[ABO*A2.08 Subtype Allele Identification and Protein Structure Analysis in Newborns].

OBJECTIVE: To study the serological characteristics of ABO*A2.08 subtype and explore its genetic molecular mechanism. METHODS: ABO blood group identification was performed on proband and her family members by routine serological methods. ABO genotyping and sequence analysis were performed by polymerase chain reaction-sequence specific primer (PCR-SSP), and direct sequencing of PCR products from exons 6 and 7 of ABO gene were directly sequenced and analyzed. The effect of gene mutation in A2.08 subtype on structural stability of GTA protein was investigated by homologous protein conserved analysis, 3D molecular modeling and protein stability prediction. RESULTS: The proband's serological test results showed subtype Ax, and ABO genotyping confirmed that the proband's genotype was ABO*A207/08. Gene sequencing of the proband's father confirmed the characteristic variation of c.539G>C in the 7th exon of ABO gene, leading to the replacement of polypeptide chain p.Arg180Pro (R180P). 3D protein molecular modeling and analysis suggested that the number of hydrogen bonds of local amino acids in the protein structure was changed after the mutation, and protein stability prediction showed that the mutation had a great influence on the protein structure stability. CONCLUSION: The mutation of the 7th exon c.539G>C of ABO gene leads to the substitution of polypeptide chain amino acid, which affects the structural stability of GTA protein and leads to the change of enzyme activity, resulting in the A2.08 phenotype. The mutated gene can be stably inherited.

Single-cell transcriptomics reveals cell atlas and identifies cycling tumor cells responsible for recurrence in ameloblastoma.

Ameloblastoma is a benign tumor characterized by locally invasive phenotypes, leading to facial bone destruction and a high recurrence rate. However, the mechanisms governing tumor initiation and recurrence are poorly understood. Here, we uncovered cellular landscapes and mechanisms that underlie tumor recurrence in ameloblastoma at single-cell resolution. Our results revealed that ameloblastoma exhibits five tumor subpopulations varying with respect to immune response (IR), bone remodeling (BR), tooth development (TD), epithelial development (ED), and cell cycle (CC) signatures. Of note, we found that CC ameloblastoma cells were endowed with stemness and contributed to tumor recurrence, which was dominated by the EZH2-mediated program. Targeting EZH2 effectively eliminated CC ameloblastoma cells and inhibited tumor growth in ameloblastoma patient-derived organoids. These data described the tumor subpopulation and clarified the identity, function, and regulatory mechanism of CC ameloblastoma cells, providing a potential therapeutic target for ameloblastoma.

Targeting BRD4 attenuates the stemness and aggressiveness of ameloblastoma.

BACKGROUND: BRD4, belonging to the bromodomain extra-terminal (BET) protein family, plays a unique role in tumor progression. However, the potential impact of BRD4 in ameloblastoma (AM) remains largely unknown. Herein, we aimed to assess the expression and functional role of BRD4 in AM. METHODS: The expression level of BRD4 was assessed by immunohistochemistry. The proliferation, migration, invasion, and tumorigenic abilities of AM cells were assessed by a series of assays. To explore the molecular expression profile of BRD4-depleted AM cells, RNA sequencing (RNA-seq) was performed. Bioinformatic analysis was performed on AM expression matrices obtained from the Gene Expression Omnibus (GEO). The therapeutic efficacy of BET-inhibitors (BETi) was assessed with AM patient-derived organoids. RESULTS: Upregulation of BRD4 was observed in conventional AMs, recurrent AMs, and ameloblastic carcinomas. Depletion of BRD4 inhibited proliferation, invasion, migration, and tumorigenesis in AM. Administration of BETi attenuated the aggressiveness of AM and the growth of AM patient-derived organoids. Bioinformatic analysis indicated that BRD4 may promote AM progression by regulating the Wnt pathway and stemness-associated pathways. CONCLUSION: BRD4 increases the aggressiveness and promotes the recurrence of ameloblastoma by regulating the Wnt pathway and stemness-associated pathways. These findings highlight BRD4 as a promising therapeutic target in AM management.

Single-cell transcriptomic analysis uncovers the origin and intratumoral heterogeneity of parotid pleomorphic adenoma.

