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Supplementary Fig S8 and legend

Supplementary Figures S10-S20 and figure legends

Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand–receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment.Significance:The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception.This article is highlighted in the In This Issue feature, p. 2355

Supplementary Methods

Summary Background The role of radiotherapy in metastatic renal cell carcinoma is controversial. We prospectively tested the feasibility and efficacy of radiotherapy to defer systemic therapy for patients with oligometastatic renal cell carcinoma. Methods This single-arm, phase 2, feasibility trial was done at one centre in the USA (The MD Anderson Cancer Center, Houston, TX, USA). Patients (aged ≥18 years) with five or fewer metastatic lesions, an Eastern Cooperative Oncology Group status of 0–2, and no more than one previous systemic therapy (if this therapy was stopped at least 1 month before enrolment) without limitations on renal cell carcinoma histology were eligible for inclusion. Patients were treated with stereotactic body radiotherapy (defined as ≤5 fractions with ≥7 Gy per fraction) to all lesions and maintained off systemic therapy. When lesion location precluded safe stereotactic body radiotherapy, patients were treated with hypofractionated intensity-modulated radiotherapy regimes consisting of 60–70 Gy in ten fractions or 52·5–67·5 Gy in 15 fractions. Additional rounds of radiotherapy were allowed to treat subsequent sites of progression. Co-primary endpoints were feasibility (defined as all planned radiotherapy completed with ClinicalTrials.gov , NCT03575611 . Findings 30 patients (six [20%] women) were enrolled from July 13, 2018, to Sept 18, 2020. All patients had clear cell histology and had a nephrectomy before enrolment. All patients completed at least one round of radiotherapy with less than 7 days of unplanned breaks. At a median follow-up of 17·5 months (IQR 13·2–24·6), median progression-free survival was 22·7 months (95% CI 10·4–not reached; 1-year progression-free survival 64% [95% CI 48–85]). Three (10%) patients had severe adverse events: two grade 3 (back pain and muscle weakness) and one grade 4 (hyperglycaemia) adverse events were observed. There were no treatment-related deaths. Interpretation Sequential radiotherapy might facilitate deferral of systemic therapy initiation and could allow sustained systemic therapy breaks for select patients with oligometastatic renal cell carcinoma. Funding Anna Fuller Foundation, the Cancer Prevention and Research Institute of Texas (CPRIT), and the National Cancer Institute.

Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand–receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment. Significance: The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception. This article is highlighted in the In This Issue feature, p. 2355

Summary The extensive clinical applications of next-generation sequence analyzers have made uncommon and compound EGFR mutations more prevalent than previously described. However, clinicopathological impacts of compound EGFR mutations in lung adenocarcinoma remain unclear. We earlier examined the presence of compound EGFR mutations primarily in the cis allele by EGFR exon sequencing and droplet digital polymerase chain reaction in 462 completely resected EGFR-mutated adenocarcinomas of the lung and identified 64 tumors with compound mutations. We evaluated clinicopathological characteristics of lung adenocarcinomas with compound EGFR mutations in comparison with cases with common or uncommon single mutations. Among 64 compound EGFR mutations, L858R/E709G (9%) was the most frequent mutation type, followed by L858R/S768I (8%), L858R/T790M (8%), and L858R/L833V (6%). We observed both single and compound mutations frequently in women, never or light smokers; their adenocarcinomas showed thyroid transcription factor-1 immunoreactivity. In contrast, compound mutations were significantly associated with lymph node metastases (p = 0.0242; single vs. compound cases) and the presence of tumor cells with clear cytoplasm (p = 0.0014; single vs. compound cases). Furthermore, patients with compound mutations had significantly poorer prognoses than cases with single EGFR mutations (p = 0.043). Overall, clinicopathological features of common, uncommon, and compound EGFR mutations are similar; however, tumors with compound mutations may be characterized by aggressive behavior and histological findings of clear cell features.

