Quantitative FISH by image cytometry for the detection of chromosome 1 imbalances in breast cancer: a novel approach analyzing chromosome rearrangements within interphase nuclei

Interphase cytogenetics have become a widespread tool for investigation of chromosome rearrangements in solid tumors. The most recurrent chromosome alteration within breast cancer affects chromosome 1, leading principally to gain of the long arm and/or loss of the short arm. We have developed a new method for detection of chromosome 1 arm imbalances in interphase nuclei. The method is based on quantitation of the fluorescence signals emitted by the hybridized two-color paintings of the short and long arms using image cytometry. The chromosome arm imbalance was determined by calculating the ratio of both fluorescence emissions of each arm. The ratio of the paintings of normal lymphocytes was used as a reference. Three breast cancer cell lines, 13 fresh tumor samples, and 6 fine-needle samplings of breast cancer were analyzed using an automated image cytometer. Whenever possible, classic cytogenetics and in situ hybridization on metaphases were performed as controls. Fluorescence ratios representing the imbalances of chromosome 1 arms with values between 1 and 3.2 were measured. Data between classic cytogenetics and interphase cytogenetics were well-correlated (r = 0.89). This method, which enables an easy detection of intrachromosomal imbalances without need of metaphase preparations, detects malignant cells and can be extended to other carcinomas for which chromosome 1 arm imbalances are recurrent or chromosome alterations specific of other malignancies. In comparison to other interphase fluorescence in situ hybridization techniques, it avoids every spot scoring problem encountered when using centromeric probes and the difficulties in interpreting structural rearrangements.     

Quantitation of HER-2/neu and c-myc gene amplification in breast carcinoma using fluorescence in situ hybridization

HER-2/neu and c-myc amplification or overexpression have been reported to be associated with poor prognosis in breast carcinoma. The prognostic significance, however, remains somewhat controversial, partly because of discrepancies among different methodologies used for detection of the oncogene amplification or overexpression. Fluorescence in situ hybridization (FISH) has recently been shown to be a useful technique for analyzing genetic alterations in interphase nuclei in various tumors. In this study, FISH was used to quantitate HER-2/ neu and c-myc gene amplification in touch preparations of frozen tissue from 100 node-negative breast carcinomas. HER-2/neu amplification was found to be associated with an abnormal DNA index (P < .001) and tumor size (P < .04). Amplification of c-myc was associated with S phase (P < .0003), abnormal DNA index (P < .003), and a negative estrogen receptor status (P < .01). The coamplification of both oncogenes was strongly associated with an abnormal DNA index (P < .0001) and with tumor size (P < .009). The use of FISH for detection of HER-2/neu gene amplification was 92% concordant with immunocytochemistry (ICC) used for detection of overexpression of HER-2/neu protein. Fifteen of the 100 cases were both amplified for HER-2/neu by FISH and positive by ICC analysis. Seven cases without HER-2/neu gene amplification demonstrated HER-2/neu protein overexpression by ICC. One HER-2/neu-amplified case was negative by ICC. Repeat analysis of a subset of cases showed FISH to be a more reproducible method than ICC in the analysis of HER-2/neu in touch preparations of breast carcinoma. FISH is a rapid and reproducible method that allows the accurate measurement of the level of oncogene amplification within interphase nuclei. The use of FISH should provide a more accurate assessment of the prognostic significance of oncogene amplification in breast carcinoma.     

Interphase cytogenetics in pathology: principles, methods, and applications of fluorescence in situ hybridization (FISH)

Characteristic chromosome aberrations have been identified in various tumors. Fluorescence in situ hybridization (FISH) using specific probes that are generated by vector cloning or in vitro amplification and labeled with fluorescent dyes allow for the detection of these genetic changes in interphase cells. This technique, that is also referred to as "interphase cytogenetics", can be performed in cytological preparations as well as in sections of routinely formaldehyde-fixed and paraffin-embedded tissue. In cancer research and diagnostics, interphase cytogenetics by FISH is used to detect numerical chromosome changes and structural aberrations, e.g., translocations, deletions, or amplifications. In this technical overview, we explain the principles of the FISH method and provide protocols for FISH in cytological preparations and paraffin sections. Moreover, possible applications of FISH are discussed.     

