speculated that the four indicators of AHRR, p16INK4a, MT1G and CLDN3 can be combined to improve the sensitivity and specificity for the diagnosis of ESCC. DNA Repair (Amst). 2019;145(3):662-670, 31. Biomarkers can overcome this bottleneck and guide us toward personalized precision medicine for oral squamous cell carcinoma. A P‐value < 0.05 and |FC| > 2 were used as the threshold to identify DEGs. 2018;39(3):407-417, 42. In China, ESCC is the fourth leading cause of cancer-related death [21]. In addition, cytoHubba (integrated into Cytoscape software) was applied to screen hub genes using the Maximal Clique Centrality (MCC) method [15]. Spondin-2 is a novel diagnostic biomarker for laryngeal squamous cell carcinoma. Despite progress in preventive and therapeutic strategies, delay in OSCC diagnosis remains one of the major causes of high morbidity and mortality; indeed the majority of OSCC has been lately identified in the advanced clinical stage (i.e., III or IV). Involvement of upregulation of DEPDC1 (DEP domain containing 1) in bladder carcinogenesis. Keywords: ESCC, biomarkers, diagnostic, poor prognosis, therapeutic targets. var thismailbody1 = ""; 2008;98(11):1824-9, 28. Bioinformatics. edu.cn. var thismailbody3 = ""; Currently, these two databases are widely used for data mining due to their large sample size and complete clinical information. DEP Domain Containing 1 is a Novel Diagnostic Marker and Prognostic Predictor for Hepatocellular Carcinoma. In the present study, we found that RAD51AP1 is an effective diagnostic biomarker for ESCC (AUC= 96.7%). In the present study, we combined six GEO microarray datasets to screen the DEGs in at least two different ESCC datasets, and further integrated with TCGA ESCC dataset to screen the possible biomarkers associated with diagnosis and prognosis for ESCC. Correlation of hub genes expression levels with immune infiltration of B cell, CD4 T cell, CD8 T cell, neutrophil, macrophage, and dendritic cell in ESCC. The aim of the study was to identify novel biomarkers for ESCC. 2018;10:3383-3392, 43. Furthermore, we performed immune infiltration analysis to gain a better understanding of the function of these genes. eCollection 2020. Kinesin Family Member 20A is a Novel Melanoma-associated Antigen. PBK/TOPK Expression Predicts Prognosis in Oral Cancer. Microbiol. 2011;104(2):300-307, 34. Mitotic centromere-associated kinesin is a novel marker for prognosis and lymph node metastasis in colorectal cancer. Fig. 2002;159(4):549-555, 30. 1. Serum HOTAIR as a novel diagnostic biomarker for esophageal squamous cellcarcinoma. Firstly, we used the “Limma” R package to standardize the data and eliminate the effects of experimental techniques on gene expression. document.write(thismailbody4 + thismailbody3+thismailbody2) Adams L, Roth MJ, Abnet CC. var thismailbody3 = ""; Liu Q, Dai SJ, Li H, Dong L, Peng YP. Substances. Chen W, Zheng R, Zeng H, Zhang S. The incidence and mortality of major cancers in China, 2012. 2016 Dec 15;4(4):41. doi: 10.3390/proteomes4040041. This site needs JavaScript to work properly. Bie L, Zhao G, Wang YP, Zhang B. Kinesin family member 2C (KIF2C/MCAK) is a novel marker for prognosis in human gliomas. *P < 0.05 represent significant differences between the indicated groups. Salivary extracellular vesicle-associated miRNAs as potential biomarkers in oral squamous cell carcinoma. As far as we know, there is no relevant research to verify the identified biomarkers at the single cell levels. Li HM, Yang H, Wen DY. Introduction. doi:10.7150/jca.50443, Liuhai Zheng1, Linzhi Li1, Jun Xie1, Hai Jin2 , Naishuo Zhu1. 5B). Terms of use. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. As we all know, there are certain technical errors in both microarray and next-generation sequencing data. Head and neck squamous cell cancer (HNSCC) Biomarkers; Human papilloma virus (HPV) Epidermal growth factor receptor (EGFR) Epstein Barr virus (EBV) Cetuximab; Nasopharyngeal carcinoma (NPC) Phosphatase and tensin homolog (PTEN) Phosphoinositide 3- kinase (PI3K) 2A-F). 