Amplification of c-myc oncogene by chromogenic and fluorescence in situ hybridization in archival breast cancer tissue array samples.

Fluorescence in situ hybridization (FISH) is currently considered to be the most specific and sensitive method for detection of oncogene amplifications in human tumor samples. However, FISH requires fluorescence microscopy, which is tedious and does not allow histopathologic evaluation of the cells and tissues examined. Here we compared FISH with the newly developed chromogenic in situ hybridization (CISH), which uses peroxidase enzyme for probe detection instead of fluorescent dyes. CISH was found to be highly concordant with FISH in a tissue array series of 177 archival breast cancer samples. This was true both when comparing CISH with single-color and two-color FISH, the latter including the chromosome 8 centromere probe as reference (the kappa coefficients were 0.67 and 0.76, respectively). Clinicopathologic correlations of c-myc amplification as detected by FISH and CISH were generally the same. By both methods, c-myc amplification was significantly associated with high histologic grade, negative progesterone receptor status, DNA aneuploidy, and high S-phase fraction. c-myc amplification was strongly associated with poor distant metastasis-free survival when amplification was detected by CISH (p = 0.0013), but this association was weaker when FISH was used (p = 0.16 for two-color FISH and p = 0.065 for single-color FISH). These data suggest that CISH is at least as sensitive and specific as FISH in the detection of oncogene amplification in human tumor samples. The possibility for concomitant tissue architecture evaluation using an ordinary transmitted light microscope may favor the use of CISH over FISH in oncogene amplification detection in large tumor series, and tissue arrays and, ultimately, in routine clinical diagnostics.

Frequent somatic loss of BRCA1 in breast tumours from BRCA2 germ-line mutation carriers and vice versa.

Breast cancer susceptibility genes BRCA1 and BRCA2 are tumour suppressor genes the alleles of which have to be inactivated before tumour development occurs. Hereditary breast cancers linked to germ-line mutations of BRCA1 and BRCA2 genes almost invariably show allelic imbalance (AI) at the respective loci. BRCA1 and BRCA2 are believed to take part in a common pathway in maintenance of genomic integrity in cells. We carried out AI and fluorescence in situ hybridization (FISH) analyses of BRCA2 in breast tumours from germ-line BRCA1 mutation carriers and vice versa. For comparison, 14 sporadic breast tumours were also studied. 8 of the 11 (73%) informative BRCA1 mutation tumours showed AI at the BRCA2 locus. 53% of these tumours showed a copy number loss of the BRCA2 gene by FISH. 5 of the 6 (83%) informative BRCA2 mutation tumours showed AI at the BRCA1 locus. Half of the tumours (4/8) showed a physical deletion of the BRCA1 gene by FISH. Combined allelic loss of both BRCA1 and BRCA2 gene was seen in 12 of the 17 (71%) informative hereditary tumours, whereas copy number losses of both BRCA genes was seen in only 4/14 (29%) sporadic control tumours studied by FISH. In conclusion, the high prevalence of AI at BRCA1 in BRCA2 mutation tumours and vice versa suggests that somatic events occurring at the other breast cancer susceptibility gene locus may be selected in the cancer development. The mechanism resulting in AI at these loci seems more complex than a physical deletion.

Amplification of c-myc by fluorescence in situ hybridization in a population-based breast cancer tissue array.

A total of 261 primary breast carcinomas were analyzed for amplification of the c-myc oncogene by fluorescence in situ hybridization performed on tumor tissue array samples. Results were compared with individual clinicopathologic and follow-up data. Thirty-eight (14.6%) of the tumors showed c-myc gene amplification (defined as two or more additional copies of c-myc gene in relation to the number of chromosome 8 centromere). The reproducibility of fluorescence in situ hybridization assay (defined by hybridization with two different myc probes) was good (kappa coefficient 0.402). Statistically significant associations were found between c-myc amplification and DNA aneuploidy (P =.0011), and progesterone receptor negativity (P =.0071), and c-myc amplification also tended to be associated with high histologic grade (P =.064), positive axillary nodal status (P =.080), and a high S-phase fraction (P =.052). c-myc amplification was not significantly associated with overall survival of patients with invasive cancer (P =.32). These data from a population-based tumor material suggest that c-myc amplification is a feature of aggressive breast cancers, but that it is unlikely to be a clinically useful prognostic factor.

