Genomic-transcriptomic evolution in lung cancer and metastasis.

Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy1. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study2,3. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic-transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary-metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis.

The evolution of non-small cell lung cancer metastases in TRACERx.

Metastatic disease is responsible for the majority of cancer-related deaths1. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse.

The evolution of lung cancer and impact of subclonal selection in TRACERx.

Lung cancer is the leading cause of cancer-associated mortality worldwide1. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource.

Antibodies against endogenous retroviruses promote lung cancer immunotherapy.

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS)1,2. Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive1,2. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma3. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response.

Lung adenocarcinoma promotion by air pollutants.

A complete understanding of how exposure to environmental substances promotes cancer formation is lacking. More than 70 years ago, tumorigenesis was proposed to occur in a two-step process: an initiating step that induces mutations in healthy cells, followed by a promoter step that triggers cancer development1. Here we propose that environmental particulate matter measuring ≤2.5 µm (PM2.5), known to be associated with lung cancer risk, promotes lung cancer by acting on cells that harbour pre-existing oncogenic mutations in healthy lung tissue. Focusing on EGFR-driven lung cancer, which is more common in never-smokers or light smokers, we found a significant association between PM2.5 levels and the incidence of lung cancer for 32,957 EGFR-driven lung cancer cases in four within-country cohorts. Functional mouse models revealed that air pollutants cause an influx of macrophages into the lung and release of interleukin-1ß. This process results in a progenitor-like cell state within EGFR mutant lung alveolar type II epithelial cells that fuels tumorigenesis. Ultradeep mutational profiling of histologically normal lung tissue from 295 individuals across 3 clinical cohorts revealed oncogenic EGFR and KRAS driver mutations in 18% and 53% of healthy tissue samples, respectively. These findings collectively support a tumour-promoting role for  PM2.5 air pollutants  and provide impetus for public health policy initiatives to address air pollution to reduce disease burden.

Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA.

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy.

Retention Rate of Free Pericardial Fat Grafts after Bronchial Stump Coverage.

The postoperative course of the graft tissue after bronchial stump coverage remains unclear. We retrospectively analyzed 44 patients who underwent anatomical lung resection followed by bronchial stump coverage using free pericardial fat grafts. All patients underwent minimally invasive video-assisted thoracoscopic surgery. Computed tomography scans showed a graft retention rate of 100% on 60 days after surgery, 61% on 180 days, and plateauing at around 20% after 1 year. Free pericardial fat grafts, harvested minimally invasively, demonstrated a promising retention rate after surgery, making them a suitable option for patients with a high risk of bronchopleural fistula.

Combination of Skeletal Muscle Mass and Density Predicts Postoperative Complications and Survival of Patients With Non-Small Cell Lung Cancer.

BACKGROUND: Few studies have assessed the comprehensive skeletal muscle depletion associated with loss of muscle quantity (sarcopenia) and reduced muscle quality in cancer patients. This study aimed to clarify the impact of skeletal muscle depletion on outcomes after non-small cell lung cancer surgery. METHODS: Data for 341 patients with pathologic stages 1 to 3A non-small cell lung cancer who underwent lobectomy and mediastinal lymph node dissection from 2009 to 2013 were retrospectively reviewed. The integrative pectoralis muscle index (IPMI) was assessed by multiplying the normalized pectoralis muscle area (area/body mass index) and mean radiodensity on chest images. Postoperative outcomes were compared among sex-specific quartiles of IPMI. The trend of continuous and categorical variables was analyzed using the Jonckheere-Terpstra test and the Cochrane-Armitage test, respectively. RESULTS: Respiratory strength declined with decreasing quartiles of IPMI (P < 0.001). The risk of major complications escalated with the decrease of IPMI among four quartiles (7.1 %, 16.7 %, 18.4 %, and 22.4 %; P = 0.008). The hospital stay was prolonged for patients with reduced IPMI (P = 0.001). Patients in the lowest and highest quartiles had the worst and best 5-year overall survival, respectively, compared with those in the two intermediate quartiles of IPMI (67.0 %, 87.9 %, and 81.2 %, respectively; P=0.001). Multivariate analysis identified the lowest quartile of IPMI as an independent poor prognostic factor (hazard ratio, 1.88; 95 % confidence interval, 1.11-3.19; P = 0.020). CONCLUSION: Comprehensive skeletal muscle profiling, including morphometric mass and componential density on chest imaging, has the potential to refine risk stratification and prognostication in non-small cell lung cancer.

