Telomere Shortening Occurs in Subsets of Normal Breast Epithelium as well as in Situ and Invasive Carcinoma
2004; Elsevier BV; Volume: 164; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63180-x
ISSN1525-2191
AutoresAlan K. Meeker, Jessica Hicks, Edward Gabrielson, William M. Strauss, Angelo M. De Marzo, Pedram Argani,
Tópico(s)Skin Protection and Aging
ResumoIn the setting of inactivated DNA damage-sensitive checkpoints, critically shortened telomeres promote chromosomal instability and the types of widespread cytogenetic alterations that characterize most human carcinomas. Using a direct telomere fluorescence in situ hybridization technique, we analyzed 114 invasive breast carcinomas, 29 carcinoma in situ lesions, 10 benign proliferative lesions, and different normal epithelial components of the male and female breast. We found marked telomere shortening in the majority (52.5%) of invasive carcinomas; smaller subsets of invasive carcinoma demonstrated moderate telomere shortening (17.5%) or normal telomere lengths (21%), while a small subgroup (5%) contained elongated telomeres. Strikingly, the majority (78%) of ductal carcinoma in situ demonstrated markedly or moderately shortened telomeres. Surprisingly, unlike all other normal epithelia studied to date, moderate telomere shortening was observed in benign secretory cells in approximately 50% of histologically-normal terminal duct lobular units (from which most breast cancer is thought to arise), while such shortening was not seen in myoepithelial cells or normal large lactiferous ducts of the female breast or male breast ducts (from which breast cancer infrequently arises). We postulate that such shortening is the result of hormonally driven, physiological proliferation, and may delineate a population of epithelial cells at risk for subsequent malignant transformation. In the setting of inactivated DNA damage-sensitive checkpoints, critically shortened telomeres promote chromosomal instability and the types of widespread cytogenetic alterations that characterize most human carcinomas. Using a direct telomere fluorescence in situ hybridization technique, we analyzed 114 invasive breast carcinomas, 29 carcinoma in situ lesions, 10 benign proliferative lesions, and different normal epithelial components of the male and female breast. We found marked telomere shortening in the majority (52.5%) of invasive carcinomas; smaller subsets of invasive carcinoma demonstrated moderate telomere shortening (17.5%) or normal telomere lengths (21%), while a small subgroup (5%) contained elongated telomeres. Strikingly, the majority (78%) of ductal carcinoma in situ demonstrated markedly or moderately shortened telomeres. Surprisingly, unlike all other normal epithelia studied to date, moderate telomere shortening was observed in benign secretory cells in approximately 50% of histologically-normal terminal duct lobular units (from which most breast cancer is thought to arise), while such shortening was not seen in myoepithelial cells or normal large lactiferous ducts of the female breast or male breast ducts (from which breast cancer infrequently arises). We postulate that such shortening is the result of hormonally driven, physiological proliferation, and may delineate a population of epithelial cells at risk for subsequent malignant transformation. Telomeres cap the ends of each human chromosome, protecting against chromosome fusions and preventing chromosome termini from being recognized as double-stranded DNA breaks.1Blasco MA Telomerase beyond telomeres.Nat Rev Cancer. 2002; 2: 627-633Crossref PubMed Scopus (153) Google Scholar, 2Kim SH Kaminker P Campisi J Telomeres, aging and cancer: in search of a happy ending.Oncogene. 2002; 21: 503-511Crossref PubMed Google Scholar, 3Hackett JA Greider CW Balancing instability: dual roles for telomerase and telomere dysfunction in tumorigenesis.Oncogene. 