Glycoengineering-based anti-PD-1-iRGD peptide conjugate boosts antitumor efficacy through T cell engagement.

Despite the important breakthroughs of immune checkpoint inhibitors in recent years, the objective response rates remain limited. Here, we synthesize programmed cell death protein-1 (PD-1) antibody-iRGD cyclic peptide conjugate (αPD-1-(iRGD)2) through glycoengineering methods. In addition to enhancing tissue penetration, αPD-1-(iRGD)2 simultaneously engages tumor cells and PD-1+ T cells via dual targeting, thus mediating tumor-specific T cell activation and proliferation with mild effects on non-specific T cells. In multiple syngeneic mouse models, αPD-1-(iRGD)2 effectively reduces tumor growth with satisfactory biosafety. Moreover, results of flow cytometry and single-cell RNA-seq reveal that αPD-1-(iRGD)2 remodels the tumor microenvironment and expands a population of "better effector" CD8+ tumor infiltrating T cells expressing stem- and memory-associated genes, including Tcf7, Il7r, Lef1, and Bach2. Conclusively, αPD-1-(iRGD)2 is a promising antibody conjugate therapeutic beyond antibody-drug conjugate for cancer immunotherapy.

bITH, a blood-based metric of intratumor heterogeneity, is associated with clinical response to immune checkpoint blockade in non-small cell lung cancer.

BACKGROUND: Intratumor heterogeneity (ITH) has been associated with poor prognosis in advanced non-small cell cancer (NSCLC) patients receiving immune checkpoint blockade (ICB) therapies. However, there is currently no evidence supporting an ITH metric as a predictor of clinical benefit from ICB. The unique advantages of blood make it a promising material for ITH estimation and relevant applications. This study aims to develop and validate a blood-based ITH index for predicting ICB response. METHODS: NSCLC patients from the OAK and POPLAR clinical trials were used as the training cohorts for algorithm development. Survival analyses with overall survival (OS) and progression-free survival (PFS) as endpoints were performed to assess clinical response. The predictive value of bITH was subsequently validated with an independent cohort of 42 NSCLC patients treated with PD-1 blockade. FINDINGS: bITH was significantly associated with the differential OS and PFS elicited by atezolizumab vs. docetaxel in both univariable and multivariable analyses in the OAK patients, suggesting bITH as an independent predictor for response to ICB. Moreover, compared with blood tumor mutation burden (bTMB), bITH enabled greater OS segregation and comparable PFS segregation, and obtained a predictive role regardless of bTMB status. Moreover, the association between bITH and PFS was validated with an independent cohort. INTERPRETATION: Patients with low blood-based ITH metric manifest significant OS and PFS benefit from immunotherapy versus chemotherapy. Future research is awaited to corroborate our findings and to enrich the clinical utility of ITH. FUNDING: This study was supported by the National Natural Science Foundation of China (Nos. 81972718 and 81572321), the Natural Scientific Foundation of Zhejiang Province, China (No. LY19H160007), the Science and Technology Program for Health and Medicine in Zhejiang Province, China (No. 2021KY541), the Scientific Research Project, Science and Technology Department of Sichuan Province (No. 21YYJC1616), the Scientific Research Project, Sichuan Medical Association (No. S20002), Wu Jieping Medical Foundation (No. 320.6750), and 2018 Entrepreneurial Leading Talent of Guangzhou Huangpu District and Guangzhou Development District (No. 2022-L023).

Comprehensive bioinformatic analyses of lncRNA-mediated ceRNA network for uterine corpus endometrial carcinoma.

