Cyclin D1 Gene Amplification Research Articles

Clinicopathological study and immunohistochemical evaluation of cyclin D1 in adenomatous polyps

Background and objective: There are many histological types of colorectal polyps. Most of these polyps are benign epithelial polyps harboring very low risks of cancerous changes. Adenomatous polyps are the most known cancer precursors. Cyclin D1 gene amplification and or overexpression occurs in many human cancers. Cyclin D1 participates potentially in the multistep process of colorectal carcinogenesis. The present study aimed to assess the clinicopathological features of colorectal polyps and evaluate the significance of immunohistochemistry expression of cyclin D1 in adenomatouse polyp as a marker for predicting malignant transformation. Methods: A total of 180 cases of colorectal polyps were collected from Rizgary teaching Hospital and some private laboratories in Erbil, Kurdistan Region, Iraq, from January 2013 to January 2018. All cases underwent polypectomy by colonoscopy for removal of the polyp. Only cases presented with a single polyp were included. The histopathological diagnosis was revised, and the polyps were divided into four subtypes; inflammatory, hyperplastic, juvenile, and adenomas. All adenoma specimens were stained using IHC technique with cyclin D1. Results: Out of the 180 cases, the results showed that 70(38.9%) were adenomatous polyps, 56(31.1%) were hyperplastic polyps,40(22.2%) were juvenile polyps, and 14(7.8%)were inflammatory polyps. Cyclin D1 nuclear staining was detected in 24 (34.2%) adenomas. Statistical significant relations between cyclin D1 expression with male gender and with high grade dysplasia were found. Conclusion: Adenoma was the most common type among colorectal polyps. Cyclin D1 was shown to be aberrantly expressed in colorectal adenomas and may play a role in the early stages of adenoma carcinoma development. Keywords: Colorectal polyp; Cyclin D1; Adenoma.

Diagnostic application of cyclin D1 fluorescent in situ hybridization for histologically undetermined early lesions of acral melanoma in situ: A case series

Histologically undetermined early acral melanoma in situ (HUAMIS) is rare but a diagnostic challenge, being clinically and dermoscopically MIS (late onset, a large size (>7 mm), parallel ridges pattern) but microscopically without recognizable cytological atypia. Cyclin D1 (CCND1) gene amplification is a genetic aberration occurring in the early radial growth phase of AMs and could thus help determine malignancy for this disease.We determine the value of CCND1 amplification by FISH as a diagnostic marker for HUAMIS. CCND1 amplification was examined in paraffin-embedded skin biopsies and excisions using a dual-probes fluorescence in situ hybridization (FISH) (11q13 and CEP11). One FISH-negative case 6 was additionally examined by Mypath Melanoma (qRT-PCR). Seventeen cases (12 dysplastic nevi, 3 AMIS, and 2 invasive AM) were served as negative controls for FISH.All six patients (4 females and 2 males) were Hispanic. Pigment lesions were on the left plantar foot (4), right third finger palm (1), and right thumb subungual (1). All cases showed similar clinical and dermoscopical characteristics, including late onset (50 to 74 years old), long duration (from 2 to 15 years), large-sized pigments (from 16 to 40 mm), and a parallel ridge pattern. Junctional melanocytes with no or minimal atypia from five cases showed CCND1 amplifications. Four of 5 cases were received 1st or/and 2nd wide excisions, which demonstrated foci of histologically overt MIS. One FISH-negative case 6 demonstrated “likely malignancy” scores (>2) by Mypath Melanoma (qRT-PCR). None of negative controls showed the amplification.We propose here a simple CCND1 FISH is a practical diagnostic test to determine the malignancy of the very early progression phase of AM preceding histopathologically defined MIS. Cases presented here could be an indolent subtype of AMIS characterized by carrying a long latent radial growth phase without vertical growth, mimicking lentigo maligna.

Cyclin D1 Gene Numerical Imbalances in Laryngeal Squamous Cell Carcinoma: A Tissue Microarray Grid Based Analysis.

Background:Deregulation of critical proteins involved in cell cycle stability, such as cyclins, is a frequent genetic event in the development and progression of solid malignancies. Concerning laryngeal squamous cell carcinoma (LSCC), cyclin D1 oncogenic transformation leads to an aberrant protein expression and seems to affect the biological behaviour of the neoplasm. The aim of this study was to determine the correlation of cyclin D1 numerical imbalances with the corresponding protein expression levels in LSCCs. Material and Method:Using tissue microarray (TMA) technology, fifty (n=50) histologically confirmed primary LSSCs were cored at a diameter of 1.5 mm. Immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) analyses were applied. Concerning the screening process in CISH slides, a novel real-time reference and calibration grid platform was implemented. Results:Protein overexpression was observed in 22/50 (44%) cases; whereas, gene amplification was seen in 13/50 (26%) cases (p=0.02). Combined protein/ gene deregulation was associated with the stage of malignancy (p= 0.0014, p=0.001), whereas overall protein expression was strongly correlated with the grade of tumour (p= 0.001). Conclusion: Cyclin D1 gene amplification led to aberrant protein expression in LSCCs and it was also correlated with an aggressive biological behaviour. To best of our knowledge, this study was the first described grid based CISH analysis under conventional bright field microscopy for detecting gene numerical imbalances while providing a novel and accurate description for screening-mapping process in the entire slide area.

