Proteomic characterization of esophageal squamous cell carcinoma response to immunotherapy reveals potential therapeutic strategy and predictive biomarkers.

Immunotherapy is the first-line therapy for esophageal squamous cell carcinoma (ESCC), yet many patients do not respond due to drug resistance and the lack of reliable predictive markers. We collected 73 ESCC patients (including discovery cohort and validation cohort) without immune thrombocytopenia and undergoing anti-PD1 immunotherapy. Proteomic and phosphoproteomic analysis of 73 ESCC treatment-naive samples by mass spectrometry-based label-free quantification were applied to explore the potential resistant and sensitive mechanisms, and identify predictive markers of ESCC immunotherapy. Comparative analysis found the pathways related to immune and mitochondrial functions were associated with ESCC immunotherapy sensitivity; while platelet activation bioprocess showed negative correlation with CD8+ T cells and related to ESCC immunotherapy non-sensitivity. Finally, we identified 10 ESCC immunotherapy predictive biomarkers with high accuracy (≥ 0.90) to predict the immunotherapeutic response, which was validated in the independent cohort.

Clonal dynamics and Stereo-seq resolve origin and phenotypic plasticity of adenosquamous carcinoma.

The genomic origin and development of the biphasic lung adenosquamous carcinoma (ASC) remain inconclusive. Here, we derived potential evolutionary trajectory of ASC through whole-exome sequencing, Stereo-seq, and patient-derived xenografts. We showed that EGFR and MET activating mutations were the main drivers in ASCs. Phylogenetically, these drivers and passenger mutations found in both components were trunk clonal events, confirming monoclonal origination. Comparison of multiple lesions also revealed closer genomic distance between lymph node metastases and the ASC component with the same phenotype. However, as mutational signatures of EGFR-positive lung squamous carcinomas (LUSCs) were more comparable to EGFR-positive ASCs than to wild-type LUSCs, we postulated different origination of these LUSCs, with ASC being the potential intermediate state of driver-positive LUSCs. Spatial transcriptomic profiling inferred transformation from adenocarcinoma to squamous cell carcinoma, which was then histologically captured in vivo. Together, our results explained the development of ASC and provided insights into future clinical decisions.

Single-cell profiling reveals the trajectory of FOLR2-expressing tumor-associated macrophages to regulatory T cells in the progression of lung adenocarcinoma.

An immunosuppressive microenvironment enriched with regulatory CD4+ T lymphocytes (Tregs) facilitates the progression of lung adenocarcinoma (LUAD). This study aims to investigate the cellular mechanism underlying the formation of the immunosuppressive microenvironment in LUAD. LUAD samples (n = 12) and normal lung samples (n = 3) were obtained from patients with different pathological stages of LUAD. Single-cell RNA sequencing was performed to classify cellular components and analyze the transcriptomes, including transcription factors/targets and chemokine ligands/receptors, followed by bioinformatics study such as pseudotime analysis. Myeloid cells and T cells were the most abundant cell types in tumors and normal lung tissues, while tumor-associated macrophage-folate receptor 2 (TAM-FOLR2) and CD4+ nuclear receptor subfamily 4 group A member 3 (NR4A3) exhibited sharp increases in invasive adenocarcinoma (IA). The enrichment of TAM-FOLR2 in IA might result from alveolar resident macrophage-resistin (ARM-RETN) transformation and recruitment of dendritic cells (DCs) and other TAMs, as evidenced by temporal trajectories and differential expression profiles of chemokine ligands/receptors versus those in the early stages of tumors. High expression of CCL17/19/22 was observed in IA as well as in DCs, along with the strong interaction of TAM-FOLR2 with DCs. The results of pseudotime analysis suggested that CD4+NR4A3 might potentially convert to CD4+FOXP3, further supported by the high expression of NR4A3 target genes in CD4+FOXP3 cells. This study provides a single-cell transcriptome atlas from preinvasive to invasive LUAD and reveals a potential ARM-RETN/TAM-FOLR2/DCs/CD4+NR4A3/CD4+FOXP3 trajectory in shaping the immune suppressive microenvironment along the pathogenesis of LUAD.