2007;26(44):6448-6455, 44. 1. Ohashi et al found that PBK overexpressed is associated with worse outcomes in ESCC [24]. 10.1016/j.fct.2016.06.010 2017;9:129, 9. 2014;15(24):10917-10922, 48. Head & Neck Squamous Cell Carcinoma is one of the highest mortality factors in the world due to the lack of potential biomarker for early detection of disease. The aim of the present study was to identify more effective molecular diagnostic biomarkers for esophageal squamous cell carcinoma (ESCC). Sci Rep. 2018;8(1):9227, 17. Clipboard, Search History, and several other advanced features are temporarily unavailable. 2007(14):14, 12. Oncol Rep. 2018;40(1):195-205, 40. The TIMER database incorporates 32 cancer types and six immune cell types (B cell, CD4 T cell, CD8 T cell, neutrophil, macrophage, and dendritic cell). Sci. With regard to biological processes, the downregulated genes were associated with muscle system processes, regulation of ion transmembrane transporter activity and actomyosin structure organization. In addition, ROC analysis also showed that PBK can effectively distinguish ESCC tissues from normal samples (AUC= 96.5%), indicating that PBK could be an independent diagnostic biomarker for ESCC. Yamashita J, Fukushima S, Jinnin M. et al. Sci Rep. 2018;8(1):8823, 4. (B) ROC analysis of hub genes in ESCC. Alix-Panabieŕes C., Pantel K. (2013). The results showed that all samples (GSE17351, GSE20347, GSE23400, GSE100942, GSE38129 and GSE77861) were clearly divided into normal and tumor groups, except for dataset GSE23400, where four normal samples (GSM573926, GSM573867, GSM573888 and GSM573889) and three tumor samples (GSM573926, GSM573935 and GSM573944) showed no difference between normal tissue and tumors; therefore, these were removed from further analyses (Fig. Pu W, Wang C, Chen S. et al. 2016;11(12):e0167449, 39. 2021 Feb 17;40(1):70. doi: 10.1186/s13046-021-01865-2. Our results revealed that low expression in ESCC is associated with worse outcomes, and NUF2 can efficiently distinguish tumor tissues from normal tissue (AUC= 99.2%), suggesting its diagnostic value for ESCC. Spector ME, Sacco AG, Bellile E, Taylor JMG, Jones T, Sun K, et al. To evaluate the clinical value of these hub genes in ESCC, Kaplan-Meier survival analysis was performed using Kaplan Meier-plotter. We hypothesized that combination of circulating miR and isomiRs detected by NGS are potentially powerful cancer biomarker. Cristaldi M (1), Mauceri R (1) (2), … 2015;15(6):789-797, 47. Recent next generation sequencer (NGS) studies have found that isoforms of micro RNA (isomiR) circulate in the bloodstream similarly to mature micro RNA (miR). Park JH, Lin ML, Nishidate T, Nakamura Y, Katagiri T. PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK) as a molecular target for breast cancer therapy. The major reason is that the poor reproducibility, small overlap between studies, and low sensitivity and specificity for diagnosis and prognosis of ESCC. 2013;8(4):e62752, 45. 3A). Cellular component analysis of the downregulated genes showed association with contractile fibers, myofibrils, and intercalated discs. Sun G(1)(2), Cao Y(2), Wang P(3), Song H(2), Bie T(2), Li M(4), Huai(2). Even if standardized protocols for isolation, characterization, and evaluation are needed, recent data suggest that saliva may be successfully included in future clinical diagnostic processes, with a considerable impact on early treatment strategies and a favorable outcome. At present, there is no effective treatment strategy together with the lack of effective diagnosis and prognosis biomarkers, the 5-year survival rate of ESCC patients is less than 30% [2]. To date, the GEO database contains > 40000 microarray gene expression datasets and > 2000 items containing the keywords “esophageal cancer”. TIMER: A Web Server for Comprehensive Analysis of Tumor-Infiltrating Immune Cells. However, there is still a great gap to put these findings into clinical application. Diagnostic value of cyclin-dependent kinase/cyclin-dependent kinase inhibitor expression ratios as biomarkers of locoregional and hematogenous dissemination risks in oral squamous cell carcinoma Mol Clin Oncol , 3 ( 2015 ) , pp. Oral squamous cell carcinoma (OSCC) is the most common epithelial malignancy affecting the oral cavity, and it is especially significant in Asian countries. 