2002; 21: 619-626Crossref PubMed Google Scholar, 4Autexier C Greider CW Telomerase and cancer: revisiting the telomere hypothesis.Trends Biochem Sci. 1996; 21: 387-391Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 5Artandi SE DePinho RA A critical role for telomeres in suppressing and facilitating carcinogenesis.Curr Opin Genet Dev. 2000; 10: 39-46Crossref PubMed Scopus (252) Google Scholar, 6Gisselsson D Jonson T Petersen A Strombeck B Dal Cin P Hoglund M Mitelman F Mertens F Mandahl N Telomere dysfunction triggers extensive DNA fragmentation and evolution of complex chromosome abnormalities in human malignant tumors.Proc Natl Acad Sci USA. 2001; 98: 12683-12688Crossref PubMed Scopus (354) Google Scholar, 7Artandi SE Chang S Lee SL Alson S Gottlieb GJ Chin L DePinho RA Telomere dysfunction promotes non-reciprocal translocations and epithelial cancers in mice.Nature. 2000; 406: 641-645Crossref PubMed Scopus (943) Google Scholar Human telomeres are dynamic structures composed of 1000–2000 tandem repeats of the hexanucleotide TTAGGG complexed to a variety of proteins. With continued cell division (the "end-replication problem") and/or oxidative damage,8von Zglinicki T Oxidative stress shortens telomeres.Trends Biochem Sci. 2002; 27: 339-344Abstract Full Text Full Text PDF PubMed Scopus (1935) Google Scholar telomeres progressively shorten until one or more telomeres become dysfunctional, which normally triggers apoptosis or cell cycle arrest. In this fashion, telomeres are thought to function as tumor-suppressive biological clocks in normal somatic cells, placing an upper limit on cell division to avoid critically shortened telomeres that would otherwise lead to end-to-end chromosome fusions and structural alterations that are associated with malignancy. Germ cells and approximately 85% to 90% of human cancers circumvent this growth limitation because they express telomerase, a reverse transcriptase that adds single-stranded telomere repeats to chromosome ends. Interestingly, most human epithelial cancers (carcinomas) possess short telomere lengths despite their having telomerase activity.9Hastie ND Dempster M Dunlop MG Thompson AM Green DK Allshire RC Telomere reduction in human colorectal carcinoma and with ageing.Nature. 1990; 346: 866-868Crossref PubMed Scopus (1504) Google Scholar, 10Furugori E Hirayama R Nakamura KI Kammori M Esaki Y Takubo K Telomere shortening in gastric carcinoma with aging despite telomerase activation.J Cancer Res Clin Oncol. 2000; 126: 481-485Crossref PubMed Scopus (43) Google Scholar, 11Mehle C Ljungberg B Roos G Telomere shortening in renal cell carcinoma.Cancer Res. 1994; 54: 236-241PubMed Google Scholar, 12Takagi S Kinouchi Y Hiwatashi N Chida M Nagashima F Takahashi S Negoro K Shimosegawa T Toyota T Telomere shortening and the clinicopathologic characteristics of human colorectal carcinomas.Cancer. 1999; 86: 1431-1436Crossref PubMed Scopus (44) Google Scholar While telomere dysfunction may play a role in the genesis of the widespread genetic alterations and complex karyotypes typical of carcinomas, it is thought that the acquired telomerase activity of carcinoma cells allows stabilization of the genetic instability effected by short telomeres.1Blasco MA Telomerase beyond telomeres.Nat Rev Cancer. 2002; 2: 627-633Crossref PubMed Scopus (153) Google Scholar, 2Kim SH Kaminker P Campisi J Telomeres, aging and cancer: in search of a happy ending.Oncogene. 2002; 21: 503-511Crossref PubMed Google Scholar Prior studies evaluating telomere lengths in human breast cancers have used bulk assessment techniques of whole tissues; such as Southern blot telomere restriction fragment (TRF) analysis or slot blot analysis.13Rogalla P Rohen C Bonk U Bullerdiek J Telomeric repeat fragment lengths are not correlated to histological grading in 85 breast cancers.Cancer Lett. 1996; 106: 155-161Abstract Full Text PDF PubMed Scopus (18) Google Scholar, 14Rha SY Park KH Kim TS Yoo NC Yang WI Roh JK Min JS Lee KS Kim BS Choi JH Lim HY Chung HC Changes of telomerase and telomere lengths in paired normal and cancer tissues of breast.Int J Oncol. 1999; 15: 839-845PubMed Google Scholar, 15Griffith JK Bryant JE Fordyce CA Gilliland FD Joste NE Moyzis RK Reduced telomere DNA content is correlated with genomic instability and metastasis in invasive human breast carcinoma.Breast Cancer Res Treat. 