Background: Given that long non-coding RNAs (lncRNAs) involved in the tumor initiation or progression of the endometrium and that competing endogenous RNA (ceRNA) plays an important role in increasingly more biological processes, lncRNA-mediated ceRNA is likely to function in the pathogenesis of uterine corpus endometrial carcinoma (UCEC). Our present study aimed to explore the potential molecular mechanisms for the prognosis of UCEC through a lncRNA-mediated ceRNA network. Methods: The transcriptome profiles and corresponding clinical profiles of UCEC dataset were retrieved from Clinical Proteomic Tumor Analysis Consortium (CPTAC) and The Cancer Genome Atlas (TCGA) databases respectively. Differentially expressed genes (DEGs) in UCEC samples were identified via "Edge R" package. Then, an integrated bioinformatics analysis including functional enrichment analysis, tumor infiltrating immune cell (TIIC) analysis, Kaplan-Meier curve, Cox regression analysis were conducted to analyze the prognostic biomarkers. Results: In the CPTAC dataset of UCEC, a ceRNA network comprised of 36 miRNAs, 123 lncRNAs and 124 targeted mRNAs was established, and 8 of 123 prognostic-related Differentially Expressed long noncoding RNAs (DElncRNAs) were identified. While in the TCGA dataset, a ceRNA network comprised of 38 miRNAs, 83 lncRNAs and 110 targeted mRNAs was established, and 2 of 83 prognostic-related DElncRNAs were identified. After filtered by risk grouping and Cox regression analysis, 10 prognostic-related lncRNAs including LINC00443, LINC00483, C2orf48, TRBV11-2, MEG-8 were identified. In addition, 33 survival-related Differentially Expressed messenger RNA (DEmRNAs) in two ceRNA networks were further validated in the Human Protein Atlas Portal (HPA) database. Finally, six lncRNA/miRNA/mRNA axes were established to elucidate prognostic regulatory roles in UCEC. Conclusions: Several prognostic lncRNAs are identified and prognostic model of lncRNA-mediated ceRNA network is constructed, which promotes the understanding of UCEC development mechanisms and potential therapeutic targets.

Increased blood-based intratumor heterogeneity (bITH) is associated with unfavorable outcomes of immune checkpoint inhibitors plus chemotherapy in non-small cell lung cancer.

BACKGROUND: The combination of immune checkpoint inhibitors (ICIs) and chemotherapy has been the standard first-line treatment for advanced non-small cell lung cancer (NSCLC) patients with driver-gene negative. However, efficacy biomarkers for ICIs-based combination therapy are lacking. We aimed to identify potential factors associated with outcomes of ICIs plus chemotherapy at baseline and dynamic changes in peripheral blood. METHODS: We collected plasma samples of 51 advanced NSCLC patients without EGFR/ALK/ROS1 alteration at baseline and/or after two treatment cycles of ICIs plus chemotherapy. A blood-based intratumor heterogeneity (bITH) score was calculated based on the allele frequencies of somatic mutations using a 520-gene panel. bITH-up was defined as a ≥ 10% increase in bITH score from baseline, with a second confirmatory measurement after treatment. RESULTS: At baseline, the number of metastatic organs and lung immune prognostic index (LIPI) were significantly associated with shorter progression-free survival (PFS) of ICIs plus chemotherapy, while bITH and other common molecular biomarkers, including ctDNA level, blood-based tumor mutational burden (bTMB), and PD-L1 expression, had no effect on PFS. LRP1B mutation at baseline was significantly associated with favorable outcomes to ICIs plus chemotherapy. There were 37 patients who had paired samples at baseline and after two cycles of treatment, with the median interval of 53 days. Intriguingly, patients with bITH-up had significant shorter PFS (HR, 4.92; 95% CI, 1.72-14.07; P = 0.001) and a lower durable clinical benefit rate (0 vs 41.38%, P = 0.036) than those with bITH-stable or down. Case studies indicated that bITH was promising to predict disease progression. CONCLUSIONS: The present study is the first to report that increased bITH is associated with unfavorable outcomes of ICIs plus chemotherapy in advanced NSCLC patients.

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.

ctDNA-adjusted bTMB as a predictive biomarker for patients with NSCLC treated with PD-(L)1 inhibitors.

