High Cyclin D1 Expression Research Articles

P16 overexpression identifies oncogenic high-risk HPV infection in non-oropharyngeal squamous cell carcinoma of the head and neck.

Human papillomavirus (HPV) is an increasing risk factor for cancer. HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) is associated with a favorable outcome. Blockstaining for p16 is a surrogate marker for HPV+ OPSCC. In oral and laryngeal squamous cell carcinoma (OSCC/LSCC), the relevance of p16 immunohistochemistry, alone or in combination with other cell cycle-related proteins, to identify HPV-driven non-OPSCC is less well understood. We stained for p16, pRb, cyclin D1, and p53 in 327 HNSCC. In 310 OPSCC, HPV-status was assessed by HPV DNA PCR. In 119 non-OPSCC, RNA in situ hybridization was additionally performed. HPV-status was correlated with staining patterns, p53 and clinical data. The OPSCC showed blockstaining for p16 in 36%, 8% were equivocal. Of these, HPV-testing was performed in 57%, and 53% were positive for HPV DNA. HPV-association correlated with absence of pRb and cyclin D1 and favorable outcome. In non-OPSCC, 18% showed p16-blockstaining, and 13% showed E6/E7 RNA. Six of seven HPV+ OSCC and 8/8 LSCC lost pRb and cyclin D1. Compared to HPV-negative counterparts, patients with HPV+ cancers had lower rates of alcohol consumption and keratinizing morphology. HPV-positive OSCC had a longer overall survival (p < 0.05). HPV subtype 16 was the most common. We conclude that HPV-positive non-OPSCC are associated with p16 overexpression and low levels of pRb and cyclin D1. High expression of pRb and cyclin D1 indicates HPV-negativity.

OVEREXPRESSION OF CYCLIN D1 IN ORAL CANCER INCREASES TPF CHEMOTHERAPEUTIC DRUGS VIA ACTIVATING THE CASPASE-3 PATHWAY

Previous studies have indicated that induction chemotherapy can limit the possibility of early lesion eradication and lessen the severity of globally advanced tumors. This study aimed to ascertain if cyclin D1 expression in OSCC patients who were qualified for TPF induction chemotherapy might be regarded as a valid prognostic indicator for long-term outcomes. This study aimed to determine whether or not there was a connection between OSCC cell intervention and the production of cyclin D1 and chemosensitivity to TPF medicines through the apoptotic mechanism. The current work used immunohistochemistry to determine the expression of cyclin D1 in various tissues. To ascertain the survival rate of 232 people diagnosed with locally advanced oral squamous cell carcinoma (OSCC), the study utilized the log-rank test and Kaplan-Meier analysis. The participants in this historical clinical trial were observed for a mean duration of five years. Cytotoxicity assays were employed to assess the therapeutic effectiveness of TPF chemotherapy drugs. The activity of caspase-3 and PARP was evaluated in the HB96, CAL27, and HN30 cell lines following cyclin D1 inhibition, respectively. Long-term clinical outcomes were significantly better for patients with OSCC whose cancer cells expressed low amounts of cyclin D1 than those whose cells expressed high protein levels. Time to initial illness occurrence, time to initial local recurrence, time to mortality, and overall survival were among the metrics evaluated. The groups with low and high expression levels of cyclin D1 showed significantly different survival rates for disease-free survival, overall survival (OS), DMFS, local recurrence-free survival, and local recurrence-free survival (p0.001, 0.003, 0.004, and 0.001, respectively). Patients with cN2 oral squamous cell carcinoma (OSCC) with higher cyclin D1 expression had better distant metastasis-free survival (OS, P=0.024; DMFS, P=0.024) and overall survival (OS, P=0.024; DMFS, P=0.024) with TPF induction chemotherapy. However, patients with cN2 OSCC and low cyclin D1 expression did not show this survival advantage (P=0.024). Research has shown that oral squamous cell carcinoma (OSCC) is more sensitive to TPF-based chemotherapeutic drugs using caspase-3-mediated apoptosis that occurs when cyclin D1 expression rises. Based on these results, TPF induction chemotherapy may provide longer-term benefits than traditional treatment approaches for patients with cN2 OSCC and high cyclin D1 expression. Through a caspase-3-dependent mechanism, oral squamous cell carcinoma (OSCC) cell lines become more susceptible to TPF chemotherapeutic drugs when cyclin D1 is overexpressed.

Upregulated TC1 and downregulated Chibby were correlated with the aberrant β-catenin expression in laryngeal squamous cell carcinoma.