Clinicopathologic Features and Frozen Diagnostic Pitfalls of Bronchiolar Adenoma/Ciliated Muconodular Papillary Tumors (BA/CMPTs).

To describe the histologic features of bronchiolar adenoma/ciliated muconodular papillary tumors (BA/CMPTs) and analyze the pitfalls in diagnosis from frozen sections. A total of 208 frozen and permanent sections of BA/CMPTs from Shanghai Chest Hospital from July 2018 to July 2021 were retrospectively analyzed. The median age of BA/CMPT patients was 65 years (15 to 79 y), and women accounted for 61.62% (122/198). The median size of BA/CMPTs was 0.6 cm (range 0.2 to 2 cm), of which 88.94% were small (≤1 cm, 185/208). In terms of location, the right lower lobe accounted for 44.23% (92/208), and the left lower lobe accounted for 33.65% (70/208). In 10 patients with 2 independent BA/CMPTs, 5 lesions were located in the left lower lobe and 4 in the right lower lobe. A total of 86.06% of the CT images of BA/CMPT showed solid/subsolid nodules (179/208). Among 208 tumors, 68.75% were distal type (143/208), and 31.25% were proximal type (65/208). The qualitative error rate of frozen sections was 21.33% (32/150), of which the distal type accounted for 75% (24/32); most of them were misdiagnosed as invasive adenocarcinoma during frozen diagnosis. The frozen diagnosis of BA/CMPTs might result in misdiagnosis as invasive adenocarcinoma. A careful search for characteristics of BA/CMPT, such as bilayer epithelial cells with basal cells and a lack of cellular atypia and invasive growth patterns, may be helpful for frozen diagnosis.

3D deep learning versus the current methods for predicting tumor invasiveness of lung adenocarcinoma based on high-resolution computed tomography images.

Background: Different pathological subtypes of lung adenocarcinoma lead to different treatment decisions and prognoses, and it is clinically important to distinguish invasive lung adenocarcinoma from preinvasive adenocarcinoma (adenocarcinoma in situ and minimally invasive adenocarcinoma). This study aims to investigate the performance of the deep learning approach based on high-resolution computed tomography (HRCT) images in the classification of tumor invasiveness and compare it with the performances of currently available approaches. Methods: In this study, we used a deep learning approach based on 3D conventional networks to automatically predict the invasiveness of pulmonary nodules. A total of 901 early-stage non-small cell lung cancer patients who underwent surgical treatment at Shanghai Chest Hospital between November 2015 and March 2017 were retrospectively included and randomly assigned to a training set (n=814) or testing set 1 (n=87). We subsequently included 116 patients who underwent surgical treatment and intraoperative frozen section between April 2019 and January 2020 to form testing set 2. We compared the performance of our deep learning approach in predicting tumor invasiveness with that of intraoperative frozen section analysis and human experts (radiologists and surgeons). Results: The deep learning approach yielded an area under the receiver operating characteristic curve (AUC) of 0.946 for distinguishing preinvasive adenocarcinoma from invasive lung adenocarcinoma in the testing set 1, which is significantly higher than the AUCs of human experts (P<0.05). In testing set 2, the deep learning approach distinguished invasive adenocarcinoma from preinvasive adenocarcinoma with an AUC of 0.862, which is higher than that of frozen section analysis (0.755, P=0.043), senior thoracic surgeons (0.720, P=0.006), radiologists (0.766, P>0.05) and junior thoracic surgeons (0.768, P>0.05). Conclusions: We developed a deep learning model that achieved comparable performance to intraoperative frozen section analysis in determining tumor invasiveness. The proposed method may contribute to clinical decisions related to the extent of surgical resection.