BACKGROUND: In non-small cell lung cancer (NSCLC) patients receiving immune checkpoint inhibitors (ICIs), higher blood tumor mutational burden (bTMB) was usually associated with better progression-free survival (PFS) and objective response rate (ORR). However, the association between bTMB and overall survival (OS) benefit remains undefined. It has been reported that patients harboring a high level of circulating tumor DNA (ctDNA) had poor survival. We hypothesized that ctDNA-adjusted bTMB might predict OS benefit in NSCLC patients receiving ICIs. METHODS: Our study was retrospectively performed in three cohorts, including OAK and POPLAR cohort (n = 853), Shanghai and Wuhan (SH&WH) cohort (n = 44), and National Cancer Center (NCC) cohort (n = 47). Durable clinical benefit (DCB) was defined as PFS lasting ≥ 6 months. The cutoff value of ctDNA-adjusted bTMB for DCB prediction was calculated based on a receiver operating characteristic curve. Interaction between treatments and ctDNA-adjusted bTMB was assessed. RESULTS: The bTMB score was significantly associated with tumor burden, while no association was observed between ctDNA-adjusted bTMB with tumor burden. In the OAK and POPLAR cohort, significantly higher ORR (P = 0.020) and DCB (P < 0.001) were observed in patients with high ctDNA-adjusted bTMB than those with low ctDNA-adjusted bTMB. Importantly, the interactions between ctDNA-adjusted bTMB and treatments were significant for OS (interaction P = 0.019) and PFS (interaction P = 0.002). In the SH&WH cohort, the interactions between ctDNA-adjusted bTMB and treatment were marginally significant for OS (interaction P = 0.081) and PFS (interaction P = 0.062). Similar result was demonstrated in the NCC cohort. CONCLUSIONS: Our study indicated that ctDNA-adjusted bTMB might predict OS benefit in NSCLC patients receiving ICIs. The potential of ctDNA-adjusted bTMB as a noninvasive predictor for immunotherapy should be confirmed in future studies.

Genomic landscape and prognosis of patients with TP53-mutated non-small cell lung cancer.

Background: The TP53 tumor suppressor gene plays an important role in preventing and inhibiting the growth of tumor by regulating cell cycle, apoptosis and DNA repair. Meanwhile, the TP53 gene is one of the most frequently altered gene in non-small cell lung cancer (NSCLC) patients. Mutant TP53 (TP53-MUT) may lose tumor suppressor activity and gain tumor promoting functions, which play an important role in cancer risk, therapy resistance and poor prognosis. The impact of TP53-MUT on the prognosis of NSCLC patients need to be further studied. Methods: We obtained genomic and clinical data from The Cancer Genome Atlas (TCGA). Mutation profiles, the TMB, disease-free survival (DFS), and overall survival (OS) were compared between patients with different TP53-MUT statuses. Results: TP53-MUTs were detected in 46.6% of patients with lung adenocarcinoma (LUAD) (264 of 566) and 82.3% of those with lung squamous cell carcinoma (LUSC) (401 of 487). The most frequently co-mutated genes in patients with LUAD carrying a TP53-MUT included classic driver genes such as epidermal growth factor receptor (EGFR) and anaplastic large-cell lymphoma kinase (ALK), while Kirsten rat sarcoma viral oncogene (KRAS) mutations and TP53-MUTs appear to be mutually exclusive. This mutual exclusivity was not observed in patients with LUSC, in whom titin (TTN) and CUB and Sushi multiple domains 3 (CSMD3) were the most frequently co-mutated genes. A higher TMB was significantly associated with TP53-MUTs in patients with LUAD but not in those with LUSC. In patients with stage I-III NSCLC who had undergone surgery, there was no significant difference in DFS between patients carrying TP53-wildtype (TP53-WT) and TP53-MUTs, irrespective of histology or mutation type. However, the presence of TP53-MUT was associated with shorter OS in patients with LUAD (49 vs. 54 months, respectively; P=0.13) and significantly longer OS in those with LUSC (62 vs. 29 months, respectively; P=0.015). Conclusions: In contrast to most previous studies, we revealed TP53-MUT characteristic in NSCLC patients according to histology-specific differences and the association between TP53-MUT and the mutation landscape, the TMB, and the OS. These findings suggest a need for individualized management for patients with LUAD and LUSC who carry a TP53-MUT, and warrant further research.