As an important member of Wnt/β-catenin signaling pathway, the aberrant expression of β-catenin has been implicated in many cancers. Chibby, a β-catenin binding partner, is an antagonist involved in this pathway. In contrast, thyroid cancer 1 (TC1) as an activator of this pathway can relieve the antagonistic activity of Chibby on the β-catenin-mediated transcription and is high expressed in human tumors. The objectives of this study were to examine the expression of TC1, Chibby, and β-catenin and investigate the association among them in laryngeal squamous cell carcinoma (LSCC). The expression of TC1, Chibby, β-catenin, c-Myc, Cyclin D1, and matrix metalloproteinase-7 (MMP-7) were examined by immunohistochemistry in samples from 53 LSCC patients. Compared with normal laryngeal squamous epithelium (NLSE), there were upregulated expression of TC1, downregulated expression of Chibby, and aberrant cytoplasmic expression of β-catenin in the LSCC tissues (P < .001). The high expression of TC1 was correlated with the tumor site, advanced TNM and T stage, lymphovascular invasion, and poor differentiation in LSCC tissues (P < .050). There were correlations between the aberrant expression of β-catenin and the tumor site, advanced TNM and T stage, lymphovascular invasion, perineurial invasion, and poor differentiation in LSCC tissues (P < .050). Upregulated TC1 and downregulated Chibby were correlated with aberrant expression of β-catenin (P < .001), but no correlation between them (P = .076). The percent of abnormal expression of β-catenin in LSCC was 96.00% in TC1+/Chibby-, 73.68% in TC1+/Chibby+, 0.00% in TC1-/Chibby-, and 0.00% in TC1-/Chibby + group (P < .001). High expression of c-Myc, Cyclin D1, and MMP-7 was observed in LSCC tissues (P < .001). There was statistically significant about the expression of Cyclin D1 and MMP-7 among the groups of TC1+/Chibby-, TC1+/Chibby+, TC1-/Chibby-, and TC1-/Chibby + (P < .001), but was not significance about the expression of c-Myc among them (P = .339). No association was found between overall survival and the expression of TC1, Chibby, and β-catenin (P > .05). The upregulated expression of TC1 and downregulated expression of Chibby were correlated with the aberrant expression of β-catenin and the high expression of Cyclin D1 and MMP-7 in LSCC tissues.

Dissecting the role of the NADPH oxidase NOX4 in TGF-beta signaling in hepatocellular carcinoma

The NADPH oxidase NOX4 has been proposed as necessary for the apoptosis induced by the Transforming Growth Factor-beta (TGF-β) in hepatocytes and hepatocellular carcinoma (HCC) cells. However, whether NOX4 is required for TGF-β-induced canonical (SMADs) or non-canonical signals is not fully understood yet, neither its potential involvement in other parallel actions induced by TGF-β. In this work we have used CRISPR Cas9 technology to stable attenuate NOX4 expression in HCC cells. Results have indicated that NOX4 is required for an efficient SMAD2/3 phosphorylation in response to TGF-β, whereas non-canonical signals, such as the phosphorylation of the Epidermal Growth Receptor or AKT, are higher in NOX4 silenced cells. TGF-β-mediated inhibition of cell proliferation and viability is attenuated in NOX4 silenced cells, correlating with decreased response in terms of apoptosis, and maintenance of high expression of MYC and CYCLIN D1. These results would indicate that NOX4 is required for all the tumor suppressor actions of TGF-β in HCC. However, analysis in human HCC tumors has revealed a worse prognosis for patients showing high expression of TGF-β1-related genes concomitant with high expression of NOX4. Deepening into other tumorigenic actions of TGF-β that may contribute to tumor progression, we found that NOX4 is also required for TGF-β-induced migratory effects. The Epithelial-Mesenchymal transition (EMT) program does not appear to be affected by attenuation of NOX4 levels. However, TGF-β-mediated regulation of cytoskeleton dynamics and focal adhesions require NOX4, which is necessary for TGF-β-induced increase in the chaperone Hsp27 and correct subcellular localization of Hic-5 within focal adhesions, as well for upregulation of the metalloprotease MMP9. All these results together point to NOX4 as a key element in the whole TGF-β signaling in HCC cells, revealing an unknown role for NOX4 as tumor promoter in HCC patients presenting activation of the TGF-β pathway.