1999; 54: 59-64Crossref PubMed Scopus (66) Google Scholar, 16Odagiri E Kanada N Jibiki K Demura R Aikawa E Demura H Reduction of telomeric length and c-erbB-2 gene amplification in human breast cancer, fibroadenoma, and gynecomastia. Relationship to histologic grade and clinical parameters.Cancer. 1994; 73: 2978-2984Crossref PubMed Scopus (85) Google Scholar These methods are confounded by variable amounts of contaminating normal tissue, which itself is heterogeneous with respect to the proportion of fibrous and fatty stroma, lymphocytic infiltration, and epithelium that it contains. Not surprisingly, these studies have yielded conflicting results; while most have found that breast cancer samples contain shorter telomeres than normal breast tissue, studies disagree as to whether there is a correlation with grade and other predictive or prognostic factors.13Rogalla P Rohen C Bonk U Bullerdiek J Telomeric repeat fragment lengths are not correlated to histological grading in 85 breast cancers.Cancer Lett. 1996; 106: 155-161Abstract Full Text PDF PubMed Scopus (18) Google Scholar, 16Odagiri E Kanada N Jibiki K Demura R Aikawa E Demura H Reduction of telomeric length and c-erbB-2 gene amplification in human breast cancer, fibroadenoma, and gynecomastia. Relationship to histologic grade and clinical parameters.Cancer. 1994; 73: 2978-2984Crossref PubMed Scopus (85) Google Scholar Additionally, no study has assessed telomere lengths in the precursors to breast carcinoma, so the timing of telomere length alterations in the evolution of breast carcinoma has not been determined. Recently, we have used an in situ method that allows telomere length assessments in archival material, with single cell resolution in intact tissue architecture.17Meeker AK Gage WR Hicks JL Simon I Coffman JR Platz EA March GE De Marzo AM Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining.Am J Pathol. 2002; 160: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 18Meeker AK Hicks JL Platz EA March GE Bennett CJ Delannoy MJ De Marzo AM Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis.Cancer Res. 2002; 62: 6405-6409PubMed Google Scholar, 19van Heek NT Meeker AK Kern SE Yeo CJ Lillemoe KD Cameron JL Offerhaus GJ Hicks JL Wilentz RE Goggins MG De Marzo AM Hruban RH Maitra A Telomere shortening is nearly universal in pancreatic intraepithelial neoplasia.Am J Pathol. 2002; 161: 1541-1547Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar This method correlates well with Southern blot TRF analysis. Using this novel technique, we now report the first in situ assessment of telomere length in human breast tissues. Surgical specimens were routinely fixed in 10% neutral buffered formalin and subjected to standard processing and paraffin embedding. The protocol for combined staining of telomeric DNA (FISH probe) and immunostaining was performed without protease digestion, as previously described.17Meeker AK Gage WR Hicks JL Simon I Coffman JR Platz EA March GE De Marzo AM Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining.Am J Pathol. 2002; 160: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar Briefly, deparaffinized 4-μm thick slides underwent heat-induced antigen retrieval followed by hybridization with a Cy3-labeled, telomere-specific peptide nucleic acid (PNA) probe having the sequence (N terminus to C terminus) CCCTAACCCTAACCCTAA with an N-terminal covalently linked Cy3 fluorescent dye (Applied Biosystems, Framingham, MA). The slides were then processed for indirect immunofluorescence using a polyclonal rabbit anti-smooth-muscle actin primary antibody (Dako, Carpinteria, CA; catalog number m0851), followed by application of a 1:100 dilution of a goat anti-rabbit IgG fraction Alexa Fluor 488 (Molecular Probes, Eugene, OR; catalog number A-11034), primarily to help distinguish myoepithelial (ME) cells (actin-positive) from adjacent secretory cells or carcinoma cells (actin-negative). Slides were counterstained with DAPI (4′−6-diamidino-2-phenylindole; Sigma Chemical Co., St. Louis, MO). Serial adjacent hematoxylin and eosin (H&E)-stained reference slides were used as a guide during simultaneous examination of the TELI-FISH slides for telomere length assessment. Telomeric staining produced a speckled pattern of widely distributed nuclear signals in all cases examined, in keeping with results previously reported for mammalian somatic cells.17Meeker AK Gage WR Hicks JL Simon I Coffman JR Platz EA March GE De Marzo AM Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining.Am J Pathol. 