Pleomorphic adenoma (PA) is the most common benign tumour in the salivary gland and has high morphological complexity. However, the origin and intratumoral heterogeneity of PA are largely unknown. Here, we constructed a comprehensive atlas of PA at single-cell resolution and showed that PA exhibited five tumour subpopulations, three recapitulating the epithelial states of the normal parotid gland, and two PA-specific epithelial cell (PASE) populations unique to tumours. Then, six subgroups of PASE cells were identified, which varied in epithelium, bone, immune, metabolism, stemness and cell cycle signatures. Moreover, we revealed that CD36+ myoepithelial cells were the tumour-initiating cells (TICs) in PA, and were dominated by the PI3K-AKT pathway. Targeting the PI3K-AKT pathway significantly inhibited CD36+ myoepithelial cell-derived tumour spheres and the growth of PA organoids. Our results provide new insights into the diversity and origin of PA, offering an important clinical implication for targeting the PI3K-AKT signalling pathway in PA treatment.

[Retrospective Analysis of Irregular Antibodies Causing Hemolytic Disease of the Fetus and Newborn in Jiangxi Province].

OBJECTIVE: To analyze the characteristics of antibody-specific distribution, laboratory detection results of hemolytic disease of the fetus and neonatal(HDFN) caused by irregular blood group antibodies other than ABO, and its correlation with the clinical situation. METHODS: The non-ABO-HDFN cases in our hospital from October 2012 to December 2021 were selected as the research objects, and the cases diagnosed with ABO-HDFN in the same period were randomly selected as the control group, and the data of antibody specific distribution, total bilirubin, direct antibodies, maternal history, age of the children, the presence or absence of combined ABO-HDFN, and whether to exchange/transfuse blood were retrospectively analyzed. The characteristics of non-ABO-HDFN in Jiangxi province were analyzed. RESULTS: The detection rate of non-ABO-HDFN in Jiangxi province increased. Among 187 non ABO-HDFN cases, the highest percentage of Rh-HDFN was detected (94.6%). Compared with the control group of ABO-HDFN, the non-ABO-HDFN had higher mean integral value of direct antibody, higher peak total bilirubin, and longer duration. Anti-M-HDFN may have severe disease but the direct antibody weak positive/negative, it was easy missed in clinical and delayed the treatment. There is no correlation between the specificity of irregular antibodies, the sex of the child, the mother's previous childbirth history, the presence or absence of combined ABO-HDFN and the need for blood exchange/transfusion(P>0.05). CONCLUSION: The irregular antibodies of causing non ABO-HDFN in Jiangxi area are mainly Rh blood group system, followed by MNS blood group system. Understanding the characteristics of HDFN disease, serological features and the correlation with clinical indexes will help to detect and treat non ABO-HDFN in time and reduce the risk of complications.

Prognostic value of tumor-infiltrating immune cells in clinical early-stage oral squamous cell carcinoma.

BACKGROUND: Tumor-infiltrating immune cells (TIICs) are critical components of tumor immune microenvironment (TIME), which play crucial roles during tumor initiation, development, and progression. However, the prognostic value of TIICs is still not well documented in clinical early-stage oral squamous cell carcinoma (OSCC). In this study, we aimed to assess the prognostic value of TIICs in clinical early-stage OSCC and develop a nomogram based on TIICs to predict the prognosis. METHODS: Eighty patients with clinical early-stage (cT1,2N0M0) OSCC were enrolled in this study. Immunohistochemical staining was performed to evaluate the infiltration of TIICs, including CD8+ T cells, CD57+ NK cells, CD163+ macrophage, and CD20+ B cells. Overall survival (OS) and disease-free survival (DFS) curves were plotted by the Kaplan-Meier method. Cox's proportional hazards regression models were performed to assess the prognostic value of TIICs. Finally, a nomogram was established to predict the OS based on TIICs infiltration and assessed by concordance index (C-index) and calibration curve. RESULTS: High infiltrations of CD57+ NK cells and CD20+ B cells indicated a better OS in clinical early-stage OSCC. Moreover, high infiltration of CD20+ B cells favored a longer DFS. Of note, low infiltrations of CD57+ NK cells and CD20+ B cells were independent prognostic factors for poor OS in clinical early-stage OSCC. The nomogram that combined CD57+ NK cells with CD20+ B cells could predict the OS in clinical early-stage OSCC, and the C-index was 0.801 (95% CI: 0.679-0.924). The calibration plot showed that prediction and observation are well matched. CONCLUSIONS: High infiltration of CD57+ NK cells and CD20+ B cells indicate a favorable OS in clinical early-stage OSCC. The nomogram constructed based on TIICs might be used for predicting the prognosis in clinical early-stage OSCC.