Tracing the genetic footprints of vertebrate landing in non-teleost ray-finned fishes.

Rich fossil evidence suggests that many traits and functions related to terrestrial evolution were present long before the ancestor of lobe- and ray-finned fishes. Here, we present genome sequences of the bichir, paddlefish, bowfin, and alligator gar, covering all major early divergent lineages of ray-finned fishes. Our analyses show that these species exhibit many mosaic genomic features of lobe- and ray-finned fishes. In particular, many regulatory elements for limb development are present in these fishes, supporting the hypothesis that the relevant ancestral regulation networks emerged before the origin of tetrapods. Transcriptome analyses confirm the homology between the lung and swim bladder and reveal the presence of functional lung-related genes in early ray-finned fishes. Furthermore, we functionally validate the essential role of a jawed vertebrate highly conserved element for cardiovascular development. Our results imply the ancestors of jawed vertebrates already had the potential gene networks for cardio-respiratory systems supporting air breathing.

Single symbiotic cell transcriptome sequencing of coral.

Zooxanthellae and coral can form an intracellular symbiotic system. Yet, little is known about the molecular mechanism underlying this symbiosis. In this study, we characterized the symbiosis based on analyses of gene expression at the single-cell level. Among 9110 single coral cells, we identified 4871 symbiotic cells based on the detection of both coral and zooxanthellae gene transcripts within a single cell. Using the bioinformatics tool Seurat, symbiotic cells were further clustered into five groups, 52 genes exhibited differential expression between groups. We proposed an index called the "symbiosis index", to indicate the degree of gene expression of both species in a single symbiotic cell. Interestingly, the index differed distinctly among the five groups. The symbiosis index was highly correlated with the expression of the coral gene gfas1.m1.6761 (ANKRD40), which encodes ankyrin repeat domain-containing protein 40 and is involved in DNA replication (r = 0.76). Two metabolism-related genes, DAGLA and betaGlu, were highly expressed in cells with a high symbiosis index. Four zooxanthellae genes, PRPF19, ATRN, aAA-ATPases and AK812-SmicGene44833, exhibited substantial changes in expression levels when zooxanthellae lived within coral. A trajectory analysis suggested that cells with a higher symbiosis index may be derived from those with a lower index during coral colony development. Taken together, our results provide evidence for zooxanthellae residing within coral, forming a symbiotic system. The symbiosis index is an effective indicator of different cell groups, with lineage relationships among groups. Additionally, we identified specific genes that exhibit expression changes in the symbiotic system.

Heterogeneous components of lung adenocarcinomas confer distinct EGFR mutation and PD-L1 expression.