Cyclin D1 differential activation and its prognostic impact in patients with advanced breast cancer treated with trastuzumab

IntroductionWe sought to determine the level of activation of the critical components of the cyclin D1-mediated pathway and to evaluate their prognostic significance across the different molecular subtypes of advanced breast cancer.Patients and methodsThe study population comprised 219 female patients with advanced breast cancer who had been found to have human epidermal growth factor receptor 2 (HER2)-positive disease by local testing and were all treated with trastuzumab-based regimens. For all tumours, central testing for HER2 was performed, and cyclin D1 gene (CCND1) amplification, mRNA and protein expression were assessed by FISH, quantitative real-time-PCR and immunohistochemistry, respectively. Prognostic impact on clinical endpoints was evaluated with Cox regression analyses.ResultsAfter central testing, only 134 (61.2%) of 219 patients were confirmed to have HER2 gene amplification by FISH and/or 3+ HER2 protein expression by immunohistochemistry. After a median follow-up time of 136.0 months (95% CI 123.3 to 148.9), 105 (78.4%) HER2-positive patients and 76 (89.4%) HER2-negative patients had died, while 80% of the former and 87.1% of the latter had experienced a disease relapse. Patients with positive oestrogen receptor/progesterone receptor status presented with higher cyclin D1 mRNA expression. In the HER2-negative subgroup, patients with negative cyclin D1 protein expression were at higher risk of progression (HR= 1.66, 95%CI 1.01 to 2.72, Wald’s p=0.045). Among de novo metastatic patients, the risk of progression was higher for patients with non-amplified CCND1 tumours (HR= 2.00, 95% CI 1.03 to 3.90, p=0.041).ConclusionAberrant activation of the cyclin D1-mediated pathway appears to reduce the risk of progression in HER2-negative tumours, but not in HER2-positive ones.

The long-term prognostic and predictive capacity of cyclin D1 gene amplification in 2305 breast tumours

BackgroundUse of cyclin D1 (CCND1) gene amplification as a breast cancer biomarker has been hampered by conflicting assessments of the relationship between cyclin D1 protein levels and patient survival. Here, we aimed to clarify its prognostic and treatment predictive potential through comprehensive long-term survival analyses.MethodsCCND1 amplification was assessed using SNP arrays from two cohorts of 1965 and 340 patients with matching gene expression array and clinical follow-up data of over 15 years. Kaplan-Meier and multivariable Cox regression analyses were used to determine survival differences between CCND1 amplified vs. non-amplified tumours in clinically relevant patient sets, within PAM50 subtypes and within treatment-specific subgroups. Boxplots and differential gene expression analyses were performed to assess differences between amplified vs. non-amplified tumours within PAM50 subtypes.ResultsWhen combining both cohorts, worse survival was found for patients with CCND1-amplified tumours in luminal A (HR = 1.68; 95% CI, 1.15–2.46), luminal B (1.37; 1.01–1.86) and ER+/LN−/HER2− (1.66; 1.14–2.41) subgroups. In gene expression analysis, CCND1-amplified luminal A tumours showed increased proliferation (P < 0.001) and decreased progesterone (P = 0.002) levels along with a large overlap in differentially expressed genes when comparing luminal A and B-amplified vs. non-amplified tumours.ConclusionsOur results indicate that CCND1 amplification is associated with worse 15-year survival in ER+/LN−/HER2−, luminal A and luminal B patients. Moreover, luminal A CCND1-amplified tumours display gene expression changes consistent with a more aggressive phenotype. These novel findings highlight the potential of CCND1 to identify patients that could benefit from long-term treatment strategies.

Prognostic significance of cyclin D1 protein expression and gene amplification in invasive breast carcinoma.

The oncogenic capacity of cyclin D1 has long been established in breast cancer. CCND1 amplification has been identified in a subset of patients with poor prognosis, but there are conflicting data regarding the predictive value of cyclin D1 protein overexpression. This study was designed to analyze the expression of cyclin D1 and its correlation with CCND1 amplification and their prognostic implications in invasive breast cancer. By using the tissue microarray technique, we performed an immunohistochemical study of ER, PR, HER2, p53, cyclin D1, Ki67 and p16 in 179 invasive breast carcinoma cases. The FISH method was performed to detect HER2/Neu and CCND1 amplification. High cyclin D1 expression was identified in 94/179 (52%) of invasive breast cancers. Cyclin D1 overexpression and CCND1 amplification were significantly associated (p = 0.010). Overexpression of cyclin D1 correlated with ER expression, PR expression and Luminal subtypes (p<0.001), with a favorable impact on overall survival in the whole series. However, in the Luminal A group, high expression of cyclin D1 correlated with shorter disease-free survival, suggesting that the prognostic role of cyclin D1 depends on the molecular subtype. CCND1 gene amplification was detected in 17 cases (9%) and correlated significantly with high tumor grade (p = 0.038), high Ki-67 protein expression (p = 0.002), and the Luminal B subtype (p = 0.002). Patients with tumors with high amplification of CCND1 had an increased risk of recurrence (HR = 2.5; 95% CI, 1.2–4.9, p = 0.01). These findings suggest that CCND1 amplification could be useful for predicting recurrence in invasive breast cancer.

