Fluorescentin situ hybridization assessment of chromosome 8 copy number in breast cancer

Summary Conventional cytogenetics of breast and other solid tumors has been hampered by a number of factors. An analysis of breast tumor tissues was therefore undertaken using fluorescentin situ hybridization (FISH). A total of 34 specimens were analyzed using a chromosome 8-specific -satellite probe. Various approaches were tested and compared. Among 30 informative samples, 11 infiltrating ductal carcinomas, not otherwise specified (NOS), 5 ductal carcinomasin situ, 5 lobular carcinomas, 3 papillary carcinomas, and 6 benign lesions were studied. Of the 11 cases of infiltrating ductal carcinomas (NOS) analyzed, four cases showed 3 signals, one case showed 4 signals, and the rest showed 2 signals. Of the 5 cases of ductal carcinomain situ samples, 1 showed 3 signals and the other 4 cases showed 2 signals. All cases of lobular carcinomas, papillary carcinomas, and benign lesions showed 2 signals. We inferred from these data that 36% of the infiltrating ductal carcinomas (NOS) were trisomic and 9% were tetrasomic, whereas 20% of the ductal carcinomasin situ were trisomic. All samples from lobular carcinomas, papillary carcinomas, and the benign lesions were disomic. From our preliminary data, it can further be concluded that a subset of breast cancer is characterized by chromosome 8 trisomy. These data are consistent with an ever-increasing database on the association of chromosomal 8 trisomy with other cancers such as leukemia, lymphoma, prostate cancer, ovarian carcinoma, salivary gland tumor, malignant melanoma, desmoid tumors, and recently gestational trophoblastic disease. It is also noted that the ability to analyze formalin-fixed, paraffin-embedded archival material will enable a more comprehensive cytogenetic study of breast cancer than is currently available.     

重组人核心蛋白聚糖基因增强多柔比星对白血病K562细胞抑制作用的实验研究

 目的　探讨慢性淋巴细胞白血病（CLL）的分子遗传学异常情况及其临床意义。方法　对17例初诊CLL患者进行常规细胞遗传学（CC）检测及应用着丝粒探针CSP12（12p11.1～12q11.1）和序列特异性探针D13s25（13q14.3）、ATM（11q22.3）、RB1（13q14）、p53（17p13.1）进行间期荧光原位杂交（I-FISH）检测。结果　CC检测18.75 %患者有核型异常,1例未见核分裂象;I-FISH检测70.6 %患者有分子遗传学异常,13q-异常47.1 %（RB1缺失23.5 %、D13S25缺失29.4 %）、+12异常29.4 %、p53基因缺失11.8 %、ATM缺失5.6 %、复杂基因组异常11.8 %。分子遗传学异常与性别、年龄、乳酸脱氢酶（LDH）、β2 -微球蛋白（β2-MG）及 Binet 分期无明显相关性。结论　I-FISH是检测CLL患者基因组异常的有效手段,与CC方法相比可明显提高CLL分子遗传学异常的检出率,分子遗传学异常与临床分期及其他临床指标无明显相关性,对患者预后的意义有待进一步研究。     

HER-2/neu amplification detected by fluorescence in situ hybridization in fine needle aspirates from primary breast cancer.

BACKGROUND: The HER-2/neu gene is amplified in 20-30% of human breast cancers and has been shown to have prognostic and predictive value for treatment with chemotherapy, hormone therapy and antibodies against the HER-2/neu domain (trastuzumab). The aim of our study was to evaluate the reliability of HER-2/neu determination by fluorescence in situ hybridization (FISH) on fine-needle aspirates (FNAs) from primary breast cancer patients by comparison with the results obtained by FISH and immunohistochemistry (IHC) on the corresponding histological sections. MATERIALS AND METHODS: HER-2/neu amplification was determined by FISH on 66 breast cancer FNAs. Twenty-three and 36 corresponding formalin-fixed, paraffin-embedded sections were assayed by FISH and by IHC, respectively, in order to detect HER-2/neu amplification and HER-2/neu protein expression. RESULTS: Twenty-seven per cent (18/66) of breast cancer FNAs showed amplification of HER-2/neu by FISH. Paired results by FISH cytology and FISH histology were available in 22 cases. Concordance was 91% (20/22). Paired results by FISH cytology and IHC were available in 36 cases. Concordance was 92% (33/36). Eighteen of 66 breast cancer FNAs were also submitted to flow cytometric DNA analysis. None of the diploid cases showed HER-2/neu amplification by FISH. Six out of the eight aneuploid cases were amplified and two were polysomic. CONCLUSIONS: HER-2/neu gene amplification can be reliably estimated by FISH on breast cancer FNAs and a good correlation has been found between FISH and IHC results from the corresponding histological sections.     

