[Clinical Features and Prognosis of Acute T-cell Lymphoblastic Leukemia in Children--Multi-Center Data Analysis in Fujian].

OBJECTIVE: To evaluate the efficacy of acute T-cell lymphoblastic leukemia (T-ALL) in children and explore the prognostic risk factors. METHODS: The clinical data of 127 newly diagnosed children with T-ALL admitted to five hospitals in Fujian province from April 2011 to December 2020 were retrospectively analyzed, and compared with children with newly diagnosed acute precursor B-cell lymphoblastic leukemia (B-ALL) in the same period. Kaplan-Meier analysis was used to evaluate the overall survival (OS) and event-free survival (EFS), and COX proportional hazard regression model was used to evaluate the prognostic factors. Among 116 children with T-ALL who received standard treatment, 78 cases received the Chinese Childhood Leukemia Collaborative Group (CCLG)-ALL 2008 protocol (CCLG-ALL 2008 group), and 38 cases received the China Childhood Cancer Collaborative Group (CCCG)-ALL 2015 protocol (CCCG-ALL 2015 group). The efficacy and serious adverse event (SAE) incidence of the two groups were compared. RESULTS: Proportion of male, age≥10 years old, white blood cell count (WBC)≥50×109/L, central nervous system leukemia, minimal residual disease (MRD)≥1% during induction therapy, and MRD≥0.01% at the end of induction in T-ALL children were significantly higher than those in B-ALL children (P <0.05). The expected 10-year EFS and OS of T-ALL were 59.7% and 66.0%, respectively, which were significantly lower than those of B-ALL (P <0.001). COX analysis showed that WBC≥100×109/L at initial diagnosis and failure to achieve complete remission (CR) after induction were independent risk factors for poor prognosis. Compared with CCLG-ALL 2008 group, CCCG-ALL 2015 group had lower incidence of infection-related SAE (15.8% vs 34.6%, P =0.042), but higher EFS and OS (73.9% vs 57.2%, P EFS=0.090; 86.5% vs 62.3%, P OS=0.023). CONCLUSIONS: The prognosis of children with T-ALL is worse than children with B-ALL. WBC≥100×109 /L at initial diagnosis and non-CR after induction (especially mediastinal mass has not disappeared) are the risk factors for poor prognosis. CCCG-ALL 2015 regimen may reduce infection-related SAE and improve efficacy.

Predicting survival of advanced laryngeal squamous cell carcinoma: comparison of machine learning models and Cox regression models.

Laryngeal squamous cell carcinoma (LSCC) is a common tumor type. High recurrence rates remain an important factor affecting the survival and quality of life of advanced LSCC patients. We aimed to build a new nomogram and a random survival forest model using machine learning to predict the risk of LSCC progress. The study included 671 patients with AJCC stages III-IV LSCC. To develop a prognostic model, Cox regression analyses were used to assess the relationship between clinic-pathologic factors and disease-free survival (DFS). RSF analysis was also used to predict the DFS of LSCC patients. The ROC curve revealed that the Cox model exhibited good sensitivity and specificity in predicting DFS in the training and validation cohorts (1 year, validation AUC = 0.679, training AUC = 0.693; 3 years, validation AUC = 0.716, training AUC = 0.655; 5 years, validation AUC = 0.717, training AUC = 0.659). Random survival forest analysis showed that N stage, clinical stage, and postoperative chemoradiotherapy were prognostically significant variables associated with survival. The random forest model exhibited better prediction ability than the Cox regression model in the training cohort; however, the two models showed similar prediction ability in the validation cohort.

Plasma Extracellular Vesicles-Derived miR-99a-5p: A Potential Biomarker to Predict Early Head and Neck Squamous Cell Carcinoma.