Abstract P4-12-07: CCND1 amplification in early breast cancer patients: Correlation with subtypes and prognosis

Abstract Background: Mechanisms of progress and recurrence of early breast cancer (BC) have gained importance since several targeted therapeutic options in metastatic breast cancer have been introduced over the last years, especially in hormone receptor positive BC. This study investigates the amplification of cell cycle regulator cyclin D1 gene (CCND1) amplification as one possible progression mechanism. Patients &amp; Methods: Patients from an unselected cohort of early BC patients were included into this study. A tissue microarray (TMA) was available for n=832 patients with early breast cancer. CCND1 amplification was assessed after FISH (Abbott Vysis LSI CCND1 SpectumOrange/CEP11 SpectrumGreen Probes Kit). A CCND1/CEP11-ratio ≥ 2.0 was considered as amplification. Staining was successful in 545 tumor cores. Amplification results were correlated with clinical patient and tumor characteristics and survival analyses were performed with regard to distant disease free survival and overall survival. Results: CCND1 amplification was found in 13.6% of patients. Triple negative, luminal A like, luminal B like and HER2 positive tumors were amplified in 7.5%, 7.8%, 18.6% and 15.7% respectively (p = 0.010). CCDN1 amplification was significantly associated with a higher grading and a higher body mass index. Furthermore an amplification was seen more frequently in lobular BC and ductal BC than other histological subtypes. Survival analysis showed a reduced DDFS for patients with CCDN1-amplification. 5 year DDFS rates were 90.6% for non-amplified tumors and 86.0% for amplified tumors (p, log-rank =0.066 ). 5 year OS rates were 93.0% for non-amplified tumors and 90.1% for amplified tumors (p, log-rank =0.119). Adjusted for age, tumor size, nodal status and molecular subtype, cox regression showed HR of 1.3 (95% CI: 0.76-2.5, p=0.46) for DDFS and a HR of 1.33 (95% CI: 0.7-2.53, p=0.38) for OS. Conclusion: With a 13.6% prevalence in all breast cancer patients, mainly present in luminal B like cancers, CCND1-amplification is a genetic aberration associated with an unfavorable prognosis. Within hormone receptor positive women CCDN1 amplification might play a role in treatment resistance mechanisms in early breast cancer patients. Citation Format: Hanf DC, Fasching PA, Villalobos IE, Gasparyan A, Wachter D, Santiago A, Guzman R, Weihbrecht S, Hanf V, Hartmann A, Beckmann MW, Press MF. CCND1 amplification in early breast cancer patients: Correlation with subtypes and prognosis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-07.

Cyclin D1 in well differentiated thyroid tumour of uncertain malignant potential.

BackgroundEncapsulated follicular tumours with equivocal papillary thyroid carcinoma (PTC) type nuclear features continue to remain a challenge despite the recent attempts to classify these borderline lesions. The term ‘well differentiated tumour of uncertain malignant potential (WDT-UMP)’ was introduced to classify these tumours. The present study aimed to evaluate the role of a cell cycle regulator like cyclin D1 in these tumours along with assessment of other well established PTC markers like galectin-3, HBME-1, CK19.MethodsThirteen cases of metastatic PTC, papillary microcarcinoma and follicular variant of PTC (FVPTC) were identified from a histological review of 510 cases. In addition, 13 cases of a subset of follicular adenomatoid nodules with focal areas showing nuclear features characteristic of PTC, identified as WDT-UMP, were also analyzed. Immunohistochemical analysis of galectin-3, HBME-1, CK19 and the proliferation markers Ki67 and cyclin D1 was performed. Lesions were analyzed for cyclin D1 gene amplification by fluorescent in-situ hybridization.ResultsAll WDT-UMP lesions showed immunolabelling of cyclin D1, Ki67; 11/ 13 cases showed immunolabelling of CK19; 10/13 cases showed immunolabelling of HBME-1 and 4/13 cases showed immunolabelling of galectin-3. Surrounding benign adenomatoid areas showed no to faint focal staining in all thirteen cases of cyclin D1, HBME-1 and galectin-3. A low rate of cyclin D1 gene amplification was identified in a significant proportion of cells in the WDT-UMP lesions as compared to surrounding benign adenomatoid areas.ConclusionsIncreased expression of cyclin D1 and amplification of its gene along with immunolabelling of HBME-1 in WDT-UMP lesions showing cytological features of papillary thyroid carcinoma within follicular adenomatoid nodules suggest that these areas could correspond to a precursor lesion of follicular variant of PTC. Overexpression of cyclin D1, associated with the amplification of the gene suggests that these WDT-UMP lesions are an intermediate between the benign and malignant groups making this group of lesions a reliable precursor of FVPTC.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1851820807142117