Abstract PD17-06: Immunohistochemical markers and determinants of clinical response in the Penelope-B trial

Abstract Background: The Penelope-B trial did not show improvement in invasive disease-free survival (iDFS) with the addition of palbociclib to endocrine therapy (ET) in patients with high-risk early breast cancer (BC) after neoadjuvant chemotherapy (NACT). Biomarkers may be able to identify subgroups of patients deriving benefit from Palbociclib and guide future studies. Estrogen-receptor (ER), progesterone-receptor (PgR) and Ki-67 might be helpful in identifying patients benefiting from palbociclib. Concordantly, tumors with elevated expression of Cyclin D1 and phosphorylated retinoblastoma protein (phospho-RB) may harbor more dependency on CDK4/6 and thus higher sensitivity to palbociclib. Methods: The percentage of positive ER and PgR cells and Ki-67 assessed in surgical specimens after NACT were combined to obtain the immunohistochemical score 3 (IHC3, Cuzick et al JCO 2011, low vs high based on the median IHC3 value). Cyclin D1 and phospho-RB Ser 807/811 immunoreactive (phospho-RB) scores were analyzed in residual tumors after NACT (range 0-12 each). Proportional hazard regression model was used to assess the predictive and prognostic value of IHC3 and treatment on iDFS. Subgroup analysis was performed according to BC intrinsic subtypes (luminal-A/normal-like, luminal-B/HER2-enriched/basal) and HER2-status (HER2 0, HER2 low). Cox/Fine-Gray regression was used to define the predictive and prognostic value of CyclinD1 (≤1, &amp;gt;1), phospho-RB (≤2, &amp;gt;2) as dichotomized and continuous variables on iDFS, distant DFS (DDFS), locoregional invasive recurrence-free interval (LRRFI) and overall survival (OS). Multivariate analyses (MVA) were adjusted for age (≤50 vs &amp;gt;50), Ki-67 (≤15 vs &amp;gt;15), region (non-Asian vs Asian), ypN (ypN0-1 vs ypN2-3), risk status (CPS-EG=2 ypN+ vs ≥3), cT (cT1-2 vs cT3-4), ypT (ypT0-2 vs ypT3-4), and grade (G1-2 vs G3). The MVA for IHC3 includes all the covariates above except Ki-67. p&amp;lt; 0.05 was defined as statistically significant. Results: Data for ER, PgR, Ki-67, HER2, Cyclin D1 and phospho-RB were available for 1250 patients. Overall, 98.9% of the patients had ER+ tumors, 75.0% PgR+, 52.2% had HER2 low, 25.5% Ki-67&amp;gt;15, 50% had IHC3 score higher than median, 93.9% had Cyclin D1 &amp;gt;1, 57.8% had phospho-RB &amp;gt;2. Patients with IHC3 score high had a worse iDFS compared to patients with IHC3 score low (MVA HR 2.28 95%CI (1.78-2.91), p&amp;lt; 0.0001). Patients with luminal-A/normal-like tumors and IHC3 low had an improved iDFS with the addition of palbociclib to ET (MVA HR 0.35 95%CI (0.14-0.90), test for interaction p=0.01). No difference was observed according to HER2 status. Cyclin D1&amp;gt;1 has no predictive value but is prognostic for better iDFS (MVA HR 0.62 95%CI (0.41-0.94), p=0.023), LRRFI (MVA HR 0.30 95%CI (0.15-0.63), p=0.001) and OS (MVA HR 0.50 95%CI (0.28-0.89), p=0.019). Similar results were obtained when Cyclin D1 was analysed as a continuous variable. Phospho-RB had neither predictive nor prognostic value. Phospho-RB highly correlates with Ki-67 (p&amp;lt; 0.001, Spearman correlation 0.248). Conclusions: Patients with high Cyclin D1 expression had a favorable prognosis independent of treatment arm, but patients with luminal-A/normal-like tumors and IHC3 low after NACT had an improved outcome when receiving palbociclib in addition to adjuvant ET. Theses exploratory studies suggest specific signatures/phenotypes could predict benefit from Palbociclib in high-risk early breast cancer. Citation Format: Erik S. Knudsen, Sivaramakrishna Rachakonda, Frederik Marmé, Miguel Martín, Michael Untch, Hervé R. Bonnefoi, Wolfgang D. Schmitt, Sung-Bae Kim, Harry D. Bear, Agnieszka Witkiewicz, Seock-Ah Im, Angela DeMichele, Laura Van’t Veer, Nicole McCarthy, Bruno V. Sinn, Karen Gelmon, José Ángel García-Sáenz, Catherine M. Kelly, Toralf Reimer, Nicholas Turner, Federico Rojo, Martin Filipits, Peter A. Fasching, Christian Schem, Lesley-Ann Martin, Yuan Liu, Masakazu Toi, Hope Rugo, Michael Gnant, Andreas Makris, Jenny Furlanetto, Karsten Weber, Carsten Denkert, Sibylle Loibl. Immunohistochemical markers and determinants of clinical response in the Penelope-B trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD17-06.

Papillomatous breast lesions with atypical columnar cell features

AimsColumnar cell lesions (CCLs) are recognised breast cancer precursor lesions. Intraductal papillomas are usually lined by benign (polyclonal) cells. Although papillomas with monoclonal lesions (atypical ductal hyperplasia (ADH)/ductal carcinoma in...