2002; 160: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar Telomere lengths were evaluated by visual assessment of the fluorescent intensities of the telomeric signals, which are proportional to the length of telomeric TTAGGG DNA repeats.17Meeker AK Gage WR Hicks JL Simon I Coffman JR Platz EA March GE De Marzo AM Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining.Am J Pathol. 2002; 160: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar Telomeres were directly compared to those within normal-appearing epithelium within the same tissue section. When normal-appearing epithelium was lacking, comparisons were made using adjacent ME cells and normal stroma. Rare cases in which detectable telomere signals were not found in normal stromal cells (fibroblasts, endothelial cells), or in which excessive autofluoresence precluded assessment of telomere signals, were excluded from the study. Digital fluorescent telomere signals in histologically normal breast terminal duct lobular units (TDLU) were quantitated using a semi-automated algorithm written with the image analysis software package IPLabs (Scanalytics, Inc., Fairfax, VA) and Microsoft Excel, as described previously.17Meeker AK Gage WR Hicks JL Simon I Coffman JR Platz EA March GE De Marzo AM Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining.Am J Pathol. 2002; 160: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar Ten to 20 representative secretory cell and ME cell nuclei per case were quantified. Nuclei were considered ME if their location was basal in the TDLU and their cytoplasm was diffusely labeled by actin. Nuclei were considered of secretory cell origin if their cytoplasm did not label at all with the actin antibody and they faced the lumen of the gland. Differences in mean telomere lengths between matched ME cells and luminal cells were tested for statistical significance by using t-test, with P values <0.01 considered significant. Each lesion was scored by each of two authors (P.A., A.K.M.), and a consensus assessment was reached when there was a discrepancy. After discovering the variable intensity of normal secretory cells (see Results), we adopted the following semiquantitative scoring system. Lesions with normal telomere intensity had signals comparable to those of normal stromal fibroblasts, endothelial cells, or normal ME cells. Lesions with (moderately) short telomeres had telomere intensities appreciably dimmer than the normal stroma, but which were still readily detectable and within the range of the intensity seen in some histologically normal secretory cells. Lesions with very short telomeres demonstrated signals so dim that they were barely perceptible, and dimmer than that of virtually any normal epithelial cell we encountered in this study. Lesions with long telomeres demonstrated telomere signals that were appreciably brighter than that of the stromal cells, and close to that of lymphocytes, which gave consistently bright signals. Lesions in which the signal varied between cells from bright to short, or very short to normal (ie, lesions with telomere signals spanning two scoring categories) were classified as heterogeneous for telomere length. To rule out differences in probe penetration or target accessibility as potential sources of observed differences in fluorescent telomere signal intensities in fixed tissue samples, we used a second fluorescently labeled PNA probe with specificity for centromeric DNA repeats.20Chen C Hong YK Ontiveros SD Egholm M Strauss WM Single base discrimination of CENP-B repeats on mouse and human Chromosomes with PNA-FISH.Mamm Genome. 1999; 10: 13-18Crossref PubMed Scopus (43) Google Scholar These control hybridizations were performed on serial sections under identical conditions as described above using a 5′ rhodamine-labeled PNA probe having the sequence ATTCGTTGGAAACGGGA with specificity for centromeric CENP-B DNA repeats.20Chen C Hong YK Ontiveros SD Egholm M Strauss WM Single base discrimination of CENP-B repeats on mouse and human Chromosomes with PNA-FISH.Mamm Genome. 