[Identification and Molecular Biology of Variant D Blood Group of RHD*95A Genotype].

OBJECTIVE: To explore the molecular biology of D variant blood group with RHD*95A genotype and the genetic mechanism of its generation. METHODS: A total of 6 samples from 3 generations of a family were analyzed. RHD blood group was identified by saline test tube and microcolumn gel card method. 10 exons of RHD gene were amplified by Polymerase Chain Reaction-Sequence Specific Primer (PCR-SSP) and analyzed by direct sequencing. Homology modeling was used to compare the structural differences between mutant RHD protein and wild-type RHD protein. RESULTS: The proband was identified as D variant by serological identification, RHD gene sequencing directly detected a c. 95 c > A mutation in exon 1 that leads to encoding the 32-bit amino acids by threonine Thr (T) into aspartic acid Asn (N), the rest of the exon sequences were normal compared with the normal RHD*01 gene. In the family, the proband's father, grandmather and uncle were all carried the same RHD*95A allele. Protein modeling results suggested that the hydrogen chain connected to the 32nd amino acid residue was changed after p.T32N mutation, which affected the structural stability of RHD protein. CONCLUSION: The first genetic lineage of the RHD*95A gene was identified in a Chinese population. The c.95C>A mutation in RHD gene was found in the family, which resulted in reduced expression of RHD antigen and showed D variant, the mutation could be stably inheritable. Gene identification and protein structure analysis of D variant population is helpful to explore the molecular mechanism of its formation and ensure the safety of blood transfusion.

[Study on BW.12 Subtype Caused by c.278C>T Mutation in Exon 6 of ABO Gene].

OBJECTIVE: To investigate the effect of ABO gene α-1,3-D galactosyl transferase mutation on B antigen expression and its molecular mechanism. METHODS: The proband and their family members were identified by routine serological methods, and ABO genotyping and sequence analysis were performed by polymerase chain reaction-sequence specificity (PCR-SSP) and direct sequencing of PCR products from exon 1-7 of ABO gene. The 3D structural simulation of mutant proteins was performed by bioinformatics software. The effect of gene mutation on protein structural stability was analyzed. RESULTS: The proband and his family members were subtype B. ABO genotyping indicated that the proband's genotype was Bw12/O. Gene sequencing results confirmed the presence of ABO*BW.12 characteristic variation c.278C>T in the 6th exon of allele B, leading to the replacement of polypeptide chain p.Pro93Leu. The 3D structure simulation analysis of the protein showed that the hydrogen bonds and water molecules connected to the protein changed after amino acid substitution. The family investigation found that the grandfather, father, uncle and brother of the proband all carried the same ABO*BW.12 allele. CONCLUSION: The mutation of the 6th exon c.278C>T of ABO gene led to the substitution of polypeptide chain amino acids, which affected the stability of α-1,3-D galactosyl transferase protein, resulting in the change of enzyme activity, and the Bw.12 phenotype, which can be stably inherited.

FOSL1 promotes tumor growth and invasion in ameloblastoma.

Background: FOSL1, a key component of the Activating protein-1 (AP-1) transcriptional complex, plays an important role in cancer cell migration, invasion, and proliferation. However, the impact of FOSL1 in ameloblastoma (AM) has not been clarified. Herein, we aimed to assess the expression of FOSL1 and investigate its functional role in AM. Methods: The expression of FOSL1 was examined based on an immunohistochemistry analysis of 96 AM samples. Cell proliferation, migration, invasion, and tumorigenesis were assessed using Cell Counting Kit-8 (CCK-8), colony formation, Transwell, and sphere formation assays. RNA sequencing (RNA-seq) was employed to investigate the molecular alterations of AM cells upon FOSL depletion. Microarrays of AMs were downloaded from the Gene Expression Omnibus (GEO) database for bioinformatics analysis. In addition, patient-derived AM organoids were used to evaluate the therapeutic value of the AP-1 inhibitor. Results: FOSL1 was detected in the nuclei of AMs and upregulated in conventional AMs compared to unicystic AMs and normal oral epithelium. Compared with primary AM, FOSL1 expression was significantly increased in recurrent AM. Genetic knockdown of FOSL1 suppressed the proliferation, migration, invasion, and sphere formation of AMs. Similar results were also observed by pharmacological inhibition of AP-1 activity. Moreover, the AP-1 inhibitor T5224 impeded the growth of organoids derived from AM patients. Mechanistically, our Ingenuity Pathway Analysis (IPA) and gene set enrichment analysis (GSEA) results revealed that depletion of FOSL1 inactivated kinetochore metaphase signaling and the epithelial-mesenchymal transition pathway and then impaired the aggressiveness of AM cells accordingly. Conclusion: FOSL1 promotes tumor recurrence and invasive growth in AM by modulating kinetochore metaphase signaling and the epithelial-mesenchymal transition pathway; thus, it represents a promising therapeutic target for AM treatment.