A melanocyte–melanoma precursor niche in sweat glands of volar skin

Determination of the niche for early-stage cancer remains a challenging issue. Melanoma is an aggressive cancer of the melanocyte lineage. Early melanoma cells are often found in the epidermis around sweat ducts of human volar skin, and the skin pigmentation pattern is an early diagnostic sign of acral melanoma. However, the niche for melanoma precursors has not been determined yet. Here, we report that the secretory portion (SP) of eccrine sweat glands provide an anatomical niche for melanocyte-melanoma precursor cells. Using lineage-tagged H2B-GFP reporter mice, we found that melanoblasts that colonize sweat glands during development are maintained in an immature, slow-cycling state but renew themselves in response to genomic stress and provide their differentiating progeny to the epidermis. FISH analysis of human acral melanoma expanding in the epidermis revealed that unpigmented melanoblasts with significant cyclin D1 gene amplification reside deep in the SP of particular sweat gland(s). These findings indicate that sweat glands maintain melanocyte-melanoma precursors in an immature state in the niche and explain the preferential distribution of early melanoma cells around sweat glands in human volar skin.

Cyclin D1 gene amplification is highly homogeneous in breast cancer.

Cyclin D1 (CCND1) gene amplification is a molecular key alteration in breast cancer and was suggested to predict resistance to antihormonal therapy. As tissue heterogeneity may affect diagnostic accuracy of predictive biomarkers, CCND1 genetic heterogeneity was assessed in this study. A novel tissue microarray (TMA) platform was manufactured for this purpose. Primary breast carcinomas from 147 patients were sampled in a "heterogeneity-TMA" by taking eight different tissue cores from 4 to 8 tumor-containing blocks per case. Additional tissue samples were taken from 1 to 4 corresponding nodal metastases in 35 of these patients. CCND1 amplification was assessed by fluorescence in situ hybridization (FISH). CCND1 amplification was seen in 28 of 133 (21.05 %) informative patients. Amplification was significantly associated with high tumor grade (p = 0.042), but unrelated to tumor type (p = 0.307), stage (p = 0.540) and ER (p = 0.061) or PR (p = 0.871) expression. A discordant Cyclin D1 amplification status was detected in 6 out of 28 (21.43 %) amplified tumors by heterogeneity-TMA analysis. Re-testing on large sections revealed three patients with true heterogeneity of high-level CCND1 amplification and another three patients with variable interpretation of borderline FISH ratios ranging between 1.7 and 2.3. No discrepancies were detected between 22 primary tumors and their matched lymph node metastases. The high degree of homogeneity seen for CCND1 amplification suggests that this alteration is an early event in the development of a small subset of breast cancers.

Cyclin D1 Amplification in Multiple Myeloma Is Associated With Multidrug Resistance Expression

Cyclin D1 is involved in normal regulation of the cell cycle and in neoplasia. Inhibition of cyclin D1 function markedly attenuates the proliferation of fibroblasts of colon, esophageal, lung, and pancreatic cancer. However, the prognostic value of overexpression of cyclin D1 in multiple myeloma is still a point of debate. This study aimed at evaluating the effect of cyclin D1 gene amplification in multiple myeloma on overall survival and response to therapy. Fifty patients with multiple myeloma were retrospectively studied. Cyclin D1 gene amplification was studied in bone marrow biopsies of these patients using FISH. An immunohistochemical study of the bone marrow biopsies was done to detect MDR1 protein expression. The correlations between the cyclin D1 gene amplification and overall survival and MDR1 expression were studied and analyzed statistically. Cyclin D1 gene amplification was found in 20% of myeloma patients and was associated with higher percentage of plasma cell infiltration of the bone marrow and increased liability for multiple osteolytic lesions. Cyclin D1-positive patients had a significantly lower progression-free and overall survival and higher levels of MDR1 compared with cyclin D1-negative patients. Cyclin D1 levels showed a highly statistically significant positive correlation with MDR1 levels (R, 0.8 and P<.0001). We suggest that there is an association between cyclin D1 gene amplification and disease severity, unfavorable prognosis, and increased expression of MDR1 in multiple myeloma patients.

RSF1 and Not Cyclin D1 Gene Amplification May Predict Lack of Benefit from Adjuvant Tamoxifen in High-Risk Pre-Menopausal Women in the MA.12 Randomized Clinical Trial