1999; 10: 13-18Crossref PubMed Scopus (43) Google Scholar Anaphase bridges were scored with the DNA-specific DAPI stain; criteria for anaphase bridges required a well-separated parallel anaphase plate displaying a perpendicularly aligned amphophilic ("stretched") connecting filament. Statistical comparison on the frequency of anaphase bridges between regions with either short or long telomeres was conducted by a test of proportions for two independent groups. In all breast samples examined microscopically, normal stromal fibroblasts and endothelial cells uniformly demonstrated strong telomeric staining intensity, in agreement with previous results.17Meeker AK Gage WR Hicks JL Simon I Coffman JR Platz EA March GE De Marzo AM Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining.Am J Pathol. 2002; 160: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 18Meeker AK Hicks JL Platz EA March GE Bennett CJ Delannoy MJ De Marzo AM Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis.Cancer Res. 2002; 62: 6405-6409PubMed Google Scholar, 19van Heek NT Meeker AK Kern SE Yeo CJ Lillemoe KD Cameron JL Offerhaus GJ Hicks JL Wilentz RE Goggins MG De Marzo AM Hruban RH Maitra A Telomere shortening is nearly universal in pancreatic intraepithelial neoplasia.Am J Pathol. 2002; 161: 1541-1547Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar This intensity was readily identifiable at low power (magnification, ×200), and was appreciably less than that of admixed stromal lymphocytes, which invariably displayed very strong telomere signals. Also in keeping with previous results, the intensity of the non-lymphocyte stromal labeling was then taken as the internal reference control to which other cells were compared. This approach has been taken by our group and by others using similar methodology.21O'Sullivan JN Bronner MP Brentnall TA Finley JC Shen WT Emerson S Emond MJ Gollahon KA Moskovitz AH Crispin DA Potter JD Rabinovitch PS Chromosomal instability in ulcerative colitis is related to telomere shortening.Nat Genet. 2002; 32: 280-284Crossref PubMed Scopus (294) Google Scholar We first examined the TDLU, which are the milk-producing component of the breast and the postulated site of origin for most breast cancers,22Rosen PP Rosen's Breast Pathology. Lippincott Williams and Wilkins, Philadelphia2001Google Scholar, 23Page DL Anderson TJ Diagnostic Histopathology of the Breast. Churchill Livingstone, Edinburgh1987Google Scholar in 12 benign breast biopsies from women who had no evidence of cancer elsewhere in the breast. These biopsies were performed in most cases for macromastia, and histologically these samples showed no specific pathological changes. In all 12 cases, the normal outer ME cells, which were clearly delineated by the actin stain, demonstrated intense telomere signals, comparable to that of the normal stroma. In five of these cases, the inner secretory cells within these TDLU uniformly demonstrated comparable intensities to those of the adjacent ME cells (Figure 1, A and B). However, in three cases, the secretory cells demonstrated clearly dimmer signals than the adjacent ME cells as judged by visual inspection (Figure 1, C and D). In four other cases, there was marked variation in secretory cell telomere staining intensity, with some cells being comparable to the ME cells and other adjacent secretory cells in the same TDLU being appreciably dimmer (Figure 1, E and F). We then examined benign breast epithelium in patients with benign proliferative disorders and breast tissue from younger patients. Among 10 breast biopsies demonstrating proliferative fibrocystic changes (age range: 29 to 71 years), two showed foci of luminal cell telomere shortening within the fibrocystic changes. Of note, the three cases in this group which showed florid usual duct hyperplasia retained strong telomere signals in these areas. Examination of the normal TDLU found within 6 of these 10 biopsies revealed focal evidence of telomere shortening in all 6 cases. Two of seven breast biopsies from women less than 20 years of age demonstrated focal luminal cell shortening within histologically normal TDLU. Four of these