Aneusomy and tumor heterogeneity in metastatic breast cancer detected by fluorescence in situ hybridization

Direct interphase cytogenetic analysis was performed on nuclei of metastatic effusions from breast cancer using fluorescence ill situ hybridization (FISH). DNA probes specific for repetitive pericentromeric regions on chromosomes 6, 8, 17, and human midi-satellite probe specific for one locus on the short arm of chromosome 1 were used to determine chromosome copy numbers in interphase tumor cells. The analysis showed cytogenetic heterogeneity in most nuclei examined with multiple subpopulations and a range of 0-6 chromosome signals per cell. The presence of subclones in these fluids, their correlation with highly malignant phenotypes and the diagnostic and prognostic applications of this method is discussed.     

Numerical aberrations of chromosome 8 in gastric cancer detected by fluorescence in situ hybridization

BACKGROUND: Limited data are available on the genetic events underlying gastric cancer. Studying a few cases by conventional cytogenetic techniques, we previously reported that chromosome 8 might be frequently involved. The aim of our study was to evaluate the numerical aberrations of chromosome 8 in gastric cancer using fluorescence in situ hybridization (FISH). MATERIALS AND METHODS: FISH, with an a-satellite DNA probe specific for chromosome 8, was applied to 37 primary gastric tumors directly processed for cytogenetic study. RESULTS: Numerical aberrations of chromosome 8 were observed in 23 out of 37 tumors (62.16%). Trisomy was detected in 16 cases (43.24%), tetrasomy in 4 cases (10.81%) and monosomy in 3 cases (8.10%). No correlation was found between polysomy 8 and the histopathologic characteristics of the tumors. CONCLUSION: An increase in the number of chromosome 8 may frequently occur in gastric cancer. Advanced and more aggressive gastric tumors did not harbor polysomy 8. Further studies at molecular and clinical level must be carried out to identify the gene alterations reflected by polysomy 8 and possibly to facilitate the detection of specific tumors subtypes.     

Prognostic value of chromosome 1 and 8 copy number in invasive ductal breast carcinoma among iranian women: An interphase FISH analysis

Breast cancer is amongst the leading causes of death in women worldwide and the most common cancer amongst Iranian women. Unfortunately, the current clinical and histological criteria can only help 60 percent of women with breast cancer in diagnosis and long-term treatment. Therefore, genetic markers both at single gene and chromosomal level can play an important role in improving the diagnosis and prognosis of breast cancer patients. The aim of this retrospective study was to investigate the role of chromosome 1 and 8 copy number assessed by interphase fluorescence in situ hybridization (FISH), as prognostic parameters in 50 Iranian women, aged 35 to 64 years, with sporadic invasive ductal breast carcinoma. Chromosome 1 and 8 copy numbers were evaluated in relation to established clinicopathological parameters, the immunohistochemical markers ER, PR, P53 and cathepsin D, DNA index by flow cytometry, age and survival status of the patients. FISH using centromeric probes for chromosomes 1 and 8 was applied to interphase cell suspensions prepared from archived, Carnoyfixed tumor cells and selected paraffin-embedded tumor sections. Aneusomy for chromosomes 1 and 8 was present in all 50 patients to different levels. The total abnormality rate for chromosome 1 was 33.92 percent (4.24 percent monosomy and 29.68 percent polysomy), whereas for chromosome 8 this rate was 28.30 percent (6.48 percent monosomy and 21.82 percent polysomy). Statistically significant association (p<0.05) was demonstrated between monosomy 1 and patients’ age below 50 years, and between monosomy 1 and poor survival, respectively. Disomy 8 was significantly associated with P53 expression. A borderline significant correlation was demonstrated between polysomy 8 and diploid DNA content, as well as between disomy 1 and disease-free status of the patients. Chromosome 1 and 8 copy numbers may be considered as useful prognostic markers in invasive ductal carcinoma of the breast.     