Aberrations of chromosome 8 in 16 breast cancer cell lines by comparative genomic hybridization, fluorescence in situ hybridization, and spectral karyotyping.

Comparative genomic hybridization (CGH) studies have shown that chromosome 8 is a frequent target for chromosomal aberrations in breast cancer. We characterized these aberrations of chromosome 8 in 16 breast cancer cell lines (BT-474, BT-549, CAMA-1, DU-4475, MCF-7, MDA-MB-134, MDA-MB-157, MDA-MB-361, MDA-MB-415, MDA-MB-436, MPE600, SK-BR-3, T-47D, UACC-812, UACC-893 and ZR-75-1) by CGH, fluorescence in situ hybridization (FISH) with arm- and locus-specific probes, and spectral karyotyping (SKY). Chromosome 8 was structurally abnormal in 13 of 16 cell lines. Loss of 8p was detected in nine cell lines, gain of entire 8q in six cell lines, 8q21-qter in three, 8q23-qter in two, and 8q12-qter and 8p21-q21 in one cell line. Extra copies of the C-MYC oncogene were found in 11 cell lines, but high-level amplification only in SK-BR-3. Derivative chromosomes including material from chromosomes 8 were complex, and the breakpoints were strikingly dissimilar. Chromosome 11 was the most frequent translocation partner with chromosome 8 (in 7 cell lines). Isochromosomes and/or isoderivative 8q were found in four cell lines. The high frequency and complexity of alterations at 8q indicate a significant pathogenetic role in breast cancer. The high-level amplification of c-myc is less common than previously thought.

Chromosome arm-specific multicolor FISH.

Several systems for 24-color fluorescence in situ hybridization (FISH) have been developed and applied to karyotyping and detection of chromosomal abnormalities. We have developed a 42-color multicolor FISH (mFISH) technique (armFISH), which permits the detection of chromosomal aberrations at the resolution of chromosome arms. The armFISH uses a commercially available mFISH reagent kit (24XCyte, MetaSystems GmbH) supplemented with a set of differentially labeled chromosome arm-specific painting probes (arm-kit, comprising either p- or q-arms of all human chromosomes, except the p-arm of the acrocentric- and Y chromosomes). The mFISH-probe cocktail and the arm-kit are combined and hybridized together to metaphase chromosomes. The armFISH is analyzed in two steps; first, the conventional mFISH image analysis is performed, followed by the arm-kit analysis to reveal the chromosome arms involved. The examples demonstrate the utility of armFISH in defining chromosomal rearrangements of human cancers.

Multiple copies of mutant BRCA1 and BRCA2 alleles in breast tumors from germ-line mutation carriers.