BACKGROUND: Lung adenocarcinoma (LAC) is composed of lepidic, papillary, mucinous, micropapillary and solid components in its parenchyma. Complex responses to therapeutics result from intratumoral heterogeneity. However, it remains confused that what components in a mixed LAC tumor are responsible to the heterogeneous EGFR mutation and PD-L1 expression. METHODS: We investigated EGFR status via laser microdissection to capture spatially separated cancer cell subpopulations and digital droplet PCR to determine the abundance of EGFR sensitizing mutation and naïve T790M. Whilst, PD-L1 expression level via tumor proportion score (TPS) was evaluated by Ventana immunohistochemistry using SP263 antibody. PD-L1 expression levels were tiered in < 1, 1-49% and > =50% groups. RESULTS: EGFR mutation harbored in 154 (59%) of 261 LAC patients and more frequently occurred in papillary, lepidic and micropapillary constituents. Higher levels of PD-L1 were found in LACs at stage III and IV (68.3%) versus those at stage I and II (31.7%) (P = 0.04). Solid predominant LACs (41.3%) expressed PD-L1 with TPS > =50%, versus mucinous and lepidic LACs (P < 0.01). LACs with solid constituents also had more positive proportion of PD-L1 protein. Cut-offs < 1, 1-49% or > =50% were associated with patients' progression-free survival and longer in the < 1% group (22.9 month, 95% CI 17.6-28.2) (P < 0.05). LACs consisting of two constituents with PD-L1 TPS < 1% had a better prognosis than the groups with single component and more than two components (P < 0.05). Eighteen LACs (6.9%) had concomitantly deletion in exon 19 or L858R and naïve T790M mutation. The abundance of T790M varied diversely with sensitizing mutation. PD-L1 expression was not concordant in same components and usually negative in the EGFR-mutated constituents. Heterogeneous PD-L1 expression occurred in the vicinity of stromal tissues. 58.8, 29.4 and 11.8% in ALK positive LACs (N = 17) were found PD-L1 expression via cutoffs of < 1, 1-49% and > =50%, respectively (P > 0.05). CONCLUSION: Intratumoral genetic heterogeneity of LACs was demonstrated associated with histological patterns. Heterogeneous PD-L1 expression in higher level usually occurred in solid component both in EGFR mutated and EGFR wild-typed LACs. EGFR mutated LACs heterogeneously had sensitizing and resistant mutation and was accompanied with PD-L1 expression, but discordant among histological constituents. Immune checkpoint inhibitor combined with third generation EGFR tyrosine kinase inhibitor should be more effective to these LACs.

DNMHMM: An approach to identify the differential nucleosome regions in multiple cell types based on a Hidden Markov Model.

Nucleosome occupancy changes across cell types and environmental conditions and such changes often have profound influence in transcription. It's of importance to identify the differential nucleosome regions (DNRs) where the nucleosome occupancy level differs across cell types. Here we developed DNMHMM, a Hidden Markov Model (HMM) based algorithm, to detect the DNRs with nucleosomal DNA sequenced dataset. The performance evaluation indicates that DNMHMM is advisable for multi-cell type comparison. Upon testing this model in yeast mutants, where the modifiable histone residues were mutated into alanine, we found that DNA sequences of the dynamic nucleosomes lack 10-11 bp periodicities and harbor binding motifs of the nucleosome remodelling complex. Moreover, the highly expressed genes have more dynamic nucleosomes at promoters. We further compared nucleosome occupancy between resting and activated human CD4+ T cells with this model. It was revealed that during the activation of CD4+ T cells, dynamic nucleosomes are enriched at regulatory sites, hence, up to some extent can affect the gene expression level. Taken together, DNMHMM offers the possibility to access precise nucleosome dynamics among multiple cell types and also can describe the closer association between nucleosome and transcription.

Well-differentiated fetal adenocarcinoma of the lung: clinicopathologic features of 45 cases in China.