patients had fibroadenomas, one of which demonstrated focal shortening of telomeres within luminal cells. The remaining three fibroadenomas demonstrated comparable signals in their ME and luminal cells. We obtained similar results when examining secretory cells from histologically normal TDLU of women with cancer elsewhere in the breast. Among 12 such cases evaluated, three demonstrated normal (ME-like) telomeres, seven demonstrated shorter telomeres than those of the ME cells, while two cases showed marked variation in secretory cell telomere staining intensity. Using digital image analysis, we quantitated telomere lengths in TDLU from six representative cases. By visual inspection, two of these cases were judged to have clearly diminished signals in the luminal cells as compared to the ME cells. Image analysis confirmed this marked difference (Figure 2, cases 5 and 6) and indicated that it was highly statistically significant (P < 0.0000001). The remaining four cases were judged to have comparable telomere signals in both secretory and ME cells. Interestingly, in three of the four cases, image quantification indicated a small, statistically significant (P < 0.01) decrease in luminal telomeric signal intensity that was not detectable by eye (Figure 2, cases 1 to 4). In contrast, we did not find luminal cell telomere shortening to be frequent in male ducts or female lactiferous ducts. First, 15 of 15 benign lactiferous ducts in women with cancer elsewhere (14 carcinoma, one angiosarcoma; patient age range: 31 to 86 years) in the breast demonstrated equally intense telomere signals in both luminal and ME cells (Figure 3). Similar results were obtained in the large ducts of 5 of 5 male breast tissues removed for gynecomastia (patient age range: 17 to 63 years). Interestingly, in the single male gynecomastia case in which true lobules were formed in the male breast, focal luminal cell telomere shortening was appreciated specifically within these lobules. In all nine cases of pure ductal carcinoma in situ (DCIS) without invasive carcinoma examined, the ME cells retained their usual intense telomeric signals. In one of these cases, the telomere intensity of the DCIS cells was comparable to that of the ME cells. However, in four cases, the DCIS cells demonstrated very short telomeres that were barely detectable with the telomere-specific probe, while in the four remaining cases, the DCIS telomere signals were moderately short. In comparison to their respective surrounding normal breast TDLU secretory cells, the DCIS cells had shorter telomeres in 7 of 9 cases. We next examined the DCIS components adjacent to 14 cases of invasive ductal carcinoma (IDC). Once again, in all of these cases, the retained normal ME cells contained robust telomere signals. In three cases, the DCIS cells had moderately short telomeres, while in seven cases the DCIS had very short (undetectable) telomeres (Figure 4). In one other case, the DCIS featured heterogeneous telomere lengths. Comparing the DCIS to their adjacent invasive ductal carcinomas, the DCIS was comparable to the IDC in telomere length in 11 of 13 cases. In the two other cases, the IDC featured shorter telomere lengths than the adjacent DCIS; these two DCIS lesions had normal-length telomeres. There was a tendency for short telomeres to be associated with high nuclear grade among the DCIS. Of 11 DCIS with very short (virtually undetectable) telomeres, eight were grade 3, while three were grade 2. Among seven DCIS with short telomeres, five were grade 3, one was grade 2, while one was grade 1. Among four DCIS with telomere lengths comparable to those of the ME cells, two were grade 1, one was grade 2, while one was grade 3. The one case with heterogeneous telomere lengths was nuclear grade 3 (Table 1).Table 1Telomere Lengths in DCIS as a Function of Nuclear GradeTelomere lengthGrade 1Grade 2Grade 3TotalNormal2114Short1157Very short03811Heterogeneous0011Total351523 Open table in a new tab Among six cases of lobular carcinoma in situ (LCIS) available for study, one featured very short telomeres, four featured short telomeres, while one had normal-length telomeres. We next examined 114 cases of invasive mammary