98(Pt A), 66–72. Wang C, Pu W, Zhao D. et al. Front. thismailbody3 = thismailbody1; For example, Takeshita et al. Subsequently, Trimmomatic tool were used to remove low quality and adapter reads, and then mapped to the human genome GRCh38 transcriptome using Bowtie. 2017;49(05):388-399, 7. Sannam Khan R, Khurshid Z, Akhbar S, Faraz Moin S. Proteomes. Silencing of NUF2 inhibits tumor growth and induces apoptosis in human hepatocellular carcinomas. 2014;12(4):539-549, 37. Herein, our results revealed that DEPDC1 is a promising diagnostic and prognostic biomarker for ESCC. BMC Bioinformatics. Salivary Biomarkers for Oral Squamous Cell Carcinoma Diagnosis and Follow-Up: Current Status and Perspectives Marta Cristaldi 1 , Rodolfo Mauceri 1,2 , Olga Di Fede 1 , Giovanna Giuliana 1 , Giuseppina Campisi 1* and Vera Panzarella 1 ESCC-related GEO datasets with primary tumor tissues and matched normal tissues, principal component analysis (PCA) analysis can well distinguish tumor samples from normal samples, expression profiling by array were enrolled into the present study. Cancer Biomarker. Our aim was to evaluate the potential of the succinate-related pathway as a diagnostic and prognostic biomarker in HNSCC. The ggplot2 package was used to display the DEGs identified from each dataset (Fig. Inhibition of DEPDC1A, a Bad Prognostic Marker in Multiple Myeloma, Delays Growth and Induces Mature Plasma Cell Markers in Malignant Plasma Cells. Moreover, silencing of RAD51AP1 can inhibit epithelial-mesenchymal transition and metastasis in non-small cell lung cancer [41, 42]. (B) GO and pathway enrichment results for down‑regulated DEGs. Esophageal cancer was the seventh most common cancer and the sixth leading cause of cancer death worldwide in 2018, with around 572,000 new cases and 509,000 deaths annually [1]. MicroRNA (miR)s are promising diagnostic biomarkers of cancer. 2017;77(21):e108-e110, 21. Cancer Lett. Cancer Manag Res. (B) GSE20347. (F) DEPDC1. Cell cluster analysis was performed using the “Seurat” R package. Cyclin-Dependent Kinase Inhibitor p16. 2014 Feb;119:299-305. doi: 10.1016/j.talanta.2013.11.008. Pathway enrichment analysis revealed that the upregulated genes were mainly involved in the cell cycle extracellular matrix organization and DNA repair (Fig. Gai C, Camussi F, Broccoletti R, Gambino A, Cabras M, Molinaro L, Carossa S, Camussi G, Arduino PG. However, biomarkers related to ESCC diagnosis and prognosis was usually identified independently, which may hinder our understanding of the process from initiation to deterioration. Public Heal. Targeted bisulfite sequencing identified a panel of DNA methylation-based biomarkers for esophageal squamous cell carcinoma(ESCC). In the present study, we found that KIF20A is overexpressed in ESCC, and low expression was associated with poor prognosis. 2020 Nov 10;12:11491-11499. doi: 10.2147/CMAR.S262442. Overlap between DEGs across different ESCC datasets. Clinicopathological and biological significance of mitotic centromere-associated kinesin overexpression in human gastric cancer. More importantly, most previous studies were mainly depended on one or two datasets, while individual dataset often can be unreliable due to bias introduced by sample processing and insufficient samples [10]. Subsequently, protein-protein interaction network analysis was performed to identify the key hub genes. thismailbody1 = thismailbody12+"3&"+"#x"; Anticancer Res. Ritchie ME, Belinda P, Di W. et al. An enzyme-linked immunosorbent assay was performed to detect … GEOquery: a bridge between the Gene Expression Omnibus (GEO) and BioConductor. To evaluate the prognostic significance of identified hub genes, a Kaplan Meier survival analysis was carried out using Kaplan Meier-plotter (https://kmplot.com/analysis/) [16]. Thereafter, a ROC analysis was conducted to investigate diagnostic value. The detailed clinic parameters of these participants are listed in Table 3. NUF2 is a valuable prognostic biomarker to predict early recurrence of hepatocellular carcinoma after surgical resection. The ethics committee at the changhai hospital, Second Military Medical University approved this study, and written informed consent on the use of clinical specimens from all participants. 