Distinct mutational features across preinvasive and invasive subtypes identified through comprehensive profiling of surgically resected lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a heterogeneous disease. Our study aimed to understand the unique molecular features of preinvasive to invasive LUAD subtypes. We retrospectively analyzed the clinical, histopathological, and molecular data of 3,254 Chinese patients with preinvasive lesions (n = 252), minimally invasive adenocarcinomas (n = 479), and invasive LUAD (n = 2,523). Molecular data were elucidated using a targeted 68-gene next-generation sequencing panel. Our findings revealed four preinvasive lesion-predominant gene mutations, including MAP2K1 insertion-deletions (indels), BRAF non-V600E kinase mutations, and exon 20 insertions (20ins) in both EGFR and ERBB2, which we referred to as mutations enriched in AIS (MEA). The detection rate of MEA in invasive tumors was relatively lower. MAP2K1 missense mutations, which were likely passenger mutations, co-occurred with oncogenic driver mutations, while small indels were mutually exclusive from other genes regardless of the invasion level. BRAF non-V600E kinase-mutant invasive adenocarcinomas (IAC) had significantly higher mutation rates in tumor suppressor genes but lower frequency of co-occurring oncogenic driver mutations than non-kinase-mutant IAC, suggesting the potential oncogenic activity of BRAF non-V600E kinase mutations albeit weaker than BRAF V600E. Moreover, similar to the extremely low frequency of MAP2K1 indels in IAC, BRAF non-V600E kinase domain mutations co-occurring with TSC1 mutations were exclusively found in preinvasive lesions. Compared with EGFR L858R and exon 19 deletion, patients with preinvasive lesions harboring 20ins in either EGFR or ERBB2 were significantly younger, while those with IAC had similar age. Furthermore, our study demonstrated distinct mutational features for subtypes of oncogene mutations favored by different invasion patterns in adenocarcinomas. In conclusion, our data demonstrate distinct mutational features between preinvasive lesions and invasive tumors with MEA, suggesting the involvement of MEA in the early stages of tumorigenesis. Further pre-clinical studies are required to establish the role of these genes in the malignant transformation of LUAD.

EGFR Thr790Leu as a Potential Resistance Mechanism to First-Generation EGFR Tyrosine Kinase Inhibitor May Respond to Osimertinib in Patients With Lung Adenocarcinoma.

INTRODUCTION: It has been well established that EGFR Thr790Met is one of the major resistance mechanisms to first- and second-generation EGFR tyrosine kinase inhibitors (TKIs). Nevertheless, whether EGFR Thr790Leu (T790L), which shares the mutation site of Thr790 with EGFR Thr790Met, mediates resistance to EGFR TKIs remains elusive. The treatment options for patients harboring this rare mutation have not been reported. METHODS: Capture-based targeted ultradeep sequencing was performed on tumor and plasma samples collected at various treatment milestones from three patients with advanced lung adenocarcinoma undergoing targeted therapy. RESULTS: Needle biopsy of lymph node metastasis from patient 1 revealed EGFR T790L at disease progression on first-line treatment of gefitinib. Patient 2 had EGFR T790L identified from needle biopsy of lung tissue at disease progression on icotinib treatment. This patient was subsequently treated with osimertinib and achieved stable disease with a progression-free survival of 9 months. For patient 3, at disease recurrence after surgery, resected lung tumor tissue was retrieved for molecular profiling and revealed EGFR exon 19 deletion and EGFR T790L. The patient subsequently received osimertinib treatment and continued to benefit for 16 months and counting. She has maintained stable disease at the time of submission of this manuscript. CONCLUSIONS: We revealed for the first time that EGFR T790L may serve as a potential resistance mechanism to first-generation EGFR TKIs. We also report the first clinical evidence of efficacy generated by osimertinib in patients with lung adenocarcinoma harboring primary or acquired EGFR T790L, shedding light on treatment options for this subset of patients.

Identification of NTRK gene fusions in lung adenocarcinomas in the Chinese population.

