detection and to predict prognosis at the time of diagnosis.

Background: There is growing appreciation for the role of tumordraining lymph nodes (TDLN) in the dynamic of immuno-editing orchestrated by non-small cell lung cancers (NSCLC). By comparing Tcell subsets and gene expression in TDLN and non-draining lymph nodes (NDLN), we aim to determine whether there is tumor-regional variation in immunophenotype. Method: Patients undergoing endobronchial ultrasound-guided transbronchial needle aspiration for the diagnosis/staging of NSCLC were recruited. Aspirates were obtained from TDLN (N1/N2 nodes with increased fluorodeoxyglucose-F-18 (FDG) avidity and/or enlarged >1cm) and NDLN (non-enlarged/non- FDG-avid N2/N3 nodes) along with peripheral blood. Samples were stained with fluorophore-conjugated antibodies (CD4-FITC, CD8-V450, CD25-PECy7, CD127-APCR700, CD45RO-PECF594) and analyzed by flow cytometry. CD4+CD25- and CD8+ effector T-cells (Teff) were sorted. Gene expression profiling was performed on sorted Teff using the NanostringTM platform to measure differential expression between TDLN and NDLNs. Result: We compared T-cell subpopulations in TDLN and paired NDLN from 16 subjects. There were significantly fewer CD4+ T-cells in TDLN vs NDLN (10.1% vs 28.9%, p=0.0039), with more Tregs (12.1% vs 7.3%, p=0.1563) suggesting a pattern of tumorregional immunosuppression in the TDLN. This was more consistent when tumor histology was adenocarcinoma compared to squamous cell cancer with respect to both depletion of Teff and higher proportion of Tregs (Fig 1). A more immunosuppressive TDLN phenotype was also observed with high tumor PD-L1 expression (>50%), with 36% fewer CD4+ T-cells in TDLN relative to paired NDLN when PD-L1 expression was high relative to just 3.2% fewer CD4+ T-cells with low PD-L1 expression. Gene expression in Teff has preliminarily demonstrated upregulation of genes mediating T-cell exhaustion (CTLA-4, PD-1, TGFb) and downregulation of co-stimulatory/recruitment factors (CD28, ICOS, ICAM2) in TDLN suggesting impaired activation of tumorregional Teff. Conclusion: Our findings suggest that TDLNs in patients with NSCLC display a tolerogenic phenotype, with more marked immunosuppression in the setting of adenocarcinoma and high tumor PD-L1 expression. (Figure Presented)

Treating KRAS-mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein. One approach to addressing this challenge is to define mutations that frequently co-occur with those in KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS-mutant LUAD tumors carry loss-of-function mutations in the KEAP1 gene encoding Kelch-like ECH-associated protein 1 (refs. 2, 3, 4), a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response. The high frequency of mutations in KEAP1 suggests an important role for the oxidative stress response in lung tumorigenesis. Using a CRISPR-Cas9-based approach in a mouse model of KRAS-driven LUAD, we examined the effects of Keap1 loss in lung cancer progression. We show that loss of Keap1 hyperactivates NRF2 and promotes KRAS-driven LUAD in mice. Through a combination of CRISPR-Cas9-based genetic screening and metabolomic analyses, we show that Keap1- or Nrf2-mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacological inhibition of glutaminase. Finally, we provide a rationale for stratification of human patients with lung cancer harboring KRAS/KEAP1- or KRAS/NRF2-mutant lung tumors as likely to respond to glutaminase inhibition.