Competing Risk Analysis in Lung Cancer Patients Over 80 Years Old Undergoing Surgery.

BACKGROUND: This study aimed to analyze cause-specific mortality in lung cancer patients over 80 years old undergoing surgery. METHODS: This retrospective, multi-institutional analysis included patients aged ≥ 80 years who underwent radical surgery for primary lung cancer from January 1998 to December 2015. Preoperative clinical data, surgical results, survival, and cause of death were evaluated. Competing risk analysis for cause-specific mortality was performed. RESULTS: Of the 337 patients (median age 82 years) enrolled and analyzed, 68.1% were male. There were 52 and 44 cancer-specific and non-cancer-specific deaths, respectively. On competing risk regression analysis, non-cancer-specific deaths were significantly associated with male sex (hazard ratio [HR]: 3.06, 95% confidence interval [CI]: 1.02-9.12, p = 0.046), coronary artery disease (HR: 2.49, 95% CI: 2.49 [1.14-5.47], p = 0.02), interstitial pneumonia (HR: 3.58, 95% CI: 1.73-7.40, p < 0.001), and pathological stage III (HR: 3.83, 95% CI: 1.44-10.13, p = 0.007). In contrast, cancer-specific deaths were significantly associated with limited resection (HR: 1.99, 95% CI: 1.02-3.89, p = 0.04) and pathological stage III (HR: 3.13, 95% CI: 1.44-6.80, p = 0.004). The 5-year cumulative incidences of lung cancer-specific and non-cancer-specific deaths were 18.0% and 15.9%, respectively. CONCLUSIONS: Prognostic factors for non-cancer-specific death were different from those of cancer-specific death, except for pathological stage. Each prognostic factor should be considered when deciding surgical indication and procedure and monitoring for pulmonary events during outpatient follow-up.

[Reexpansion Pulmonary Edema and Atrial Fibrillation after Resection of a Giant Solitary Fibrous Tumor of the Pleura].

We report a case of giant solitary fibrous tumor( SFT) of the pleura postoperatively complicated with probable reexpansion pulmonary edema and atrial fibrillation. An 85-year-old woman was diagnosed to have a 13 cm sized intrathoracic neoplasm. Upon thoracotomy, the tumor was found to pedunculate from the right lung with no direct invasion to the surrounding structures. Complete resection of the tumor and expansion of the right lung was obtained. Histopathology revealed the tumor was a benign SFT arising from the visceral pleura. An infiltrative shadow in the right lower lung field soon after the surgery suggested a reexpansion pulmonary edema, which eventually recovered within a week. The patient suffered from a refractory atrial fibrillation that led to a congestive heart failure requiring an intensive medical treatment. It is emphasized that thoracic surgeons should be aware of these postoperative complications in treating such a case like this.

NK cell and IFN signatures are positive prognostic biomarkers for resectable pancreatic cancer.

To establish prognostic biomarkers and to identify potential novel therapeutic targets, we performed integrative immunomonitoring of blood and tumor in patients with resectable pancreatic cancer. Flow cytometry (FC) was employed for phenotyping immune cells, multiplex bead assays for plasma cytokine and chemokine determination, and RNA-Seq for the analysis of gene expression in the tumor. Nineteen pancreatic cancer patients were stratified into those with longer or shorter than median recurrence-free survival after surgery (median, 426 days). There were no significant differences between the two groups for clinical parameters including age, sex, surgical procedure, stage, or postoperative adjuvant therapy. However, we found that the percentages of NK cells as assessed by FC in peripheral blood mononuclear cells were higher in patients with late recurrence (P = .037). RNA-Seq data indicated no differences in the amount of immune cells or stromal cells between the two groups, although NK cells in the tumor did tend to be higher in patients with late recurrence (P = .058). Type I and II IFN signatures were enriched in late-recurring tumors (FDR q-value <0.001), while genes related to KRAS signaling and the epithelial mesenchymal transition (EMT) were enriched in early recurrence. We conclude that tumor-intrinsic properties of metastasis and recurrence influence prognosis, whereas NK cells that might contribute to prevent metastasis are associated with longer recurrence-free survival. Therefore, enhancement of NK cell activity and inhibition of the EMT and KRAS signaling might represent appropriate therapeutic targets following surgical resection of pancreatic cancer.