Expression of Cell-Cycle Regulatory Proteins pRb, Cyclin D1, and p53 Is Not Associated with Recurrence Rates of Equine Sarcoids.

Simple SummaryExpression of cell-cycle regulatory proteins pRB, Cyclin D1, and p53 is not associated with recurrence rates of equine sarcoids. Sarcoids are common tumors of equids, and since the pathogenesis and Bovine Papillomavirus (BPV) mechanism of tumorigenesis is not fully understood yet, we investigated the expression of cell cycle regulatory proteins pRB, Cyclin D1, and p53 to understand whether BPV and Human Papillomavirus (HPV) share similar mechanisms of action. The expression pattern of these proteins in a series of BPV-positive equine sarcoids is different from HPV-induced tumors. Moreover, their expression is not correlated with the recurrence or occurrence of sarcoids in new body sites.Sarcoids are among the most common tumors diagnosed in equids; their association with bovine papillomaviruses (BPV) infection has been widely reported, but the mechanism of carcinogenesis has not been fully elucidated. To verify whether BPV infection causes dysregulation of the pRb-Cyclin D1-p16CDKN2A-p53 pathway as reported for human papillomavirus (HPV), the study employed immunohistochemistry to test 55 equine sarcoid biopsies for the expression of pRb, Cyclin D1, and p53 cell cycle regulatory proteins and to evaluate the proliferative rate through Ki67. High Cyclin D1 and pRb expression were observed in 51% and 80% of cases, respectively, while low expression was observed in 49% and 20% of cases, respectively. Significantly higher Ki67 proliferation indexes were observed in fibroblastic, nodular, and mixed sarcoids compared to the occult and verrucous. High proliferation was significantly associated with high Cyclin D1 expression. In contrast with previous studies, p53 positivity was not observed in the cases examined in this study. Moreover, follow-up analysis revealed that fibroblastic, mixed sarcoids were associated with significantly higher local recurrence rates while the verrucous subtype was associated with higher rates of new sarcoid development at distant sites.

Expression of Cyclin D1 in Colorectal Cancer and Its Association With Clinicopathologic Parameters in Patients Underwent Colectomy

Colorectal cancer (CRC) is common cancer with a high mortality rate worldwide. Cyclin D1 is a gene that regulates cell cycle passage from stage G1 to S (G1/S checkpoint) and has recently been linked to the prognosis of a variety of cancers. Therefore, the aim of this study was to investigate the expression of cyclin D1 in colorectal cancers and its relationship with clinicopathologic factors. In this retrospective study, paraffin blocks of tumors of consecutive CRC patients registered in the histopathology laboratory of hospitals affiliated with Ahvaz Jundishapur University of Medical Sciences were used. Patients' clinicopathologic findings were extracted from their files, and using paraffin blocks, specific staining for cyclin D1 was performed using the immunohistochemistry method. Data were analyzed by SPSS software. In terms of staining, 11 samples (28.9%) scored 4, 11 samples (28.9%) scored 3, 8 samples (1/21%) scored 2, 3 samples (7.9%) scored 1, and 5 samples (2/13٪) scored zero. Staining intensity was severe in 10 cases (26.3%), moderate in 14 cases (36.8%), mild in 8 cases (21.1%), and negative in 6 cases (15.8%). The severity and extent of staining had no significant relationship with sex, age, tumor location, degree of differentiation (grade), depth of invasion, tumor size, lymph node involvement, and vascular and perineural invasion (P&gt;0.05). Despite the high expression of cyclin D1 in colorectal carcinoma, no significant relationship was observed between its expression and prognostic factors, which is probably due to the small sample size.

Immunohistochemical Expression of Cyclin D1 in Oral Lichen Planus

Background: Many studies have reported that a condition initially classified as oral lichen planus (OLP) has a variable chance of developing cancer progression over time, although these results are still questionable. Cyclin D1 controls the mitotic cell cycle's transition from G1 to S. Cyclin D1 has a significant function in carcinogenesis due to its critical involvement in cell cycle control. Cyclin D1 deregulation or overexpression may result in shortened G1 phase, enhanced cell replication, and decreased reliance on growth regulators, causing disruption in conventional cell cycle progression and the progression of cancer. The aim of the current study was to evaluate the expression of Cyclin D1 in histopathologically confirmed OLP samples and to predict the rate of malignant transformation in these lesions by comparing the level of expression with some clinical parameters. Materials and Methods: The immunohistochemical procedure was used to examine the expression of cyclin D1 on paraffin-embedded sections of (40) OLP lesions and (10) specimens of normal oral mucosa. We used the SPSS software version (18.0) to do the statistical analysis in this work. Results: There is a very significant difference between the people investigated (P = 0.01), which is statistically significant. CyclinD1 was found in all of the oral lichen planus cases in the research (100%), but it was also found (80%) in the normal oral mucosa. There was also no statistically significant difference between the clinical types of OLP and the Cyclin D1 scoring (P = 0.942), which revealed that the reticular type was the most frequent (7) in mild scoring, whereas the erosive type was the most frequent in severe scoring, which accounted for the majority of the cases. Conclusion: This research found that higher cyclinD1 expression increases the risk of cancer and many of the markers should be tested. Furthermore, larger sample numbers, improved designs, and gene-reduction studies should be performed. OLP patients should be continuously monitored for OSCC progression.

