Cervical Intraepithelial Neoplasia grade 2 biopsy: Do p16INK4a and Ki-67 biomarkers contribute to the decision to treat? A cross-sectional study

ABSTRACT BACKGROUND: Managing cervical intraepithelial neoplasia grade 2 (CIN2) is challenging, considering the CIN2 regression rate, perinatal risks associated with excisional procedures, and insufficient well-established risk factors to predict progression. OBJECTIVES: To determine the ability of p16INK4a and Ki-67 staining in biopsies diagnosed with CIN2 to identify patients with higher-grade lesions (CIN3 or carcinoma). DESIGN AND SETTING: Cross-sectional study conducted at a referral center for treating uterine cervical lesions. METHODS: In 79 women, we analyzed the correlation of p16INK4a and Ki-67 expression in CIN2 biopsies with the presence of a higher-grade lesions, as determined via histopathology in surgical specimens from treated women or via two colposcopies and two cytological tests during follow-up for untreated women with at least a 6-month interval. The expression of these two biomarkers was verified by at least two independent pathologists and quantified using digital algorithms. RESULTS: Thirteen (16.8%) women with CIN2 biopsy exhibited higher-grade lesions on the surgical excision specimen or during follow-up. p16INK4a expression positively and negatively predicted the presence of higher-grade lesions in 17.19% and 86.67% patients, respectively. Ki-67 expression positively and negatively predicted the presence of higher-grade lesions in 40% and 88.24% patients, respectively. CONCLUSIONS: Negative p16INK4a and Ki67 immunohistochemical staining can assure absence of a higher-grade lesion in more than 85% of patients with CIN2 biopsies and can be used to prevent overtreatment of these patients. Positive IHC staining for p16INK4a and Ki-67 did not predict CIN3 in patients with CIN2 biopsies.

Therapeutic effect of Rab11 inhibitor cyclin-dependent kinase inhibitor-73 on liver fibrosis and its related mechanisms / 中华肝胆外科杂志

Objective:To investigate the therapeutic effect and potential molecular mechanisms of cyclin-dependent kinase inhibitor-73 (CDKI-73), the Rab11 inhibitor, on liver fibrosis.Methods:Human LX2 cells were divided into four groups: negative control group, transforming growth factor-β (TGF-β) group, CDKI-73 group and TGF-β+ CDKI-73 group. Fifteen 5-week-old female C57 mice with body weight of (18.04±0.62) g were divided into 3 groups with 5 mice in each group: control group (intraperitoneal injection of olive oil + vehicle gavage), carbon tetrachloride (CCl 4) group (intraperitoneal injection of CCl 4 + vehicle gavage) and CCl 4+ CDKI-73 group (intraperitoneal injection of CCl 4+ CDKI-73 gavage). Another 15 5-week-old female C57 mice with body weight of (18.06±0.34) g were divided into 3 groups with 5 mice in each group: sham operation group (Sham), bile duct ligation (BDL) group + vehicle group (BDL+ vehicle gavage) and bile duct ligation+ CDKI-73 group (BDL+ CDKI-73 gavage). The expression of α-smooth muscle actin (α-SMA) and fibronectin(FN)in LX2 cells were analyzed by Western blot. Masson and Sirius red were used to examine the liver fibrosis after CDKI-73 treatment in vivo. Immunohistochemistry (IHC) was utilized to examine the expression of α-SMA in mice liver. Results:Collagen content assessed by Sirius red and Masson staining and α-SMA expression evaluated by IHC were all increased in CCl 4 group compared with control group ( q=38.47, 24.99, 36.79). Moreover, the collagen content and α-SMA expression in CCl 4 + CDKI-73 treatment group were obviously decreased compared with CCl 4 group ( q=24.72, 14.87, 27.50), and the differences were statistically significant (all P<0.001). Compared with Sham group, collagen content and α-SMA expression in bile duct ligation group were increased ( q=28.23, 41.01, 44.16). Furthermore, in BDL group, after treatment with CDKI-73, the collagen content and α-SMA expression were notably decreased ( q=22.88, 34.31 and 33.97, all P<0.001). Consistent with in vivo results, the relative expression levels of α-SMA and FN protein in TGF-β group were higher than those in TGF-β+ CDKI-73 group (α-SMA: 3.71±0.34 vs. 1.28±0.31; FN: 3.21±0.39 vs. 0.83±0.06, all P<0.001). The mRNA relative expression levels of α-SMA and FN in TGF-β group were higher than those in TGF-β+ CDKI-73 group, and the differences were statistically significant ( P<0.001). However, the relative expression of TGF-β receptor Ⅱ protein in CDKI-73 group was higher than those in negative control group (4.68±0.63 vs. 1.00±0.22, P=0.004). The relative expression level of phosphorylated SMAD2 in TGF-β+ CDKI-73 group was lower than those in TGF-β group (1.67±0.24 vs. 3.99±0.44, P<0.001). Transwell assay showed that 0.5 μmol/L CDKI-73 could effectively inhibit the migration of LX2 cells, and the inhibitory ability became stronger with the increase of CDKI-73 concentration. Conclusion:CDKI-73 can inhibit the activation of hepatic stellate cells and liver fibrosis by inhibiting Rab11-dependent TGF-β signaling pathway both in vivo and in vitro.