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.

[Neoantigens and Whole-Exome Sequencing].

During cancer progression, many somatic mutations accumulate in cancer cells. Antigens derived from tumor-specific mutated genes are primary sources ofneoantigens in cancer immunology. As compared with non-mutated self-antigens, neoantigens are thought to have higher antigenicity, and are expected to be ideal targets for tumor rejection. Recent studies demonstrate that T cells recognize neoantigens and elicit immune responses against tumor cells. The importance ofneoantigens in cancer immunity has been well acknowledged, and development ofneoantigen -targeted cancer immunotherapy is in progress worldwide. High-throughput detection ofsomatic mutations by whole-exome sequencing is combined with computational algorithm for MHC-peptide binding affinity to predict potential neoantigens. After the antigenicity is confirmed by immunological assays, personalized vaccines targeting the identified neoantigens will be generated with peptides, dendritic cells, or RNAs. Such neoantigen-targeted cancer vaccines are being developed for practical use. In fact, several clinical trials have already been initiated in Europe and the US.

Outcomes of outpatient treatment for primary spontaneous pneumothorax using a small-bore portable thoracic drainage device.

BACKGROUND: There is no consensus regarding the initial intervention for primary spontaneous pneumothorax. We report the outcomes of outpatient treatment for primary spontaneous pneumothorax using a portable thoracic drainage device. PATIENTS AND METHODS: Between April 2007 and December 2011, 99 consecutive patients with a first episode of primary spontaneous pneumothorax were indicated for insertion of a portable thoracic drainage device. All patients were treated with a small-bore portable thoracic drainage device that consists of a flexible 9F silicone catheter with one-way valves and a small plastic chamber. Successful treatment was defined as when the pneumothorax was resolved after the insertion of a portable thoracic drainage device solely on an outpatient basis. Demographic data and treatment outcomes were obtained by a retrospective chart review. RESULTS: Ninety-seven patients (98%) with a first primary spontaneous pneumothorax were discharged from the emergency department after insertion of a portable thoracic drainage device. Ninety-three patients (94%) resolved with outpatient treatment. The median duration of portable thoracic drainage device insertion was 4 days (range, 0-33 days). The recurrence rate after treatment with a portable thoracic drainage device was 34% (32/93). There were two infections (2.0%), two instances of hemothoraces (2.0%), and one severe discomfort at the insertion site (1.0%). There were no cases of tension pneumothorax or reexpansion edema. CONCLUSION: Outpatient treatment for primary spontaneous pneumothorax using a portable thoracic drainage device had a high success rate with few serious complications and an acceptable recurrence rate.

Sciatic hernia: is it really rare?

PURPOSE: It is generally believed that sciatic hernia is extremely rare; however, asymptomatic sciatic hernia is occasionally found in patients with an obturator hernia. We investigated the frequency, risk factors, and prognosis of asymptomatic sciatic hernia, which have never been discussed in a published report. METHODS: We retrospectively reviewed multidetector-row computed tomography (MDCT) images of 38 consecutive cases of new-onset obturator hernia. The co-existence of sciatic hernia was diagnosed from the MDCT findings of some of these patients. The clinical characteristics and clinical courses were compared between the sciatic hernia group and the non-sciatic hernia group. RESULTS: Nine patients (24 %) had concomitant asymptomatic sciatic hernias, five (13 %) of which were bilateral.The body mass index (BMI) was significantly lower in the patients with a concomitant sciatic hernia (17.2 ± 2.4 kg/m(2)) than in those without a sciatic hernia (19.6 ± 2.6 kg/m(2); P = 0.02). All patients received treatment for incarcerated obturator hernias, but none underwent repair of the concomitant sciatic hernia because all were non-incarcerated and asymptomatic. None of the patients has had trouble with their untreated sciatic hernia after the obturator hernia treatment. CONCLUSIONS: Up to 24 % of these obturator hernia patients had a concomitant sciatic hernia. A low BMI was a risk factor for concomitant sciatic hernia. Immediate surgical repair of the sciatic hernia may not be needed, unless it is symptomatic.