Comparison of fluorescence in situ hybridization with flow cytometry and static image analysis in ploidy analysis of paraffin-embedded prostate adenocarcinoma

Nuclear DNA ploidy has been shown to have an important prognostic association for patients with adenocarcinoma of the prostate. Flow cytometry and static image analysis are ploidy methods that have been used in prostate carcinoma. Fluorescence in situ hybridization (FISH) using chromosome-specific probes can be used to evaluate the ploidy of interphase nuclei. In this study FISH was compared with flow cytometry and static image analysis in determining ploidy in paraffin-embedded tissue from 34 prostatic adenocarcinomas. Ploidy status using FISH was determined by enumerating centromeres of two chromosomes (8 and 12) by use of directly-labeled alpha-satellite DNA probes in isolated whole nuclei obtained by the Hedley technique. All three methods identified 11 of 34 cases as diploid and 17 of 34 cases as nondiploid (82% concordance). Six cases were discordant; two cases had discrepant results by each method. Ploidy classification as determined by FISH had an 88% concordance with ploidy classification by either flow cytometry or static image analysis. In conclusion, FISH was found to be a sensitive method of ploidy analysis in isolated paraffin-embedded nuclei from prostate adenocarcinomas. When the chromosomes commonly involved in aneuploidy have been identified in prostate adenocarcinoma, FISH has the potential to provide greater sensitivity for aneuploidy detection compared with currently available methods.     

Interphase cytogenetic diagnosis of bladder cancer on cells from urine and bladder washing

In order to determine the value of fluorescence in situ hybridization (FISH) in the diagnosis and follow-up of bladder cancer interphase cytogenetics was performed on cells from urine and bladder washings. 50 ml of urine or bladder washings were collected. FISH was carried out using centromere probes for chromosomes 7, 8, 9 and 12 according to standard protocols. In each case 100 cell nuclei were analysed. Fifty-four samples from urine and 67 samples from bladder washing were analysed by FISH in comparison with results obtained by conventional cytology. Sensitivity of detection of tumor cells by FISH was 68.5% in urine and 63% in bladder washings regardless of tumor stage and grade. Sensitivity obtained by conventional cytology was 50% in urine and 77.3% in bladder washings. FISH on cells from urine samples is an effective complement to the standard urine cytology. Using centromere probes this approach is characterized by high specificity and sensitivity in tumors with T-category higher than pTa and grade higher than G1.     

Detection of deletions in the short arm of chromosome 3 in uncultured renal cell carcinomas by interphase cytogenetics

PURPOSE: Analysis of genetic alterations may facilitate the differential diagnosis of renal cell carcinoma (RCC) subtypes. For genetic classification, deletion of the short arm of chromosome 3 (3p), the hallmark of nonpapillary/clear cell RCC, is a major diagnostic criterion. Because of the limited routine applicability of cytogenetics and molecular genetic techniques we investigated interphase fluorescence in situ hybridization (FISH) for the detection of this aberration in RCC. MATERIALS AND METHODS: Using seven chromosome 3 specific probes FISH was performed on isolated nuclei from 26 uncultured sporadic RCC. RESULTS: Alterations of chromosome 3 were identified in 19 RCC (73%). Monosomy and/or 3p-deletions were observed in 15 of 19 (79%) non-papillary/clear cell RCC but not in other morphologic subgroups. The median percentage of cells in a specimen containing loss of 3p was 45%. Deletion mapping indicated that large deletions affecting different regions in 3p are predominant. Chromosomal region 3p24 was recurrently involved in all RCC with a deletion in 3p. CONCLUSION: Interphase FISH for the detection of loss in 3p provides a sensitive and feasible method for the genetic classification of kidney tumors and the delineation of recurrently deleted regions in 3p.     