BET inhibition triggers antitumor immunity by enhancing MHC class I expression in head and neck squamous cell carcinoma.

BET inhibition has been shown to have a promising antitumor effect in multiple tumors. However, the impact of BET inhibition on antitumor immunity was still not well documented in HNSCC. In this study, we aim to assess the functional role of BET inhibition in antitumor immunity and clarify its mechanism. We show that BRD4 is highly expressed in HNSCC and inversely correlated with the infiltration of CD8+ T cells. BET inhibition potentiates CD8+ T cell-based antitumor immunity in vitro and in vivo. Mechanistically, BRD4 acts as a transcriptional suppressor and represses the expression of MHC class I molecules by recruiting G9a. Pharmacological inhibition or genetic depletion of BRD4 potently increases the expression of MHC class I molecules in the absence and presence of IFN-γ. Moreover, compared to PD-1 blocking antibody treatment or JQ1 treatment individually, the combination of BET inhibition with anti-PD-1 antibody treatment significantly enhances the antitumor response in HNSCC. Taken together, our data unveil a novel mechanism by which BET inhibition potentiates antitumor immunity via promoting the expression of MHC class I molecules and provides a rationale for the combination of ICBs with BET inhibitors for HNSCC treatment.

Prognostic and clinicopathological significance of cytocapsular tubes in oral squamous cell carcinoma.

BACKGROUND: Cytocapsular tubes (CTs) provide membranous channels for cancer cells interconnection and multidirectional locomotion, which facilitate cancer cell transportation and metastasis. However, the clinicopathological significance of CTs has not been documented in oral squamous cell carcinoma (OSCC). Herein, we aimed to identify CTs and assess their clinicopathological significance in OSCC. METHODS: Operetta CLS™ high-content analysis system was used to detect the CTs originated from OSCC cells cultured in a 3D Matrigel matrix. Then, pan-cadherin and γ-actin immunostaining were performed to identify CTs in 4NQO-induced murine OSCC tissues, OSCC xenografts and 88 human primary OSCC samples. Finally, the prognostic value and clinicopathological significance of CTs in OSCC were further examined by using the Kaplan-Meier method and Cox regression analysis. RESULTS: CTs were observed in OSCC cells in a 3D Matrigel matrix. In vivo, CTs were frequently identified in 4NQO-induced murine OSCC tissues, OSCC xenografts and human primary OSCC samples. CTs density was significantly associated with T stage, lymph node metastasis, differentiation, invasive depth, tumor budding, TNM stage and tumor recurrence. Importantly, the high-CTs density indicated a decreased overall survival (OS) and progression-free survival (PFS) in OSCC patients. Cox regression models showed that CTs could serve as a prognostic factor for OS and PFS. CONCLUSION: CTs, which are correlated with the cell migration and invasion, can be readily identified in OSCC and appear to be a novel biomarker for patients at risk of metastasis.

Sox9+ cells are required for salivary gland regeneration after radiation damage via the Wnt/ß-catenin pathway.

Radiotherapy for head and neck cancer can cause serious side effects, including severe damage to the salivary glands, resulting in symptoms such as xerostomia, dental caries, and oral infection. Because of the lack of long-term treatment for the symptoms of xerostomia, current research has focused on finding endogenous stem cells that can differentiate into various cell lineages to replace lost tissues and restore functions. Here, we report that Sox9+ cells can differentiate into various salivary epithelial cell lineages under homeostatic conditions. After ablating Sox9+ cells, the salivary glands of irradiated mice showed more severe phenotypes and the reduced proliferative capacity. Analysis of online single-cell RNA-sequencing data reveals the enrichment of the Wnt/ß-catenin pathway in the Sox9+ cell population. Furthermore, treatment with a Wnt/ß-catenin inhibitor in irradiated mice inhibits the regenerative capability of Sox9+ cells. Finally, we show that Sox9+ cells are capable of forming organoids in vitro and that transplanting these organoids into salivary glands after radiation partially restored salivary gland functions. These results suggest that regenerative therapy targeting Sox9+ cells is a promising approach to treat radiation-induced salivary gland injury.