Inactivation of the BRCA1 and BRCA2 breast cancer susceptibility genes has been reported to occur via a germ-line mutation of one allele and a somatic loss of the remaining wild-type allele. We investigated the genetic mechanisms behind the second event in breast tumors from 17 BRCA1 and eight BRCA2 germ-line mutation carriers, as compared with 21 sporadic breast tumors. Microsatellite markers intragenic or in close proximity to both genes were used to analyze imbalances between the mutant and wild-type alleles. The actual and relative gene copy numbers were scored by fluorescence in situ hybridization (FISH) analysis of tumor cells using locus and centromere specific probes. All but one of the informative BRCA1 and BRCA2 tumors exhibited allelic imbalance and loss of the corresponding wild type allele. In contrast to sporadic tumors, however, where allelic imbalance at the BRCA1 and BRCA2 loci correlated well with relative copy number losses by FISH, a simple reduction to a single copy (average copy number ratio 2:1) was found in only two BRCA1 (12%) and four BRCA2 (50%) tumors. The majority of BRCA1 and BRCA2 tumors showed a copy number reduction (relative to reference probe with ratios 4:2, 3:2, 4:3) at corresponding loci, suggesting that a specific physical deletion of the wild-type BRCA gene allele has been followed by a duplication of the remaining mutant allele via polyploidization. Several tumors contained multiple copies of BRCA1 and BRCA2 genes without relative copy number changes, implying that loss of wild-type alleles is executed by alternative mechanisms such as mitotic recombination, non-disjunctional chromosomal loss with or without reduplication, or by gene conversion. A paradoxical relative copy number gain of the mutant allele was evident in three BRCA1 tumors (18%), which could be of biological relevance if a dominant negative or gain-of-function model was ascribed for certain BRCA1 mutants. Our results indicate that complex genetic alterations are operational at the BRCA1 and BRCA2 loci in tumors from genetically predisposed individuals.

Frequent amplification of chromosomal region 20q12-q13 in ovarian cancer.

DNA amplification at chromosomal region 20q12-q13, which is common in breast cancer, has recently been described also in ovarian tumors. We studied the amplification of the recently identified candidate oncogenes in this region in 24 sporadic, 3 familial and 4 hereditary ovarian carcinomas, and in 8 ovarian cancer cell lines. High-level amplification of at least one of the five nonsyntenic regions at 20q12-q13.2 was found in 13 sporadic (54%) and in all four hereditary tumors. Typically, two or more distinct amplicons (separated by nonamplified DNA) were found coamplified in various combinations. The regions defined by the AIB1 and PTPN1 genes (at 20q12 and 20q13.1, respectively) were amplified in 25% and 29% of the sporadic tumors, also without simultaneous coamplification of other regions. Amplification of AIB1 (a steroid receptor coactivator gene) was associated with estrogen receptor positivity in sporadic ovarian carcinomas (P = 0.01) and showed a tendency to correlate with poor survival of patients. Of the genes amplified in breast cancer, the BTAK gene was amplified in 21%, the MYBL2 gene in 17%, and the ZNF217 gene in 12.5% of the sporadic tumors. The high frequency of gene amplification at 20q12-q13.2 suggests that the genes amplified therein may play a central role in the pathogenesis of sporadic and hereditary ovarian carcinoma.

Predictive value of topoisomerase IIalpha and other prognostic factors for epirubicin chemotherapy in advanced breast cancer.

Although cytotoxic chemotherapy is widely used in advanced breast cancer, there are no powerful predictors for the therapy response. Because topoisomerase IIalpha (Topo IIalpha) is the molecular target for the anthracycline class of anti-cancer drugs, we compared the immunocytochemical assay of Topo IIalpha with other biomarkers in the prediction of clinical response to Topo II inhibitor chemotherapy. Fifty-five patients with advanced breast cancer were treated with a single cytotoxic drug, Topo II-inhibitor, epirubicin (30 mg m(-2) weekly up to 1000 mg m(-2)), as first line cytotoxic chemotherapy. Objective response to treatment was analysed according to UICC criteria. The predictive value of Topo IIalpha expression, c-erbB2 oncoprotein, p53 tumour-suppressor protein, oestrogen (ER) and progesterone receptor (PR), S-phase fraction and DNA ploidy were analysed from representative formalin-fixed paraffin-embedded primary tumour samples. The proportion of Topo IIalpha-positive cells (Topo IIalpha index) failed to predict response to epirubicin therapy. Mean Topo IIalpha scores in 29 responding patients were similar when compared with those with no change in disease progression (n = 13) and those with progressive disease (n = 13) (14.9% +/- 11.4% vs 15.5% +/- 7.6% vs 17.3% +/- 13.2%, not significant). Among the other biomarkers tested, overexpression of c-erbB2 oncoprotein and hormone receptor negativity were significantly associated with poor response. Response rate in patients with c-erbB2-overexpressing tumours was 32% compared with 65% in patients with no c-erbB2 overexpression (P = 0.0058). Accordingly, the response rate for ER-positive patients was 67% compared with 26% in ER-negative patients (P = 0.0021). Although both negative ER status and c-erbB2 overexpression are associated with high Topo IIalpha expression in breast cancer, step-wise logistic regression analysis showed that ER and c-erbB2 were associated with therapy response independent of Topo IIalpha expression. Histological grade, p53, DNA-ploidy, tumour proliferation rate (S-phase fraction), stage of the disease at diagnosis, age of the patient, previous anti-oestrogen therapy or site of metastasis did not predict the response to epirubicin therapy. In conclusion, despite extensive in vitro evidence, expression of Topo IIalpha is unlikely to predict the response to Topo II inhibitor chemotherapy in advanced breast cancer. Among the prognostic biomarkers, overexpression of c-erbB2 oncogene and negative ER may have predictive value in epirubicin therapy in patients with advanced breast cancer.