Programmed death ligand 1 and 2 (PD-L1 and PD-L2) bind programmed death 1 (PD-1). PD-L1 is an established predictive biomarker of response to immunotherapies targeting PD-1 and PD-L1 in lung adenocarcinoma (LUAD). However, the clinical relevance of PD-L2 expression in patients with LUAD remains unclear; we aimed to examine this aspect using LUAD specimens.PD-L2 expression status was immunohistochemically evaluated in 980 surgically resected LUAD specimens. PD-L2 expression status was classified based on the tumor proportion score (TPS) as negative (1%), weakly positive (1-49%), or strongly positive (≥50%). Correlations between PD-L2 and PD-L1 expression status, clinicopathological features, driver oncogene alterations (EGFR, KRAS, ALK, ROS1, and RET), and prognosis were also analyzed.PD-L2 expression was negative in 720 (73%) of 980 LUADs, weakly positive in 190 (19%), and strongly positive in 70 (7%). The concordance rate between PD-L1 and PD-L2 expression was 60%. Male sex, smokers, tumors 3 cm in size, high-grade tumors, tumors without EGFR mutation or ALK fusion, and tumors with KRAS mutation were more common in patients with PD-L2-positive tumors (TPS ≥ 1%) than in patients with PD-L2-negative tumors (TPS 1%). PD-L2 expression was not associated with overall survival (OS) or relapse-free survival (RFS). However, positive PD-L2 expression tended to be associated with better OS/RFS in PD-L1-positive patients and worse OS/RFS in PD-L1-negative patients.PD-L2-positive LUADs showed biologically aggressive characteristics. PD-L2 expression status was not associated with survival outcomes, but tended to show contrasting prognostic impacts based on PD-L1 expression status.

701 Background: In patients with clear cell renal cell carcinoma (ccRCC), progressive local dysfunction in the tumor immune microenvironment (TIM) has been shown to play a significant role in clinical outcomes and response to therapy. Patients with oligometastatic ccRCC may particularly benefit from localized treatment such as radiation therapy. However, immune cell dysfunction in TIM and its influence on patient outcomes in oligometastatic ccRCC has yet to be defined. To address this unmet need we conducted spatially resolved immune profiling on longitudinally collected patient samples from our ongoing prospective phase II trial (NCT03575611) of radiation therapy in oligometastatic ccRCC. This methodology offers a unique multidimensional evaluation of TIM in relation to histological architecture. Methods: Thirty-seven FFPE tumor samples from 32 patients with ccRCC (primary, 21; oligometastatic, 16) were obtained to construct a tissue microarray (TMA) and assess tumor immune activation biomarkers using the nanoString GeoMx Digital Spatial Profiler (DSP). We used the pan-cytokeratin, CD3, CD68, and SYTO13 as morphology biomarkers to profile, in each TMA core, a panel of 49 protein immune biomarkers in each of these compartments (DSP areas of illumination/AOI): tumor (panCK+), T-cell (CD3+), and macrophage (CD68+). In addition, DSP Immunofluorescence images were used to calculate cell density, and cell-to-cell distances of T cell and Macrophage. A median of 7 AOIs (range 3-9) were assessed in each tissue. Comparisons were made between primary and metastatic tissues by Mann-Whitney test with correction false discovery rate of 1%. Regression models were used to evaluate association between protein counts within each compartment and clinical outcomes. Results: In T-cells, CD127 was significantly higher in primary ccRCC tissue (p=0.00009) while FOXP3 and LAG3 were significantly increased in oligometastatic tissue (p=0.001). Macrophages in oligometastatic tissue expressed higher CD163 and ARG1 (p=0.01 and 0.02, respectively), compared to those in primary tissue. Average distances between macrophage and the nearest T cell were significantly shorter in oligometastatic tumor tissue compared to those in primary ccRCC (22.9 vs 28.8 µm, p=0.04). Higher PD-1 (R2=0.26) with lower CD127 (R2=0.22) and CD27 (R2=0.21) expression in T cells in primary tumor predicted earlier time to first diagnosis of metastases after nephrectomy (all p

Programmed death-ligand 1 (PD-L1) expression is a predictive biomarker of response to immunotherapies targeting programmed death-1/PD-L1 in advanced-stage lung adenocarcinoma. The aim of this study was to investigate the associations between PD-L1 expression and clinicopathological features, prognosis, and driver oncogene alterations in patients with lung adenocarcinoma.We evaluated PD-L1 expression in 1,005 surgically resected lung adenocarcinoma specimens, by immunohistochemistry using the 22C3 antibody. PD-L1 positivity was defined based on the proportion of stained tumor cells (TPS) on tissue microarrays:1% (negative), 1-49% (weakly positive), and ≥ 50% (strongly positive). Correlations between PD-L1 expression and clinicopathological features, prognosis, and driver oncogene (EGFR, KRAS, ALK, ROS1, and RET) alterations in lung adenocarcinoma were analyzed.PD-L1 expression was negative in 726 (72%) of 1,005 tumors, weakly positive in 161 (16%), and strongly positive in 118 (12%). Male sex, smoking, elevated serum carcinoembryonic antigen levels, advanced pathological stages, high-grade tumors, predominantly solid tumors, tumors with lymphatic permeation or vascular or pleural invasion, tumors without EGFR mutations, and tumors with KRAS mutations were more common in patients with PD-L1-positive tumors (TPS ≥ 1%) than in those with PD-L1-negative tumors (TPS 1%). PD-L1 positivity was not associated with ALK, ROS1, or RET fusion status. Although PD-L1 positivity was associated with poor overall survival and poor relapse-free survival in all patients, this was not statistically significant after adjusting for prognostic factors in the multivariate analysis. In the subgroup analysis according to driver oncogene alterations, PD-L1 positivity was associated with poor relapse-free survival only in patients with EGFR-mutated tumors.Surgically resected lung adenocarcinomas with increased PD-L1 expression were biologically aggressive tumors that frequently occurred in male smokers. PD-L1 expression and its prognostic significance differed according to driver oncogene alterations.