3. Circulating tumor cells: liquid biopsy of cancer. Six patients with ESCC who underwent surgical resection of tumor tissues without any treatment were enrolled for qPCR assay. Chang CF, Chen SL, Sung WW. Genital squamous cell carcinoma is linked with p16 (in less aggressive form of vulvar SCC), p53 (weaker prognosis), cofilin-1, galectin-7 and weel. Menini M, De Giovanni E, Bagnasco F, Delucchi F, Pera F, Baldi D, Pesce P. J Pers Med. Therefore, a serum metabolomic strategy … 2017;59:76-81, 41. Patients diagnosed with OSCC have an unfavorable prognosis and additional prognostic markers would help improve therapeutic strategies. Of the 10 genes, we found that PBK (hazard ratio [HR]=0.25, logrank P= 0.00062), KIF2C (HR=0.37, logrank P= 0.05), NUF2 (HR=0.37, logrank P= 0.011), KIF20A (HR=0.4, logrank P= 0.024), RAD51AP1 (HR=0.42, logrank P= 0.033), and DEPDC1 (HR=0.41, logrank P= 0.037) were significantly correlated with overall survival (Fig. Diagnostic and therapeutic biomarkers useful for esophageal squamous cell carcinoma (ESCC) have the ability to increase the long term survival of cancer patients. Here, we present a systematic review to identify, evaluate and summarize the evidence for OSCC reported markers. Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies with poor diagnosis. Patients typically present with advanced, incurable disease, so new means of detecting early disease are a research priority. Ishikawa S, Ishizawa K, Tanaka A, Kimura H, Kitabatake K, Sugano A, Edamatsu K, Ueda S, Iino M. In Vivo. 4C). (C) Venn diagram displaying the overlap between DEGs from the GEO and TCGA datasets. Cancer Manag Res. Chin J Cancer. 2013;381(9864):400-412, 3. Therefore, to obtain more convincing results, more datasets from multiple platforms are needed. //--> thismailbody3 = thismailbody1; Then, the following cycling conditions were applied: 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds and 62°C for 30 seconds. (E) GSE38129. HR: Hazard Ratio. Talanta. UALCAN (http://ualcan.path.uab.edu/), which contains TCGA and MET500 transcriptome sequencing data, was used to validate mRNA expression of the hub genes [18]. MicroRNAs: genomics, biogenesis, mechanism, and function. Serum microRNA expression profile: miR-1246 as a novel diagnostic and prognostic biomarker for oesophageal squamous cell carcinoma. Author information: (1)Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Department of Thoracic surgery, Sun yat-sen memorial hospital, Sun yat-sen University, NO.107 of … In contrast, our study showed that low expression levels of KIF2C correlated with worse survival (HR=0.37, logrank P= 0.05). Br J Cancer. var thismailbody2 = ""; Song et al. 2017;45(D1):D362-D368, 15. Lee LT, Wong YK, Hsiao HY, Wang YW, Chan MY, Chang KW. Li T, Fan J, Wang B. et al. circulating tumor DNA; early diagnosis; extracellular vesicles; liquid biopsy; microRNAs; oral squamous cell carcinoma; prognosis; salivary biomarkers. Zhang W, He W, Shi Y. et al. Neoplasia. Sean D, Meltzer PS. Bardou P, Mariette J, Escudié F, Djemiel C, Klopp C. jvenn: an interactive Venn diagram viewer. We aimed to explore the molecular mechanism of this CSCC and screen feature genes that can function as the biomarker of CSCC and thus provide a theoretical basis for the … The GEO datasets (GSE17351, GSE20347, GSE23400, GSE100942, GSE38129 and GSE77861) and TCGA datasets were processed using the “Limma” R package [12]. High Expression of KIF20A Is Associated with Poor Overall Survival and Tumor Progression