Enhanced Myogenesis by Silencing Myostatin with Nonviral Delivery of a dCas9 Ribonucleoprotein Complex.

Stress urinary incontinence (SUI) and pelvic floor disorder (PFD) are common conditions with limited treatment options in women worldwide. Regenerative therapy to restore urethral striated and pelvic floor muscles represents a valuable therapeutic approach. We aim to determine the CRISPR interference-mediated gene silencing effect of the nonviral delivery of nuclease-deactivated dCas9 ribonucleoprotein (RNP) complex on muscle regeneration at the cellular and molecular level. We designed four myostatin (MSTN)-targeting sgRNAs and transfected them into rat myoblast L6 cells together with the dCas9 protein. Myogenesis assay and immunofluorescence staining were performed to evaluate muscle differentiation, while CCK8 assay, cell cycle assay, and 5-ethynyl-2'-deoxyuridine staining were used to measure muscle proliferation. Reverse transcription-polymerase chain reaction and Western blotting were also performed to examine cellular signaling. Myogenic factors (including myosin heavy chain, MSTN, myocardin, and serum response factor) increased significantly after day 5 during myogenesis. MSTN was efficiently silenced after transfecting the dCas9 RNP complex, which significantly promoted more myotube formation and a higher fusion index for L6 cells. In cellular signaling, MSTN repression enhanced the expression of MyoG and MyoD, phosphorylation of Smad2, and the activity of Wnt1/GSK-3β/β-catenin pathway. Moreover, MSTN repression accelerated L6 cell growth with a higher cell proliferation index as well as a higher expression of cyclin D1 and cyclin E. Nonviral delivery of the dCas9 RNP complex significantly promoted myoblast differentiation and proliferation, providing a promising approach to improve muscle regeneration for SUI and PFD. Further characterization and validation of this approach in vivo are needed.

G870A Polymorphic Variants of CCND1 Gene and Cyclin D1 Protein Expression as Prognostic Markers in Laryngeal Lesions.

CCND1 gene encodes Cyclin D1 protein, the alternations and overexpression of which are commonly observed in human cancers. Cyclin D1 controls G1-S transition in the cell cycle. The aim of the study was to assess utility of the genotyping and protein expression in predicting the susceptibility of transformation from normal tissue to precancerous laryngeal lesions (PLLs) and finally to laryngeal cancer (LC). Four hundred and thirty-five patients (101 with LC, 100 with PLLs and 234 healthy volunteers) were enrolled in the study. Cyclin D1 expression was examined by immunohistochemistry and G870A polymorphism of gene CCND1 by PCR-RFLP technique. We confirmed association between the A allele and risk of developing LC from healthy mucosa (p = 0.006). Significantly higher expression of Cyclin D1 was observed in LC compering with PLLs (p < 0.0001) and we found that it could be a predictive marker of shorter survival time. To sum up, in the study population CCND1 gene polymorphism A870G and Cyclin D1 expression have a significant impact on the risk of developing PLLs and LC, and, therefore, Cyclin D1 could be a useful marker for the prediction of survival time in LC, whereas CCND1 gene polymorphism does not have a direct impact on patients’ outcome.

The Relationship between MACC1/c-Met/Cyclin D1 Axis Expression and Prognosis in ESCC.

Background Esophageal cancer is one of the most common malignant tumors of the digestive system, with high incidence and mortality. Methods Immunohistochemical method was used to detect the expression of MACC1, c-Met, and cyclin D1 in ESCC and its adjacent tissues. Statistical analysis was done by SPSS 23.0. Results The high expression of MACC1 and cyclin D1 was significantly correlated with tumor size. High c-Met expression was associated with patient ethnicity. MACC1 expression was positively correlated with both c-Met and cyclin D1. c-Met expression was also positively correlated with cyclin D1. Patients with high expression of MACC1 and c-Met had worse OS; patients with high c-Met expression also had worse PFS. Conclusion MACC1, c-Met, and cyclin D1 proteins are closely related to the occurrence and development of esophageal squamous cell carcinoma. MACC1 may affect the prognosis of ESCC by regulating the expression of the c-Met/cyclin D1 axis.

Thyroid Hormone Receptor Agonist Promotes Hair Growth in Mice.