Advances of the regulatory mechanism of cyclin, cyclin- dependent kinases and related kinase inhibitors in cell cycle progression / 生物工程学报

Cell cycle plays a crucial role in cell development. Cell cycle progression is mainly regulated by cyclin dependent kinase (CDK), cyclin and endogenous CDK inhibitor (CKI). Among these, CDK is the main cell cycle regulator, binding to cyclin to form the cyclin-CDK complex, which phosphorylates hundreds of substrates and regulates interphase and mitotic progression. Abnormal activity of various cell cycle proteins can cause uncontrolled proliferation of cancer cells, which leads to cancer development. Therefore, understanding the changes in CDK activity, cyclin-CDK assembly and the role of CDK inhibitors will help to understand the underlying regulatory processes in cell cycle progression, as well as provide a basis for the treatment of cancer and disease and the development of CDK inhibitor-based therapeutic agents. This review focuses on the key events of CDK activation or inactivation, and summarizes the regulatory processes of cyclin-CDK at specific times and locations, as well as the progress of research on relevant CDK inhibitor therapeutics in cancer and disease. The review concludes with a brief description of the current challenges of the cell cycle process, with the aim to provide scientific references and new ideas for further research on cell cycle process.

Study on the mechanism of miRNA expression profile and enrichment analysis on p21 in oxygen-induced retinopathy mice / 中华眼底病杂志

Objective:To observe the expression of miRNA in retinal tissue of mice with oxygen-induced retinopathy (OIR), and screen miRNAs related to p21 and retinal neovascularization (RNV) formation.Methods:A experimental study. Forty healthy 7-day-old C57BL/6J mice were randomly divided into normal group and OIR group, with 20 mice in each group. The oxygen induced RNV model was constructed in the OIR group, and no treatment was performed in the normal group. At the age of 17 days, the mice were killed and the RNV of mice was observed by retinal fluorescence; the nuclei of vascular endothelium that broke through the inner limiting membrane of retina were counted under light microscope. The retinal tissues were taken for miRNA chip analysis to detect the differentially expressed miRNAs between the normal group and the OIR group. The resulting differential miRNA target genes were subjected to enrichment analysis based on gene annotation (GO) and Kyoto Encyclopedia of genes and genomes (KEGG); miRNAs and pathways that may be related to p21 were screened through Targetscan, MiRanda and MicroT-CDs database alignment. Independent sample t-test was used for pairwise comparison between groups. Results:Compared with the normal group, the area of nonperfusion area, RNV and the number of vascular endothelial nuclei that broke through the inner limiting membrane of the retina in the OIR group increased significantly, differences were statistically significant ( t=18.800, 9.025; P<0.05). Compared with the normal group, there were 54 miRNAs that were statistically differentially expressed in the OIR group, of which 47 were up-regulated and 7 were down-regulated. A total of 13 miRNAs related to p21 were screened from the alignment results of the three databases with the obtained differential miRNAs. According to the difference multiples, they were miR-7218-5p, miR-322-5p, miR-224-5p, miR-335-5p, miR-329-3p, miR-362-3p, miR-532-5p, miR-20b-5p, miR-20a-5p, miR-195a-5p, miR-423-5p, miR-497a-5p, and miR-129-5p. Differential miRNA target gene enrichment analysis yielded 1 112 go entries and 50 KEGG pathways, of which 50 go entries and 13 KEGG pathways were related to p21. Conclusion:13 miRNAs related to p21 were screened out in the OIR model.

Study on the mechanism of miRNA expression profile and enrichment analysis on p21 in oxygen-induced retinopathy mice / 中华眼底病杂志