in Early-Stage Cervical Squamous Cell Carcinoma. Saliva is a biofluid containing factors such as cytokines, DNA and RNA molecules, circulating and tissue-derived cells, and extracellular vesicles (EVs) that may be used as biomarkers; their analysis may give us useful information to do early diagnosis of OSCC and improve the prognosis. Paired adjacent nontumor tissues from the proximal resection margins (>5 cm away from the ESCC sample) were also collected for RNA extraction and qPCR assay. The detailed reasons need to be further validated. et al. Yukinaga M, Kenichi K, Tomoko N. et al. However, their expression had no obvious correlation with infiltration of other immune cells (B cell, CD4 T cell, CD8 T cell, neutrophil, macrophage) (Fig. 2015;8(3):2638-2648, 33. Results and Discussion. We found 132 downregulated and 48 upregulated genes (Fig. Please enable it to take advantage of the complete set of features! 2021 Jan-Feb;35(1):541-547. doi: 10.21873/invivo.12289. 2006;66(18):9186-95, 23. Chandrashekar DS, Bashel B, Balasubramanya SAH, Creighton CJ, Varambally S. UALCAN: A Portal for Facilitating Tumor Subgroup Gene Expression and Survival Analyses. Ohashi T, Komatsu S, Ichikawa D. et al. Our results indicate that these genes may also be potential targets for ESCC therapy. 2005 Songhu Road, Yangpu District, Shanghai, China. 6B). Food Chem. Li CY, Zhang WW, Xiang JL, Wang XH, Wang JL, Li J. The details of these datasets are listed in Table 1. J Cell Mol Med. Subsequently, the PPI network information was imported into Cytoscape and the MCC method in cytoHubba was applied to screen hub genes. Indian J … Br J Cancer. document.write(thismailbody4 + thismailbody3+thismailbody2) Moreover, after primary treatment, recurrences and/or metastases are found in more than half of the patients (80% of cases within the first 2 years) and the 5-year survival rate is still lower than 50%, resulting in a serious issue for public health. (A) Expression levels of PBK, KIF2C, NUF2, KIF20A, RAD51AP1 and DEPDC1 in UALCAN database. thismailbody4 = thismailbody4 + "4;"; Esophageal squamous cell carcinoma (ESCC) remains one of the leading causes of cancer-related mortality around the world. Medicina (Kaunas). BMC Syst Biol. We found that all hub genes mRNA expression levels were significantly elevated in tumor tissues compared to adjacent normal tissues (P < 0.05) (Fig. Growing evidence has indicated that tumor biomarkers, including cytokeratin 19 fragment antigen 21–1 (Cyfra21–1), carbohydrate antigen 19–9 (CA19–9), carbohydrate antigen 72–4 (CA72–4), carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC-Ag) were reported to be commonly used in diagnosis and prognosis in esophageal squamous cell carcinoma … Saliva liquid biopsy for point-of-care applications. ESCC has a generally poor prognosis and there is a lack of available biomarkers for diagnosis and prognosis. Our study could provide novel biomarkers for ESCC diagnosis and prognosis, and potential targets for ESCC therapy. Validation of miRNA prognostic power in hepatocellular carcinoma using expression data of independent datasets. 2019;9:262, 46. Currently, histological investigation represents the "gold standard" of OSCC diagnosis; however, recent studies have evaluated the potential use of non-invasive methods, such as "liquid biopsy," for the detection of diagnostic and prognostic biomarkers in body fluids of oral cancer patients. We screened the overlapping differentially expressed genes (DEGs) acquired from six Gene Expression Omnibus (GEO) ESCC datasets and The Cancer Genome Atlas (TCGA) ESCC datasets. Data are presented as the means ± SEM. hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells. See http://ivyspring.com/terms for full terms and conditions. Received 2020-7-8 Accepted 2020-11-16 Published 2021-1-1, ©2021 Ivyspring International Publisher. Oral tongue squamous cell carcinoma (OTSCC) is the most common malignancy of the oral cavity. We aimed to identify new biomarkers or potential therapeutic targets for OSCC. DCs are the most important antigen presenting cells, which play a key role in connecting innate immunity with acquired immunity. Br J Cancer. 