Purpose: This study aimed to investigate the applicability of plasma extracellular vesicles (EVs) miR-99a-5p as a potential head and neck squamous cell carcinoma (HNSCC) diagnostic biomarker. Methods: The miRNA expression of HNSCC tissue and plasma EVs were profiled by small RNA sequencing. qRT-PCR was performed to detect miR-99a-5p expression in HNSCC (n = 93) and benign disease (n = 39) plasma EVs and formalin-fixed and paraffin-embedded (FFPE) tissue (n = 110). We constructed receiver-operating characteristic curves to investigate the diagnostic efficiency of plasma EVs miR-99a-5p. Results: Tumor tissue exhibited lower miR-99a-5p than para-tumor tissue. Patients with high miR-99a-5p expression exhibited significantly more p16 positive status. In contrast, HNSCC plasma EVs harbored more miR-99a-5p than the benign disease group. Plasma EVs miR-99a-5p distinguished HNSCC with area under the curve (AUC) of 0.7494 (95% CI: 0.6692-0.8296; p < 0.0001), with 61.54% sensitivity and 75.27% specificity, respectively. Furthermore, plasma EVs miR-99a-5p also distinguished early HNSCC with AUC of 0.7394 (95% CI: 0.6284-0.8504; p = 0.0002), with 79.07% sensitivity and 61.54% specificity, respectively. Conclusion: Plasma EVs miR-99a-5p is a potential biomarker for predicting early HNSCC.

Cancer-associated fibroblasts in a human HEp-2 established laryngeal xenografted tumor are not derived from cancer cells through epithelial-mesenchymal transition, phenotypically activated but karyotypically normal.

Cancer-associated fibroblasts (CAFs) play a crucial role in cancer progression and even initiation. However, the origins of CAFs in various cancer types remain controversial, and one of the important hypothesized origins is through epithelial-mesenchymal transition (EMT) from cancer cells. In this study, we investigated whether the HEp-2 laryngeal cancer cells are able to generate CAFs via EMT during tumor formation, which is now still unknown. The laryngeal xenografted tumor model was established by inoculating the HEp-2 laryngeal cancer cell line in nude mice. Primary cultured CAFs from the tumor nodules and matched normal fibroblasts (NFs) from the adjacent connective tissues were subcultured, purified, and verified by immunofluorescence. Migration, invasion, and proliferation potentials were compared between the CAFs and NFs. A co-culture of CAFs with HEp-2 cells and a co-injection of CAFs with HEp-2 cells in nude mice were performed to examine the cancer-promoting potential of CAFs to further verify their identity. Karyotypic analyses of the CAFs, NFs, and HEp-2 cells were conducted. A co-culture of NFs with HEp-2 cells was also performed to examine the expression of activated markers of CAFs. A pathological examination confirmed that the laryngeal xenografted tumor model was successfully established, containing abundant CAFs. Immunocytochemical staining verified the purities and identities of the CAFs and NFs. Although the CAFs manifested higher migration, invasion, proliferation, and cancer-promoting capacities compared with the NFs, an analysis of chromosomes revealed that both the CAFs and NFs showed typical normal mouse karyotypes. In addition, the NFs co-cultured with HEp-2 cells did not show induced expressions of activated markers of CAFs. Our findings reveal that the CAFs in the HEp-2 established laryngeal xenografted tumor are not of laryngeal cancer origin but of mouse origin, indicating that the HEp-2 laryngeal cancer cells cannot generate their own CAFs via EMT in this model.

Hypoxia promotes stem-like properties of laryngeal cancer cell lines by increasing the CD133+ stem cell fraction.