Genetic aberrations in hypodiploid breast cancer: frequent loss of chromosome 4 and amplification of cyclin D1 oncogene.

The evolution of somatic genetic aberrations in breast cancer has remained poorly understood. The most common chromosomal abnormality is hyperdiploidy, which is thought to arise via a transient hypodiploid state. However, hypodiploidy persists in 1 to 2% of breast tumors, which are characterized by a poor prognosis. We studied the genetic aberrations in 15 flow cytometrically hypodiploid breast cancers by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). Surprisingly, numerous copy number gains were detected in addition to the copy number losses. The number of gains per tumor was 4.3 +/- 3.2 and that of losses was 4.5 +/- 3.3 (mean +/- SD), which is similar to that previously observed in hyperdiploid breast cancers. Gains at chromosomes or chromosomal regions at 11q13, 1q, 19, and 16p and losses of 2q, 4, 6q, 9p, 13, and 18 were most commonly observed. Compared with unselected breast carcinomas, hypodiploid tumors showed certain differences. Loss of chromosome 4 (53%) and gain of 11q13 (60%) were significantly more common in hypodiploid tumors. The gain at 11q13 was found by FISH to harbor amplification of the Cyclin D1 oncogene, which is therefore three to four times more common in hypodiploid than in unselected breast cancers (15 to 20%). Structural chromosomal aberrations (such as Cyclin D1 amplification) were present both in diploid and hypodiploid tumor cell populations, as assessed by FISH and CGH after flow cytometric sorting. Together these results indicate that hypodiploid tumors form a distinct genetic entity of invasive breast cancer, although they probably share a common genetic evolution pathway where structural chromosomal aberrations precede gross DNA ploidy changes.

Vitamin D receptor alleles and bone's response to physical activity.

The objective of this prospective controlled study was to determine whether the osteogenic response of bone to mechanical loading is dependent on the vitamin D receptor (VDR) polymorphism. Thirty-five healthy premenopausal women took part in a progressive, high-impact exercise three times a week for a period of 18 months and 45 women served as nonexercising controls. The trainees were divided into three groups: bb (n = 12, 34%); Bb (n = 16, 46%); BB (n = 7, 20%) according to polymorphism at the gene encoding the VDR (BB representing subjects without the restriction enzyme BsmI sites on the two VDR gene alleles). Bone mineral content (BMC) and areal bone mineral density (BMD) were measured at the lumber spine, proximal femur, knee, calcaneus, and dominant distal radius before the beginning of the exercise regimen and at 12 and 18 months of training using dual-energy x-ray absorptiometry (DXA). As an indicator of the total osteogenic effect of the training, SigmaBMC was derived by summing up the BMC values of the loaded sites (i.e., the lower limb sites and the lumbar spine). The mean SigmaBMC increased 2.0% in the bb group, 3.0% in the Bb group, and 2.8% in the BB group (P = 0.184 for the intergroup difference), but only 0. 8% in the controls (exercisers versus controls, P < 0.001). Individuals with the BB genotype of the VDR gene, subjects with whom the BMC can be lower than normal and whose bones can be less responsive to pharmacological therapies than bones of the other individuals, seem to have as good osteogenic response to mechanical loading as subjects with other VDR genotypes. Thus, irrespective of the VDR genotype, physical activity seems to be beneficial for bones of premenopausal women.