Identification of eicosanoids in the red algae, Gracilaria asiatica, using high-performance liquid chromatography and electrospray ionization mass spectrometry

The "gold standard" inflow cytometric DNA analysis of breast cancer uses fresh tumor cells simultaneously labeled for cytokeratin (CK) and DNA. We developed a 2-parameter CK-DNA flow assay suitable for archival, paraffin-embedded tissue (PT). Six anti-CK monoclonal antibodies were tested by immunocytochemistry and our assay for staining of nuclei extracted from PT breast cancers by combination pepsin-trypsin digestion. Clone CAM 5.2 was inadequate for PT nuclear suspensions, but a cocktail of 2 anti-CK clones (AE1/AE3 and KL-1) distinguished epithelial from nonepithelial nuclei in 2-parameter flow dot plots. We studied 82 routine PT breast tumors by our assay and used a univariate flow DNA histogram based on fresh biopsy tissue for comparison. Three histogram data quality indicators were improved. A trend toward higher S-phase fractions was found for DNA diploid PT tumors, although when inflammation was evident histologically, the increment in S-phase fraction with gating was often marked. CK gating identified PT tumors containing concurrent CK-positive DNA diploid and nondiploid populations (27 of 56 DNA nondiploid histograms). By excluding nonepithelial nuclei, 2-color CK-DNA flow methods may increase the accuracy of ploidy and S-phase fraction measurements. Our method appears superior to previous techniques using clone CAM 5.2 for labeling of archival breast cancers.     

Sequential combined therapy with thalidomide and narrow-band (TL01) UVB in the treatment of prurigo nodularis

Karyotypic analysis by direct demonstration of DNA sequences in interphase nuclei has been termed interphase cytogenetics and can be applied to a wide variety of cellular material, including paraffin-embedded tissue, allowing detection of both numerical and structural chromosome aberrations. The principal established method in the fluorescence in situ hybridization (FISH) technique, but more recently primed in situ labelling (PRINS) has been employed, as illustrated in an accompanying paper in this issue of the Journal. Where there are defining cytogenetic abnormalities, as is the case for the detection of fetal numerical chromosome abnormalities and in some paediatric and soft tissue tumours, this approach has clear diagnostic applicability. In other circumstances, such as the investigation of most solid tumours, this technique is largely of research interest but, particularly with application to paraffin sections, in providing valuable information on the morphological distribution of molecular changes in both invasive and 'pre-invasive' lesions. Continued technical refinement and research application of this methodology will lead not only to greater clinical applicability but also to improved understanding of the pathobiology of tumours.     

Monitoring of remission status by fluorescence in situ hybridisation in chronic myeloid leukaemia patients treated with interferon-alpha

Interferon-alpha (IFN-alpha) can be considered as treatment of choice for patients with chronic myeloid leukaemia (CML) in chronic phase. With this treatment major cytogenetic responses can be achieved in 30% to 50% of patients. Regular monitoring of cytogenetic response is essential for the therapeutic management of these patients. As conventional cytogenetics is not always successful, especially under IFN-alpha treatment, molecular cytogenetic methods have been established for the examination of interphase nuclei for the presence of the BCR-ABL fusion gene, the molecular counterpart of the Philadelphia chromosome. To demonstrate the value of these new methods we have analysed interphase nuclei from sequentially cultured bone marrow cells from 14 CML patients who were treated with IFN-alpha and whose bone marrow was investigated regularly during therapy. Dual-colour FISH with a breakpoint spanning BCR-YAC and a flanking cosmid from the ABL region was applied. When compared with conventional cytogenetics the results achieved by FISH were favourable. The most evident advantage of FISH analysis is that in case of failure of conventional cytogenetics a reliable determination of the remission status can be done. Together with other recent studies our results illustrate the advantages and limitations of the interphase FISH method for monitoring CML patients.     

Chromosomal imbalances in primary and metastatic pancreatic carcinoma as detected by interphase cytogenetics: basic findings and clinical aspects

To date, cytogenetic studies on pancreatic carcinoma are rare, and little is known about the frequency of cytogenetic aberrations in primary carcinomas compared with metastatic tumour cells. We therefore evaluated the frequency of chromosomal aberrations in 12 primary pancreatic carcinomas and in effusion specimens from 25 patients with pancreatic cancer by using interphase fluorescence in situ hybridization (FISH) and a panel of four centromeric probes. Hyperdiploidy and chromosomal imbalances, predominantly affecting chromosome 8, were a constant finding in metastatic effusion cells, whereas concordant gain of chromosomes or relative loss of chromosome 18 characterized primary pancreatic carcinomas. The potential role of oncogenes located on chromosome 8 for pancreatic cancer progression was further investigated by double-hybridization studies of aneuploid effusion cells with a probe to 8q24 (MYC) and a centromeric probe to chromosome 8, which demonstrated amplification of the MYC oncogene in two of ten cases (20%). Finally, a potential application of basic findings in the clinical setting was tested by searching for micrometastatic cells in effusions from pancreatic cancer patients primarily negative by FISH. Two-colour FISH in combination with extensive screening (>10,000 nuclei) seems to be a useful tool to unequivocally identify micrometastatic cells by demonstrating hyperdiploidy and intranuclear chromosomal heterogeneity.     