Objective:To observe the expression of miRNA in retinal tissue of mice with oxygen-induced retinopathy (OIR), and screen miRNAs related to p21 and retinal neovascularization (RNV) formation.Methods:A experimental study. Forty healthy 7-day-old C57BL/6J mice were randomly divided into normal group and OIR group, with 20 mice in each group. The oxygen induced RNV model was constructed in the OIR group, and no treatment was performed in the normal group. At the age of 17 days, the mice were killed and the RNV of mice was observed by retinal fluorescence; the nuclei of vascular endothelium that broke through the inner limiting membrane of retina were counted under light microscope. The retinal tissues were taken for miRNA chip analysis to detect the differentially expressed miRNAs between the normal group and the OIR group. The resulting differential miRNA target genes were subjected to enrichment analysis based on gene annotation (GO) and Kyoto Encyclopedia of genes and genomes (KEGG); miRNAs and pathways that may be related to p21 were screened through Targetscan, MiRanda and MicroT-CDs database alignment. Independent sample t-test was used for pairwise comparison between groups. Results:Compared with the normal group, the area of nonperfusion area, RNV and the number of vascular endothelial nuclei that broke through the inner limiting membrane of the retina in the OIR group increased significantly, differences were statistically significant ( t=18.800, 9.025; P<0.05). Compared with the normal group, there were 54 miRNAs that were statistically differentially expressed in the OIR group, of which 47 were up-regulated and 7 were down-regulated. A total of 13 miRNAs related to p21 were screened from the alignment results of the three databases with the obtained differential miRNAs. According to the difference multiples, they were miR-7218-5p, miR-322-5p, miR-224-5p, miR-335-5p, miR-329-3p, miR-362-3p, miR-532-5p, miR-20b-5p, miR-20a-5p, miR-195a-5p, miR-423-5p, miR-497a-5p, and miR-129-5p. Differential miRNA target gene enrichment analysis yielded 1 112 go entries and 50 KEGG pathways, of which 50 go entries and 13 KEGG pathways were related to p21. Conclusion:13 miRNAs related to p21 were screened out in the OIR model.

Diagnostic value of combined detection of p16 and human papillomavirus typing in cervical intraepithelial neoplasia / 肿瘤研究与临床

Objective:To explore the application value of combined detection of p16 and human papillomavirus (HPV) typing in the diagnosis of cervical intraepithelial neoplasia (CIN).Methods:A total of 8 346 patients aged between 25 years old and 65 years old at Baoji Central Hospital of Shaanxi Province from February 2019 to February 2020 were selected. There were 2 882 patients with cervical lesions diagnosed by colposcopy biopsy. Patients were divided into the different groups based on the age range, and then the condition of HPV infection in all age groups was analyzed. Taking biopsy as the gold standard and according to the pathological results, the detection rate of p16 and HPV typing and the diagnostic value of the single and combined detection in CIN were also analyzed.Results:The age group with the highest positive rate of p16 and HPV was 31-40 years old [47.42% (1 014/2 427) and 36.84% (894/2 427), respectively], followed by 41-50 years old group [30.15% (907/2 942) and 28.11% (827/2 942)], and there were statistically significant differences in positive rate of p16 and HPV in all age groups (all P < 0.05). Among 2 882 patient with cervical lesions diagnosed by pathological examination, there were 2 572 cases (89.24%) of p16 positive, and 2 169 cases (75.26%) of HPV positive. With the disease progression of cervical lesions, the positive rate of p16 and HPV was gradually increased, and the positive rate of p16 of inflammation, CINⅠ, CINⅡ, CIN Ⅲ, cervical squamous cell carcinoma (SCC) was 11.68% (23/197), 94. 85% (1 105/1 165), 93.57% (771/824), 96.76% (538/556), 96.43% (135/140), respectively; the positive rate of HPV was 17.77% (35/197), 77.60% (904/1 165), 80.22% (661/824), 80.40% (447/556), 87.14% (122/140), respectively, and HPV infection was mostly HPV16/18 infection type with the disease progression of cervical lesions. The sensitivity, specificity, positive predictive value and negative predictive value in detecting CIN of HPV was 75.26%, 81.13%, 67.78% and 86.14%, respectively; the sensitivity, specificity, positive predictive value and negative predictive value in detecting CIN of p16 was 89.24%, 84.74%, 75.51% and 93.72%, respectively; the diagnostic efficacy of p16 was higher than that of HPV in detecting CIN, and the difference was statistically significant ( P < 0.05). The sensitivity, specificity, positive predictive value and negative predictive value of HPV combined with p16 in detecting CIN was 94.10%, 91.33%, 85.12%, 96.71%, which were higher compared with those of single detection (all P < 0.05). Conclusions:HPV infection mainly occurs in women aged 31-40 years old followed by 41-50 years old, and the infected population of CIN tends to be younger. p16 is superior to HPV in detecting the diagnostic efficacy of CIN; combined detection of p16 and HPV can increase the sensitivity and specificity, reduce the rate of misdiagnosis, and can play a key clinical value in early diagnosis and treatment of CIN.

Research progress of radiotherapy and CDK4/6 inhibitors in metastatic breast cancer / 国际肿瘤学杂志

Cyclin-dependent kinase (CDK) 4/6 inhibitors are a new class of molecular targeted drugs, which can enhance radiotherapy sensitivity by anti angiogenesis, inhibiting DNA damage repair and inhibiting mammalian target of rapamycin signal transduction. Existing clinical trials have confirmed that radiotherapy combined with CDK4/6 inhibitors can effectively control the local symptoms of breast cancer metastases and prolong progression-free survival. Compared with CDK4/6 inhibitors alone, the combination with radiotherapy does not significantly increase the incidence and severity of adverse reactions. However, there are also reports about severe adverse reactions of normal tissue happened in the radiation field in individual cases of combined treatment, and its efficacy and safety need to be clarified by more basic and clinical observational researches.