Background: Blood biomarkers are only infrequently used for the diagnosis of malignant pleural mesothelioma. Most of these biomarkers are single marker proteins relying on antibody assays and with limited accuracy for mesothelioma detection in the blood. In our study, we apply targeted proteomics technologies to investigate novel diagnostic strategies based on multiplexed protein biomarkers for mesothelioma detection in serum. Method: We studied more than 400 serum samples of early (I/II) and late stage (III/IV) mesothelioma and asbestos exposed donors collected in USA, Australia and Europe. For quantitative proteomics, 50 ml of serum were processed on 96-well plates over different days to enrich for N-linked glycoproteins based on hydrazide chemistry. After tryptic digestion, serum peptides were analyzed in replicates, separated by ultra-performance liquid chromatography followed by selected reaction monitoring on a triple quadrupole mass spectrometer (LC-SRM). Isotopically labeled peptides were spiked in each sample for quantification and to assess the performance of the LCSRM platform. Two non-human N-linked glycoproteins were spiked in each serum sample before processing to monitor the performance of the targeted proteomics workflow across samples and plates. The software package MSstats was used for large scale quantitative data analysis. Result: We processed and quantified over 400 serum samples analyzed in LC-SRM replicates. We assessed the performance of the targeted proteomics platform for large scale quantification of a multiplexed six peptide signature (including peptides from the established mesothelin biomarker). The coefficient of variation (CV) for parallel peptide quantification on LC-SRM ranged from 2% to 11.4% with CVs below 8% for all peptides but for one. Based on quantification of the two non-human spiked-in glycoproteins, average standard deviation of the targeted proteomics workflow was 0.42 over all samples. We investigated the performance of the multiplexed six peptide signature in discriminating mesothelioma from asbestos exposed donors. For the signature we fit a multiple logistic regression model on a training set of 212 patients and a validation set of 193 mesothelioma and asbestos exposed donors. The multiplexed biomarker signature discriminated mesothelioma from asbestos exposed with AUC of 0.72 in the validation set. Here, compared with performance of the single marker mesothelin (assessed by LC-SRM), the multiplexed biomarker signature separated early stage mesothelioma from asbestos exposed with AUC of 0.74, with sensitivity of 37.8% at 90% specificity, whereas the single mesothelin peptide had AUC of 0.66 and sensitivity of 22.2%. Conclusion: Multiplexed biomarker strategies based on targeted proteomics technologies can improve mesothelioma diagnosis in blood samples

Background: Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer associated with exposure to asbestos, and most patients die within 24 months of diagnosis. There is an urgent need to identify new therapies for treating MPM patients. Targeting "addicted" receptor tyrosine kinase (RTK) signaling networks has become a critical therapy option in cancer therapy. RTK hetero-dimerization may however, be a key element in the development of resistance to such therapy. As such Tyrosine kinase inhibitors (TKIs) with the ability to target multiple receptors may have superior efficacy to those targeting individual receptors. We and others have identified c-MET, MST1R (also known as RON), Axl and Tyro3 as RTKs frequently overexpressed and activated in MPM, making these attractive candidate targets. Several agents have been developed which target these. LCRF0004 specifically targets MST1R, whereas BMS-777607, RXDX-106 or Merestinib (LY2801653) are orally bioavailable small molecule inhibitors which inhibit c-MET, MST1R, Axl and Tyro3 at nM concentrations. These drugs may therefore have clinical utility in the treatment/management of MPM. Method: Expression of RON/MET/TAM and associated ligands were assessed in a cohort of patient samples and MPM cell lines comprising benign, epithelial, biphasic, and sarcomatoid histologies. In vitro and in vivo experiments were undertaken to determine the efficacy of single and multi RTK targeting agents (LCRF0004, RXDX- 106, BMS-777607). The effects of LCRF0004 and BMS-777607 were subsequently examined in an in vivo SQ xenograft tumor model. Result: mRNA expression of the RON/MET/TAM family and associated ligands (MSP, GAS6) was detected in a large panel of normal pleural and MPM cell lines. In a cohort of patient samples, mRNA levels of c-MET, Axl, Tyro3 and various isoforms of MST1R (flRON, sfRON, t-DELTARON) and MSP but not Gas6 or MERTK were increased in tumors compared with benign pleural samples (p<0.05). No MET Exon 14 skipping mutations were detected. RTK targeting agents displayed in vitro efficacy in terms of reduced proliferation. In vivo, the multitarget TKI (BMS-777607) demonstrated superior anti-tumor activity compared with LCRF0004 (MST1R specific compound). IHC analysis of the xenograft tumors showed high cytoplasmic expression of Vimentin, Cytokeratin and Calretinin, with significant necrosis in many. Conclusion: Our data suggests that a multi-TKI, targeting the RON/MET/TAM signaling network, is superior to selective RTK inhibition as an interventional strategy in MPM