4B). The diagnostic role of KIF2C in ESCC has not been previously reported. Cancer Prev Res (Phila). Our results highlight that saliva liquid biopsy has several promising clinical uses in OSCC management; it is painless, accessible, and low cost and represents a very helpful source of diagnostic and prognostic biomarker detection. Int J Cancer. (A) PBK. Prevention and treatment information (HHS). GO, pathways enrichment, and PPI of overlap DEGs in ESCC. Prognostic Significance of Cytoplasmic SPNS2 Expression in Patients with Oral Squamous Cell Carcinoma. Ádám N, András L, Otília M, Balázs Gr. 2015;43(7):e47, 13. Single-cell RNA sequencing reveals diverse intratumoral heterogeneities and gene signatures of two types of esophageal cancers. KIF2C, a member of the motor proteins family, functions as a microtubule-dependent molecular motor [25]. Wu H, Yu J, Li Y. et al. Chin CH, Chen SH, Wu HH, Ho CW, Ko MT, Lin CY. qPCR was performed to quantify the hub genes mRNA expression level using the LightCycler® 480 SYBR Green I Master kit (Roche Molecular Diagnostics Inc., South Branchburg, NJ, USA). (C) The relative mRNA expression of PBK, KIF2C, NUF2, KIF20A, RAD51AP1 and DEPDC1 were confirmed by qPCR (n=6). Prognostic and diagnostic potential of miR-146a in oesophageal squamous cell carcinoma. Lack of biomarkers for discriminating ESCC and ESD from healthy subjects limits the early diagnosis and treatment of ESCC. In addition, scRNA-seq results showed that the hub genes is mainly expressed on carcinoma cells, and qPCR results also indicated that the expression of the hub genes in tumor tissues is significantly higher than normal tissues. The authors have declared that no competing interest exists. Previous studies have demonstrated that high KIF2C expression can serve as an independent marker of poor prognosis in several tumors, including glioma, colorectal cancer, and gastric cancer [26-28]. var thismailbody11 = ""; Thus, screening for more efficient biomarkers for ESCC diagnosis and prognosis is urgently required. For GEO datasets, each set of DEGs was screened separately. et al. var thismailbody12 = "fuda"; Background. Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide. High Expression of DEPDC1 Promotes Malignant Phenotypes of Breast Cancer Cells and Predicts Poor Prognosis in Patients With Breast Cancer. Zhao H, Yu M, Sui L. et al. (C) PPI network of overlap DEGs from GEO data and TCGA datasets. Systematic profiling identifies PDLIM2 as a novel prognostic predictor for oesophageal squamous cell carcinoma (ESCC). Next, an online tool (jvenn) was used to identify overlapping DEGs in at least two GEO datasets. In our study, we integrated six GEO microarray datasets with TCGA ESCC dataset to screen the possible biomarkers for ESCC. 6A). (F) GSE77861. Ni H (1), Ni T (2), Feng J (3), Bian T (3), Liu Y (4), Zhang J (5). Extracellular miRNAs as biomarkers in cancer. (A) Kaplan Meier survival analysis of PBK, KIF2C, NUF2, KIF20A, RAD51AP1 and DEPDC1 in ESCC. A receiver operating curve (ROC) analysis was conducted using the “pROC” R package to explore the diagnostic value of these hub genes [17]. (D) GSE100942. To obtain biological changes in gene expression in ESCC, all data were pre-processed. 2016;114(3):290-7, 50. Wang C, Guan S, Liu F. et al. Single strand complementary DNA (cDNA) was synthesized from 0.5 μg of total RNA using the transcriptor first strand cDNA synthesis kit (Roche Molecular Diagnostics Inc., South Branchburg, NJ, USA). To explore the function of the common DEGs, GO, pathway enrichment and PPI analysis were conducted using the STRING database. identified PDLIM2 as a novel prognostic predictor for ESCC, which also associated with nodal invasion. Wang W(1), He X(1), Zheng Z(1), Ma X(1), Hu X(1), Wu D(2), Wang M(3). The pathologic diagnosis of NSCLC subtypes, which include SqCLC, adenocarcinoma, and large-cell carcinoma, is a multistep process.