Evidence indicates that a hypoxic micro-environment plays an essential role in the regulation of cancer stem cells (CSCs). However, whether hypoxia is able to regulate the stem-like biological properties of laryngeal cancer cells remains unknown. In this study, we investigated the influence of hypoxia on the stemness of two laryngeal cancer cell lines, Hep-2 and AMC-HN-8. We cultured the two cell lines under hypoxia and normoxia and examined the influence of hypoxia on the expression of hypoxia-inducible factors (HIFs) and the cancer stem-like properties of these cells, including cell cycle distribution, expression of stem cell genes (OCT4, SOX2 and NANOG) and laryngeal CSC surface marker (CD133), proliferation, invasion, colony formation and sphere formation capacity. We determined that both of these cell lines, when maintained under hypoxic conditions, showed expanded cells in the G0/G1 phase, exhibited preferential expression of stem cell genes and CD133, and manifested upregulation of HIFs. When treated with hypoxia followed by normoxia exposure, the two cell lines exhibited enhanced capacities for proliferation, invasion, and sphere and colony formation compared with cells maintained consistently under normoxia. Our findings indicate that a hypoxic microenvironment may upgrade the stem-like biological properties of laryngeal cancer cell lines by the expansion of the CD133(+) stem cell fraction.

Establishment and characterization of a novel HPV-negative laryngeal squamous cell carcinoma cell line, FD-LSC-1, with missense and nonsense mutations of TP53 in the DNA-binding domain.

Laryngeal squamous cell carcinoma (LSCC) is a common malignancy in China; however, publically available LSCC cell lines are few and not established from Chinese populations. Hence, novel and well-characterized LSCC cell lines of Chinese origin are urgently needed to provide researchers with a comprehensive database for LSCC research. From 40 cases of LSCC, we established a novel cell line that was maintained for more than 100 passages in vitro and was found to have typical epithelial morphology and ultrastructure. In-depth characterization analysis revealed polyploidy in DNA content; a doubling time of some 24h; high tumorigenicity in immunodeficient mice; higher invasive potential and more sensitive to radiation and cisplatin compared with HeLa cell line; upregulated Ki67, Notch1, EGFR, and CK5 protein levels; negative infection of human papillomavirus (HPV) and mycoplasma; expression of head and neck squamous cell carcinoma (HNSCC) biomarkers; mutations of TP53 in exons 5 and 8; a near-triploid karyotype with complex structural aberrations; and dozens of dysregulated genes and miRNAs. Cell authentication testing by the American Type Culture Collection (ATCC) confirmed the human origin of this cell line. Our findings indicate that a novel and well-differentiated LSCC cell line recapitulating the primary tumor's malignant characteristics is established and well characterized. It does not match any cell lines within the ATCC database and helps to elucidate the molecular pathogenesis of LSCC.

The Composition of Microbiome in Larynx and the Throat Biodiversity between Laryngeal Squamous Cell Carcinoma Patients and Control Population.

The throat is an ecological assemblage involved human cells and microbiota, and the colonizing bacteria are important factors in balancing this environment. However, this bacterial community profile has thus been poorly investigated. The purpose of this study was to investigate the microbial biology of the larynx and to analyze the throat biodiversity in laryngeal carcinoma patients compared to a control population in a case-control study. Barcoded pyrosequencing analysis of the 16S rRNA gene was used. We collected tissue samples from 29 patients with laryngeal carcinoma and 31 control patients with vocal cord polyps. The findings of high-quality sequence datasets revealed 218 genera from 13 phyla in the laryngeal mucosa. The predominant communities of phyla in the larynx were Firmicutes (54%), Fusobacteria (17%), Bacteroidetes (15%), Proteobacteria (11%), and Actinobacteria (3%). The leading genera were Streptococcus (36%), Fusobacterium (15%), Prevotella (12%), Neisseria (6%), and Gemella (4%). The throat bacterial compositions were highly different between laryngeal carcinoma subjects and control population (p = 0.006). The abundance of the 26 genera was significantly different between the laryngeal cancer and control groups by metastats analysis (p<0.05). Fifteen genera may be associated with laryngeal carcinoma by partial least squares discriminant analysis (p<0.001). In summary, this study revealed the microbiota profiles in laryngeal mucosa from tissue specimens. The compositions of bacteria community in throat were different between laryngeal cancer patients and controls, and probably were related with this carcinoma. The disruption of this bio-ecological niche might be a risk factor for laryngeal carcinoma.