Selective inhibition of CDK4/6: A safe and effective strategy for developing anticancer drugs

The sustained cell proliferation resulting from dysregulation of the cell cycle and activation of cyclin-dependent kinases (CDKs) is a hallmark of cancer. The inhibition of CDKs is a highly promising and attractive strategy for the development of anticancer drugs. In particular, third-generation CDK inhibitors can selectively inhibit CDK4/6 and regulate the cell cycle by suppressing the G1 to S phase transition, exhibiting a perfect balance between anticancer efficacy and general toxicity. To date, three selective CDK4/6 inhibitors have received approval from the U.S. Food and Drug Administration (FDA), and 15 CDK4/6 inhibitors are in clinical trials for the treatment of cancers. In this perspective, we discuss the crucial roles of CDK4/6 in regulating the cell cycle and cancer cells, analyze the rationale for selectively inhibiting CDK4/6 for cancer treatment, review the latest advances in highly selective CDK4/6 inhibitors with different chemical scaffolds, explain the mechanisms associated with CDK4/6 inhibitor resistance and describe solutions to overcome this issue, and briefly introduce proteolysis targeting chimera (PROTAC), a new and revolutionary technique used to degrade CDK4/6.

Comparison of senescence progression in mesenchymal cells from human umbilical cord walls measured by immunofluorescence and flow cytometry of p16 and p21 / Comparação da progressão da senescência em células mesenquimais de parede de cordão umbilical humano medida por imunofluorescência e citometria de fluxo de p16 e p21

ABSTRACT Objective To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory. Methods Mesenchymal stem cells from umbilical cords were isolated from Wharton's jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence). Results A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence. Conclusion Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.

RESUMO Objetivo Acompanhar a expansão de células-tronco mesenquimais de cordão umbilical por dois marcadores clássicos de senescência, p16 (INK4A) e p21 (CDKN1A), usando métodos práticos, rápidos e com custo menor do que a técnica padrão-ouro de Western blotting, para avaliar sua aplicabilidade em laboratório. Métodos Células-tronco mesenquimais de cordão umbilical foram isoladas da geleia de Wharton e, após controle de qualidade e caracterização morfológica e imunofenotípica por citometria de fluxo, foram expandidas em cultura, até chegarem próximas à parada do ciclo celular (senescência replicativa). Resultados Foi feita a comparação entre células jovens, na passagem 5, e células pré-senescentes, na passagem 10, avaliando a expressão proteica dos marcadores clássicos de senescência celular p16 e p21, comparando os resultados obtidos por Western blotting com os obtidos por citometria de fluxo e imunofluorescência indireta. Conclusão O seguimento de culturas celulares, por meio da imunofluorescência indireta de p16, permite identificar as culturas de células-tronco mesenquimais de cordão umbilical em risco de atingirem a senescência replicativa.

MicroRNA-186 targets SKP2 to induce p27(Kip1)-mediated pituitary tumor cell cycle deregulation and modulate cell proliferation

Pituitary tumors are usually benign but can occasionally exhibit hormonal and proliferative behaviors. Dysregulation of the G1/S restriction point largely contributes to the over-proliferation of pituitary tumor cells. F-box protein S-phase kinase-interacting protein-2 (SKP2) reportedly targets and inhibits the expression of p27(Kip1), a well-known negative regulator of G1 cell cycle progression. In this study, SKP2 expression was found to be upregulated while p27(Kip1) expression was determined to be downregulated in rat and human pituitary tumor cells. Furthermore, SKP2 knockdown induced upregulation of p27(Kip1) and cell growth inhibition in rat and human pituitary tumor cells, while SKP2overexpression elicited opposite effects on p27(Kip1) expression and cell growth. The expression of microRNA-186 (miR-186) was reported to be reduced in pituitary tumors. Online tools predicted SKP2 to be a direct downstream target of miR-186, which was further confirmed by luciferase reporter gene assays. Moreover, miR-186 could modulate the cell proliferation and p27(Kip1)-mediated cell cycle alternation of rat and human pituitary tumor cells through SKP2. As further confirmation of these findings, miR-186 and p27(Kip1) expression were downregulated, while SKP2 expression was upregulated in human pituitary tumor tissue samples; thus, SKP2 expression negatively correlated with miR-186 and p27(Kip1) expression. In contrast, miR-186 expression positively associated with p27(Kip1) expression. Taken together, we discovered a novel mechanism by which miR-186/SKP2 axis modulates pituitary tumor cell proliferation through p27(Kip1)-mediated cell cycle alternation.