Aims In the evolving era of precision medicine, increasing emphasis is placed on detecting molecular alterations driving the development of specific cancers and targeting them with matched therapies that can yield the best outcomes for patients. Lung adenocarcinomas with uncommon actionable alterations, including MET exon 14 skipping (METex14), ERBB2 and BRAF mutations, are rare and poorly characterised cancers. Methods and results To more clearly understand the histopathological features of lung adenocarcinoma with uncommon actionable alterations, we compared the histological features of 678 cases with mitogenic driver alterations from 996 surgically resected lung adenocarcinomas. Genomic data from our cohort revealed METex14, ERBB2 and BRAF mutations in 13, 16 and 15 cases, respectively. Patients who had lung adenocarcinoma with METex14 were often elderly females. Histological features such as clear cell features (23%), hyaline globules (31%) and nuclear pleomorphism (39%) were the most frequently identified in METex14-positive cases; among those, three cases (23%) had tumour cells with bizarre giant or multilobulated nuclei. Additionally, the micropapillary pattern was the most frequently identified in ERBB2-mutated lung adenocarcinoma (31%). Lung adenocarcinoma with BRAF mutations tended to be less invasive, and the BRAF V600E mutation was identified in only one case with lepidic adenocarcinoma. Immunohistochemically, all METex14, ERBB2 and BRAF-positive tumours, except for invasive mucinous adenocarcinoma, were positive for thyroid transcription factor 1 (TTF-1). Conclusions Our data from Japanese patients showed that lung adenocarcinoma with METex14 had unique clinicopathological characteristics: tumour cells with marked nuclear pleomorphism, hyaline globules and expression of TTF-1 in elderly women who never or lightly smoked.

Simple Summary The coronavirus disease 2019 (COVID-19) pandemic continues to spread rapidly on a global scale. When presenting with severe respiratory complications, COVID-19 results in markedly high death rates, particularly among patients with comorbidities such as cancer. Motivated by the ongoing global health crisis, we leveraged a growing in-house cohort of pulmonary tissues from lung cancer patients to analyze, at high resolution, the expression of host proteins implicated in the entryway of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into lung epithelial cells. Our results identify key pathways in lung pathobiology and inflammation that offer the potential to identify novel markers and therapeutic targets that can be repurposed for clinical management of COVID-19, particularly among lung cancer patients, a population that represents over half a million individuals in the United States alone. Abstract The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-associated mortality among lung cancer patients. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. The single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. We sought to delineate single-cell expression profiles of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung adenocarcinoma (LUAD) patients. We examined the expression levels and cellular distribution of ACE2 and SARS-CoV-2-priming proteases TMPRSS2 and TMPRSS4 in 5 LUADs and 14 matched normal tissues by single-cell RNA-sequencing (scRNA-seq) analysis. scRNA-seq of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2, and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, the expression of TMPRSS4 was highest and most frequently detected (75%) in lung cells with malignant features. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. We describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, thus comprising potential treatment targets for COVID-19 particularly among lung cancer patients.