OBJECTIVE: Well-differentiated fetal adenocarcinoma (WDFA) is a rare pulmonary carcinoma with low malignancy and favorable prognosis. All cases were collected, analyzed and summarized to better understand this disease. METHODS: We used the keywords "fetal adenocarcinoma" and "epithelial pulmonary blastoma (EPB)" to search WANFANG MED ONLINE, CNKI and NCBI PUBMED for cases reported by Chinese authors from 1987 to July 2015. RESULTS: A total of 64 cases reported in China were reviewed, and the details of the clinicopathological features of 45 cases were summarized. Among these 45 patients, 23 (23/45, 51.1%) patients were male and 22 (22/45, 48.9%) patients were female. The mean age at diagnosis was 35 ± 15 years old (range, 6-72 years old) with a bimodal peak in the second and third decades. Furthermore, 24 tumors (24/31, 77.4%) were found to have progressed past stage I, while only three (3/45, 6.7%) tumors had lymph nodes metastases. These tumor cells were 100% reactive for keratin, ß-catenin, Napsin A and PDGFRα when stained by these antibodies. Better survival could be obtained if the metastatic tumor is removed in some patients with metastases. Four (4/31, 12.9%) patients died due to their tumors. CONCLUSIONS: WDFA is very different to conventional adenocarcinoma in clinicopathology. It prefers to occur in the second and third decades. Lymph node metastasis is infrequent. Beta-catenin may be a potential marker for disease. Surgery is the best therapy method if the technology is feasible.

Chaperonin containing TCP1 subunit 5 is a tumor associated antigen of non-small cell lung cancer.

Novel tumor antigens and their related autoantibodies have tremendous potential for early diagnosis of non-small cell lung cancer (NSCLC). In this study, we identify antigens from NSCLC tissue and autoantibodies in sera of patients with NSCLC using a modified proteomics-based approach. We seperated and identified four NSCLC-associated proteins extracted from the cytosol in tumor tissues by mini-two-dimensional gel electrophoresis, followed by Western blot and hybridization with individual sera for confirmation of antibody binding. Of the proteins we identified, we selected 58 kDa chaperonin containing TCP1(T-Complex Protein 1) subunit 5 (CCT5) for validation. Serum levels of carcinoembryonic antigen (CEA) and cytokeratin 19 fragments (CYFRA 21-1) were measured in all serum samples with an immunoluminometric assay and a receiver operating characteristic (ROC) curve was analyzed for autoantibodies against CCT5, CEA and CYFRA 21-1. The results show that CCT5 can induce an autoantibody response in NSCLC sera and show higher expression in NSCLC tissues by immunohistochemistry and Western blot. Anti-CCT5 autoantibody was found in 51% (23/45) of patients with NSCLC, but only 2.5% (1/40) in non-tumor individual controls. A receiver operating characteristic curve constructed with a panel of autoantibodies against CCT5 (AUC=0.749), CEA (AUC=0.6758), and CYFRA 21-1(AUC=0.760) show a sensitivity of 51.1% and 97.5% specificity in discriminating NSCLC from matched controls. These results indicate the potential utility of screening autoantibodies in sera, show that CCT5 could be used as a biomarker in cancer diagnosis.

Analysis of clinical characteristics and prognosis of patients with anaplastic lymphoma kinase-positive and surgically resected lung adenocarcinoma.

BACKGROUND: Recent research into lung cancer-related driver genes has identified a distinctive molecular subtype of non-small cell lung cancer (NSCLC) - anaplastic lymphoma kinase (ALK)-positive NSCLC. We evaluated the clinical features and survival rates of ALK-positive lung adenocarcinoma patients who had undergone surgery but had not received ALK inhibitor therapy, along with the characteristics of patients with distant metastases. METHODS: Clinical data of 40 patients with ALK-positive, postsurgical lung adenocarcinoma were retrospectively analyzed. Relationships between the patients' clinical characteristics, distant metastases, and their disease-free survival (DFS) and overall survival (OS) rates were assessed. RESULTS: Most patients were relatively young, never-smokers, had peripheral tumors, and the tumors were either moderately or poorly differentiated. The most common organ of distant metastases was the brain. The median time from surgery to brain metastasis was 17.2 months. The median OS following brain metastasis was 9.4 months. DFS in patients with early stage disease, peripheral tumors, no lymph node metastases, and treated with adjuvant therapy was significantly longer than for those with late stage disease (P = 0.015), central tumors (P = 0.000), lymph node metastases (P = 0.026), and not treated with adjuvant therapy (P = 0.000). Patients with early stage disease, peripheral tumors, and treated with adjuvant therapy obtained markedly longer OS than those with late stage disease (P = 0.021), central tumors (P = 0.003), and not treated with adjuvant therapy (P = 0.006). CONCLUSION: Patients with ALK-positive surgically resected lung adenocarcinoma have distinctive clinical characteristics. The brain is the most common site of extrapulmonary metastasis. Survival is associated with stage, tumor location, and the administration of adjuvant therapy.