Thyroxine is important to maintain the normal operation of the body. Both clinical and experimental results show thyroxine is closely related to hair growth, the mechanism of which is not fully understood. Investigate the effect of thyroxine receptor agonist, TDM10842, for dorsal hair growth in C3H mice and explore its underlying mechanism. Depilated mice were applied with the TDM10842, vehicle of this drug and without any materials on dorsal skin. RNA-sequencing (RNA-seq) was employed to identify the change in gene expression of skin tissues. Quantitative real-time PCR (rt-PCR) and immunoblotting were conducted to validate key differentially expressed genes (DEGs) between different groups. The TDM group showed early induction of anagen. 857, 782, and 276 differentially expressed genes were identified between 3 groups. As a critical DEG in group TDM, Pclaf was positively related to the motivation of Wnt/beta-catenin and Hedgehog signaling pathways, with a high expression of Ki67 and cyclinD1. TDM10842 accelerates the anagen entrance and the potential mechanism might be the activation of Wnt/beta-catenin and Hedgehog pathways. Pclaf serves as a critical molecule involved in pathway activation, and cyclinD1 is an important effector protein downstream of the pathways.

Local drug delivery system for the treatment of tongue squamous cell carcinoma in rats.

The present study describes a local drug delivery system with two functions, which can suppress tumor growth and accelerate wound healing. Thе system consists of a two-layer multicomponent fibrin-based gel (MCPFTG). The internal layer of MCPFTG, which is in direct contact with the wound surface, contains cisplatin placed on a CultiSpher-S collagen microcarrier. The external layer of MCPFTG consists of a CultiSpher-S microcarrier with lyophilized bone marrow stem cells (BMSCs). The efficacy of MCPFTG was evaluated in a rat model of squamous cell carcinoma of the tongue created with 4-nitroquinoline 1-oxide. The results of the study showed that, within 20–25 days, a non-healing wound of the tongue was formed in animals that underwent only 85% resection of squamous cell carcinoma, while rapid progression of the residual tumor was concomitantly observed. Immunohistochemical methods revealed high expression of cyclin D1 and low expression of E-cadherin in these animals. Additionally, high expression of p63 and Ki-67 was noted. In 80% of animals with squamous cell carcinoma of the tongue that were treated with MCPFTG after 85% tumor resection, a noticeable suppression of tumor growth was evident throughout 150 days, and tumor recurrence was not detected. Immunohistochemistry revealed low or moderate expression of cyclin D1, and high expression of E-cadherin throughout the whole observation period. The MCPFTG-based local drug delivery system was shown to be effective in suppressing tumor growth and preventing recurrence. MCPFTG decreased the toxicity of cisplatin and enhanced its antitumor activity. In addition, lyophilized paracrine BMSC factors present in MCPFTG accelerated wound healing after tumor removal. Thus, the present study suggests novel opportunities for the development of a multifunctional drug delivery system for the treatment of squamous cell carcinoma.

Prognostic significance of E-Cadherin, β-Catenin and cyclin D1 in oral squamous cell carcinoma: a tissue microarray study.

To study the prognostic significance of E-cadherin, β-catenin, and cyclin D1 expression in oral squamous cell carcinoma. The study included 65 subjects with histologically confirmed squamous cell carcinoma. TMA blocks were prepared for immunohistochemical quantification of the expression of the three markers using IHC profiler and Immune ratio plugin of Image J. E-cadherin expression was significantly correlated with histological grades and the metastasis status (p<0.05), whereas β-catenin expression was significantly correlated with smoking and tumor recurrence (P<0.05). Cyclin D1 expression was significantly correlated with depth of invasion and tumor recurrence. (p<0.05). Advanced tumor stage and depth of tumor invasion increases the risk of recurrence or death by 2.5 times (OR= 2.53 and 0.84 respectively). High expression of β-Catenin and Cyclin D1 are significantly correlated with tumor recurrence and old age. Depth of invasion, low histological grade and old age were a significant predictor for the risk of having tumor recurrence and cancer related death.

Epithelial\u2013mesenchymal transition (EMT) in vulvar cancer with and without inguinal lymph node involvement