Since its first report by Churchill and Belsey in 1939, and its evolution in lung cancer management described by Jensik in 1973 (Figure), pulmonary segmentectomy use for early lung cancer diagnosis and treatment has remained controversial. Definitive answers must wait regarding the results of the JCOG and Alliance randomized trials before true standards for surgical care for the lung cancer less than 2 cm are determined. Nevertheless, sublobar resections including wedge resection and segmentectomy are being adopted at an increasing rate compared to its use in previously published large databases (SEER, 5%; ACS NSQIP, 4%). Proper preoperative teaching and intraoperative performance of the technical aspects of sublobar resection are now becoming priorities for general thoracic resident and fellow training in an era where minimally invasive techniques are becoming increasingly the standard of care. General thoracic surgeons must have meticulous attention to detail in performing these resections in order to decrease the likelihood of collateral damage to neighboring segments as well as to minimize local recurrence whether there are performing the operation open, hybrid, standard VATS, uniportal VATS, or robotic. The technical aspects of sublobar resection begin before the patient goes to the operating room, and prime objectives in planning these resections include: 1) expertise in the segmental anatomy for that particular patient, 2) location and size of the nodule with relation to adjoining segmental bronchovascular components, 3) careful study and possible supplementation of high resolution computerized tomography with newer 3-D methods to define the spatial relationships of the nodule and segments, 4) pre- and intraoperative methods for locating the nodule if there is suspicion that parenchymal palpation will fail, 5) whether to perform wedge resection first or proceed directly to anatomic segmentectomy, 6) defining and managing the fissure between segments and recognizing when extended segmentectomy is possible or whether to convert to lobectomy and, 7) to use other intraoperative strategies to avoid technique related complications. Preoperative planning includes careful examination of the CT scan in the axial, coronal and sagittal plans in order to get a first appreciation of the depth, size, segmental anatomy and relationship of the nodule bronchovascular elements. Three-dimensional reconstruction can be as simple as navigational bronchoscopy planning images, or newer techniques for total 3-D pulmonary reconstruction which are in development.1 When there is a question of whether up front segmentectomy is to be performed and a part solid or non-solid nodule may not be palpable, intraoperative localization techniques such as navigation bronchoscopy2 or placement of fiducials/microcoils3 can be very useful. When there is no preoperative histologic diagnosis, whether a wedge or segmentectomy is performed initially will depend on the location and depth of the lesion as well as the fitness of the patient. Segmentectomy for initial diagnosis with intraoperative frozen section of both the primary lesion and suspicious level 13 and 14 stations can be prudent, especially if wedge resection could compromise performing the segmentectomy.4 In order to avoid positive margins, meticulous attention to detail with compulsive dissection and skeletonizing of the bronchovascular elements must be performed. If it is difficult to preserve the margin in a single segment resection, an extended resection of the parenchyma of adjacent segments or bisegmentectomy can be performed.5 There is controversy regarding the chance for loco-regional recurrence for segmentectomy especially in cases of pure solid lesions or segmentectomies which involve portions of the basilar segments or right upper lobe.6-8 Defining the fissure and the method with which it is divided can be one of the most important yet challenging portions of the operation. A variety of methods to define the fissure have evolved including inflation of the residual lung after segment occlusion, selective inflation of the segment to be removed, or the use of indocyanine green to define the intersegmental vein,4,5 and the fissure can be divided either with stapling alone or in combination with harmonic scalpel.9 A variety of fibrin sealants are available to decrease postoperative fistulae. With regard to the optimal approach, to date there have been no studies which show any superiority regarding conventional VATs or uniportal VATs for segmental resection, or any difference between the VATs approaches and robotic segmentectomy (10). A recent meta-analysis of over 7438 patients revealed a trend towards increased conversion to open with VATs, while postoperative complications, operation time, length of stay, chest tube duration, and number of lymph nodes were comparable. (Figure Presented)