Generation of knockout mouse models of cyclin-dependent kinase inhibitors by engineered nuclease-mediated genome editing / 한국실험동물학회지

Cell cycle dysfunction can cause severe diseases, including neurodegenerative disease and cancer. Mutations in cyclin-dependent kinase inhibitors controlling the G1 phase of the cell cycle are prevalent in various cancers. Mice lacking the tumor suppressors p16(Ink4a) (Cdkn2a, cyclin-dependent kinase inhibitor 2a), p19(Arf) (an alternative reading frame product of Cdkn2a,), and p27(Kip1) (Cdkn1b, cyclin-dependent kinase inhibitor 1b) result in malignant progression of epithelial cancers, sarcomas, and melanomas, respectively. Here, we generated knockout mouse models for each of these three cyclin-dependent kinase inhibitors using engineered nucleases. The p16(Ink4a) and p19(Arf) knockout mice were generated via transcription activator-like effector nucleases (TALENs), and p27(Kip1) knockout mice via clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9). These gene editing technologies were targeted to the first exon of each gene, to induce frameshifts producing premature termination codons. Unlike preexisting embryonic stem cell-based knockout mice, our mouse models are free from selectable markers or other external gene insertions, permitting more precise study of cell cycle-related diseases without confounding influences of foreign DNA.

Clinical application of STR genotyping diagnosis for hydatidiform mole and nonmolar gestation / 中华病理学杂志

Objective@#To investigate the value of short tandem repeat (STR) genotyping in the diagnostic workup of molar and non-molar gestations with correlation of histological characteristics.@*Methods@#Six hundred and fifty-six cases were selected based on clinically suspected hydropic abortion and/or molar pregnancy from July 2015 to September 2017 at Beijing Obstetrics and Gynecology Hospital. DNA was extracted from dissected chorionic villi and paired maternal endometrial FFPE tissue samples by Simplex OUP™ FFPE DNA Tissue Kit. STR genotyping was performed by PowerPlex 16 HS system.@*Results@#DNA genotyping was informative in 649 of 656 cases, leading to identification of 215 hydatidiform mole gestations and 434 non-molar gestations. Most of non-molar gestations (375 cases, 86.4%) were diploid hydropic abortion. Various trisomy syndromes were found (53 cases, 12.2%), including trisomy 2, 3, 4, 7, 8, 13, 16 and 21. Only 2(0.5%) digynic triploid gestations were detected. Moreover, 4 cases (0.9%) of uniparental disomies (homologous or heterologous) were found. There were 196 cases with histologic diagnostic suspicious of hydatidiform moles were accurate sub-classified. Among them, 59 cases hydatidiform moles were under-diagnosed as diploid hydropic abortions, and 28 cases diploid hydropic abortions were over-diagnosed as hydatidiform moles.Compared with partial moles(PHM), there were no specific histomorphological features between the various types of non-molar gestations and partial moles for definitive diagnostic separation. There was no significant difference in the expression of p57kip2 among PHM, trisomy and diploid hydropic abortions group (P=0.247).@*Conclusions@#STR genotyping can distinguish non-molar gestations from early hydatidiform moles, and efficiently avoid misdiagnosis based only on histological evaluation. Therefore, using STR genotyping, not only can the overdiagnosis of non-molar pregnancy be avoided, but also individualized management can be offered to patients including monitoring of serum hCG.

The role of p21/CIP1/WAF1 (p21) in the negative regulation of the growth hormone/growth hormone receptor and epidermal growth factor/epidermal growth factor receptor pathways, in growth hormone transduction defect

PURPOSE: Growth hormone transduction defect (GHTD) is characterized by severe short stature, impaired STAT3 (signal transducer and activator of transcription-3) phosphorylation and overexpression of the cytokine inducible SH2 containing protein (CIS) and p21/CIP1/WAF1. To investigate the role of p21/CIP1/WAF1 in the negative regulation of the growth hormone (GH)/GH receptor and Epidermal Growth Factor (EGF)/EGF Receptor pathways in GHTD. METHODS: Fibroblast cultures were developed from gingival biopsies of 1 GHTD patient and 1 control. The protein expression and the cellular localization of p21/CIP1/WAF1 was studied by Western immunoblotting and immunofluorescence, respectively: at the basal state and after induction with 200-μg/L human GH (hGH) (GH200), either with or without siRNA CIS (siCIS); at the basal state and after inductions with 200-μg/L hGH (GH200), 1,000-μg/L hGH (GH1000) or 50-ng/mL EGF. RESULTS: After GH200/siCIS, the protein expression and nuclear localization of p21 were reduced in the patient. After successful induction of GH signaling (control, GH200; patient, GH1000), the protein expression and nuclear localization of p21 were reduced. After induction with EGF, p21 translocated to the cytoplasm in the control, whereas in the GHTD patient it remained located in the nucleus. CONCLUSIONS: In the GHTD fibroblasts, when CIS is reduced, either after siCIS or after a higher dose of hGH (GH1000), p21’s antiproliferative effect (nuclear localization) is also reduced and GH signaling is activated. There also appears to be a positive relationship between the 2 inhibitors of GH signaling, CIS and p21. Finally, in GHTD, p21 seems to participate in the regulation of both the GH and EGF/EGFR pathways, depending upon its cellular location.