Amplification of c-myc, K-sam, and c-met in gastric cancers: detection by fluorescence in situ hybridization

Gene amplifications of c-myc, K-sam, and c-met were examined in cancer nuclei isolated from 154 primary gastric adenocarcinomas by fluorescence in situ hybridization (FISH) using cosmid probes for 8q24 (c-myc locus) and 7q31 (c-met), as well as a DNA probe for K-sam synthesized by PCR. The results were compared with those of Southern blot analysis. Dual-color FISH using gene locus and chromosome-specific probes detected gene amplifications of c-myc in 24 tumors (15.5%), c-met in 6 tumors (3.9%), and K-sam in 3 tumors (2.9%). The six tumors with c-myc amplification had also been found to have amplified c-erbB-2 in our previous study, and coamplification of c-myc and c-met was found in two other tumors. This technique also differentiated the amplified genes on the homogeneous staining region (HSR) and on double minute chromosomes (DMs) in metaphase spreads and interphase nuclei of cell lines established from poorly differentiated adenocarcinomas, KATO III, SNU 16, and HSC 39. Examination of FISH images of these cell lines suggested that the high-level amplifications of c-myc found in primary tumors occurred mainly on DM in four tumors and on HSR in one, and those of K-sam occured on DM in two tumors and on HSR in one. No high-level amplification of c-met was found. These high-level amplifications were also detected in formalin-fixed, paraffin-embedded tissues from primary gastric tumors and metastatic lymph nodes, in some of which heterogeneity of gene amplification was demonstrated within the same tumor. We conclude that FISH is an important tool for examining the proto-oncogene aberrations in intact cells in solid tumors.     

Acute coronary vascular and myocardial perfusion effects of conjugated equine estrogen in the anesthetized dog

OBJECTIVE: To describe the laser scanning cytometry (LSC) processing and analysis developed for the quantitative analysis of estrogen receptor (ER) content in routine paraffin sections of breast carcinomas. STUDY DESIGN: Histologic sections of archival, paraffin-embedded tissues from 30 breast carcinomas were labeled for ER with fluoresceinated monoclonal antibody. ER expression was quantified by LSC and expressed as percent positive tumor cells and as histogram distributions of receptor expression per cell. Duplicate sections of the same tumors were stained for ER by a conventional immunoperoxidase reaction and percent positive tumor cells counted visually. RESULTS: Percent ER-positive tumor cells by LSC of immunofluorescence-stained sections correlated well with conventional (visual) counts of immunoperoxidase-stained duplicate sections when the latter were categorized as low, intermediate or high percent of positive cells. In addition, the marked variation in relative number of ER binding sites per cell could be quantified by LSC and displayed in histogram distribution. CONCLUSION: LSC measurements are fast and objective and can be carried out on sections of paraffin-embedded tissue after routine processing in the pathology laboratory. In addition, LSC data provide the relative number of ER binding sites per unit of DNA; that may reveal clinically significant skewed distributions or subpopulations of tumor cells.     