Reduced expression of mutS homolog 2 and mutL homolog 1 affects overall survival in laryngeal squamous cell carcinoma patients: Investigation into a potential cause.

The risk factors affecting the survival rates of laryngeal carcinoma are not well understood. In this study, we investigated the expression status of mutS homolog 2 (MSH2) and mutL homolog 1 (MLH1) and examined the relationship between these two molecules and overall survival rates in laryngeal cancer. We also explored the potential reason for the altered expression of these two genes. Using real-time polymerase chain reaction and western blotting, we detected MSH2 and MLH1 expression in laryngeal cancer tissue samples. We collected a retrospective cohort with 180 laryngeal cancer patients, and inspected MSH2 and MLH1 staining with tissue microarray immunohistochemistry. Prognostic value of clinicopathological characteristics was evaluated by statistical analysis. Laryngeal carcinoma cells were co-cultured with Helicobacter pylori (H. pylori) bacteria. MSH2 and MLH1 were expressed at lower levels compared to those of adjacent tissues in 21 laryngeal carcinoma patients. Patients with negative expression of MSH2 and MLH1 tended to have a higher risk of mortality compared to patients with positive expression (HR=4.38; HR=3.0, respectively). Cigarette smoking rate was higher in the MLH1 expression positive group. H. pylori infection reduced the MSH2 and MLH1 expression levels of laryngeal carcinoma cell lines within co-culture conditions. It is suggested that the altered expression levels of MSH2 and MLH1 probably affect the overall survival of laryngeal carcinoma patients. H. pylori infection may have an effect on the expression of MSH2 and MLH1 in laryngeal carcinoma patients.

Identification of cancer stem-like side population cells in purified primary cultured human laryngeal squamous cell carcinoma epithelia.

Cancer stem-like side population (SP) cells have been identified in many solid tumors; however, most of these investigations are performed using established cancer cell lines. Cancer cells in tumor tissue containing fibroblasts and many other types of cells are much more complex than any cancer cell line. Although SP cells were identified in the laryngeal squamous cell carcinoma (LSCC) cell line Hep-2 in our pilot study, it is unknown whether the LSCC tissue contains SP cells. In this study, LSCC cells (LSCCs) were primary cultured and purified from a surgically resected LSCC specimen derived from a well-differentiated epiglottic neoplasm of a Chinese male. This was followed by the verification of epithelium-specific characteristics, such as ultrastructure and biomarkers. A distinct SP subpopulation (4.45±1.07%) was isolated by Hoechst 33342 efflux analysis from cultured LSCCs by using a flow cytometer. Cancer stem cell (CSC)-associated assays, including expression of self-renewal and CSC marker genes, proliferation, differentiation, spheroid formation, chemotherapy resistance, and tumorigenicity were then conducted between SP and non-SP (NSP) LSCCs. In vitro and in vivo assays revealed that SP cells manifested preferential expression of self-renewal and CSC marker genes, higher capacity for proliferation, differentiation, and spheroid formation; enhanced resistance to chemotherapy; and greater xenograft tumorigenicity in immunodeficient mice compared with NSP cells. These findings suggest that the primary cultured and purified LSCCs contain cancer stem-like SP cells, which may serve as a valuable model for CSC research in LSCC.

Separation and cultivation of laryngeal carcinoma-associated fibroblasts and biological influence on a laryngeal carcinoma cell line.