Most women with estrogen receptor expressing breast cancers receiving anti-estrogens such as tamoxifen may not need or benefit from them. Besides the estrogen receptor, there are no predictive biomarkers to help select breast cancer patients for tamoxifen treatment. CCND1 (cyclin D1) gene amplification is a putative candidate tamoxifen predictive biomarker. The RSF1 (remodeling and spacing factor 1) gene is frequently co-amplified with CCND1 on chromosome 11q. We validated the predictive value of these biomarkers in the MA.12 randomized study of adjuvant tamoxifen vs. placebo in high-risk premenopausal early breast cancer. Premenopausal women with node-positive/high-risk node-negative early breast cancer received standard adjuvant chemotherapy and then were randomized to tamoxifen (20 mg/day) or placebo for 5 yrs. Overall survival (OS) and relapse-free survival (RFS) were evaluated. Fluorescent in-situ hybridization was performed on a tissue microarray of 495 breast tumors (74% of patients) to measure CCND1 and RSF1 copy number. A multivariate Cox model to obtain hazard ratios (HR) adjusting for clinico-pathologic factors was used to assess the effect of these biomarkers on Os and RFS. 672 women were followed for a median of 8.4 years. We were able to measure the DNA copy number of CCND1 in 442 patients and RSF1 in 413 patients. CCND1 gene amplification was observed in 8.7% and RSF1 in 6.8% of these patients, preferentially in estrogen receptor-positive breast cancers. No statistically significant interaction with treatment was observed for either CCND1 or RSF1 amplification, although patients with high RSF1 copy number did not show benefit from adjuvant tamoxifen (HR = 1.11, interaction p = 0.09). Unlike CCND1 amplification, RSF1 amplification may predict for outcome in high-risk premenopausal breast cancer patients treated with adjuvant tamoxifen.

Florid lobular carcinoma in situ: molecular profiling and comparison to classic lobular carcinoma in situ and pleomorphic lobular carcinoma in situ

We evaluated genomic alterations and biomarker expression in 20 florid lobular carcinomas in situ using array-based comparative genomic hybridization and immunohistochemical analysis. The genetic characteristics of florid lobular carcinoma in situ were compared with 20 classic lobular carcinomas in situ and 21 pleomorphic lobular carcinomas in situ (which included 8 apocrine variants), from our previously published data performed on a similar array-based comparative genomic hybridization platform. All 20 florid lobular carcinoma in situ cases were E-cadherin negative, and 92% were positive for estrogen receptor. Cyclin D1 expression correlated significantly negatively with estrogen receptor expression and was higher in cases with cyclin D1 (CCND1) gene amplification. Compared with classic lobular carcinoma in situ, florid lobular carcinoma in situ displayed significantly more fraction genome alteration (mean, 0.109 versus 0.072; P=.007), fraction genome loss (mean, 0.06 versus 0.03; P=.007), numbers of breakpoints (mean, 11.55 versus 6.95; P=.002), numbers of chromosome with breakpoints (mean, 5.85 versus 3.8; P=.004), and higher numbers of amplifications (mean, 2.10 versus 0.25; P=.03). Interestingly, florid lobular carcinoma in situ had the same genetic complexity as apocrine pleomorphic lobular carcinoma in situ. Our study demonstrated that florid lobular carcinoma in situ shares the cytologic features, E-cadherin loss, and the lobular genetic signature of 1q gain and 16q loss found in classic lobular carcinoma in situ. However, this variant demonstrates more genomic alterations than classic lobular carcinoma in situ and shares the same genetic complexity as apocrine pleomorphic lobular carcinoma in situ. Our data support the conclusion that florid lobular carcinoma in situ is genetically more advanced compared with the indolent phenotype of classic lobular carcinoma in situ. This may explain the greater frequency of concurrent invasive carcinoma in florid lobular carcinoma in situ compared with classic lobular carcinoma in situ.

Differential Expression of p16INK4A and Cyclin D1 in Benign and Malignant Salivary Gland Tumors: A Study of 44 Cases

Salivary gland tumors (SGT) are a heterogeneous group of lesions. There is conflicting data concerning the molecular events involving the tumour suppressor retinoblastoma protein (pRb) pathway in these tumors. Few studies examined the alterations in components of the Rb pathway by immunohistochemical (IHC) methods in benign and malignant SGTs. Furthermore, recent evidence implicates human papillomavirus (HPV) in mucoepidermoid carcinoma (MEC) carcinogenesis. The purpose of our study is to examine p16(INK4A) and cyclin D1 expression in a variety of benign and malignant salivary gland tumors, and to investigate p16(INK4A) expression as a surrogate marker for HPV infection in MEC. Our series includes 30 malignant tumors [14 MEC, 6 acinic cell carcinomas (ACC), 5 polymorphous low grade adenocarcinomas (PLGA), 5 (AdCC)] and 14 benign tumors (4 benign cysts, 5 Warthin tumors and 5 pleomorphic adenomas (PA). All cases were tested by IHC for p16(INK4A) and cyclin D1. Testing for HPV wide spectrum (HPV-WS) was performed by in situ hybridization in all MEC cases. Staining intensity was recorded semi quantitatively (on a scale from 0 to 4+). Fisher's exact test and Pearson X2 test with a p < 0.05 were used. Cyclin D1 and p16(INK4A) are expressed similarly in malignant and benign tumors (p = 0.146 and p = 0.543, respectively). None of the MEC cases showed nuclear reactivity for HPV-WS. Statistical analysis showed positive correlation between cyclin D1 and p16(INK4A) expression. Our findings suggest that p16(INK4A) overexpression is likely secondary to cyclin D1 gene upregulation or amplification. Further molecular studies are warranted.