Methyltransferase like 13 mediates the translation of Snail in head and neck squamous cell carcinoma.

Methyltransferase like 13 (METTL13), a kind of methyltransferase, is implicated in protein binding and synthesis. The upregulation of METTL13 has been reported in a variety of tumors. However, little was known about its potential function in head and neck squamous cell carcinoma (HNSCC) so far. In this study, we found that METTL13 was significantly upregulated in HNSCC at both mRNA and protein level. Increased METTL13 was negatively associated with clinical prognosis. And METTL13 markedly affected HNSCC cellular phenotypes in vivo and vitro. Further mechanism study revealed that METTL13 could regulate EMT signaling pathway by mediating enhancing translation efficiency of Snail, the key transcription factor in EMT, hence regulating the progression of EMT. Furthermore, Snail was verified to mediate METTL13-induced HNSCC cell malignant phenotypes. Altogether, our study had revealed the oncogenic role of METTL13 in HNSCC, and provided a potential therapeutic strategy.

Sox9-expressing cells promote regeneration after radiation-induced lung injury via the PI3K/AKT pathway.

BACKGROUND: Radiation-induced lung injury (RILI) is considered one of the most common complications of thoracic radiation. Recent studies have focused on stem cell properties to obtain ideal therapeutic effects, and Sox9 has been reported to be involved in stem cell induction and differentiation. However, whether Sox9-expressing cells play a role in radiation repair and regeneration remains unknown. METHODS: We successfully obtained Sox9CreER, RosatdTomato and RosaDTA mice and identified Sox9-expressing cells through lineage tracing. Then, we evaluated the effects of the ablation of Sox9-expressing cells in vivo. Furthermore, we investigated the underlying mechanism of Sox9-expressing cells during lung regeneration via an online single-cell RNA-seq dataset. RESULTS: In our study, we demonstrated that Sox9-expressing cells promote the regeneration of lung tissues and that ablation of Sox9-expressing cells leads to severe phenotypes after radiation damage. In addition, analysis of an online scRNA-Seq dataset revealed that the PI3K/AKT pathway is enriched in Sox9-expressing cells during lung epithelium regeneration. Finally, the AKT inhibitor perifosine suppressed the regenerative effects of Sox9-expressing cells and the AKT pathway agonist promotes proliferation and differentiation. CONCLUSIONS: Taken together, the findings of our study suggest that Sox9-expressing cells may serve as a therapeutic target in lung tissue after RILI.

A Super-Enhancer Driven by FOSL1 Controls miR-21-5p Expression in Head and Neck Squamous Cell Carcinoma.

MiR-21-5p is one of the most common oncogenic miRNAs that is upregulated in many solid cancers by inhibiting its target genes at the posttranscriptional level. However, the upstream regulatory mechanisms of miR-21-5p are still not well documented in cancers. Here, we identify a super-enhancer associated with the MIR21 gene (MIR21-SE) by analyzing the MIR21 genomic regulatory landscape in head and neck squamous cell carcinoma (HNSCC). We show that the MIR21-SE regulates miR-21-5p expression in different HNSCC cell lines and disruption of MIR21-SE inhibits miR-21-5p expression. We also identified that a key transcription factor, FOSL1 directly controls miR-21-5p expression by interacting with the MIR21-SE in HNSCC. Moreover, functional studies indicate that restoration of miR-21-5p partially abrogates FOSL1 depletion-mediated inhibition of cell proliferation and invasion. Clinical studies confirmed that miR-21-5p expression is positively correlated with FOSL1 expression. These findings suggest that FOSL1-SE drives miR-21-5p expression to promote malignant progression of HNSCC.

Corrigendum: Deficiency of Mettl3 in Bladder Cancer Stem Cells Inhibits Bladder Cancer Progression and Angiogenesis.

[This corrects the article DOI: 10.3389/fcell.2021.627706.].

