Analysis of different deleted regions in chromosome 11 and their interrelations in early and late onset breast tumors: association with cyclin D1 amplification and survival

Previous studies have shown that younger women exhibit more aggressive pathologic features of breast cancer (BC) in comparison to older women; young age could be an independent predictor of adverse prognosis. In order to find any existing differences in the molecular progression of BC in both younger and older women, chromosome 11 (chr.11) was taken as a tool, due to its frequent deletion and amplification, particularly of CyclinD1 (CCND1) locus in BC. In the present work, the comparative analysis in the frequency of deletion in different regions in chr.11 and CCND1 amplification in BC in the two age groups was studied, as well as the interrelation and prognostic significance of these chromosomal alterations. The chr. 11 alterations were also studied in types of breast lesions other than carcinoma to see the prevalence of the alterations in these diseases. For this purpose, comparative deletion mapping of chr.11 using 17 microsatellite markers and CCND1 amplification was examined in 30 early-onset (≤40 years) and 33 late-onset (>40 years) breast carcinomas, as well as 11 other types of breast lesions. The frequency of deletion and CCND1 amplification was much higher in carcinomas than with other types of breast lesions. A total of six highly deleted regions, namely, 11p15.5, 11p11.2, 11q13.2, 11q22.3-23.1, 11q23.3-24.1, and 11q25, were identified in carcinomas of the two age groups. The 11q13.2 deletion and CCND1 amplification was comparatively higher in the carcinoma of younger women. The following significant associations were observed for (a) LOH at 11q25 with LOH at 11q13.2, 11q22.3-23.1, 11q23.3-24.1 and CCND1 amplification, respectively, and (b) LOH at 11p15.5 with LOH at 11q22.3-23.1 in carcinoma of younger women. On the other hand, the significant associations in older women were (a) LOH at 11q25 with LOH at 11q22.3-23.1, 11q23.3-24.1, respectively, and (b) LOH at 11q22.3-23.1 with LOH at 11q23.3-24.1. Deletion at 11q13.2 was also associated with reduced overall survival in the younger group, indicating its prognostic significance. It is evident from our data that the pattern of chromosomal alterations are not exactly same in the carcinomas in the two age groups. Differential interrelationship of the chromosomal alterations and prognosis in these two age groups indicate that the molecular pathogenesis of the carcinomas is not similar

Molecular study of clonality in multifocal and bilateral breast tumors

The clonal origin of multiple tumors in the same individual has long been debated. The main aim of this study is to find out whether multiple tumors in same individuals originated from a single clone. In our previous work (Pathol. Res. Pract. 199 (2003) 313-321), the deletion at chromosome1p36 was found to occur early because of common allelic loss in the bilateral tumors. In order to further investigate the findings about the clonality of tumors, eight tumors from four patients (two synchronous bilateral breast carcinoma [biBC], one case with breast carcinoma in one breast and multiple calcified fibroadenoma nodules in another breast, and one case with multifocal fibroadenosis in one breast) were subjected to polymerase chain reaction (PCR) to detect (a) loss of heterozygosity (LOH) and microsatellite size alterations (MA) using microsatellite markers distributed over five chromosomal arms 11p/q, 13q and 17p/q, and (b) Cyclin D1 amplification. Some markers were intragenic for BRCA1, BRCA2, BRCAX, ATM, TP53, and RB1. Although a few cases were studied, our findings suggest that in at least a proportion of patients multiple tumors may arise from a single clone

Differential Association of BRCA1 and BRCA2 Genes with Some Breast Cancer–Associated Genes in Early and Late Onset Breast Tumors

Accumulating evidence indicating more aggressive features of breast carcinoma (BC) in young women than their older counterparts have raised the question of whether these differences are present at the genetic level. Methods: For this purpose, we performed a comparative analysis of the frequency of deletions of BRCA1, BRCA2, BRCAX, TP53, ATM, and RB1 and amplification of Cyclin D1 and also studied the interrelation and prognostic significance of these genetic alterations in 30 early onset (�40 years) and 33 late onset (�40 years) cases of BC. These gene alterations were also studied in 11 other types of breast lesions. Results: A differential pattern of alterations (deletion/amplification) was observed in the two age groups, with the sequence in younger women being BRCA1 (72%), TP53 (71%), ATM (64%), BRCA2 (62%), RB1 (60%), Cyclin D1 (43%), and BRCAX (24%) and that in the older group being TP53 (66%), RB1 (63%), BRCA1 (56%), ATM (53%), BRCA2 (45%), Cyclin D1 (24%), and BRCAX (23%). Similar, differential correlations were also seen with several clinicopathological parameters, prognosis, and combinations of alterations among these genes in the two age groups. Conclusions: Differential frequencies and interrelationships of genetic alterations and prognoses in these two age groups indicate that the molecular pathways for the development of tumors in both age groups may not be similar, though the ultimate effect is deregulation of cell cycle checkpoints and defects in the DNA repair pathway

Regulation of T cell differentiation and alloimmunity by the cyclin-dependent kinase inhibitor p18ink4c.