Background: The role of definitive radiation therapy (RT) in metastatic renal cell carcinoma (RCC) is controversial. We prospectively tested the feasibility, efficacy, and safety of definitive RT to defer systemic therapy for patients with oligometastatic RCC. Methods: This single-arm phase 2 study (NCT03575611) enrolled patients with histologically confirmed RCC, ≤5 metastatic lesions, and ≤1 prior systemic therapy. Patients were treated with definitive RT to all lesions and maintained off systemic therapy. Additional rounds of RT were allowed to treat new sites of progression. The co-primary endpoints were progression-free survival (PFS) and feasibility. Criteria for initiating systemic therapy were progression in >3 lesions, toxicity, local progression, or patient/provider preference. Findings: Thirty patients were enrolled in July 2018–September 2020 (median follow-up time 17.5 months). All patients had clear cell histology and had undergone nephrectomy before enrollment. Nine patients (30%) had received one prior systemic therapy. All patients completed initial RT and 13 (43%) received a second round. Median PFS was 22·7 months (1-year PFS rate 64%). All patients were alive at last follow-up, and one local failure was observed. The 1-year systemic therapy–free survival was 82%, with 7 patients (23%) initiating systemic therapy at a median 8.1 months from enrollment. Adverse events were grade ≥2 in six patients and ≥3 in three patients. At first follow-up, no viable tumor cells were detected in 5 of 14 biopsy samples obtained after RT (36%). For patients in whom viable tumor was present, the Ki-67 labeling index was significantly reduced (P =0·0068). Interpretation: Sequential definitive RT is a feasible and effective strategy for patients with oligometastatic RCC seeking to defer systemic therapy. Clinical Trial Registration: This study is registered with ClinicalTrials.gov as NCT03575611. Funding: Grant support from the Anna Fuller Foundation, the Cancer Prevention and Research Institute of Texas (CPRIT), and the National Cancer Center, National Institutes of Health (P30 CA016672, PI P. Pisters). Declaration of Interest: PM has received honoraria for service on scientific advisory boards for Mirati Therapeutics, Bristol- Myers Squibb, and Exelixis; consulting for Axiom Healthcare Strategies; non-branded educational programs supported by Exelixis and Pfizer; and research funding for clinical trials from Takeda, Bristol- Myers Squibb, Mirati Therapeutics, Gateway for Cancer Research, and UT MD Anderson Cancer Center. INCOMPLETE Ethical Approval: This protocol underwent full approval by the Institutional Review Board at MD Anderson. All patients provided written informed consent to participate.

Rationale: Early pathogenesis of lung adenocarcinoma (LUAD) remains largely unknown. We found that, relative to wild-type littermates, the innate immunomodulator Lcn2 (lipocalin-2) was increased in...

Invasive mucinous adenocarcinoma (IMA) of the lung is a unique variant of lung adenocarcinoma. Aberrant mucin expression is associated with cancer development and metastasis. However, the clinicopathological significance of mucin expression in IMA is not fully understood. Herein, we evaluated the clinicopathological, immunohistochemical, and molecular characteristics of 70 IMA tumors. EGFR, KRAS, GNAS, and TP53 mutations were assessed by PCR-based sequencing. Next-generation sequencing was used to assess cases without EGFR/KRAS mutations. A NanoString-based screening for fusions was performed in all IMAs without mitogenic driver mutations. Expression of mucins (MUC1, MUC2, MUC4, MUC5AC, and MUC6) was evaluated by immunohistochemistry and categorized as follows: negative (

Lung adenocarcinoma (LUAD) is the most commonly diagnosed histological subtype of lung cancer. While earlier work has underscored genomic and immune alterations in LUAD, the roles of individual cell populations in early-stage human LUAD evolution in space remain unknown. Here, we provide a detailed cellular atlas of early-stage LUAD and its spatial ecosystem along the peripheral lung. We performed single-cell RNA sequencing of 186,916 cells including enriched epithelial fractions from five early-stage LUADs with fourteen multi-region normal lung tissues of defined spatial proximities from the primary LUADs. We show that major epithelial and immune cellular lineages, states, and transcriptomic features geospatially and progressively evolve across normal regions and with increasing LUAD proximity. Analysis of 70,030 lung epithelial cells unraveled diverse lineage trajectories, transcriptional lineage plasticity programs underlying KRAS-mutant cells, and intratumoral heterogeneity within single sites. T regulatory cell programs including multiple immune checkpoints increased in tissues with closer proximity to LUADs, in sharp contrast to signatures of CD8+ cytotoxic T cells, antigen presentation by macrophages, and inflammatory dendritic cells. We found that some spatial signatures (e.g. a B cell signature score) were increased along the pathologic spectrum of normal lung, preneoplastic lesions, and matched invasive LUADs. LUAD cell-cell communication networks were enriched with ligand-receptor interactions involving CD24, LGALS9 and TIM3 immune checkpoints, including crosstalk between CD24 antigen in LUAD epithelial cells and SIGLEC10 in myeloid subsets. CD24 was markedly increased in preneoplasias relative to normal lung and further in LUAD, and its expression was highly positively correlated with immunosuppressive phenotypes. These data provide an atlas of cellular states and phenotypes underlying early-stage LUAD evolution in space, and a scalable resource for identification of targets for early treatment. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Kieko Hara, Patrick Brennan, Minghao Dang, Dapeng Hao, Ruiping Wang, Enyu Dai, Hitoshi Dejima, Jiexin Zhang, Elena Bogatenkova, Beatriz Sanchez-Espiridion, Kyle Chang, Danielle R. Little, Samer Bazzi, Linh Tran, Kostyantyn Krysan, Carmen Behrens, Dzifa Duose, Edwin R. Parra, Maria Gabriela Raso, Luisa M. Solis, Junya Fukuoka, Jianjun Zhang, Boris Sepesi, Tina Cascone, Lauren A. Byers, Don L. Gibbons, Jichao Chen, Seyed Javad Moghaddam, Edwin J. Ostrin, Daniel G. Rosen, John V. Heymach, Paul Scheet, Steven Dubinett, Ignacio I. Wistuba, Junya Fujimoto, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. Resolving the spatial and cellular architecture of lung adenocarcinoma by multi-region single-cell sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 130.