Effects of cyclin D1 gene amplification and protein expression on time to recurrence in postmenopausal breast cancer patients treated with anastrozole or tamoxifen: a TransATAC study.

IntroductionGene amplification of CCND1 is observed in a subgroup of breast cancers with poor prognosis, whereas overexpression of the protein cyclin D1 has been linked to both worse and better clinical outcome. CCND1 amplification and protein overexpression have also been associated with resistance to treatment with tamoxifen or even to a potentially detrimental effect of tamoxifen.MethodsTo clarify these challenging and partly contrasting treatment predictive and prognostic links for cyclin D1 we analysed a large cohort of postmenopausal breast cancer patients randomised to receive either adjuvant anastrozole or tamoxifen, as part of the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial. The CCND1 amplification status and protein expression of cyclin D1 were assessed by chromogenic in situ hybridisation and immunohistochemistry, respectively, in 1,155 postmenopausal, oestrogen-receptor-positive breast cancer patients included in the TransATAC substudy.ResultsAmplification of CCND1 was observed in 8.7% of the tumours and was associated with increased risk of disease recurrence (hazard ratio = 1.61; 95% confidence interval, 1.08 to 2.41) after adjustment for other clinicopathological parameters. In contrast, nuclear expression of cyclin D1 protein was associated with decreased recurrence rate (hazard ratio = 0.6; 95% confidence interval, 0.39 to 0.92). The intensity of nuclear or cytoplasmic expression was not of prognostic value. There was no significant interaction between cyclin D1 status and treatment efficacy, ruling out any major detrimental effect of tamoxifen in CCND1-amplified postmenopausal breast cancer.ConclusionsIn summary, CCND1 amplification and low nuclear expression of cyclin D1 predicted poor clinical outcome in postmenopausal breast cancer patients treated with either anastrozole or tamoxifen.Trial RegistrationCurrent Controlled Trials ISRCTN18233230.

Cyclin D1 gene amplification in multiple myeloma and its impact on the disease outcome and drug resistance

Background Cyclin D1 is involved in the normal regulation of the cell cycle and in neoplasia. Inhibition of cyclin D1 function markedly attenuates the proliferation of fibroblasts of colon, esophageal, lung, and pancreatic cancer. However, the prognostic value of overexpression of the cyclin D1 gene in multiple myeloma (MM) is still a point of debate. This study aims at evaluating the prognostic significance of cyclin D1 gene amplification in MM and whether it is correlated with MDR1 expression as an indicator of chemoresistance. Patients and methods Twenty-five patients with MM were studied retrospectively. Cyclin D1 gene amplification was studied in bone marrow biopsies of these patients by fluorescence in-situ hybridization. Immunohistochemical study of the bone marrow biopsies was carried out to detect multidrug resistance (MDR1) gene expression. The correlations between the cyclin D1 gene amplification on the one hand and overall survival and MDR1 expression on the other were studied and analyzed statistically. Results Cyclin D1 gene amplification was found in 20% of patients with myeloma and was associated with a higher percentage of plasma cell infiltration of the bone marrow and increase liability for multiple osteolytic lesions. Cyclin D1-positive patients had a significantly lower progression-free and overall survival and higher levels of MDR1 compared with cyclin D1-negative patients. Cyclin D1 levels showed a statistically highly significant positive correlation with MDR1 levels (r: 0.802 and P Conclusion We suggest that there is an association between cyclin D1 gene amplification and higher disease severity, unfavorable prognosis, and increased expression of MDR1 in patients with MM.

P3-05-05: Cyclin D1 Gene Amplification Is Rarely Heterogeneous in Breast Cancer.

Abstract Background: Amplification of Cyclin D1 (CCND1) occurs in about 10–20% of breast cancers and has been suggested to predict resistance to anti-hormonal therapy. As the diagnostic accuracy of predictive biomarkers can be substantially limited by regional expression differences within tumors, heterogeneity of CCND1 amplification was assessed in this study. To assess heterogeneity, a novel tissue microarray based analysis platform was developed. Material and Methods: To comprehensively asses the three-dimensional molecular composition of breast cancers, a “heterogeneity TMA” was constructed containing 8 different tissue cylinders from as many different cancer containing tumor blocks as possible (at least 4) from 147 primary breast cancers. Additional tissue samples were taken from 1–4 corresponding nodal metastases from 35 of these patients. Dual labeling fluorescence in situ hybridization (FISH) with probes for CCND1 and centromere 11 was applied. Results: The analysis revealed amplification in 29 of 133 (21.8%) patients with interpretable FISH data. CCND1 amplification was more frequently seen in ductal (22 of 87; 25.29%) than in lobular type (5 of 32; 15.63%) (p=0.251). CCND1 amplification was also associated with high tumor grade with amplification rates of 1 of 18 (5.56%) in grade 1, 15 of 72 (20.83%) in grade 2 and 12 of 40 (30%) in grade 3 carcinoma (p=0.075). CCND1 amplification was more frequently seen in ER positive cases (27 of 110; 24.55%) than in ER negative cases (1 of 17; 5.88%) (p=0.052). No association could be found between CCND1 amplification and tumor stage (p=0.445) and CCND1 amplification and PR status (p=0.752). Heterogeneous amplification status was detected in 9 of 29 (31.0%) amplified tumors, i.e. in 6.8% of all informative cases. Heterogeneity was successfully validated on large sections in all 4 heterogeneous cases with high level amplification. In the remaining 5 “heterogeneous cases” discordant results were due to variable interpretation of borderline amplification results with CCND1/centromer 11 ratios between 1.7 and 2.3. There were no discrepancies seen between primary tumors and matched lymph node metastases. Discussion: The high degree of homogeneity seen for CCND1 amplification suggests that this alteration represents an early event in tumor development/progression in a subset of breast cancers. CCND1 status determined in a small biopsy will be highly representative of the entire tumor and will thus be appropriate for predicting treatment outcome. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-05-05.