Cervical squamous intraepithelial lesions grades 1 and 2: value of an alternative morphological classification and of p16 immunohistochemistry in the prediction of clinical outcome / Lesões intraepiteliais escamosas cervicais graus 1 e 2: valor de uma classificação morfológica alternativa e do estudo imuno-histoquímico com p16 na predição do desfecho clínico

ABSTRACT Introduction: Diagnostic reproducibility and determination of prognostic factors in cervical intraepithelial neoplasias grades 1 and 2 are still relevant problems in the daily practice of gynecological histopathology. Objective: To correlate the value of morphological reclassification and of p16 immunoexpression in cervical intraepithelial neoplasias grades 1 and 2 with clinical outcome. Materials and methods: Sixty-six patients were included (34 with cervical intraepithelial neoplasia grade 1, and 32 with grade 2); an immunohistochemical study with p16 and reclassification according to the Lower Anogenital Squamous Terminology (LAST) Consensus and by the alternative proposal of Herfs and Crum were done; unfavorable outcome was defined as a subsequent histologic diagnosis of cervical intraepithelial neoplasia grade 3 or invasive squamous cell carcinoma. Results: We observed superior performance of the alternative morphological classification (p = 0.002) to determine unfavorable outcome. We also detected superior performance of p16 in the same determination (p = 0.002). Conclusion: The use of an alternative morphological classification is promising; in the context of the use of immunohistochemical antibodies as biomarkers, p16 showed good sensitivity and negative predictive value in the determination of cases in which the outcome was unfavorable.

RESUMO Introdução: A reproducibilidade diagnóstica e a determinação de fatores prognósticos em neoplasias intraepiteliais cervicais graus 1 e 2 ainda são problemas relevantes na prática diária da histopatologia ginecológica. Objetivo: Correlacionar o valor da reclassificação morfológica e da imunoexpressão do marcador p16 em neoplasias intraepiteliais cervicais graus 1 e 2 com o desfecho clínico. Materiais e métodos: Incluídas 66 pacientes (34 com neoplasia intraepitelial cervical grau 1 e 32 com grau 2); realizou-se estudo imuno-histoquímico com p16 e reclassificação segundo o Consenso Lower Anogenital Squamous Terminology (LAST) e a proposta alternativa de Herfs e Crum; desfecho desfavorável foi definido como diagnóstico histológico subsequente de neoplasia intraepitelial cervical grau 3 ou carcinoma de células escamosas invasivo. Resultados: Observamos performance superior da classificação morfológica alternativa (p = 0,002) para determinação de desfecho desfavorável. Também detectamos performance superior do marcador p16 na mesma determinação (p = 0,002). Conclusão: A utilização de uma classificação morfológica alternativa é promissora. No âmbito da utilização dos anticorpos imuno-histoquímicos como biomarcadores, o p16 apresentou boa sensibilidade e valor preditivo negativo na determinação dos casos em que o desfecho foi desfavorável.

Significance of p16/Ki-67 double immunocytochemical staining in cervical cytology ASCUS, LSIL,and ASC-H / 中华妇产科杂志

Objective To investigate the application value of p16/cell proliferation associated nuclear antigen (Ki-67) double-staining and human papillomavirus mRNA in the cytological screening.Methods Two hundred and fifty-one cases who suffered from atypical squamous cell of undetermined significance (ASCUS),low-grade squamous intraepithelial lesion (LSIL),atypical squamous cell-cannot exclude high-grade squamous intraepithelial lesion (ASC-H) in ThinPrep cytologic test (TCT) were collected in Peking University First Hospital between October 2015 and March 2016.And p16/Ki-67 double-staining and hybrid capture Ⅱ (HC-Ⅱ) detection were performed on the cervical cells.The result was compared with the pathological result of colposcope guided biopsy.All statistical analysis was completed by Stata 12.0 statistical software analysis.The results of diagnostic tests were described by using the sensitivity,specificity,positive predictive value,negative predictive value,and the area under the receiver operating characteristic (ROC) curve.Results (1) One hundred and eight cases of liquid based cytology diagnosis of ASCUS patients,the positive rate of p16/Ki-67 was 13.9％ (15/108),102 cases of liquid based cytology diagnosis of LSIL patients,the positive rate of p16/Ki-67 was 21.6％ (22/102),41 cases of liquid based cytology diagnosis of ASC-H patients,the positive rate of p16/Ki-67 was 39.0％ (16/41),compared amongthree groups,the difference was statistically significant (x2=78.516,P＜0.05);cervical exfoliated cells p16/Ki-67 expression rate was 13.0％ (28/215) in cervical low-grade lesions [cervical intraepithelial neoplasia (CIN)Ⅰ],which was 69.4％ (25/36) in high level lesions (CIN Ⅱ-Ⅲ),the difference was statistically significant (x2=7.932,P＜0.05).(2) The specificity of p16/Ki-67 detection and diagnosis were higher than those of HC-Ⅱ in ASCUS,LSIL,and ASC-H (89.8％ vs 71.4％,83.3％ vs 15.6％,88.9％ vs 40.7％;all P＜0.05),meanwhile,the positive predictive value of p16/Ki-67 detection and diagnosis exceed those of HC-Ⅱ in ASCUS,LSIL,and ASC-H (33.3％ vs 26.3％,31.8％ vs 12.6％,81.3％ vs 38.5％;all P＜0.05).Moreover,the ROC curve of p16/Ki-67 were bigger than those of HC-Ⅱ in ASCUS,LSIL,and ASC-H (0.799 vs 0.696,0.708 vs 0.531,0.909 vs 0.561;all P＜0.05).Conclusion For patients with cytological diagnosis of ASCUS,LSIL,and ASC-H,p16/Ki-67 double staining method could be used as an effective method to assist in the diagnosis of high-grade cervical lesions,and the screening efficiency is superior to that of high-rist HPV.