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-asociated mortality among lung cancer patients. Angiotensin converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. Yet, the single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. To fill these voids, we leveraged our ongoing efforts in single-cell transcriptomic analysis of 186,916 cells including a large number of epithelial cells (n=70,030) derived from 5 LUADs and 14 matching uninvolved normal lung tissues, to delineate expression levels and cellular distribution of ACE2 and SARS-CoV-2 priming proteases TMPRSS2 and TMPRSS4. Single-cell RNA sequencing of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2 and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, expression of TMPRSS4 was highest and most frequently detected (75%) in malignant lung cells. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. In conclusion, we describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, and that thus comprise potential treatment targets for COVID-19 particularly among lung cancer patients. Citation Format: Guangchun Han, Ansam Sinjab, Kieko Hara, Warapen Treekitkarnmongkol, Patrick Brennan, Kyle Chang, Elena Bokatenkova, Beatriz Sanchez-Espiridion, Carmen Behrens, Luisa M. Solis, Boning Gao, Luc Girard, Jianjun Zhang, Boris Sepesi, Tina Cascone, Lauren A. Byers, Don L. Gibbons, Jichao Chen, Seyed Javad Moghaddam, Edwin J. Ostrin, Paul Scheet, Junya Fujimoto, Jerry Shay, John V. Heymach, John D. Minna, Steven Dubinett, Ignacio I. Wistuba, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. Single-cell expression landscape of SARS-CoV-2 receptor ACE2 and host proteases in human lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 702.

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-associated mortality among lung cancer patients. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. The single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. We sought to delineate single-cell expression profiles of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung adenocarcinoma (LUAD) patients. We examined the expression levels and cellular distribution of ACE2 and SARS-CoV-2-priming proteases TMPRSS2 and TMPRSS4 in 5 LUADs and 14 matched normal tissues by single-cell RNA-sequencing (scRNA-seq) analysis. scRNA-seq of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2, and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, the expression of TMPRSS4 was highest and most frequently detected (75%) in lung cells with malignant features. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. We describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, thus comprising potential treatment targets for COVID-19 particularly among lung cancer patients.

The prognostic value of BRAF V600E and TERT promoter mutation in papillary thyroid carcinoma (PTC) is controversial. We examined alterations in BRAF V600E and TERT promoter by PCR-direct sequencing in PTC of 144 Japanese patients. Alternative lengthening of telomeres was examined as another mechanism of telomere maintenance by immunohistochemical staining for ATRX and DAXX. Of the clinicopathological characteristics, regional lymph node metastasis, extra-thyroid extension, multifocality/intrathyroidal spread, and advanced stage (III/V) were associated with shorter disease-free survival rate (DFSR). TERT promoter mutation was found in eight patients (6 %), and this was significantly associated with total thyroidectomy, multifocality/intrathyroidal spread, lymph node metastasis and advanced stage. The BRAF V600E mutation was found in 53 patients (38.2 %) but was not associated with any clinicopathological factors. TERT mutations were not correlated with BRAF V600E mutation status. TERT mutation-positive tumors (TERT+) showed lower DFSR than BRAF V600E -mutation-positive tumors (BRAF V600E +), and TERT+/BRAF V600E + tumors showed lower DFSR than BRAF V600E + tumors. No cases showed loss of ATRX/DAXX expression by immunohistochemistry. TERT promoter mutations showed a lower prevalence in our series and appeared to be associated with aggressive behavior. In PTCs, telomerase activation by TERT promoter mutation might be more important than alternative lengthening of telomeres.