Chromosome-specific aneusomy in carcinoma of the breast

Fluorescence in situ hybridization was performed on touch preparations from 55 primary infiltrating ductal carcinomas of the breast to determine numeric chromosome abnormalities. The frequency of aneusomy, measured by both nondisomy and chromosomal gain, was determined for chromosomes X, 4, 6-12, 17, and 18 with the use of chromosome-specific, alpha-satellite DNA probes. The presence of chromosome-specific numeric abnormalities was correlated with established clinicopathological parameters, including tumor size, lymph node involvement, tumor grade, estrogen receptor level, and menopause status. In addition, a case-control study was performed to explore a possible association between chromosome-specific aneusomy and recurrence in lymph-node-negative patients. Although chromosomes 8 and 6 were most frequently aneusomic, numeric abnormalities of chromosomes 4 and 11 were most strongly associated with established prognostic factors. For chromosomes 4 and 11, strong associations were found with tumor involvement of lymph nodes and increased tumor size, along with a weaker association with tumor grade. In addition, numeric abnormalities of the following chromosomes were associated with the corresponding prognostic factors: chromosomes X, 7, and 12 with lymph node status; chromosomes 10, 17, and 6 with tumor size; and chromosomes 7, 12, 17, and X with tumor grade. No correlations were observed with estrogen receptor level or menopause status. In the case-control study performed on isolated nuclei of paraffin-embedded tissue from lymph node-negative breast cancer patients (19 cases and 19 controls), the gain of chromosome 4 was correlated with disease progression. These findings suggest that chromosome-specific aneusomy is associated with certain established prognostic factors and may be associated with disease progression.     

Characterization of Y3 receptor-mediated synaptic inhibition by chimeric neuropeptide Y-peptide YY peptides in the rat brainstem

Archival material from primary and metastatic renal clear cell carcinomas of 25 patients was studied by comparative genomic hybridization. Copy number changes of entire chromosomes or chromosomal subregions were detected in 22 primary and 21 metastatic tumors. Copy number changes affected the following chromosomes in at least 20% of the 25 primary tumors (minimal common region given in parentheses): gains were noted for chromosomes 1 (1q21-->q23), 5 (5q31-->q34), 7 (7p), 8 (8q), 16 (16p), 17 (17q12-->qter), 19, and 22 (22q12-->qter); losses were revealed for chromosomes 3 (3p21-->pter), 8 (8p23-->pter), 14(14q21-->qter), and Y. The same chromosomal regions that were involved in primary renal clear cell carcinomas were also found in the respective metastatic tumors but with strikingly different frequencies for a few regions. Metastatic tumors showed a significantly higher frequency of complete or partial gains of the long arm of chromosome 1, in particular at 1q21-->q23 than primary tumors (16 cases versus 6 cases; P < 0.005). These data suggest a correlation of metastatic events in renal clear cell carcinomas with an increase in the copy number of genes located at 1q, in particular at 1q21-->q23. In contrast, the entire or partial loss of the short arm of chromosome 3 was significantly less frequent in metastatic tumors (8 cases versus 15 cases; P < 0.025). The validity of 1q and 3p copy number changes detected by comparative genomic hybridization was confirmed by interphase cytogenetics with region-specific yeast artificial chromosomes to paraffin-embedded tumor tissue sections.     

Analysis of telomeric length in epithelial carcinoma of the ovary

BACKGROUND: As a consequence of the high cell division rate, telomeric repeat reduction in human tumor cells, giving rise to genetic instability, has recently been described. The aim of this study was to analyze by Southern blot telomeric length alterations in a retrospective group of patients with ovarian epithelial carcinoma. PATIENTS AND METHODS: Tumor and corresponding normal DNA were isolated from paraffin-embedded tissue of 16 patients with ovarian epithelial carcinoma. Telomeric Restriction Fragments (TRF) were studied by Southern blot and densitometric analysis. RESULTS: No telomere alterations were detected in 37.5% of patients (6/16). Of the remaining ten, 5 were found to have telomere reduction and five telomere elongation. No significant correlation was found between clinicopathological variables, response to chemotherapy, survival rate or time to progression, and telomere length alterations. CONCLUSIONS: In ovarian epithelial carcinoma telomere elongation may be a marker of the presence of immortal cells within the tumor, but telomere or the absence of telomeric alterations do not rule out the presence of these cells. Although TRF analysis can be performed in paraffin-embedded tissues, it is not the best indicator of telomerase activity and thus of tumor aggressiveness in early stages of this carcinoma.     