The molecular profile of neurotrophic tyrosine kinase receptor (NTRK) gene fusions in lung adenocarcinoma (LUAD) is not fully understood. Next-generation sequencing (NGS) and pan-tyrosine kinase receptor (TRK) immunohistochemistry (IHC) are powerful tools for NTRK fusion detection. In this study, a total of 4,619 LUAD formalin-fixed, paraffin-embedded tissues were collected from patients who underwent biopsy or resection at the Shanghai Chest Hospital during 2017-2019. All specimens were screened for NTRK1 rearrangements using DNA-based NGS. Thereafter, the cases with NTRK1 rearrangements and cases negative for common driver mutations were analyzed for NTRK1/2/3 fusions using total nucleic acid (TNA)-based NGS and pan-TRK IHC. Overall, four NTRK1/2 fusion events were identified, representing 0.087% of the original sample set. At the DNA level, seven NTRK1 rearrangements were identified, while only two TPM3-NTRK1 fusions were confirmed on TNA-based NGS as functional. In addition, two NTRK2 fusions (SQSTM1-NTRK2 and KIF5B-NTRK2) were identified by TNA-based NGS in 350 'pan-negative' cases. Two patients harboring NTRK1/2 fusions were diagnosed with invasive adenocarcinoma, while the other two were diagnosed with adenocarcinoma in situ and minimally invasive adenocarcinoma. All four samples with NTRK fusions were positive for the expression of pan-TRK. The two samples with NTRK2 fusions showed cytoplasmic staining alone, while the other two samples with NTRK1 fusions exhibited both cytoplasmic and membranous staining. In summary, functional NTRK fusions are found in early-stage LUAD; however, they are extremely rare. According to this study's results, they are independent oncogenic drivers, mutually exclusive with other driver mutations. We demonstrated that NTRK rearrangement analysis using a DNA-based approach should be verified with an RNA-based assay.

Challenging of frozen diagnoses of small sclerosing pneumocytoma.

AIMS: An increasing number of small pulmonary nodules are being screened by CT, and an intraoperative diagnosis is necessary for preventing excessive treatment. However, there is limited literature on the frozen diagnosis of small sclerosing pneumocytomas (SPs). In particular, tumours smaller than 1 cm are challenging for pathologists performing intraoperative frozen diagnosis. METHODS: In total, 230 cases of SP were surgically resected between January 2015 and March 2019 at Shanghai Chest Hospital, and of them, 76 cases were smaller than 1 cm. The histology and clinical information of these 76 cases (33.0%, 76/230) were reviewed retrospectively, 54 cases of which were diagnosed intraoperatively, and the pitfalls were summarised. All diagnoses were confirmed on permanent sections and immunohistochemical sections. RESULTS: Histologically, 78.9% (60/76) of the small SP was dominated by one growth pattern, and solid and papillary growth pattern were the most commonly misdiagnosed circumstances. The rate of intraoperative misdiagnosis of these SP smaller than 1 cm was 11.1% (6/54). CONCLUSIONS: The main reason for misdiagnosis was failure to recognise the dual cell populations and the cellular atypia. Diagnostic clues include the gross morphology, the presence of dual-cell populations and a hypercellular papillary core, foam cell accumulation in glandular spaces and haemorrhage and haemosiderin on the periphery. In spite of awareness of pitfalls some cases may still be essentially impossible to diagnose on frozen section.

Clinical characteristics and prognostic factors of surgically resected combined small cell lung cancer: a retrospective study.