Lung adenocarcinoma (LUAD) is the most frequently diagnosed histological subtype of lung cancer and accounts for most smoking-related cancer deaths, warranting strategies for early intervention. Earlier work revealed genome-wide aberrations in LUADs and the adjacent premalignant field, known as “field carcinogenesis”, that are pertinent to LUAD pathogenesis. Yet, we still poorly understand the cellular and molecular architecture of LUAD and its nearby “field”. To fill this void, we performed, using the 10X Genomics system and NovaSeq 6000 platform, single-cell RNA sequencing (scRNA-seq) analysis of 4 early-stage resected LUADs as well as 11 matched normal lung tissues with differing spatial proximity from the tumors. We first analyzed 15,739 cells from a LUAD and matched tumor-adjacent and -distant normal tissues from patient 1. The resulting fraction of EPCAM+ cells from this analysis was approximately 4%. For deeper single-cell resolution of LUAD, we thus performed scRNA-seq of separately sorted/enriched epithelial cells (EPCAM+; n = 50,883) and non-epithelial cells (EPCAM-; n= 91,093) from patients 2, 3 and 4, each with an early-stage LUAD and three matched spatially-distributed normal-appearing tissues. Overall, we achieved on average approximately 150,000 reads and 1,956 genes detected per cell. Divergent populations of malignant and non-malignant cells and multiple epithelial and immune subsets clustered in an overall spatially modulated pattern according to proximity to the tumor. Hierarchical clustering revealed multiple distinct populations of airway lineage cells, including alveolar type 1 (AT1), alveolar type 2 (AT2), AT1/AT2 bipotential cells, club, goblet, basal and ciliated cells, with cells from the resected LUAD present in some but not all clusters. By analysis of epithelial-enriched fractions, we were also able to identify and interrogate rare cell subpopulations including CFTR-expressing ionocytes. By inference of copy number variation along with analysis of key oncogene expression patterns, we also identified epithelial cells that represent potential tumor cells-of-origin. Spatial reprogramming of field carcinogenesis encompassed immune cell populations including tumor-exclusive FOXP3+ regulatory T cells with marked over-expression of the major immune checkpoints CTLA4 and TIGIT. In contrast, for at least some of the cases, tumors were nearly devoid of natural killer cells, and abundance of CD8+ T cells dampened with increasing proximity to the tumor. Our single-cell surveys offer insights into novel cues in LUAD pathogenesis. Efforts are underway to interrogate the single-cell epithelial and immune landscape of additional LUADs and matched nearby normal-appearing lung, including those from non-smokers, to better understand the evolution of the disease and, thus, identify a low-hanging fruit of targets for early management of this fatal malignancy. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Patrick Brennan, Kieko Hara, Kyle Chang, Elena Bogatenkova, Beatriz Sanchez-Espiridion, Carmen Behrens, Jianjun Zhang, Boris Sepesi, Tina Cascone, Don L. Gibbons, Jichao Chen, George Eapen, Edwin J. Ostrin, Junya Fujimoto, Avrum E. Spira, Paul A. Scheet, Ignacio I. Wistuba, Linghua Wang, Humam Kadara. A single-cell transcriptomic atlas of lung adenocarcinoma and adjacent normal-appearing tissue [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1518.

Adipophilin, a lipid droplet-associated protein that regulates lipid droplet structure and formation, is expressed in a wide variety of tumors. The aims of this study were to evaluate the frequency and distribution pattern of adipophilin expression in gastric epithelial neoplasia and to correlate these variables with clinicopathological features and the mucin phenotype. We retrospectively examined 159 cases of gastric epithelial neoplasia, which were classified according to the Vienna classification system as 52 noninvasive low-grade adenoma (category 3), 65 noninvasive high-grade neoplasia (category 4), and 42 invasive neoplasia (category 5). Immunohistochemistry for adipophilin was performed, and phenotypic marker expression was determined by immunohistostaining with MUC2, MUC5AC, CD10, MUC6, and villin. Adipophilin was expressed in 41 of the 52 (79 %) category 3 cases, in 42 of the 65 (65 %) category 4 cases, and in 23 of the 42 (55 %) category 5 cases. Expression of adipophilin was only present in lesions with complete or incomplete intestinal phenotypes. Adipophilin was expressed more frequently in the surface epithelium in category 3, whereas there was a stepwise increase in cryptal staining of adipophilin from category 3 to category 5. In conclusion, adipophilin expression is closely related to the intestinal differentiation of the tumor. The pattern of immunostaining for adipophilin might be a useful new marker for discriminating adenomas from adenocarcinomas.