The case for case studies in nursing research: the problem of generalization

The development and progression of human breast neoplasia is characterized by the accumulation of numerous somatic genetic alterations. Although mutation of the p53 tumor suppressor gene is one of the most common alterations identified in invasive carcinomas, it is not clear whether mutations usually occur in noninvasive lesions before the development of invasion. To investigate the presence and heterogeneity of p53 mutations in breast neoplasia, we studied a morphological spectrum of paired lesions including invasive carcinomas and adjacent noninvasive epithelial lesions. Using 18 invasive ductal carcinomas with known p53 mutations, tissue samples were microdissected from formalin-fixed, paraffin-embedded tissue sections, and mutations in exons 4-8 of the p53 gene were identified by PCR amplification, single-strand conformational polymorphism, and direct sequencing. Multiple geographically distinct foci of invasive carcinoma were microdissected from six different invasive carcinomas, and all samples from each case had the same mutation. The absence of mutation heterogeneity provides evidence that p53 mutations occurred at the time of, or before, the clonal selection of the dominant component of the invasive carcinoma. To delineate the timing of p53 inactivation further in these cases, histological slides were reviewed to identify all noninvasive epithelial lesions. There were eight cases with associated ductal carcinoma in situ (DCIS), and in total, 27 distinct tissue samples representing a spectrum of histological subtypes and grades of DCIS were microdissected. In all 27 samples, the identical p53 mutation was identified in the DCIS as was present in the invasive carcinoma. In contrast, no p53 mutations were identified in any of the 21 microdissected foci of epithelial hyperplasia analyzed, including one sample with atypia. Together, these findings provide support that p53 mutations commonly occur early in breast neoplasia, usually at the stage of DCIS, but are not often identified in foci of hyperplasia. These findings support an important biological role for p53 mutation in progression from noninvasive precursors in breast neoplasia and provide further evidence that p53 mutation could have potential use as a molecular marker.     

Evidence of clonal divergence in colorectal carcinoma

BACKGROUND: The aim of this study was to investigate the generation of DNA ploidy diversity in different stages of colorectal carcinoma development. METHODS: DNA flow cytometry was performed on tissue samples from 20 colorectal adenomas, 38 colorectal carcinomas, 30 lymph node metastases, and 70 hematogenous metastases. RESULTS: DNA aneuploidy was detected in 30% of the adenomas, 82% of the primary colorectal tumors, 57% of the lymph node metastases, 92% of the liver metastases, and 100% of the other distant hematogenous metastases. Multiple DNA tumor stemlines were found in 10%, 39%, 29%, 24%, and 40%, respectively. Sixty-two percent of the DNA tumor stemlines detected in the lymph node or liver metastases were also present in the primary tumors. In primary carcinomas and lymph node metastases, the DNA index distribution had a bimodal shape with a minimum at the 1.2-1.4 region. In the hematogenous metastases, a higher percentage of hypertetraploid stemlines was found. CONCLUSIONS: The emergence of DNA aneuploidy as well as clonal divergence seems to take place during the transition from adenoma to carcinoma. The DNA aneuploid stemlines formed during this phase remain relatively stable over time, although ongoing clonal evolution at distant metastatic tumor sites cannot be completely ruled out.     

Hormone receptor determination in breast carcinoma tissue. Comparison of recent immunohistologic techniques with biochemical receptor testing

For evaluation of the hormone receptor status in breast cancer tissues two methods are mainly used: immunohistochemical detection by monoclonal antibodies on frozen sections (ER-ICA, PgR-ICA) and the biochemical radioligand-binding assay (DCC) of fresh tissue. Using new antibodies makes it possible to evaluate the estrogen and progesterone receptor status in formalin-fixed and paraffin-embedded tissue. In the present retrospective study, tissues from 223 primary breast carcinomas or breast carcinoma recurrences were re-evaluated with the three methods mentioned above and the results were compared. We used antibody 1D5.26 reacting with the estrogen receptor and mPR1 specific for the progesterone receptor in paraffin-embedded tissue. The agreement of positive and negative cases between these two immunohistochemical procedures was 97.8% for the estrogen receptor and 85.7% for the progesterone receptor. Comparison of immunohistochemistry on paraffin-embedded tissue and biochemical evaluation showed an agreement of 74.7% for the estrogen receptor and 68.7% for the progesterone receptor. These results are comparable to the correspondence between ER-ICA and PgR-ICA and the DCC method. This study proves that the prognostically and therapeutically important hormone receptors can be reliably determined in formalin-fixed and paraffin-embedded tissues. These results are not only important for the evaluation of hormone receptors of a small breast carcinoma that is not found in the frozen section, but for the considerable difference in costs among the different methods.