Reduced E-cadherin expression is associated with invasiveness and unfavorable prognosis in breast cancer.

E-cadherin (E-cad) is a calcium-dependent, epithelial cell adhesion molecule whose reduced or lost expression has been associated with tumor dedifferentiation and increased metastatic potential in human carcinomas. The authors studied immunohistochemically E-cad expression in frozen sections of 362 breast carcinomas using a monoclonal antibody (HECD-1). The immunohistochemical detection of reduced E-cad expression was confirmed by mRNA in situ hybridization with two different oligonucleotide probes. THe proportion of tumors with reduced or lost E-cad expression increased significantly from pure intraductal carcinomas (20%, 4 of 20) through invasive ductal (IDCs; 52%, 124 of 239) to recurrent carcinomas (64%, 18 of 28; chi square test for trend, P = .004). Invasive lobular carcinomas (ILCs) and IDCs differed from each other in their E-cad expression. None of the ILCs (n=55) retained normal E-cad expression in contrast to 48% (115 of 239) of the IDCs. In 259 primary IDCs, reduced E-cad expression was associated with high histologic grade (chi square test for trend, P < .001), negative estrogen receptor status (ER; Fisher's exact test; P = .042), and marginally with axillary node involvement (Fisher's exact test, P = .063). In a subset of 109 primary IDC patients whose clinical follow-up was available (median follow-up 51 months), reduced E-cad expression was associated with shortened disease-free survival (DFS; Mantel-Cox test, P = .027). In Cox's multivariate regression analysis, progesterone receptor status (P = .018) and E-cad expression (P = .072) were selected as independent predictors of DFS. Our findings provide clinical evidence that loss of normal E-cad expression is an indicator of increased invasiveness and dedifferentiation in breast carcinoma. E-cad is a potentially important prognostic factor in primary IDCs.

Anti-oestrogen stimulation of ERBB2 ectodomain shedding from BT-474 human breast cancer cells with ERBB2 gene amplification.

Oestrogen has previously been shown to downregulate the expression of ERBB2 oncogene in human breast cancer cells, which contain a normal non-amplified ERBB2 gene. However, amplified ERBB2 seems to escape from hormonal regulation. We studied shedding of the extracellular domain (ectodomain, ECD) of the ERBB2 encoded protein in BT-474 human breast cancer cells treated with oestrogen or anti-oestrogen. Oestrogen-responsiveness of these cells has been previously demonstrated by stimulation of cell growth and expression of pS2, a marker gene known to be regulated by oestrogen receptor at transcriptional level. The concentration of the soluble ECD in the culture medium was increased by the anti-oestrogen toremifene as a function of time. In contrast, the level of ERBB2 mRNA and protein in cell lysates was not stimulated, but was transiently suppressed by toremifene. In the presence of oestrogen, the level of ECD remained low. The increased shedding of ECD in the presence of toremifene, without parallel change in ERBB2 transcripts (4.8 and 2.3 kb) and in cellular ERBB2 protein level, suggests that toremifene specifically contributes to the shedding of the ERBB2 ectodomain. These results show that shedding of ECD is an additional level of regulation of ERBB2 by the anti-oestrogen toremifene. This may contribute to resistance to growth inhibition by anti-oestrogens of breast cancers which overexpress ERBB2.

Genetic aberrations detected by comparative genomic hybridization predict outcome in node-negative breast cancer.