Expression level of CRKL and AXL combined with exon 19 deletion in EGFR and ALK status confer differential prognosis of lung adenocarcinoma subtypes.

Non-small cell lung cancer (NSCLC) is a lethal cancer-related disease in population. Adenocarcinoma (AC) is subclassified into several subtypes based on the new classification by the International Association for the Study of Lung Cancer, American Thoracic Society and European Respiratory Society in 2011. Correlation between original expression of Crk-like (CRKL) and anaplastic lymphoma receptor tyrosine kinase in diverse histological components of AC and epidermal growth factor receptor (EGFR) or ALK status was evaluated by immunohistochemistry and sequencing in present study. A total of 106 cases, including 83 patients (78.3%) with mixed-type ACs, were assessed in the present study using eligible follow-up data. The ACs consisted of 32 acinar, 12 papillary, 5 mucinous, 11 micropapillary and 46 solid-predominant ACs. In total, 69.8% samples were composed of 2 or 3 histological components, with different expression levels of CRKL and AXL. ACs with EGFR mutation had a higher level of AXL expression compared with ACs without mutation (P=0.019). Multivariate survival analysis showed that AC subtypes and EGFR mutation subtypes were significantly associated with the progression-free survival (PFS) time. Acinar AC was the subtype with the most notable PFS time (30.6 months), which was significantly different from the PFS time of papillary, mucinous, micropapillary and solid-predominant ACs (hazard ratio, 0.4; 95% CI, 0.21-0.75; P=0.005). Among the ACs with exon 19 mutation, the median PFS time (28.8 months) of patients with a lower level of AXL protein expression was increased compared with the PFS time of patients with the L858R mutation and wild-type EGFR (9.1 months and 11 months, respectively; P=0.03), whereas no significant difference in ACs with an increased level of AXL expression. However, AC patients with higher level of CRKL expression had better PFS (28.8 months) than patients with the L858R mutation and wild-type EGFR (9.1 months and 11.3 months, respectively). Exon 19 deletion is an important status that is associated with an improved response to conventional chemotherapy. The identification of EGFR mutations combined with CRKL and AXL status may potentially alter the way that lung AC is treated.

Topoisomerase I expression is associated with prognosis in postoperative non-small cell lung cancer patients.

BACKGROUND: Biomarkers may help to improve non-small cell lung cancer (NSCLC) prognosis. However, the prognostic effect of topoisomerase I (Topo I) on NSCLC is unknown. We evaluated the clinicopathologic and prognostic significance of tumor Topo I and thymidylate synthase (TS) protein expression in postoperative NSCLC patients. METHODS: One hundred and fifteen patients with postoperative NSCLC were enrolled. Topo I and TS protein were detected in removed tumors by immunohistochemistry. The correlations between Topo I/TS protein expression and clinicopathologic characters and outcomes of patients were analyzed. RESULTS: Increased expression of Topo I was found in 57 (49.6%) tumors. The largest diameter of the tumor was significantly different between patients with high and low Topo I expression (P = 0.035). TS staining showed that 35 (30.4%) carcinomas were TS positive. The level of TS expression was correlated with tumor differentiation (P = 0.037). Patients with low Topo I expression had significantly longer overall survival (OS) than those with high expression (P = 0.004). The correlation between Topo I expression and OS was demonstrated among patients with squamous cell carcinoma (P = 0.030) and patients in pathological tumor node metastasis stage I (P = 0.027). Topo I expression was positively correlated with TS expression in tumor tissue (R = 0.251, P = 0.007). CONCLUSIONS: Low Topo I expression is an independent favorable prognostic factor for longer OS in postoperative NSCLC patients, especially in squamous cell carcinoma. There is a correlation between the expression of TS and Topo I in removed tumor tissue.