carcinoma. Among these cases, 60 (52.5%) had very short (barely detectable) telomere lengths, 20 (17.5%) had short telomere lengths, and 24 (21%) had normal (ME-like) telomere lengths. six (5%) cases demonstrated abnormally long telomeres, similar in intensity to that of adjacent lymphocytes (Figure 5). In four (4%) cases, marked intratumoral telomere length heterogeneity was observed. The results of telomere length assessment in these lesions are summarized in Table 2.Table 2Summary of Telomere Length Results for Breast Luminal CellsTelomere lengthNormal lobules (n = 24)Normal lactiferous ducts (n = 15)Male breast (n = 5)Normal pediatric lobules (n = 7)Fibrocystic changes (n = 10)DCIS (n = 23)LCIS (n = 6)Invasive mammary carcinoma (n = 114)Very short0000111160 (52.5%)Short10002*Luminal shortening was focal in these cases.17420 (17.5%)Normal8155†Focal luminal telomere shortening was seen in a lobule of a male breast with gynecomastia.584124 (21%)Long00000006 (5%)Heterogeneous60000104 (4%)* Luminal shortening was focal in these cases.† Focal luminal telomere shortening was seen in a lobule of a male breast with gynecomastia. Open table in a new tab The relationship between telomere length and Elston grade of the invasive carcinoma is shown in Table 3. There appeared to be a tendency for high-grade tumors (Elston grade 3) to demonstrate non-normal (non-ME-like) telomere lengths; however, these differences were not statistically significant (data not shown). Of note, all of the cases with marked heterogeneity in telomere length and all cases with abnormally long telomeres were high-grade.Table 3Telomere Length as a Function of Elston Grade in IDCTelomere lengthGrade 1Grade 2Grade 3Very short3 (33%)20 (57%)37 (53%)Short3 (33%)6 (17%)11 (16%)Normal3 (33%)9 (26%)12 (17%)Long006 (9%)Heterogeneous004 (6%) Open table in a new tab When telomeres shorten to a critical length, telomeric fusions between chromosome ends may occur and lead to ring and dicentric chromosomes that form so-called "anaphase bridges" during mitosis. Anaphase bridges are therefore chromosomal bridges that are not resolved during anaphase,24Gisselsson D Bjork J Hoglund M Mertens F Dal Cin P Akerman M Mandahl N Abnormal nuclear shape in solid tumors reflects mitotic instability.Am J Pathol. 2001; 158: 199-206Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar and are correlated with telomere dysfunction. Among tumors with telomeres scored as either very short or short, 18 of 26 anaphases demonstrated bridging (69%). In contrast, only seven anaphases among the 68 counted in tumors with either normal length or long telomeres demonstrated anaphase bridges (10%) (Figure 6, A and C). This difference was highly statistically significant (P < 0.0001). We then evaluated an invasive tumor with heterogeneous telomere lengths that contained 5 anaphases. Three anaphases were normal, and these three cells had normal telomere lengths. The two other anaphases demonstrated anaphase bridges; these two cells had short telomeres. Hence, the presence of anaphase bridges in invasive carcinomas correlated with the presence of shortened telomeres. We were unable to find adequate mitotic figures to formally study the effect of telomere lengths on anaphase bridges in DCIS. However, we noted no anaphase bridges in seven anaphases in one case of DCIS with normal telomere lengths. In the DCIS case that showed variable telomere lengths, anaphase bridges were noted, but only in the mitoses occurring in areas with shortened telomeres (Figure 6, B and D). The FISH-based telomere length assessment method used in this study was previously validated by comparing quantitative telomere length data obtained by TELI-FISH to data obtained by an independent method of telomere length measurement (Southern blotting), using a panel of formalin-fixed paraffin-embedded human cell lines. To rule out differences in either probe penetration or target accessibility as potential sources of the differences we observe in fluorescent telomeric signal intensities in fixed tissue samples, we used a second fluorescently labeled PNA probe with specificity for centromeric DNA repeats.20Chen C Hong YK Ontiveros SD Eghol
Referência(s)