Immunohistochemical markers in the identification of metastatic breast cancer

Summary Fluorescentin situ hybridization techniques have provided an important tool for interphase cytogenetic studies of human neoplasms. However, these techniques are difficult to use on formalin-fixed archival tissue sections. We describe here a non-fluorescent, non-isotopicin situ hybridization (ISH) approach that is easily applicable to paraffin-embedded breast tissue sections. The technical steps that must be monitored and individualized to optimize signal generation and detection are discussed. This ISH technique has several advantages over fluorescent detection methods. The signal obtained can be viewed using an ordinary light microscope and does not fade with time. More importantly, the signal is observed and analyzed in the context of tissue morphology. The technique permits detection of numerical chromosomal abnormalities not only in malignant but also in apparently normal and potentially premalignant mammary tissue. This may allow identification of focal genetic abnormalities as well as field-defects and enable analysis of their evolution during the multistep transformation to mammary neoplasm. This technique is also suitable for analysis of tumor heterogeneity and the correlation of numerical chromosomal aberrations with histologic, immunocytochemical, and clinical features of breast tumors.     

Numeric chromosome aberrations in prostate cancer detected by in situ hybridization

Background This retrospective study was designed to examine the applicability of in situ hybridization to the study of numeric chromosome aberrations in conventionally fixed, embedded tissue sections of human prostate cancers. Methods By use of in situ hybridization with chromosome-specific DNA probes, the copy number of pericentromeric sequences on chromosomes 7, 11, 17 X, and Y was detected within interphase nuclei in formalin-fixed and paraffin-embedded tissue specimens from 21 patients with adenocarcinoma of the prostate. The percentage of hyperdiploid cells (3 or more spots) was estimated by using light microscopy. Results The percentage of hyperdiploid cells for chromosomes 7 and 17 was highly correlated with increasing tumor Mostofi grade (P<0.05, Spearman rank correlation) or increasing Gleason score (P<0.05). The percentage of hyperdiploid cells for chromosome 11 was not correlated with either tumor Mostofi grade or Gleason score (P>0.05). Conclusion Since high tumor grade is indicative of more aggressive tumor behavior and a worse prognosis, these findings suggest that the percentage of hyperdiploid cells may be highly predictive of prostate tumor aggressiveness. Our preliminary results suggest that measurement of numeric chromosome aberrations using in situ hybridization in prostate cancer may serve as a predictive factor for the prognosis of prostate cancer.     

Osteoblastic metastatic disease as a therapeutic response to adjuvant chemotherapy in breast cancer

Included in this study were 43 breast cancer patients treated with adjuvant chemotherapy who presented metastasis only in bone. Forty-two percent had metastasis during chemotherapy, and 58% had metastasis after the completion of chemotherapy. In 66% of the patients, the lesions were osteolytic. Twenty-nine percent had osteoblastic lesions, and 5% had mixed lesions. Of the patients with osteoblastic metastatic disease, five showed asymptomatic, osteoblastic disease; this response was considered to be a healing response to chemotherapy. These five patients were continued on the same therapy. The median duration of this response to the adjuvant chemotherapy was 29 months (range 6 to 62+). In one patient, osteoblastic disease gradually faded, and skeletal radiographs reverted to normal.     

间期荧光原位杂交技术(FISH)在急性淋巴细胞性白血病(ALL)8号染色体三体检测中的应用

目的　探讨间期荧光原位杂交 (FISH)技术对于ALL三体 8检测的价值。方法　采用荧光素SpectrumRed直接标记 8号染色体着丝粒探针 ,检测了 5 9例ALL病例和 8例正常对照组骨髓细胞 ,并与细胞遗传学 (CG)结果相比较。结果　 5 9例ALL病例中 ,8例FISH检测为三体 8阳性 ;CC检测仅 3例为阳性。结论　间期FISH对ALL三体 8检测有重要意义 ,是CG的重要补充。     

Deletion of chromosome 17p loci in breast cancer cells detected by fluorescence in situ hybridization.