Single Nucleotide Polymorphisms (SNPs) in Genes for Glutathione-Related Metabolism, Cyclin D1, and DNA Repair As Predictive Biomarkers in Mantle Cell Lymphoma Patients Treated with R-HyperCVAD with Ten Year Clinical Follow-up,

Abstract 3650 Background:Mantle cell lymphoma (MCL) is an aggressive lymphoma with poor prognosis. Cyclin D1 (CCND1) gene amplification is a marker of MCL. Glutathione (GSH) S-transferase pi (GSTP1) is a gene on the same amplicon as CCND1. Alternative splicing of CCND1 is mediated by an A/G single nucleotide polymorphism (SNP) in the exon 4 splice donor region. Inhibition of GSTP1 activity increased the sensitivity of MCL cell lines to cisplatin, cytarabine and bortezomib, but not doxorubicin suggesting its importance for drug resistance (Paret et al, 2005, ASH Abst #2806). Other GSH-related proteins were hypothesized to potentially be predictive biomarkers. We retrospectively analyzed DNA samples from MCL patients in a clinical trial with uniform first line chemoimmunotherapy treatment in order to study potential prognostic and predictive molecular markers including: GST M1/T1 gene copy number as well as SNPs for CCND1, GSTP1 and other GSH metabolism family of genes (described in Moyer et al, 2010 CEBP). Methods:DNA was available from 89 MCL patients treated with R-HyperCVAD from 1999 to 2002 at MD Anderson Cancer Center (MDACC). The CCND1 exon 4 (codon 242) SNP was evaluated using pyrosequencing. GST copy numbers were determined as previously described (Moyer et al., 2007 CCR). GSH-related gene SNP testing was performed at the Mayo Clinic Advanced Genomic Technology Center using an Illumina Golden Gate 384 plex panel [290 GSH-related SNPs (30 genes) and 94 additional SNPs in DNA damage repair and other pathways (19 genes] for GSTs and related GSH metabolism genes including GSH synthetase and GSH transporters. Comparison to ten year clinical data with an 8 year median follow-up was used under an IRB approved protocol (Romaguera et al., 2010, Br J Haematol).For overall survival (OS) and failure free survival (FFS), each SNP was first examined by the method of Kaplan and Meier and the log-rank test. To account for multiple testing (of 300+ SNP), we used a beta-uniform mixture model to model the sets of p-values for OS and FFS separately. SNPs that had a false discovery rate (FDR) adjusted p-value of less than 0.2 were evaluated with Cox proportional hazard models to examine the association between SNP and time to event. When there was evidence of non-proportional hazards, we fitted Bayesian parametric accelerated failure regression model. All tests are two-sided. All analyses were done using SAS (v 9.1, Cary, NC) and the R statistical project (http://www.r-project.org/). Results:Fifteen SNPs were significantly associated with FFS or OS in univariable analysis using a FDR q-value cut off of 0.2 (Table). Ten of these SNPs remained significant with FFS or OS after adjustment for age. Prognostic SNPs included GST (GSTO1, GSTO2, GSTA5) and DNA repair (LIG1, ERCC2, RAD52) genes. GST M1/T1 gene copy numbers and SNPs for GSTP1 and CCND1 were not found to be significantly associated with FFS and OS. Conclusion:DNA repair and GSH-associated gene SNPs correlated with OS and FFS in MCL patients treated with R-HyperCVAD at ten years follow-up. This is the largest MCL molecular marker study of which we are aware in a uniformly treated patient group and includes over 300 SNPs including CCND1, GSTP1, and GSH-associated metabolism pathway genes. Additional clinical trials may look at these SNPs in correlative studies.Statistically Significant SNPsSNPPrognostic for:GeneFunctionMed OS (mon)Med FFS (mon)rs3730913OS*, FFS*LIG1IntronAA: 44.215.4AG: NA111GG: 10053.6rs3916874OS*, FFS*ERCC2IntronCC: 32.318.9CG: NA72.6GG: 10250.8rs1147611OS*GSTO1IntronAA: 58.8—AC: 85.4CC: NArs12664895OSGCLCIntronAA: NA—GG: 32.3rs156697OS*GSTO2Exon: AAT-> GATAA: NA—AA142: N-> DAG: NAGG: 58.8rs157080OS*GSTO2IntronTT: 56.2—TG: 102GG: NArs2164624OS*GSTO2IntronTT: 78—TC: 62.5CC: NArs9634161OSRAD52IntronAA: 102—AG: NAGG: 18.4rs568526OSGSTO2IntronTT: NA—TC: NACC: 58.8rs4925OS*GSTO1Exon: GCT-> GATAA: 85.3—AA140: A-> DAC: 68.8CC: NArs416844OS*LIG1IntronTT: 51.4—TG: NAGG: 78rs717620OS*ABCC25'UTRAA: NA—AG: 50.7GG: NArs7755335OSGSTA5IntronAA: NA—AG: 31.8rs8187689OSABCC2Exon: T->GAA: NA—AA: L->GAC: 23.1rs6088659FFS*MYH7BNear gene 5'—AA: 9.38AG: 50.8GG: 62.1NA = Not Available.*Ten of these SNPs are still associated with FFS or OS after adjusted for age. Disclosures:No relevant conflicts of interest to declare.