Background Nrf2 is a transcription factor associated with cancer growth and chemotherapy resistance. Nrf2 expression levels may be associated with prognosis of lung cancer, but previous results have been inconsistent. Methods The expression of Nrf2 in 1037 surgically resected lung adenocarcinoma (LAD) specimens was evaluated by immunohistochemistry. Nrf2 positivity was defined based on the intensity of stained nucleus of tumor cells on the tissue microarray: negative, weakly positive and strongly positive. The correlations between the Nrf2 expression and the prognosis, clinicopathological features and driver oncogene alterations were analyzed. Results The Nrf2 expression was negative in 236 (23%) of 1037 tumors, weakly positive in 444 (43%) and strongly positive in 357 (34%). The proportion of Nrf2-positive tumors was significantly higher in patients with pathological stage I (81%), tumors with EGFR mutations (87%) or KRAS mutations (87%), tumors without ALK fusions (78%) or ROS1 fusions (78%). The Nrf2 expression was not associated with the age, gender and smoking status. In multivariate analysis (Logistic regression model), pathological stage I, positive EGFR or KRAS mutation status were significant predictors of Nrf2 positivity. The increased expression of Nrf2 was associated with longer overall survival regardless of driver oncogene status. In multivariate analysis (Cox proportional hazards model), advanced age, male gender, advanced pathological stage and negative EGFR mutation status were significant unfavorable prognostic factors. Conclusions LADs that positively express Nrf2 have unique clinicopathological features. The increased expression of Nrf2 is a favorable prognostic marker in surgically resected LAD. Legal entity responsible for the study The authors. Funding AMED. Disclosure All authors have declared no conflicts of interest.

Intraductal papillary neoplasm of the bile duct (IPNB) is a grossly visible, preinvasive neoplasm of the bile duct that characteristically shows an intraductal predominant growth in dilated bile duct(s). IPNB is composed of a well-differentiated papillary or villous neoplasm covering delicate and ramifying fibrovascular stalks. Tubular components are usually admixed, although they usually constitute less than 50% of the neoplasm. IPNB is regarded as the counterpart of pancreatic intraductal papillary mucinous neoplasm (IPMN). As in IPMN, IPNB is divided into the intestinal, gastric, pancreatobiliary, and oncocytic subtypes and further classified into low-intermediate-grade and high-grade intraepithelial neoplasms. The cases of IPNB with invasion are called “IPNB with an associated invasive carcinoma.” IPNBs arising in the intrahepatic bile duct and right or left hepatic bile ducts are quite similar to IPMNs, whereas those arising in the extrahepatic bile duct, particularly in the distal bile ducts, show more aggressive features and more tubular components in comparison to IPMNs. Ordinary cholangiocarcinoma with an inconspicuous or low-height intraluminal papillary component, mucinous cystic neoplasms (MCNs), biliary intraepithelial neoplasia (BilIN), and intraductal tubulopapillary neoplasm should be distinguished from IPNB. Molecular and genetic analyses of more cases of IPNB with reference to IPMN are promising for the evaluation of pathologic and biologic heterogeneities associated with IPNB.

The prognostic value of BRAF

Solitary fibrous tumors (SFTs), initially observed in the pleura, were later found to develop in almost any extrapleural site. Dedifferentiation within SFTs was characterized only recently. We report a case of dedifferentiated SFT arising within the pelvis of a 70-year-old Japanese woman. Macroscopically, the resected tumor measured 17 × 17 × 13 cm. Histologically, the tumor displayed distinct heterologous osteosarcomatous and chondrosarcomatous components on a background of conventional SFT. Immunohistochemistry uncovered a loss of CD34 expression in the dedifferentiated area, whereas the nuclear expression of signal transducer and activator of transcription-6 (STAT6) and NGFI-A-binding protein 2 (NAB2) was maintained in both components. The p53 mutation 158 CGC > CAC (A158H) was found only in the dedifferentiated component. Furthermore, a fusion gene of NAB2(exon6)-STAT6(exon18) was detected in both the conventional and dedifferentiated components. The patient died of the disease 4 months after surgery. This case identifies a possible role of p53 dysfunction in the dedifferentiation process of SFT as reported in other sarcomas.