OBJECTIVES: Small cell lung cancer (SCLC) is the most malignant lung cancer. Some of them are mixed with non-small cell lung cancer(NSCLC, Non SCLC),which are called combined small cell lung cancer (C-SCLC).Due to the difficulty of pathological diagnosis and the complexity of treatment, studies of C-SCLC have just been rising in recent years. This study is to evaluate the clinical and pathologic characteristics of C-SCLC. METHODS: Stage Ⅰ-Ⅲa C-SCLC patients who received radical R0 surgery between 2009-2018 in Shanghai Chest Hospital were enrolled. Clinical characteristics and prognosis were analyzed. RESULTS: Totally 181 patients were included, most of them were small cell combined with large cell neuroendocrine components(SCLC/LCNEC,58.0 %,N = 105),then with adenocarcinoma(SCLC/ADC:13.8 %,N = 25),and finally with squamous cell carcinoma(SCLC/SCC:13.3 %,N = 24).Median DFS and OS of C-SCLC patients underwent radical surgery were 32.5 and 49.7 months.1,3 and 5 years DFS rates of the entire cohort were 68.5 %,32.6 % and 16.0 %,respectively. Patients with SCLC/LCNEC had longer DFS (44.1 m vs. 20.4 m, p = 0.040) and longer OS trend (62.1 m vs. 33.2 m, p = 0.122).Groups of whether tumor invaded the pleura(p = 0.028 and p = 0.050),lymph node stage(p = 0.029 and p = 0.010) and the courses of adjuvant chemotherapy(p = 0.011 and p = 0.001) had statistical differences on DFS and OS. CONCLUSIONS: SCLC/LCNEC was the most common type of C-SCLC. Patients' DFS and OS were also longer than other combined types. Adjuvant chemotherapy for SCLC is still the main treatment for surgical C-SCLC. Further studies are needed to clarify the clinical characteristics and prognosis of C-SCLC.

Altered Clock and Lipid Metabolism-Related Genes in Atherosclerotic Mice Kept with Abnormal Lighting Condition.

Background. The risk of atherosclerosis is elevated in abnormal lipid metabolism and circadian rhythm disorder. We investigated whether abnormal lighting condition would have influenced the circadian expression of clock genes and clock-controlled lipid metabolism-related genes in ApoE-KO mice. Methods. A mouse model of atherosclerosis with circadian clock genes expression disorder was established using ApoE-KO mice (ApoE-KO LD/DL mice) by altering exposure to light. C57 BL/6J mice (C57 mice) and ApoE-KO mice (ApoE-KO mice) exposed to normal day and night and normal diet served as control mice. According to zeitgeber time samples were acquired, to test atheromatous plaque formation, serum lipids levels and rhythmicity, clock genes, and lipid metabolism-related genes along with Sirtuin 1 (Sirt1) levels and rhythmicity. Results. Atherosclerosis plaques were formed in the aortic arch of ApoE-KO LD/DL mice. The serum lipids levels and oscillations in ApoE-KO LD/DL mice were altered, along with the levels and diurnal oscillations of circadian genes, lipid metabolism-associated genes, and Sirt1 compared with the control mice. Conclusions. Abnormal exposure to light aggravated plaque formation and exacerbated disorders of serum lipids and clock genes, lipid metabolism genes and Sirt1 levels, and circadian oscillation.

Effect of H2S on the circadian rhythm of mouse hepatocytes.

BACKGROUND: Dysregulation of circadian rhythms can contribute to diseases of lipid metabolism. NAD-dependent deacetylase sirtuin-1(SIRT1) is an important hub which links lipid metabolism with circadian clock by its deacetylation activity depends on intracellular NAD+/NADH content ratio. Hydrogen sulfide (H2S) is an endogenous reductant which can affect the intracellular redox state. Therefore, we hypothesized that exogenous H2S can affect the expression of circadian clock genes mediated by sirt1 thereby affecting body's lipid metabolism. And also because the liver is a typical peripheral circadian clock oscillator that is intimately linked to lipid metabolism. Thus the effect of H2S were observed on 24-hour dynamic expression of 4 central circadian clock genes and sirt1gene in primary cultured hepatocytes. RESULTS: We established a hepatocyte model that showed a circadian rhythm by serum shock method. And detected that the expression level and the peak of circadian clock genes decreased gradually and H2S could maintain the expression and amplitude of circadian clock genes such as Clock, Per2, Bmal1 and Rev-erbαwithin a certain period time. Accordingly the expression level of sirt1 in H2S group was significantly higher than that in the control group. CONCLUSION: Exogenous reductant H2S maintain the circadian rhythm of clock gene in isolated liver cells. We speculated that H2S has changed NAD+/NADH content ratio in hepatocytes and enhanced the activity of SIRT1 protein directly or indirectly, so as to maintain the rhythm of expression of circadian clock genes, they play a role in the prevention and treatment of lipid metabolism-related disease caused by the biological clock disorders.