PurposeEpithelial-mesenchymal transition (EMT) is associated with increased metastatic spread and poor prognosis. Data on vulvar carcinoma are limited.MethodsThirty-two cases of squamous cell carcinoma of the vulva (16 with and 16 without inguinal lymph node metastases) and their lymph node deposits were evaluated for immunohistochemical expression of EMT markers (vimentin, cyclin D1, e-cadherin), p16, p53 and Ki-67. Results of EMT-immunostainings were compared to lymph node involvement and expression of p53 and p16. The micro-anatomical staining pattern for EMT markers comparing the tumor center with the front of invasion was analysed in each tumor.ResultsThere was no difference in the expression of EMT markers between node negative and node positive tumors. Staining for vimentin and cyclin D1 was seen within tumor cells at the front of invasion in 100 and 84.4% of the tumors, respectively. The majority of cases (68.7%) showed negative or reduced staining for e-cadherin in this micro-anatomical localization. Tumor cells within the lymph node metastases showed positive staining for e-cadherin in 75% and for cyclin D1 in 49% of the cells but were negative for vimentin in 13 out of 16 cases (81.3%). Tumors with aberrant p53 staining represented a non-significant higher vimentin but significantly higher cyclin D1 expression at the front of invasion than those with p53 wild-type pattern.ConclusionThe present study shows no differences in the expression of EMT markers between node positive and node negative vulvar cancers. The evaluation of immunostaining within the micro-anatomical context indicates that an EMT-phenotype is restricted to the tumor cells at the front of invasion. Paired analyses of vulvar carcinomas and their lymph node deposits suggest mesenchymal-epithelial transition (MET) in the metastatic deposits. Immunohistochemical staining results may suggest that EMT is more prevalent in vulvar cancer with aberrant p53 staining.

Expression of Ki-67, Cyclin D1, P53, and P16 in patients with oral leukoplakia and leukoplakia cancerization with spicy diet in Chengdu.

To investigate the expression of Ki-67, Cyclin D1, P53, and P16 in patients with oral leukoplakia (OLK) and OLK cancerization who have aspicy diet in Chengdu. Thirtypatients with OLK andspicy diet and 15 patients with OLK without spicy diet in Chengdu were divided into three groups: hyperplastic OLK (OLK-), OLK with mild to moderate dysplasia (OLK+), and severe dysplastic OLK or oral squamous cell carcinoma (OSCC) transforming from OLK (OLK++/OSCC). The expression of Ki-67, Cyclin D1, P53, and P16 were detected by immunohistochemistry and statistically analyzed. The expression of Ki-67 and P53 in patients with or without spicy diet in the OLK+and OLK++/OSCC groups were stronger than that of the OLK- group (P<0.05). The OLK++/OSCC group showed a higher expression of Cyclin D1 and lower expression of P16 than the OLK- group (P<0.05). The expression of Ki-67, Cyclin D1, P53, and P16 in patients with spicy diet and without spicy diet had no substantial difference. The expression of Ki-67 and Cyclin D1 showed a positive correlation (r=0.439, P=0.015). Spicy diet did not have an influence on the expression of Ki-67, Cyclin D1, P53, and P16 in patients with OLK and OSCC. The expression of Ki-67, Cyclin D1, and P53 increased with the development of OLK, whereas P16 showed opposite expression trend.

CCND1 copy number increase and cyclin D1 expression in acral melanoma: a comparative study of fluorescence in situ hybridization and immunohistochemistry in a Chinese cohort

BackgroundCCND1 copy number increase is characteristic of acral melanoma and is useful in distinguishing benign and malignant acral melanocytic lesions. Increase of the gene copy number may result in protein overexpression. This raises the possibility that detection of high expression of cyclin D1 by immunohistochemistry (IHC) may be used as a surrogate for direct evaluation of increase in the CCND1 gene copy number.MethodsWe examined increases in CCND1 copy number with fluorescence in situ hybridization (FISH), and examined cyclin D1 protein expression with IHC in 61 acral melanomas.ResultsUsing FISH, 29 acral melanomas (29/61, 47.5%) showed increase in the CCND1 copy number, including 8 (8/61, 13.1%) which showed low-level increase in the CCND1 copy number and 21 (21/61, 34.4%) with high-level increase in the CCND1 copy number. By analysis of IHC, the median IHC score was 15% (range: 1–80%) in acral melanomas with no CCND1 copy number alteration. In acral melanomas with low-level CCND1 copy number increase, the median IHC score was 25% (range: 3–90%). In acral melanomas with high-level CCND1 copy number increase, the median IHC score was 60% (range: 1–95%). Comparing FISH and IHC, cyclin D1 protein expression level has no corelation with the CCND1 copy number in acral melanomas which have no CCND1 copy number alteration and low-level CCND1 copy number increase (P = 0.108). Cyclin D1 protein expression level correlated positively with CCND1 copy number in acral melanomas with high-level CCND1 copy number increase (P = 0.038). The sensitivity, specificity and positive predictive value of using cyclin D1 IHC to predict CCND1 FISH result was 72.4, 62.5 and 63.6%. Increase in CCND1 copy number was associated with Breslow thickness in invasive acral melanoma.ConclusionHigh-level increase in the CCND1 copy number can induce high cyclin D1 protein expression in acral melanomas. However low-level increase and normal CCND1 copy number have no obvious correlation with protein expression. Cyclin D1 IHC cannot serve as a surrogate for CCND1 FISH in acral melanomas.