Background: While patient-derived xenografts (PDXs) offer a powerful modality for translational cancer research, a precise evaluation of how accurately patient responses correlate with matching PDXs in a large, heterogeneous population is needed for assessing the utility of this platform for preclinical drug-testing and personalized patient cancer treatment. Patients and methods: Tumors obtained from surgical or biopsy procedures from 237 cancer patients with a variety of solid tumors were implanted into immunodeficient mice and whole-exome sequencing was carried out. For 92 patients, responses to anticancer therapies were compared with that of their corresponding PDX models. Results: We compared whole-exome sequencing of 237 PDX models with equivalent information in The Cancer Genome Atlas database, demonstrating that tumorgrafts faithfully conserve genetic patterns of the primary tumors. We next screened PDXs established for 92 patients with various solid cancers against the same 129 treatments that were administered clinically and correlated patient outcomes with the responses in corresponding models. Our analysis demonstrates that PDXs accurately replicate patients' clinical outcomes, even as patients undergo several additional cycles of therapy over time, indicating the capacity of these models to correctly guide an oncologist to treatments that are most likely to be of clinical benefit. Conclusions: Integration of PDX models as a preclinical platform for assessment of drug efficacy may allow a higher success-rate in critical end points of clinical benefit.

Carriers of heterozygous germline BAP1 mutations (BAP1^+/-) develop cancer. We studied plasma from 16 BAP1^+/- individuals from 2 families carrying different germline BAP1 mutations and 30 BAP1 wild-type (BAP1^WT) controls from these same families. Plasma samples were analyzed by liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS), ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-TQ-MS), and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). We found a clear separation in the metabolic profile between BAP1^WT and BAP1^+/- individuals. We confirmed the specificity of the data in vitro using 12 cell cultures of primary fibroblasts we derived from skin punch biopsies from 12/46 of these same individuals, 6 BAP1^+/- carriers and 6 controls from both families. BAP1^+/- fibroblasts displayed increased aerobic glycolysis and lactate secretion, and reduced mitochondrial respiration and ATP production compared with BAP1^WT. siRNA-mediated downregulation of BAP1 in primary BAP1^WT fibroblasts and in primary human mesothelial cells, led to the same reduced mitochondrial respiration and increased aerobic glycolysis as we detected in primary fibroblasts from carriers of BAP1^+/- mutations. The plasma and cell culture results were highly reproducible and were specifically and only linked to BAP1 status and not to gender, age or family, or cell type, and required an intact BAP1 catalytic activity. Accordingly, we were able to build a metabolomic model capable of predicting BAP1 status with 100% accuracy using data from human plasma. Our data provide the first experimental evidence supporting the hypothesis that aerobic glycolysis, also known as the 'Warburg effect', does not necessarily occur as an adaptive process that is consequence of carcinogenesis, but rather that it may also predate malignancy by many years and facilitate carcinogenesis.

Purpose The panel updated the American Society of Clinical Oncology (ASCO) adjuvant therapy guideline for resected non-small-cell lung cancers. Methods ASCO convened an update panel and conducted a systematic review of the literature, investigating adjuvant therapy in resected non-small-cell lung cancers. Results The updated evidence base covered questions related to adjuvant systemic therapy and included a systematic review conducted by Cancer Care Ontario current to January 2016. A recent American Society for Radiation Oncology guideline and systematic review, previously endorsed by ASCO, was used as the basis for recommendations for adjuvant radiation therapy. An update of these systematic reviews and a search for studies related to radiation therapy found no additional randomized controlled trials. Recommendations Adjuvant cisplatin-based chemotherapy is recommended for routine use in patients with stage IIA, IIB, or IIIA disease who have undergone complete surgical resections. For individuals with stage IB, adjuvant cisplatin-based chemotherapy is not recommended for routine use. However, a postoperative multimodality evaluation, including a consultation with a medical oncologist, is recommended to assess benefits and risks of adjuvant chemotherapy for each patient. The guideline provides information on factors other than stage to consider when making a recommendation for adjuvant chemotherapy, including tumor size, histopathologic features, and genetic alterations. Adjuvant chemotherapy is not recommended for patients with stage IA disease. Adjuvant radiation therapy is not recommended for patients with resected stage I or II disease. In patients with stage IIIA N2 disease, adjuvant radiation therapy is not recommended for routine use. However, a postoperative multimodality evaluation, including a consultation with a radiation oncologist, is recommended to assess benefits and risks of adjuvant radiation therapy for each patient with N2 disease. Additional information is available at www.asco.org/lung-cancer-guidelines and www.asco.org/guidelineswiki .