The purpose of this study was to determine whether a high NaCl intake impairs endothelium-dependent and -independent vasodilation of forearm circulation in salt sensitive (SS) patients with essential hypertension. We evaluated the effects of intra-arterial acetylcholine (ACh) and isosorbide dinitrate (ISDN) on forearm hemodynamics in 29 patients with essential hypertension, while consuming a low NaCl (50 mmol/d) or high Na Cl (340 mmol/d) diet for 1 week. The forearm blood flow (FBF) was measured by strain-gauge plethysmography. Patients were classified as SS (n=12) or salt resistant (SR; n=17) based on salt-induced changes in blood pressures. The FBF responses of ACh and ISDN were similar in the SS and SR patients while on either NaCl diet, and was not altered by salt loading (ACh, SS: low NaCl 22.8+/-4.3 vs. high NaCl 21.1+/-3.6 ml/min per 100 ml, SR: low NaCl 22.5+/-4.0 vs. high NaCl 23.3+/-4.1 ml/min per 100 ml; ISDN, SS: low NaCl 13.9+/-2.1 vs. high NaCl 14.1+/-2.2 ml/min per 100 ml, SR: low NaCl 13.8+/-2.3 vs. high NaCl 14.0+/-2.2 ml/min per 100 ml). There were no significant differences in the vascular responses to ACh and ISDN in the presence of N(G)-monomethyl-L-arginine, a nitric oxide synthase inhibitor, in either group for either NaCl diet. These findings suggest that forearm resistance artery endothelial function may not be influenced by salt loading in either SS patients which finding may play a role in determining salt sensitivity in patients with essential hypertension or SR patients.

Metastasis is still a major issue in cancer, and the discovery of biomarkers predicting metastatic capacity is essential for the development of better therapeutic strategies for treating lung adenocarcinoma. By using a proteomic approach, we aimed to identify novel predictors for lymph node metastasis in lung adenocarcinoma. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed 6 spots differentially expressed between lymph node metastasis-positive and lymph node metastasis-negative groups in a discovery set. Subsequent mass spectrometry showed that 2 of these spots were derived from galectin-4, and western blot analysis confirmed the overexpression of galectin-4 in metastatic samples. The predictive value of galectin-4 was confirmed by immunohistochemical analysis for a validation set consisting of 707 surgically resected specimens of lung adenocarcinomas (stages I to IV). We observed that 148 lung adenocarcinomas (20.9%) expressed galectin-4, which was significantly associated with variables of disease progression such as tumor size (p

Colonoscopy in an 11-year-old boy identified a large long pedunculated colonic duplication originating from the mesenteric side of the cecum extending to the sigmoid colon that did not share a common wall with the native colon and had become intussuscepted. Following uneventful excision, normal growth resumed after resolution of all symptoms.

Background Targeted therapies based on the molecular and histological features of cancer types are becoming standard practice. The most effective regimen in lung cancers is different between squamous cell carcinoma (SCC) and adenocarcinoma (AD). Therefore a precise diagnosis is crucial, but this has been difficult, particularly for poorly differentiated SCC (PDSCC) and AD without a lepidic growth component (non-lepidic AD). Biomarkers enabling a precise diagnosis are therefore urgently needed. Methods Cap Analysis of Gene Expression (CAGE) is a method used to quantify promoter activities across the whole genome by determining the 5’ ends of capped RNA molecules with next-generation sequencing. We performed CAGE on 97 frozen tissues from surgically resected lung cancers (22 SCC and 75 AD), and confirmed the findings by immunohistochemical analysis (IHC) in an independent group (29 SCC and 45 AD). Results Using the genome-wide promoter activity profiles, we confirmed that the expression of known molecular markers used in IHC for SCC (CK5, CK6, p40 and desmoglein-3) and AD (TTF-1 and napsin A) were different between SCC and AD. We identified two novel marker candidates, SPATS2 for SCC and ST6GALNAC1 for AD, as showing comparable performance and complementary utility to the known markers in discriminating PDSCC and non-lepidic AD. We subsequently confirmed their utility at the protein level by IHC in an independent group. Conclusions We identified two genes, SPATS2 and ST6GALNAC1, as novel complemental biomarkers discriminating SCC and AD. These findings will contribute to a more accurate diagnosis of NSCLC, which is crucial for precision medicine for lung cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2792-1) contains supplementary material, which is available to authorized users.