Breast cancer progression is determined by a complex pattern of multiple genetic aberrations the association of which with patient prognosis is unknown. In this study, we have undertaken a genome-wide screening to detect genetic changes associated with clinical outcome in node-negative breast cancer. Comparative genomic hybridization was used to screen for DNA sequence gains and losses across all human chromosomes in 23 tumors from node-negative breast cancer patients with no disease recurrence after at least 5 years of follow-up and in 25 node-negative patients with recurrence during the first 5 years of follow-up. The total number of genetic aberrations (copy number gains and losses) per tumor was significantly greater in the recurrence group (P = 0.019) and in the subgroup of these patients who died as a result of breast cancer (P = 0.0022). When copy number losses and gains were analyzed separately, only losses were significant (P = 0.013 for recurrence and P = 0.002 for overall survival). Of the individual loci involved, a high level gain of the long arm of chromosome 8 was significantly associated with recurrence (P = 0.01, Fisher's exact test). Furthermore, amplification of DNA sequences at chromosome 20q12-13 was found in 7 cases (15%), 6 of which had early recurrence within 32 months of diagnosis. This genome-wide overview by comparative genomic hybridization suggests that genetically advanced node-negative breast cancers having a high overall number of genetic aberrations may have a poor prognosis and that increased copy number of two specific regions, 8q and 20q13, may confer a more aggressive phenotype. Results of this pilot study suggest that determination of the total number of DNA sequence copy number aberrations may help therapeutic decision making. Specific probes should be developed to test the prognostic value of 8q and 20q12-13 amplifications in large numbers of patients.

Comparison of different immunohistochemical methods in the assessment of angiogenesis: lack of prognostic value in a group of 77 selected node-negative breast carcinomas.

There is evidence that tumor angiogenesis, as detected by immunohistochemical staining of endothelium, is of prognostic significance in breast cancer. However, little attention has been paid to possible differences between antibodies or to quantitation of the stained microvessels. We compared three endothelial cell antibodies [anti-human von Willebrand factor (anti-VWF, also termed factor VIII), anti-CD31, and anti-CD34] in archival paraffin-embedded specimens. Anti-CD34 and anti-VWF showed better staining performances than anti-CD31, although the staining results with different antibodies were comparable. Two different methods of microvessel quantitation (the highest microvessel count and percentage microvessel area) were evaluated and also showed significant correlation. From a retrospective database (n = 1000), 77 axillary node-negative invasive ductal breast carcinomas were selected on the basis of clinical outcome to maximize the prognostic power of the sample set (37 died due to a metastatic breast carcinoma, 40 showed no recurrence during 8-yr follow-up). Microvessel quantitations were related to flow cytometric DNA ploidy, c-erb-B-2 overexpression, and estrogen receptor status of the tumor. Surprisingly, neither highest microvessel counts nor microvessel area measurements quantitated with anti-CD34 or anti-VWF immunohistochemistry were able to discriminate between favorable and unfavorable outcome patients. Thus, our results suggest that further evidence is still needed on tumor angiogenesis immunohistochemistry before it can be adopted as a prognostic marker in routine, clinical practice.

Genetic changes in primary and recurrent prostate cancer by comparative genomic hybridization.