Micropapillary: A component more likely to harbour heterogeneous EGFR mutations in lung adenocarcinomas.

The micropapillary (MP) subtype has recently been established to be a distinct marker of poor prognosis in lung adenocarcinomas (LACs). According to the 2015 WHO classification system, LAC constituents are required to be precisely reported. T790M mutation and an insertion in exon 20 (E20ins) are associated with EGFR-TKI resistance. A total of 211 LAC patients were involved in this study, and EGFR mutations were determined using an amplification refractory mutation system (ARMS). Sex, smoking history, lymph node status, and clinical stage differed significantly between the EGFR wild type and mutant groups (p < 0.05). The EGFR mutation occurred more frequently in female, non-smokers, ACs with papillary (85.7%) or MP components (91.4%) (p < 0.001). Twenty ACs with naïve T790M or E20ins were microdissected. The AC constituents metastasizing to lymph nodes exhibited a phenotype and EGFR status that was consistent with the primary loci constituents. Glomerulus-like solid components exhibited the same EGFR status as the surrounding T790M-mutated MP components. The MP and glomerulus-like portions in AC tumours exhibited a congenial EGFR status, but the acinar cells with papillary cells were heterogeneous. The naïve T790M mutants, although minor in the MP component, dramatically increased after EGFR-TKI therapy and indicate that the MP components feature intrinsic heterogeneity.

Association between the histological subtype of lung adenocarcinoma, EGFR/KRAS mutation status and the ALK rearrangement according to the novel IASLC/ATS/ERS classification.

The present study aimed to investigate the association between epidermal growth factor receptor (EGFR)/Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangements and the morphological characteristics of lung adenocarcinoma (LAC), according to the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) classification in a large group of patients with primary LAC. A total of 200 patients with invasive LAC who had undergone complete resections at the Beijing Chest Hospital (Beijing, China) were randomly selected. The morphology of the samples was reassessed in 5% increments by two pathologists, according to the IASLC/ATS/ERS scheme. EGFR and KRAS mutations were tested by direct DNA sequencing. ALK rearrangements were screened by immunohistochemistry on a Benchmark XT stainer. The data revealed that EGFR and KRAS mutations, and ALK rearrangements were identified in 46.0% (92/200), 9.0% (18/200) and 11.5% (23/200) of the patients, respectively. The EGFR/KRAS mutations and ALK rearrangements were mostly exclusive. However, 1 patient exhibited the coexistence of the EGFR (at exon 20) and KRAS (codon 12) mutations, and another patient exhibited the coexistence of the EGFR mutation (at exon 21) and the ALK gene fusion. EGFR mutations were indicated to be closely associated with the acinar predominant (43/77; 55.8%; P=0.030) and papillary predominant (26/49; 53.1%; P=0.006) subtypes. KRAS mutations were more commonly associated with the solid predominant subtype (9/52; 17.3%; P=0.023) and invasive mucinous LAC (5/10; 50.0%; P=0.004), and less commonly associated with the acinar predominant subtype (1/77; 1.3%; P=0.002). ALK rearrangements more commonly occurred in the solid predominant subtype compared with other subtypes (13/52; 25%; P=0.002), and less commonly occurred in the papillary predominant subtype (1/49; 2.0%; P=0.004). Tumors harboring ALK rearrangements were characterized by signet-ring cell (7/9; 77.8%; P<0.0001) and cribriform (7/12; 58.3%; P<0.0001) patterns. The association between the mutation status and histological subtype in LAC was distinct. The predominant subtype according to the IASLC/ATS/ERS classification provided important information for gene mutations and integrated clinical findings to improve the treatment of LAC patients.