Abstract 1963: High expression of Cyclin D1 is an independent marker for favorable prognosis in Middle Eastern breast cancer

Abstract Cyclin D1 protein regulates cell cycle progression which is mediated by its interactions with cyclin-dependent kinases. Over-expression of Cyclin D1 has been observed in several human cancers. This study was conducted to evaluate Cyclin D1 expression in a large cohort of Middle Eastern breast cancers and determine its prognostic significance. Cyclin D1 expression was assessed immunohistochemically and its association with clinico-pathological parameters was analyzed. Cyclin D1 was over-expressed in 59.4% (596/1003) of cases and significantly associated with a subset of breast cancers having favorable prognostic features such as low grade (p &amp;lt; 0.0001), low stage (p = 0.0276), estrogen receptor positive (p &amp;lt; 0.0001) and progesterone receptor positive (p &amp;lt; 0.0001) tumors. An inverse association was found with triple negative breast cancers (p &amp;lt; 0.0001). More importantly, Cyclin D1 expression was an independent predictor of favorable overall survival in our cohort (Hazard ratio = 0.69; 955 confidence interval = 0.48 - 0.98; p = 0.0405). Also, tumors that highly expressed cyclin D1 had a longer recurrence free survival. However, recurrence free survival was only significant in univariate analysis. In conclusion, our results reinforced the role of cyclin D1 in breast cancer pathology and revealed its expression as a valuable independent prognostic indicator for breast cancer from Middle Eastern ethnicity. Citation Format: Khawla S. Al-Kuraya, Abdul K. Siraj, Sandeep K. Parvathareddy, Sarah Siraj, Saud Azam, Padmanaban Annaiyappanaidu, Maria Angelita Sabido, Mark Ranier Diaz. High expression of Cyclin D1 is an independent marker for favorable prognosis in Middle Eastern breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1963.

Abstract 3111: Notch3 functions as a tumor suppressor in ER-positive breast cancer

Abstract The Notch signaling pathway has been implicated in the development as well as tumorigenesis of the mammary gland. Notch3 promotes luminal cell specification and marks a highly clonogenic luminal progenitor population. Aberrant Notch3 activation has been shown to promote the growth of basal breast cancer as well as resistance to hormonal therapy in metastatic luminal breast cancer. Interestingly, recent studies also suggest a tumor-suppressive function of Notch3 in mammary epithelium through regulation of estrogen receptor α (ER). Here we report the expression and functional analysis of Notch3 in the mouse mammary gland. X-gal staining of the Notch3β-Geo/+ mammary gland showed that Notch3 was highly expressed in luminal cells throughout mammary development with exception at the initiation of post-lactational involution, when Notch3 expression was restricted to the basal cells. Deletion of Notch3 caused decreased Notch activation in the CD24HiCD49fLo subpopulation of the pubescent mammary gland, accompanied by a significant decrease in the number of CD24HiCD49fLoCD61+ luminal progenitor cells. Whole-mount preparation of the Notch3 knockout mammary glands showed normal development during puberty, pregnancy, lactation and involution. Surprisingly, parous Notch3 knockout mice developed mammary ductal hyperplasia by 10 months of age, some of which progressed to ductal carcinoma in situ, and ultimately to invasive and metastatic cancer. Parous Notch3 knockout mice exhibited an expansion of the CD24HiCD49fLo subpopulation, and mammary tumors from these mice were composed predominantly of CD24HiCD49fLo cells. The vast majority of these tumors were ER-positive and relatively well differentiated, with a papillary and/or glandular pattern. They expressed the luminal marker cytokeratin 8, and some of them co-expressed cytokeratin 14, a basal marker. All Notch3 knockout mammary tumors showed high level expression of Cyclin D1. These results suggest that Notch3 may prevent ER-positive breast cancer through suppression of CD24HiCD49fLo progenitor cell self-renewal in the postpartum mammary gland. In addition to its impact on mammary epithelium, deletion of Notch3 altered the mammary microenvironment, in particular, brown adipocyte differentiation in the mammary fat pad. Further experiments will be performed to define a potential role for Notch3-regulated brown adipose tissue in breast cancer initiation and progression. Finally, analysis of TCGA and METABRIC human breast cancer data sets revealed a negative correlation between expressions of NOTCH3 and ESR1 (encoding estrogen receptor α), and association of high NOTCH3 expression with enhanced survival of patients with luminal A and luminal B subtype breast tumors. Taken together, Notch3 can act as a tumor suppressor in the mammary epithelium, and possibly in the mammary microenvironment as well, to prevent development and metastasis of ER-positive breast cancer. Citation Format: Wen-Cheng Chung, Sean E. Egan, Keli Xu. Notch3 functions as a tumor suppressor in ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3111.