Genetic changes leading to the development of prostate cancer and factors that underlie the clinical progression of the disease are poorly characterized. Here, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes along all chromosomes in 31 primary and 9 recurrent uncultured prostate carcinomas. The aim of the study was to identify those chromosome regions that contain genes important for the development of prostate cancer and to identify genetic markers of tumor progression. CGH analysis indicated that 74% of primary prostate carcinoma showed DNA sequence copy number changes. Losses were 5 times more common than gains and most often involved 8p (32%), 13q (32%), 6q (22%), 16q (19%), 18q (19%), and 9p (16%). Allelic loss studies with 5 polymorphic microsatellite markers for 4 different chromosomes were done from 13 samples and showed a 76% concordance with CGH results. In local recurrences that developed during endocrine therapy, there were significantly more gains (P < 0.001) and losses (P < 0.05) of DNA sequences than in primary tumors, with gains of 8q (found in 89% of recurrences versus 6% of primary tumors), X (56% versus 0%), and 7 (56% versus 10%), as well as loss of 8p (78% versus 32%), being particularly often involved. In conclusion, our CGH results indicate that losses of several chromosomal regions are common genetic changes in primary tumors, suggesting that deletional inactivation of putative tumor suppressor genes in these chromosomal sites is likely to underlie development of prostate cancer. Furthermore, the pattern of genetic changes seen in recurrent tumors with the frequent gains of 7, 8q, and X suggests that the progression of prostate cancer and development of hormone-independent growth may have a distinct genetic basis. These chromosome aberrations may have diagnostic utility as markers of prostate cancer progression.

Prognostication of astrocytoma patient survival by Ki-67 (MIB-1), PCNA, and S-phase fraction using archival paraffin-embedded samples.

The prognostic power of three proliferation estimation methods, Ki-67 (MIB-1) and PCNA immunohistochemistry, and flow cytometry (S-phase and S + G2/M fractions, respectively), were evaluated in 50 cases of astrocytoma. Each proliferation index showed a strong association with the grade of malignancy (grades I-IV). The MIB-1 labelling index (LI) provided additional information, as it could be used for the discrimination of grade II and grade III astrocytomas (P = 0.0357). All three proliferation estimation methods also had strong prognostic potential (MIB-1 LI: P < 0.0001; PCNA Li: P < 0.0001; S-phase: P = 0.0004; S + G2/M: P = 0.0124). According to the receiver operating characteristics (ROC) curve, the MIB-1 LI showed generally the best sensitivity and specificity in placing the patients correctly into groups of survivors and non-survivors, which was further confirmed in the multivariate analysis. Only 4 per cent of the patients having high MIB-1 scores (> 15.3 per cent) were alive after 2-years' follow-up. In contrast, 72 per cent of patients with tumours of low proliferation activity survived. It appears that Ki-67 (MIB-1) immunolabelling using archival paraffin-embedded samples is of value in predicting prognosis in astrocytic tumours.

Automated peak detection and cell cycle analysis of flow cytometric DNA histograms.

We describe an algorithm for fully automated flow cytometric DNA histogram classification and analysis that provides rapid, reproducible determination of DNA index and S-phase fraction (SPF). Automated classification agreed with subjective assessment of DNA ploidy in 96-98% of DNA histograms. Automated and conventional analyses of DNA index (r = 0.95) and SPF (r = 0.89) were also highly correlated with one another. In a series of 86 node-negative breast carcinomas, SPF calculated with the fully automated method was a significant predictor of 10 year survival (p = 0.009). Automation greatly increased the speed of DNA histogram analysis, allowing evaluation of the same set of histograms with different methods. In a preliminary study exploring the optimization of DNA histogram analysis, the best association between SPF and prognosis of breast cancer patients was achieved using sliced nuclei debris modeling, reporting only the aneuploid SPF (in aneuploid histograms), while excluding small aneuploid clones (< 15% of total cell count) from evaluation. In conclusion, automated DNA histogram analysis does not replace the need for close human supervision but provides a useful guideline for less experienced users, facilitates interlaboratory comparisons, and makes possible extensive reanalyses of large data sets.

Improved technique for analysis of formalin-fixed, paraffin-embedded tumors by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) and specific DNA probes for peri-centromeric repeat regions and unique sequence loci have made it possible to study chromosomal aberrations from interphase tumor nuclei. Large-scale retrospective studies on the prognostic value of interphase cytogenetics would become feasible if these techniques were readily applicable to nuclei from archival formalin-fixed tumor tissues. We describe here an improved technique for interphase FISH analysis of tumors that have been extensively fixed in formalin. The protocol aims at improving probe penetration and hybridization efficiency by inducing chromatin decondensation and swelling of the nuclei with a heat treatment in a 90 degrees C glycerol solution prior to hybridization. Using this cell pretreatment, FISH results on the detection of chromosome copy number aberrations and amplification of the c-erbB-2 oncogene from formalin-fixed, paraffin-embedded tissues were highly concordant with those from fresh tissues. In contrast to previously described methods, separate adjustments of denaturation or proteinase K digestion are not required for each sample. This method facilitates retrospective analyses of large series of tumors and is also useful for applying FISH to routine diagnostic purposes using formalin-fixed material.