Cellular proliferation in response to mitogenic stimuli is negatively regulated by the Cip/Kip and the Ink4 families of cyclin-dependent kinase (CDK) inhibitors. Several of these proteins are elevated in anergic T cells, suggesting a potential role in the induction or maintenance of tolerance. Our previous studies showed that p27kip1 is required for the induction of T cell anergy and transplantation tolerance by costimulatory blockade, but a role for Ink4 proteins in these processes has not been established. Here we show that CD4+ T cells from mice genetically deficient for p18ink4c divide more rapidly than wild-type cells in response to antigenic, costimulatory and growth factor signals. However, this gain of proliferative function was accompanied by a moderate increase in the rate of cell death, and was accompanied by an overall defect in the generation of alloreactive IFNγ-producing effector cells. Consistent with this, p18ink4c-deficient T cells were unable to induce graft-vs-host disease in vivo, and p18ink4c deficiency cooperated with costimulatory blockade to significantly increase the survival of fully mismatched allografts in a cardiac transplantation model. While both p18ink4c and p27kip1 act to restrict T cell proliferation, p18ink4c exerts an opposite effect from p27kip1 on alloimmunity and organ transplant rejection, most likely by sustaining T cell survival and the development of effector function. Our studies point to additional important links between the cell cycle machinery and the processes of T cell differentiation, survival and tolerance

Deletion mapping of chromosome 1 in early onset and late onset breast tumors - a comparative study in Eastern India

Younger women exhibit more aggressive pathologic features of breast cancer (BC) compared to their older counterparts. Young age has been shown to be an independent predictor of adverse prognosis. These findings have raised the question of whether these differences are also present at the genetic level. Twenty-five early onset (age ≤ 40 years) tumors including 4 bilateral tumors, and 26 late onset(> 40 years) breast tumors, including 2 bilateral tumors, were xamined for loss of heterozygosity (LOH) at chromosome 1 using 11 polymorphic microsatellite markers. A comparative study revealed high frequencies of LOH in chr.1p36 (61%), 1p31.3 (40%), 1p21.3 (50%) and 1q22–23.2 (56%) in a younger group, and chr. 1p36 (46%), 1p34.2 (48%), and 1q22–23.2 (52%) in an older group. These differences in LOH frequency in these two age groups were significant for chr. 1p21.3(p = 0.025) only. These data suggest that the deletion pattern in early onset breast tumors is not fully identical to late onsetbreast tumors. Similar differential deletion patterns of LOH in the 5 highly deleted regions were seen in premenopausal and postmenopausal groups. An association was seen between LOH at chr.1p34.2 and chr. 23.2 and higher grade of the tumors in older women. Among the highly deleted regions, the deletion at chr.1p36 was found to occur early in both groups because of common allelic loss in the bilateral tumors

Ikaros Silences T-bet Expression and Interferon-γ Production during T Helper 2 Differentiation*

CD4+ T cells can be instructed by nonantigen-specific signals to differentiate into functionally distinct lineages with mutually exclusive patterns of cytokine production. The molecular events that drive interferon-γ (IFNγ) production during Th1 development are well understood, but mechanisms that silence this cytokine during Th2 polarization are not clear. In this study, we find that the tbx21 gene encoding the Th1 master regulator T-bet is a direct target of the transcriptional repressor Ikaros. In Th2 cells, which do not express T-bet, strong Ikaros binding could be detected at the endogenous tbx21 promoter, whereas this gene was not occupied by Ikaros in T-bet-expressing Th1 cells. Inhibition of Ikaros DNA binding activity during Th2 polarization resulted in loss of Ikaros promoter occupancy, increased T-bet expression, and inappropriate T-bet-dependent production of IFNγ. Ikaros was also required for epigenetic imprinting of the ifnγ locus during Th2 polarization, and loss of Ikaros function in vivo led to an inappropriate Th1 response to the parasite Shistosoma mansoni. These studies demonstrate that Ikaros, a factor with an established role in lymphocyte development, also regulates the development of peripheral T helper responses

Differential alterations of the genes in the CDKN2A-CCND1-CDK4-RB1 pathway are associated with the development of head and neck squamous cell carcinoma in Indian patients

Purpose: The aim of this study was to analyse the alterations of the genes in the CDKN2A/CCND1/CDK4/RB1 pathway in the G1-S phase of the cell cycle during development of head and neck squamous cell carcinoma (HNSCC). Methods: The alterations of these genes were analysed in 22 dysplastic lesions, 26 stage-I/II and 33 stage-III/IV HNSCC tumours of Indian patients. Results: The alterations [mutation, hypermethylation, homozygous deletion and loss of heterozygosity/microsatellite size alteration (LOH/MA)] in the CDKN2A were found to be highest in 57% of the samples, followed by CCND1 amplification and LOH/MA at the RB1 locus in 14% and 8.5% of the samples, respectively. No dominant CDK4 Arg24Cys mutation was seen in our samples. Comparatively high frequency of CDKN2A alterations (except homozygous deletion) was found in dysplastic head and neck lesions and remained almost constant or increased during progression of the tumour, whereas the homozygous deletion of CDKN2A and the alterations in CCND1 and RB1 genes were seen mainly in the later stages of the tumour. Conclusions: Our study suggested that mutation/hypermethylation/allelic alterations (LOH/MA) of CDKN2A were associated with the development of dysplastic head and neck lesions. All the other alterations might provide some cumulative effect during progression of later stages of the tumour to have selective growth advantages

Reduced cytokine production by p18^ink4c-deficient T cells.

<p>T cells from p18^ink4c+/+ (dark gray symbols) or p18^ink4c−/− (light gray symbols) mice were stimulated with soluble anti-CD3 Ab (5 µg/mL) or anti-CD3 plus CTLA-4Ig (10 µg/mL). IL-2 (<b>A</b> and <b>B</b>) and IFNγ (<b>C</b>) in the supernatants of primary cultures was measured by ELISA at the indicated time points in <b>A</b>, at 24 hours in <b>B</b>, and at 72 hours in <b>C</b>. CD4+ T cells purified from the primary cultures in <b>A–C</b> were rested, restimulated with plate-bound anti-CD3 (1 µg/mL), and IL-2 secretion was measured by ELISA at 18 hours post-stimulation (<b>D</b>). Data are plotted as the mean +/−SEM of duplicate cultures, and are representative of 2–3 independent experiments.</p