The artificial intelligence-based model ANORAK improves histopathological grading of lung adenocarcinoma.

The introduction of the International Association for the Study of Lung Cancer grading system has furthered interest in histopathological grading for risk stratification in lung adenocarcinoma. Complex morphology and high intratumoral heterogeneity present challenges to pathologists, prompting the development of artificial intelligence (AI) methods. Here we developed ANORAK (pyrAmid pooliNg crOss stReam Attention networK), encoding multiresolution inputs with an attention mechanism, to delineate growth patterns from hematoxylin and eosin-stained slides. In 1,372 lung adenocarcinomas across four independent cohorts, AI-based grading was prognostic of disease-free survival, and further assisted pathologists by consistently improving prognostication in stage I tumors. Tumors with discrepant patterns between AI and pathologists had notably higher intratumoral heterogeneity. Furthermore, ANORAK facilitates the morphological and spatial assessment of the acinar pattern, capturing acinus variations with pattern transition. Collectively, our AI method enabled the precision quantification and morphology investigation of growth patterns, reflecting intratumoral histological transitions in lung adenocarcinoma.

Spatial Architecture of Myeloid and T Cells Orchestrates Immune Evasion and Clinical Outcome in Lung Cancer.

Understanding the role of the tumor microenvironment (TME) in lung cancer is critical to improving patient outcomes. We identified four histology-independent archetype TMEs in treatment-naïve early-stage lung cancer using imaging mass cytometry in the TRACERx study (n = 81 patients/198 samples/2.3 million cells). In immune-hot adenocarcinomas, spatial niches of T cells and macrophages increased with clonal neoantigen burden, whereas such an increase was observed for niches of plasma and B cells in immune-excluded squamous cell carcinomas (LUSC). Immune-low TMEs were associated with fibroblast barriers to immune infiltration. The fourth archetype, characterized by sparse lymphocytes and high tumor-associated neutrophil (TAN) infiltration, had tumor cells spatially separated from vasculature and exhibited low spatial intratumor heterogeneity. TAN-high LUSC had frequent PIK3CA mutations. TAN-high tumors harbored recently expanded and metastasis-seeding subclones and had a shorter disease-free survival independent of stage. These findings delineate genomic, immune, and physical barriers to immune surveillance and implicate neutrophil-rich TMEs in metastasis. SIGNIFICANCE: This study provides novel insights into the spatial organization of the lung cancer TME in the context of tumor immunogenicity, tumor heterogeneity, and cancer evolution. Pairing the tumor evolutionary history with the spatially resolved TME suggests mechanistic hypotheses for tumor progression and metastasis with implications for patient outcome and treatment. This article is featured in Selected Articles from This Issue, p. 897.

Representation of genomic intratumor heterogeneity in multi-region non-small cell lung cancer patient-derived xenograft models.

Patient-derived xenograft (PDX) models are widely used in cancer research. To investigate the genomic fidelity of non-small cell lung cancer PDX models, we established 48 PDX models from 22 patients enrolled in the TRACERx study. Multi-region tumor sampling increased successful PDX engraftment and most models were histologically similar to their parent tumor. Whole-exome sequencing enabled comparison of tumors and PDX models and we provide an adapted mouse reference genome for improved removal of NOD scid gamma (NSG) mouse-derived reads from sequencing data. PDX model establishment caused a genomic bottleneck, with models often representing a single tumor subclone. While distinct tumor subclones were represented in independent models from the same tumor, individual PDX models did not fully recapitulate intratumor heterogeneity. On-going genomic evolution in mice contributed modestly to the genomic distance between tumors and PDX models. Our study highlights the importance of considering primary tumor heterogeneity when using PDX models and emphasizes the benefit of comprehensive tumor sampling.

Obturator hernia of the fallopian tube.

Obturator hernia of the fallopian tube is extremely rare. Multidetector computed tomography of a 43-year-old nulliparous woman with sudden onset lower right abdominal pain showed a low-density mass in the right obturator canal suspected of being an obturator hernia of the uterine adnexa. She was diagnosed as having an incarcerated obturator hernia of the fallopian tube at operation and treated with prosthetic mesh. Obturator hernia of the fallopian tube is very rare, and all cases reported in the literature were localized on the right side, perhaps due to the lesser mobility of the left than the right fallopian tube.