Tumor microenvironment in head and neck squamous cell carcinoma: Functions and regulatory mechanisms.

The tumor microenvironment has been recently reported to play a pivotal role in sustaining tumor cells survival and protecting them from immunotherapy and chemotherapy-induced death. It remains largely unknown how the specific signaling pathway exerts the tumor microenvironment in head and neck squamous cell carcinoma though previous studies have elucidated the regulatory mechanisms involve in tumor immune microenvironment, stromal cells, tumor angiogenesis and cancer stem cell. These components are responsible for tumor progression as well as anti-cancer therapy resistance, leading to rapid tumor growth and treatment failure. In this review, we focus on discussing the interaction between tumor cells and the surrounding components for better understanding of anti-cancer treatment ineffectiveness and its underlying molecular mechanisms.

Deficiency of Mettl3 in Bladder Cancer Stem Cells Inhibits Bladder Cancer Progression and Angiogenesis.

RNA N6-methyladenosine is a key step of posttranscriptional modulation that is involved in governing gene expression. The m6A modification catalyzed by Mettl3 has been widely recognized as a critical epigenetic regulation process for tumorigenic properties in various cancer cell lines, including bladder cancer. However, the in vivo function of Mettl3 in bladder cancer remains largely unknown. In our study, we found that ablation of Mettl3 in bladder urothelial attenuates the oncogenesis and tumor angiogenesis of bladder cancer using transgenic mouse model. In addition, conditional knockout of Mettl3 in K14+ bladder cancer stem cell population leads to inhibition of bladder cancer progression. Coupled with the global transcriptome sequencing and methylated RNA immunoprecipitation sequencing results, we showed that deletion of Mettl3 leads to the suppression of tyrosine kinase endothelial (TEK) and vascular endothelial growth factor A (VEGF-A) through reduced abundance of m6A peaks on a specific region. In addition, the depletion of Mettl3 results in the decrease in both messenger RNA (mRNA) and protein levels of TEK and VEGF-A in vitro. Taken together, Mettl3-mediated m6A modification is required for the activation of TEK-VEGF-A-mediated tumor progression and angiogenesis. Our findings may provide theoretical basis for bladder cancer treatment targeting Mettl3.

Distribution of human papilloma virus genotype prevalence in invasive cervical carcinomas and precancerous lesions in the Yangtze River Delta area, China.

BACKGROUND: This study aimed to provide more information for cancer prevention strategies by determining the distribution of human papilloma virus (HPV) genotype prevalence in invasive cervical carcinoma (ICC) and precancerous lesion patients in the Yangtze River Delta area in China. METHODS: This multi-centre descriptive cross-sectional study involves four university hospitals in the Jiangzhehu area. Women with histologically confirmed cervical intraepithelial neoplasia (CIN) 1, CIN2, CIN3 or ICC who were diagnosed and treated in the four selected hospitals between February 2012 and April 2014 were eligible for recruitment. The average age of the patients was 40.93 ± 11.87 years old, among whom the youngest was 17 years old and the oldest was 76 years old.Those with immunodeficiency diseases or a previous history of cancer or CIN were excluded. HPV genotyping was performed by a central laboratory. The distribution and age and disease specificity of the HPV genotype prevalence were analysed. RESULTS: Of the 2181 collected samples, 251 were ICC and 1930 were CIN. The mean age of cervical cancer and CIN patients was 40.93 ± 11.8 years (range, 17-76 years). The five most commonly identified HPV types in each lesion class were as follows: CIN1: 52, 58, 16, 33, and CP; CIN2: 16, 58, 52, 33, and 31; CIN3: 16, 58, 33, 52, and 31; and ICC: 16, 58, 18, 52, and 33. CIN1 had an earlier age of onset (30-40 years) than CIN2, CIN3, and cervical cancer. The age of onset of cervical cancer exhibited two peaks at 40-44 and 50-54 years of age. In all infected patients, the frequency of HPV infection with a single type was 62.9%, and with multiple types, it was 38.1%. There was no difference in the frequencies of multiple types amongst the different cervical lesions. CONCLUSIONS: The most prevalent genotypes in the investigated area (52, 58, 16 and 18) justify the necessity of anti-HPV vaccination in teenagers and young girls under 24 years old in the Yangtze River Delta area in China. Infection with multiple high-risk HPV types versus single infection does not increase the risk for ≥ CIN2 in ICC development.