Hideki Kuwano

An Immunogram for the Cancer-Immunity Cycle: Towards Personalized Immunotherapy of Lung Cancer.

Introduction: The interaction of immune cells and cancer cells shapes the immunosuppressive tumor microenvironment. For successful cancer immunotherapy, comprehensive knowledge of antitumor immunity as a dynamic spatiotemporal process is required for each individual patient. To this end, we developed an immunogram for the cancer‐immunity cycle by using next‐generation sequencing. Methods: Whole exome sequencing and RNA sequencing were performed in 20 patients with NSCLC (12 with adenocarcinoma, seven with squamous cell carcinoma, and one with large cell neuroendocrine carcinoma). Mutated neoantigens and cancer germline antigens expressed in the tumor were assessed for predicted binding to patients’ human leukocyte antigen molecules. The expression of genes related to cancer immunity was assessed and normalized to construct a radar chart composed of eight axes reflecting seven steps in the cancer‐immunity cycle. Results: Three immunogram patterns were observed in patients with lung cancer: T‐cell–rich, T‐cell–poor, and intermediate. The T‐cell–rich pattern was characterized by gene signatures of abundant T cells, regulatory T cells, myeloid‐derived suppressor cells, checkpoint molecules, and immune‐inhibitory molecules in the tumor, suggesting the presence of antitumor immunity dampened by an immunosuppressive microenvironment. The T‐cell–poor phenotype reflected lack of antitumor immunity, inadequate dendritic cell activation, and insufficient antigen presentation in the tumor. Immunograms for both the patients with adenocarcinoma and the patients with nonadenocarcinoma tumors included both T‐cell–rich and T‐cell–poor phenotypes, suggesting that histologic type does not necessarily reflect the cancer immunity status of the tumor. Conclusions: The patient‐specific landscape of the tumor microenvironment can be appreciated by using immunograms as integrated biomarkers, which may thus become a valuable resource for optimal personalized immunotherapy.

Identification of Individual Cancer-Specific Somatic Mutations for Neoantigen-Based Immunotherapy of Lung Cancer

Introduction: Two strategies for selecting neoantigens as targets for non–small cell lung cancer vaccines were compared: (1) an “off‐the‐shelf” approach starting with shared mutations extracted from global databases and (2) a personalized pipeline using whole‐exome sequencing data on each patients tumor. Methods: The Catalogue of Somatic Mutations in Cancer database was used to create a list of shared missense mutations occurring in more than 1% of patients. These mutations were then assessed for predicted binding affinity to HLA alleles of 15 lung cancer patients, and potential neoantigens (pNeoAgs) for each patient were selected on this basis. In the personalized approach, pNeoAgs were selected from missense mutations detected by whole‐exome sequencing of the patients own samples. Results: The list of shared mutations included 22 missense mutations for adenocarcinoma and 18 for squamous cell carcinoma (SCC), resulting in a median of 10 off‐the‐shelf pNeoAgs for each adenocarcinoma (range 5–13) and 9 (range 5–12) for each SCC. In contrast, a median of 59 missense mutations were identified by whole‐exome sequencing (range 33–899) in adenocarcinoma and 164.5 (range 26–232) in SCC. This resulted in a median of 46 pNeoAgs (range 13–659) for adenocarcinoma and 95.5 (range 10–145) for SCC in the personalized set. We found that only one or two off‐the‐shelf pNeoAgs were included in the set of personalized pNeoAgs—and then in only three patients, with no overlap seen in the remaining 12 patients. Conclusions: Use of an off‐the‐shelf pipeline is feasible but may not be satisfactory for most patients with non–small cell lung cancer. We recommend identifying personal mutations by comprehensive genome sequencing for developing neoantigen‐targeted cancer immunotherapies.

Prediction and prioritization of neoantigens: integration of RNA sequencing data with whole-exome sequencing

The importance of neoantigens for cancer immunity is now well‐acknowledged. However, there are diverse strategies for predicting and prioritizing candidate neoantigens, and thus reported neoantigen loads vary a great deal. To clarify this issue, we compared the numbers of neoantigen candidates predicted by four currently utilized strategies. Whole‐exome sequencing and RNA sequencing (RNA‐Seq) of four non‐small‐cell lung cancer patients was carried out. We identified 361 somatic missense mutations from which 224 candidate neoantigens were predicted using MHC class I binding affinity prediction software (strategy I). Of these, 207 exceeded the set threshold of gene expression (fragments per kilobase of transcript per million fragments mapped ≥1), resulting in 124 candidate neoantigens (strategy II). To verify mutant mRNA expression, sequencing of amplicons from tumor cDNA including each mutation was undertaken; 204 of the 207 mutations were successfully sequenced, yielding 121 mutant mRNA sequences, resulting in 75 candidate neoantigens (strategy III). Sequence information was extracted from RNA‐Seq to confirm the presence of mutated mRNA. Variant allele frequencies ≥0.04 in RNA‐Seq were found for 117 of the 207 mutations and regarded as expressed in the tumor, and finally, 72 candidate neoantigens were predicted (strategy IV). Without additional amplicon sequencing of cDNA, strategy IV was comparable to strategy III. We therefore propose strategy IV as a practical and appropriate strategy to predict candidate neoantigens fully utilizing currently available information. It is of note that different neoantigen loads were deduced from the same tumors depending on the strategies applied.