Centrosome clustering and cyclin D1 gene amplification in double minutes are common events in chromosomal unstable bladder tumors

BackgroundAneuploidy, centrosome abnormalities and gene amplification are hallmarks of chromosome instability (CIN) in cancer. Yet there are no studies of the in vivo behavior of these phenomena within the same bladder tumor.MethodsTwenty-one paraffin-embedded bladder tumors were analyzed by conventional comparative genome hybridization and fluorescence in situ hybridization (FISH) with a cyclin D1 gene (CCND1)/centromere 11 dual-color probe. Immunofluorescent staining of α, β and γ tubulin was also performed.ResultsBased on the CIN index, defined as the percentage of cells not displaying the modal number for chromosome 11, tumors were classified as CIN-negative and CIN-positive. Fourteen out of 21 tumors were considered CIN-positive. All T1G3 tumors were included in the CIN-positive group whereas the majority of Ta samples were classified as CIN-negative tumors. Centrosome clustering was observed in six out of 12 CIN-positive tumors analyzed. CCND1 amplification in homogeneously staining regions was present in six out of 14 CIN-positive tumors; three of them also showed amplification of this gene in double minutes.ConclusionsComplex in vivo behavior of CCND1 amplicon in bladder tumor cells has been demonstrated by accurate FISH analysis on paraffin-embedded tumors. Positive correlation between high heterogeneity, centrosome abnormalities and CCND1 amplification was found in T1G3 bladder carcinomas. This is the first study to provide insights into the coexistence of CCND1 amplification in homogeneously staining regions and double minutes in primary bladder tumors. It is noteworthy that those patients whose tumors showed double minutes had a significantly shorter overall survival rate (p < 0.001).

Nuclear EGFR in ductal invasive breast cancer: correlation with cyclin-D1 and prognosis

The epidermal growth factor receptor (EGFR)-family and cyclin-D1 have been extensively studied in breast cancer; however systematic studies that examine protein expression and gene status in the same cohort of patients are lacking. Also emerging evidences suggest existence of a direct EGFR-signaling pathway, which involves cellular transport of EGFR from cell membrane to the nucleus, and transcriptional regulation of the target genes. Thus, we examined the protein expression of membrane EGFR, nuclear EGFR, cyclin-D1 and the corresponding gene status in 113 breast carcinomas by immunohistochemistry and fluorescence in situ hybridization using tissue microarrays. Membrane EGFR overexpression and EGFR gene amplification were detected in 2% cases, while nuclear EGFR was detected in 40% of cases, with 12% having high nuclear EGFR staining. Nuclear EGFR correlated with tumor size (P=0.0005), lymph node metastasis (P=0.0288), Nottingham prognostic index (P=0.0011) and estrogen receptor (ER) expression (P=0.0258) but the letter correlation was observed only in premenopausal group of patients. Strong cyclin-D1 expression and cyclin-D1 gene (CCND1) amplification were found in 64 and 13% of the cases, respectively. Cyclin-D1 expression showed positive correlation with ER (P=0.0113) and inverse correlation with Nottingham prognostic index (P=0.0309) and membrane EGFR (P=0.0201). CCND1 amplification also showed inverse correlation with membrane EGFR (P=0.0420). A strong correlation between membrane EGFR expression and gene amplification (P=0.0035), as well as cyclin-D1 overexpression and gene amplification (P=0.0362), was demonstrated. On univariate analysis cyclin-D1 expression showed a correlation with longer overall survival in the premenopausal group and nuclear EGFR correlated with shorter overall survival in whole cohort as well in the premenopausal group of patients. Multivariate analysis revealed nuclear EGFR to be an independent prognostic factor and showed 3.4 times greater mortality risk for nuclear EGFR+++ patients as compared with nuclear EGFR negative patients (hazard ratio =3.402; P=0.0026).