CONCLUSIONS: Carcinoma-associated fibroblasts (CAFs) can influence the biological characteristics of a laryngeal carcinoma cell line. These results could lay the foundation for further studies on the role of CAFs in the laryngeal tumor-host microenvironment. OBJECTIVE: CAFs are important contributors to the microenvironment in determining the fate of tumors. The aim of this study was to separate, culture, and identify laryngeal CAFs and investigate their biological influence on the laryngeal carcinoma cell line. METHODS: The primary CAFs and normal fibroblasts (NFs) of the larynx were obtained by tissue culture. The cells were verified according to immunohistochemical and immunofluorescence staining of certain proteins. Conditioned medium (CM) from CAFs and NFs was obtained. Functional assays were performed to test the influence of each CM on laryngeal carcinoma cell lines. RESULTS: Third-passage purified laryngeal CAFs and NFs were successfully attained. The CAFs showed positive staining for vimentin, α-smooth muscle actin (α-SMA), and fibroblast activation protein (FAP). The migration ability of the CAFs increased significantly compared with that of NFs (p < 0.05). CM from CAFs (compared with CM from NFs) stimulated proliferation, migration, and invasion to a greater extent.

[Isolation and in vivo tumorigenicity assay of CD133+ side population cells from laryngeal cancer cell line].

OBJECTIVE: To investigate a valuable strategy for further purifying cancer stem cells (CSCs) from laryngeal cancer cell line. METHODS: CD133+ side population (SP) and CD133-SP cells were detected and isolated from laryngeal cancer Hep-2 cell line with SP discrimination and CD133 surface marker, assisted by fluorescence activated cell sorting technology. Freshly sorted CD133+SP and CD133-SP cells were xenografted into the subcutaneous space of the right axillary fossa of NOD/SCID mice and tumorigenic capacity of the cells from two subgroups were examine. Cell cycle distributions of the two cell populations were detected. RESULTS: CD133+SP and CD133-SP cells accounted for (0.30±0.12)% and (17.52±1.59)% in Hep-2 cell line, respectively. CD133+SP cells formed tumor nodules in 15 of 16 mice and CD133-SP cells in 7 of 16 mice (Fisher's exact test, P<0.05). The mean weight of CD133+SP tumor nodules was (0.36±0.15)g and that of CD133-SP tumor nodules was (0.08±0.04) g. The difference was significant (t=4.64, P<0.01). Cell cycle analysis revealed similar cycle distributions between the two subgroups. CONCLUSIONS: CD133+SP cells harbored much more cancer stem-like tumorigenic potential in NOD/SCID mice than CD133-SP cells. The combination of SP discrimination and surface marker selection helped to purify CSCs further from laryngeal cancer cell line.

[Isolation and in vitro characterization of CD133(+) side population cells from laryngeal cancer cell line].

OBJECTIVE: To investigate an approach enriching cancer stem cells (CSCs) more effectively from laryngeal cancer cell line. METHODS: CD133(+)SP and CD133(-)SP subpopulation was detected and isolated from Hep-2 cell line using Hoechst33342 dye and phycoerythrin (PE)-conjugated CD133 monoclonal antibody assisted by fluorescence activated cell sorting technology. Sorted CD133(+)SP and CD133(-)SP cells were compared in CSCs-related assays including proliferation, differentiation, spheroid formation and drug sensitivity. RESULTS: CD133(+)SP cells accounted for a very small fraction of (0.30 ± 0.12)% in Hep-2 cell line, far less than the proportion of CD133(+) subgroup and side population subgroup, which were (3.15 ± 0.83)% and (17.1 ± 2.0)% respectively. Intriguingly, CD133(+)SP cells proliferated much faster than CD133(-)SP cells in RPMI1640 and gave rise to CD133(-)SP cells and other heterogeneous cells that formed the bulk of the tumor. In contrast, CD133(-)SP cells were not able to differentiate into CD133(+)SP cells. In serum-free medium CD133(+)SP cells grew as spherical clusters and remained floating. In addition, CD133(+)SP cells manifested the marked resistance to chemotherapy than CD133(-)SP cells. CONCLUSIONS: Compared with CD133(-)SP cells, CD133(+)SP subpopulation exhibited extraordinary cancer stem-like properties, were enriched for cancer stem cells more effectively and might serve as an ideal putative candidate for CSCs research in laryngeal cancer.