Effects of 12-lipoxygenase and angiotensin Ⅱ on p21, p27 and p57 in rat diabetic glomeruli / 中华肾脏病杂志

Objective To investigate the effects of 12-lipoxygenase (12-LO) and angiotensin Ⅱ (Ang Ⅱ) on the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs) p21,p27 and p57 related to cell hypertrophy.Methods Mesangial cells were treated with high glucose for 24 hours and 48 hours respectively.12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and Ang Ⅱ were infused to rats by osmotic mini-pump for 1 week and 2 weeks respectively.Rats fed high fat diet were received low dose streptozotocin (STZ) to make type 2 diabetes (DN).The rats were divided into normal control group,DN group,DN+Ang Ⅱ type 1 receptor blocker (ARB) group or 12-LO inhibitor (CDC) group.DN+ARB rats were treated by losartan for 6 weeks,and DN+CDC rats were treated for 8 weeks.Urine albumin and protein expressions of p21,p27 and p57 were detected by ELISA and Western blotting respectively.Glomeruli injury and expressions of p21 and p27 were detected by PAS staining and immunohistochemistry respectively.Results High glucose increased p21 and p27 protein expression in mesangial cells significantly compared with the relative control (all P ＜ 0.05),but had no effect on p57.Ang Ⅱ increased p27 protein expression in gloneruli significantly (P ＜ 0.05),but had no effect on p21 and p57 protein expression.12(S)-HETE increased both p21 and p27 protein expression in glomeruli significantly (all P ＜ 0.05),but had no effect on p57 protein expression.Blood glucose,kidney/body weight,urinary protein,and glomerular p21 and p27 protein expressions were increased in DN group (all P ＜ 0.05) compared with those in control group,with little change of p57 protein expression (P ＜ 0.05).Moreover,glomerular hypertrophy and extra cellular matrix accumulation were observed in DN group.However,urine protein,kidney/body weight,renal injury,but not blood glucose,were decreased in DN+ARB group and DN+CDC group compared with DN group respectively (P＜ 0.05).Further DN+CDC rats had decreased both p21 and p27 protein expressions in glomeruli,but DN+ ARB rats only had decreased p27 protein expression (all P ＜ 0.05).Conclusions 12-LO may induce both p21 and p27 protein expression in DN glomeruli,but Ang Ⅱ may induce only p27 expression.

Application of p16/Ki-67 immunocytochemistry in triage of patients with atypical squamous cells of undetermined significance / 中华病理学杂志

Objective@#To investigate the clinical value of p16/Ki-67 immunocytochemistry in patients with atypical squamous cells of undetermined significance(ASC-US).@*Methods@#One hundred and seventy-one cases of thin-prep cytology test (TCT) diagnosed as ASC-US underwent p16/Ki-67 immunocytochemistry. All patients had colposcopy and biopsy from March 2015 to January 2016. Ninety of the 171 cases underwent high-risk HPV test at the same time.@*Results@#p16/Ki-67 immunocytochemistry was positive in 43.9% (75/171) of the 171 cytology samples; the sensitivity and specificity of p16/Ki-67 immunocytochemistry were 77.6%(52/67) and 77.9%(81/104) in detecting CIN2+ , and the positive and negative predictive value were 69.3%(52/75) and 84.4%(81/96), respectively. The sensitivity and specificity in diagnosing CIN2+ were 100.0%(34/34) and 10.7%(6/56) for HPV test, and the positive and negative predictive value were 40.5%(34/84) and 6/6. p16/Ki-67 immunocytochemistry showed lower sensitivity but obviously higher specificity than high-risk HPV detection.@*Conclusion@#p16/Ki-67 immunocytochemistry is a good triage test for identifying CIN2+ in ASC-US specimens.