Role of post-mapping computed tomography in virtual-assisted lung mapping

Background Virtual-assisted lung mapping is a novel bronchoscopic preoperative lung marking technique in which virtual bronchoscopy is used to predict the locations of multiple dye markings. Post-mapping computed tomography is performed to confirm the locations of the actual markings. This study aimed to examine the accuracy of marking locations predicted by virtual bronchoscopy and elucidate the role of post-mapping computed tomography. Methods Automated and manual virtual bronchoscopy was used to predict marking locations. After bronchoscopic dye marking under local anesthesia, computed tomography was performed to confirm the actual marking locations before surgery. Discrepancies between marking locations predicted by the different methods and the actual markings were examined on computed tomography images. Forty-three markings in 11 patients were analyzed. Results The average difference between the predicted and actual marking locations was 30 mm. There was no significant difference between the latest version of the automated virtual bronchoscopy system (30.7 ± 17.2 mm) and manual virtual bronchoscopy (29.8 ± 19.1 mm). The difference was significantly greater in the upper vs. lower lobes (37.1 ± 20.1 vs. 23.0 ± 6.8 mm, for automated virtual bronchoscopy; p < 0.01). Despite this discrepancy, all targeted lesions were successfully resected using 3-dimensional image guidance based on post-mapping computed tomography reflecting the actual marking locations. Conclusions Markings predicted by virtual bronchoscopy were dislocated from the actual markings by an average of 3 cm. However, surgery was accurately performed using post-mapping computed tomography guidance, demonstrating the indispensable role of post-mapping computed tomography in virtual-assisted lung mapping.

Low truncal muscle area on chest computed tomography: a poor prognostic factor for the cure of early-stage non-small-cell lung cancer†

OBJECTIVES Depletion in skeletal muscle is closely associated with limited physical ability and high mortality. In this study, we evaluated the prognostic significance of skeletal muscle depletion in patients with early-stage non-small-cell lung cancer. METHODS A retrospective analysis of patients with pathological stages I-II lung cancer, who underwent curative resection between 2009 and 2013, was conducted. The truncal muscle index (TMI) (area/height2) at the first lumbar vertebral level was measured by preoperative axial computed tomography. Overall survival and recurrence-free survival were compared between the lowest gender-specific quartile of the TMI and the other quartiles. RESULTS A total of 314 subjects were included in the study. The cumulative 5-year recurrence-free and overall survival rates were significantly shorter in patients with lower TMIs (69% vs 83.5%, P = 0.028; 64.8% vs 80.1%, P = 0.003, respectively). In multivariable models, the TMI was identified as an independent prognostic factor for overall survival (P =  0.017, hazard ratio 1.84, 95% confidence interval 1.12-3.05), after adjusting for age, gender, preoperative serum albumin, carcinoembryonic antigen, neutrophil to lymphocyte ratio and pathological stage. CONCLUSIONS A low preoperative TMI was associated with a poor postoperative outcome in patients with early-stage non-small-cell lung cancer. This factor may be included in the preoperative assessment of patients, for whom surgical intervention is considered.

Management of lung nodules newly found by virtual-assisted lung mapping: a case report

BackgroundVirtual-assisted lung mapping is a novel bronchoscopic lung marking technique that uses virtual images to perform multiple concurrent dye marking of barely palpable pulmonary tumors. Subsequent chest computed tomography is required to confirm the locations marked. We here report a patient in whom computed tomography after virtual-assisted lung mapping unexpectedly revealed additional tiny pulmonary nodules.Case presentationA 64-year-old woman with a history of renal cell carcinoma presented with two pulmonary nodules suspicious of metastases from renal cell carcinoma. Because we anticipated that the nodules would be difficult to palpate intraoperatively, we performed virtual-assisted lung mapping prior to attempting to resect them. Computed tomography after mapping unexpectedly detected two additional nodules. Although the existing markings did not relate to the newly found nodules, we used imaginary auxiliary lines and anatomical landmarks to extend the lung map to incorporate the unexpected nodules. The additional nodules were successfully resected by thoracoscopic wedge resection. Pathologic examination identified all nodules as metastases from renal cell carcinoma, and the surgical margins were negative.ConclusionsImaginary auxiliary lines and anatomical landmarks extended the existing lung map of virtual-assisted lung mapping, enabling resection of unexpected pulmonary nodules found in post-mapping computed tomography images.

A deep azygoesophageal recess may increase the risk of secondary spontaneous pneumothorax

PurposeThe azygoesophageal recess (AER) is known as a possible cause of bulla formation in patients with spontaneous pneumothorax. However, there has been little focus on the depth of the AER. We evaluated the relationship between the depth of the AER and pneumothorax development.MethodsWe conducted a retrospective study of 80 spontaneous pneumothorax patients who underwent surgery at our institution. We evaluated the depth of the AER on preoperative computed tomography scans.ResultsRuptured bullae at the AER were found in 12 patients (52.2%) with secondary spontaneous pneumothorax (SSP) and 8 patients (14.0%) with primary spontaneous pneumothorax (PSP) (p < 0.001). In patients with ruptured bullae at the AER, 10 SSP patients (83.3%) had a deep AER while only 2 PSP patients (25%) had a deep AER (p = 0.015).ConclusionsA deep AER was more frequently associated with SSP than with PSP. A deep AER may contributes to bulla formation and rupture in SSP patients.

Flat Chest of Pleuroparenchymal Fibroelastosis Reversed by Lung Transplantation

A patient with pleuroparenchymal fibroelastosis (PPFE) was successfully treated with living-donor lobar lung transplantation. A 27-year-old woman with a 3-month history of dyspnea received a diagnosis of PPFE. Her chest wall was extremely flattened over time, and her respiratory condition progressively deteriorated. She underwent semielective bilateral living-donor lobar lung transplantation. Her chest wall rigidity, which was secondary to PPFE, required intensive pulmonary rehabilitation postoperatively. By 6 months after transplantation, the flattening of her chest wall was reversed. Living-donor lobar lung transplantation was a life-saving procedure for this patient and improved the chest wall deformity of PPFE.