Allelic loss of tumor suppressor genes on chromosome 17p has been implicated in the progression of breast cancer. This is in principle detectable by fluorescence in situ hybridization if the loss occurs by deletion. In order to determine if detectable deletions occur in primary breast cancer, we used dual-color hybridization with chromosome 17 pericentromeric and region-specific DNA probes to study 19 primary breast cancers. The copy numbers of 17 centromere and 17p13.1 sequences were compared with the loss of heterozygosity (LOH) for probe YNZ22 at 17p13.3 detected by restriction fragment length polymorphism. Nine of 11 cases showing LOH also showed the major population of nuclei with a deletion. The remaining two tumors with LOH were trisomic for both the centromere and 17p13.1 cosmid. In contrast, seven of eight tumors without LOH had no deletions by fluorescence in situ hybridization. These data suggest that the dominant mechanism of allelic loss at 17p in breast cancer is a physical deletion and that analysis of deletions by fluorescence in situ hybridization is a rapid and sensitive approach to studying chromosomal aberrations.     

Male breast cancer,age and sex chromosome aneuploidy

Background:

In cultured, dividing transformed T lymphocytes and in dividing bone marrow cells from normal men and those with a haematological malignancy, sex chromosome aneuploidy has been found to increase in prevalence and degree with age. This has rarely been investigated in non-dividing uncultured blood samples. The loss and gain of the X chromosome in dividing transformed lymphocytes in women with age is much more frequent than that of the Y chromosome in males. However, paradoxically X chromosome aneuploidy is rarely seen in the dividing cells of bone marrow of females.

Methods:

In blood samples from 565 men with breast cancer and 54 control men from the England and Wales general population, 80 cell nuclei per sample were scored for presence of X and Y chromosomes using fluorescent centromeric probes.

Results:

Sex chromosome aneuploidy, largely Y chromosome loss, was present in 63% of cases and 57% of controls, with the prevalence and degree of aneuploidy increasingly sharply and highly significantly with age. At ages 65–80 years, 71% of cases and 85% of controls showed aneuploidy and 15% and 25%, respectively, had ⩾10% of cells aneuploid. Allowing for age, aneuploidy was less prevalent (P=0.03) in cases than controls.

Conclusion:

Sex chromosome aneuploidy in non-dividing nuclei of peripheral blood cells is frequent in adult men, the prevalence and degree increasing sharply with age. The possible relation of sex chromosome aneuploidy to breast cancer risk in men, and to cancer risk generally, needs further investigation, ideally in cohort studies.     

复发转移性三阴性乳腺癌临床特征及总生存分析

目的：分析复发转移性三阴性乳腺癌患者的治疗方式及总生存情况。方法回顾性分析复发转移后来我院治疗,并随访至病亡的78例三阴性乳腺癌患者临床资料,根据其临床特征、复发情况及治疗方式评价其总生存情况。结果78例患者总生存时间（OS）为6～236个月,中位OS为32.1个月。1年生存率92.3%,5年生存率28.2%,10年生存率6.4%。71例（91.0%）三阴性乳腺癌患者行根治术或改良根治手术,其无病生存时间（DFS）为1～184个月,中位DFS为15.0个月,7例（9.0%）患者为初治Ⅳ期。Ⅰ期三阴性乳腺癌患者OS为19.7～236个月,中位OS为90.0个月。复发转移后OS为3.0～93.3个月,中位OS为14.4个月。13例（16.7%）患者局部复发或单纯部位转移的患者行局部手术治疗联合全身治疗者中位OS为26.5月,65例（83.3%）仅行全身治疗者中位OS为12.2个月（P=0.034）。一线化疗方案含紫杉类药物的患者中位OS为14.6个月,未予紫杉类药物的患者中位OS为11.0个月（P=0.048）。结论复发转移性三阴性乳腺癌整体预后较差,生存期短,但异质性较高,且早期三阴性乳腺癌患者具有明显的生存优势。值得一提的是本文只针对已经有生存节点的患者进行分析,这组患者乳腺癌的恶性程度相对较高。也间接提示三阴性乳腺癌的治疗也不能一概而论。对这些早期复发的患者,目前的辅助治疗可能是不足够的。     