Application of immunohistochemistry for p16 and GATA3 and molecular HPV typing in diagnosis of secondary bladder involvement by cervical carcinoma / 中华病理学杂志

Objective@#To investigate the expression of p16 and GATA3 and the detection of human papillomavirus (HPV) in secondary bladder involvement by cervical carcinomas.@*Methods@#Sixteen cases of cervical carcinoma with bladder involvement diagnosed from December 2008 to March 2016 were collected and evaluated by light microscopy, immunohistochemistry for p16 and GATA3 detection and PCR-reverse dot blot for molecular typing of HPV.@*Results@#The age of the patients ranged from 25 to 76 years with median of 52 years. Morphologically, 14 cases(14/16) showed tumor nests infiltrating lamina propria or muscle bundles of the bladder. By immunohistochemistry, 15 cases (15/16) were found to be diffusely and strongly positive for p16, and 1 showed patchy staining pattern. Seven cases (7/7) of corresponding original cervical cancers were also diffusely and strongly positive for p16. GATA3 staining was negative in 13 cases (13/16), and focal weak to moderate positivity was detected in 3 cases.Three cases (3/7) of corresponding original cervical cancers showed focal weak to moderate positivity of GATA3. Fifteen cases (15/16) showed concordant high risk HPV-positivity, including HPV16 in 8 cases and HPV31 in one case. Five cases showed co-infection of HPV16 and HPV18. One case showed co-infection with HPV18 and HPV45.@*Conclusion@#Differential diagnosis by p16 or GATA3 alone is of limited value. Combination of immunohistochemistry for p16 and GATA3 and molecular typing for HPV detection are useful to distinguish primary bladder carcinoma from the secondary involvement by cervical carcinoma.

Multiple cutaneous melanomas associated with gastric and brain metastases

Abstract The occurrence of multiple primary melanomas in a single individual is rare. Most commonly, malignant melanocytic lesions subsequent to the initial diagnosis of melanoma are secondary cutaneous metastases. We report a patient with gastrointestinal bleeding from gastric metastasis of cutaneous melanoma. During clinical evaluation and staging, we discovered a brain metastasis associated with 3 synchronous primary cutaneous melanomas. We suggest the research on the mutation in the cyclin-dependent kinase inhibitor 2A (CDKN2A) (INK4a) in such cases. We also emphasize the importance of clinical examination and dermoscopy of the entire tegument, even after a malignant melanocytic lesion is identified.

Placental expression of CDKN1C imprinted gene and birth weight of appropriate and small for gestational age neonates / 中华围产医学杂志

Objective To investigate the relationship between the expression of imprinted gene CDKN1C in placenta and the birth weight of neonates.Methods Twenty-nine term small for gestational age (SGA) neonates admitted to Peking University Third Hospital from January 1,2014 to December 31,2014 were recruited,and 29 appropriate for gestational age (AGA) neonates with a difference of not more than one week in gestational age served as controls.Fresh placental tissue was collected and the expression of imprinted gene CDKN1C mRNA in the placenta were detected by real-time fluorescence quantitative-polymerase chain reaction,and its protein expression was estimated by Western-blot.Chi-square test,independent-sample t test,Pearson's correlation analysis were used for statistical analysis.Results The CDKN1C mRNA expression level in SGA was significantly higher than that in AGA (0.133± 0.059 vs 0.100±0.046,t=2.401,P=0.020),so was the CDKN1C protein expression (0.280±0.043 vs 0.190±0.041,t=8.410,P=0.000).The CDKN1C mRNA expression levels were negatively correlated with birth weight in both groups (SGA group,r=-0.587,P=0.001;AGA group,r=-0.569,P=0.001),and the correlation was slightly stronger in SGA (r2=0.344) than in AGA (r2=0.324).The CDKN1C protein expression levels of the two groups were negatively correlated with birth weight (SGA group,r=-0.579,P=0.001;AGA group,r=-0.497,P=0.006),the correlation being stronger in SGA group (r2=0.335) than in AGA group (r2=0.247).The CDKN1C mRNA and protein expression levels of the two groups were negatively correlated with birth weight for gender,especially in males [mRNA:r2=0.293(male)vs r2=0.185(female);protein:r2=0.730 (male) vs r2=0.601(female)].Neither CDKN1C mRNA nor protein expression level was correlated to the placenta weight (mRNA:SGA group,r=0.119,P=0.540;AGA group,r=-0.069,P=0.722;protein:SGA group,r=0.126,P=0.515;AGA group,r=-0.247,P=0.196).Conclusions The expressions of CDKN1C mRNA and protein may be related to birth weight of term SGA neonates,especially in male infants.