Elevated erbB-2 oncoprotein levels in preoperative and follow-up serum samples define an aggressive disease course in patients with breast cancer.

BACKGROUND: Recent evidence indicates that a soluble fragment of the erbB-2 oncogene product may be released from cell surface and become detectable in the serum of patients with breast cancer. METHODS: To study the diagnostic utility of this phenomenon, the authors measured serum erbB-2 levels with a quantitative enzyme-linked immunosorbent assay in 227 preoperative samples from women who underwent breast surgery and in 339 samples from 225 patients with breast cancer during follow-up. RESULTS: Eleven (9%) of 114 preoperative samples from patients with a histologically verified breast cancer and 2 of 113 (1.8%) from patients with benign breast tumors had elevated (greater than 20 U/ml) serum erbB-2 antigen levels. Ten (91%) of the 11 carcinomas and one of the benign tumors from patients with elevated serum erbB-2 levels also showed overexpression of the erbB-2 protein in immunohistochemical analysis of tissue sections. Elevated preoperative serum erbB-2 levels were predominantly found in patients with large tumors, and those with axillary lymph node or distant metastases. Sixty-three of the 339 (19%) follow-up samples had elevated serum erbB-2 antigen levels. Approximately one-third (30.9%) of the samples taken during recurrent disease were serum erbB-2 positive, which is close to the overall overexpression rate of this oncogene. Elevated erbB-2 levels were more common in patients whose disease was not responsive to treatment. Patients with distant metastases had elevate erbB-2 levels more often (40%) than did those with locoregional recurrence (20%). Elevated erbB-2 levels predicted the appearance of metastases within the next 6 months in 10 of 27 (37%) patients. CONCLUSION: The study's results suggest that assay serum erbB-2 levels may be valuable in the follow-up and monitoring of patients with breast cancer whose primary tumors show erbB-2 overexpression by immunohistochemistry.

Prognostic value of cells with more than 5c DNA content in node-negative breast cancer as determined by image cytometry from tissue sections.

The aim of this investigation was to study the prognostic significance of 5c cells (presence of cancer cells with > 5c DNA content; ie, over 18 pg of DNA per nucleus) in axillary node-negative breast cancer. Tissue sections (3 microns) from 134 tumors were stained for DNA using the Feulgen method and screened for the percentage of 5c cells with the CAS 200 image analysis system (Cell Analysis System, Inc, Lombard, IL). Cancer cells with a DNA content exceeding the 5c level were found in 45% (60 of 134) of the cases, accounting for a median of 0.2% (range, 0.05% to 1.05%) of all cells. The presence of 5c cells was associated with a high histologic grade of the tumor (P = .0001), a large number of mitoses (P < .0001), flow cytometric DNA aneuploidy and high S-phase fraction (P = .0002 and P < .0001, respectively), and c-erbB-2 oncoprotein and p53 tumor suppressor gene product overexpression (P = .0002 and P = .0006, respectively). Patients with 5c cell-positive tumors had a significantly worse 8-year survival rate (P = .003) than those with 5c cell-negative tumors. Subgroup analysis showed that the presence of 5c cells had a prognostic impact in low malignancy tumors, ie, in well-differentiated (grade I or II) and slowly proliferating tumors. Our findings suggest that determination of 5c cells may be a useful additional prognostic factor in axillary node-negative breast cancer. It adds prognostic information, especially in cases that are otherwise thought to have a favorable course.