8p deletion is strongly linked to poor prognosis in breast cancer

Deletions of chromosome 8p occur frequently in breast cancers, but analyses of its clinical relevance have been limited to small patient cohorts and provided controversial results. A tissue microarray with 2,197 breast cancers was thus analyzed by fluorescence in-situ hybridization using an 8p21 probe in combination with a centromere 8 reference probe. 8p deletions were found in 50% of carcinomas with no special type, 67% of papillary, 28% of tubular, 37% of lobular cancers and 56% of cancers with medullary features. Deletions were always heterozygous. 8p deletion was significantly linked to advanced tumor stage (P < 0.0001), high-grade (P < 0.0001), high tumor cell proliferation (Ki67 Labeling Index; P < 0.0001), and shortened overall survival (P < 0.0001). For example, 8p deletion was seen in 32% of 290 grade 1, 43% of 438 grade 2, and 65% of 427 grade 3 cancers. In addition, 8p deletions were strongly linked to amplification of MYC (P < 0.0001), HER2 (P < 0.0001), and CCND1 (p = 0.001), but inversely associated with ER receptor expression (p = 0.0001). Remarkably, 46.5% of 8p-deleted cancers harbored amplification of at least one of the analyzed genes as compared to 27.5% amplifications in 8p-non-deleted cancers (P < 0.0001). In conclusion, 8p deletion characterizes a subset of particularly aggressive breast cancers. As 8p deletions are easy to analyze, this feature appears to be highly suited for future DNA based prognostic breast cancer panels. The strong link of 8p deletion with various gene amplifications raises the possibility of a role for regulating genomic stability.     

HER-2 status discrepancy between primary breast cancer and metastatic sites. Impact on target therapy

In this prospective study, we determined HER-2 status in primary breast invasive carcinomas and in the paired lymph node metastases (synchronous and metachronous), local recurrence and metachronous distant metastases, to verify the percentage of discordant cases. HercepTest and Fluorescence in situ hybridization (FISH) were used to determine HER-2 status on 119 cases of primary infiltrating breast carcinoma and paired metastases (45 cases with synchronous lymph node metastases, 9 cases with metachronous lymph node metastases, 30 cases with local recurrence, and 35 cases with metachronous distant metastases). A therapeutically significant HER-2 status discordance was demonstrated between primary carcinoma and synchronous lymph node metastases (6.7%), local recurrence (13.3%) and metachronous distant metastases (28.6%). In the first comparison, there was a normal HER-2 status in primary tumours and HER-2 amplification in paired metastases, in the second the opposite phenomenon was present, and both types of discordance were evident in the third comparison. Considering the cases of local recurrences and metachronous distant metastases all together, 14 out of 65 cases (21.5%) showed a therapeutically significant discordance of HER-2 status between the primary tumour and the paired metachronous recurrence or metastasis (p < 0.001), the 15.4% of cases showing normal HER-2 status in the primary tumour and HER-2 amplification in the neoplastic relapse. For the treatment of metastatic patients, the evaluation of HER-2 status should be performed in neoplastic tissue from metastatic site, whenever possible. This procedure could be also suggested in the patients that are metastatic at the time of diagnosis.     

Immunohistochemical detection of ornithine-decarboxylase in primary and metastatic human breast cancer specimens

Increased ornithine decarboxylase (ODC) activity in human breast cancer specimens has recently been shown to be an independent adverse prognostic factor for recurrence and death. Biochemical measurement of ODC, however, is not practical for routine clinical use. Furthermore, it does not take into account the heterogeneous composition of human breast cancers which contain variable proportions of epithelial and stromal elements. Therefore, we developed an immunohistochemical method for ODC determination which can be applied to formalin-fixed, paraffin-embedded tissue sections. We report here our results in a series of 30 human breast cancer samples. ODC expression was detected most consistently in the malignant epithelial component of the tumors. Twenty-seven of 30 samples stained positive with intensities ranging from 1+ to 3+. The fraction of malignant epithelial cells expressing ODC varied among specimens between 10% and >90%. When quantitated by H-SCORE, ODC expression was significantly higher in the malignant epithelial component than in normal appearing epithelial cells and stroma admixed within the tumor. Normal mammary tissue adjacent to the cancer was available for analysis in six cases. ODC expression was absent in two (while both cancers were positive) but present in four to a degree which was overall comparable to that observed in the corresponding tumors. We believe that this technique will be useful for future studies aimed at expanding our knowledge of the role of ODC and polyamines (PA) in breast cancer biology.     

The prognostic impact of loss of chromosome 7 material detected by fluorescence in situ hybridization (FISH) in myeloid malignancies

Background

Monosomy 7 (?7) or deletion in its long arm [del(7q)] is among the most common chromosomal abnormalities in myeloid malignancies. There are prognostic variations between ?7 and del(7q) in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).