Ductal lavage for detection of cellular atypia in women at high risk for breast cancer.

BACKGROUND: Breast cancer originates in breast epithelium and is associated with progressive molecular and morphologic changes. Women with atypical breast ductal epithelial cells have an increased relative risk of breast cancer. In this study, ductal lavage, a new procedure for collecting ductal cells with a microcatheter, was compared with nipple aspiration with regard to safety, tolerability, and the ability to detect abnormal breast epithelial cells. METHODS: Women at high risk for breast cancer who had nonsuspicious mammograms and clinical breast examinations underwent nipple aspiration followed by lavage of fluid-yielding ducts. All statistical tests were two-sided. RESULTS: The 507 women enrolled included 291 (57%) with a history of breast cancer and 199 (39%) with a 5-year Gail risk for breast cancer of 1.7% or more. Nipple aspirate fluid (NAF) samples were evaluated cytologically for 417 women, and ductal lavage samples were evaluated for 383 women. Adequate samples for diagnosis were collected from 111 (27%) and 299 (78%) women, respectively. A median of 13,500 epithelial cells per duct (range, 43-492,000 cells) was collected by ductal lavage compared with a median of 120 epithelial cells per breast (range, 10-74,300) collected by nipple aspiration. For ductal lavage, 92 (24%) subjects had abnormal cells that were mildly (17%) or markedly (6%) atypical or malignant (<1%). For NAF, corresponding percentages were 6%, 3%, and fewer than 1%. Ductal lavage detected abnormal intraductal breast cells 3.2 times more often than nipple aspiration (79 versus 25 breasts; McNemar's test, P<.001). No serious procedure-related adverse events were reported. CONCLUSIONS: Large numbers of ductal cells can be collected by ductal lavage to detect atypical cellular changes within the breast. Ductal lavage is a safe and well-tolerated procedure and is a more sensitive method of detecting cellular atypia than nipple aspiration.

Diagnostic accuracy of fine-needle aspiration biopsy is determined by physician training in sampling technique.

BACKGROUND: Fine-needle aspiration biopsy (FNAB) has been used with variable success as a diagnostic test for benign and malignant breast lesions. The goal of this study was to examine the effects of training physicians in the fine-needle aspiration sampling-technique on the diagnostic accuracy of FNAB of palpable breast masses. The settings for this study were private physicians' offices and university clinics of primary care physicians, surgeons, and cytopathologists. METHODS: We reviewed 1043 consecutive FNAB specimens of the breast obtained during 1 year (1992): 729 FNABs were performed by formally trained physicians (at least 150 FNABs performed previously under supervision during fellowship training or the equivalent) who had done at least 100 FNABs during the year; 314 FNABs were performed by physicians without formal training who had done a median of only 2 FNABs during the year (range, 1-43 FNABs). All FNAB specimens were reviewed microscopically and evaluated for cellularity and type of material present, for diagnostic accuracy, and for the rate of surgical intervention. A minimum of 2 years of follow-up was obtained by matching all cases to the population-based Northern California Cancer Registry. FNAB specimens were correlated with histologic specimens when they were available. RESULTS: Using FNAB, the formally trained physicians missed 2% of cancers, whereas the physicians without formal training missed 25%. Among the patients with benign lesions seen by the formally trained physicians, 8% went on to surgery, whereas 30% of those seen by physicians without formal training did so. Specimens obtained by the formally trained physicians were significantly more cellular and were significantly less likely to be nondiagnostic. CONCLUSIONS: FNAB, when performed by physicians who are well trained in the technique, is a highly accurate, cost-effective diagnostic method that carries minimal morbidity and could replace a large number of surgical biopsies. When performed by physicians without adequate training, FNAB is often misleading and potentially harmful.

Genetic changes in paired atypical and usual ductal hyperplasia of the breast by comparative genomic hybridization.

PURPOSE: Breast cancer is thought to develop from noninvasive precursor lesions, although the earliest steps of neoplastic transformation are still undefined. Usual ductal hyperplasia (UDH) is considered to represent a benign proliferation of ductal epithelial cells, whereas atypical ductal hyperplasia (ADH) may represent the first clonal neoplastic expansion of these cells. The aim of this study was to examine genetic alterations in UDH and ADH and to determine the relationship between these lesions in the same breast biopsy. EXPERIMENTAL DESIGN: Comparative genomic hybridization analysis was used to define copy number alterations in DNA extracted from archival sections of 18 patients. Nine patients showed ADH with adjacent UDH, and nine showed pure UDH. None showed evidence of invasive cancer or ductal carcinoma in situ. RESULTS: Five of the nine ADH lesions showed chromosome copy number alterations. 16q loss (five cases) and 17p loss (two cases) were the most frequent changes. The associated UDH lesions in these five patients also showed copy number alterations, always a subset of the changes present in the paired ADH. In one other patient, the UDH showed eight chromosomal alterations, whereas the paired ADH showed no changes. Only one of nine cases with pure UDH showed comparative genomic hybridization abnormalities. CONCLUSIONS: These data support the likelihood that UDH is a precursor of ADH, at least in some cases representing neoplastic growth. The frequencies of 16q and 17p losses suggest that alterations of candidate genes located in these chromosomal regions may play a role early in breast carcinogenesis.

Bone mass and breast cancer risk in older women: differences by stage at diagnosis.

BACKGROUND: Older women with low bone mineral density (BMD) have a decreased incidence of breast cancer. It is not known whether this association is confined to early-stage, slow-growing tumors. METHODS: We prospectively studied 8905 women who were 65 years of age or older during the period from 1986 through 1988 and had no history of breast cancer. At study entry, we used single-photon absorptiometry to measure each woman's BMD at three skeletal sites: the wrist, forearm, and heel. The women were followed for a mean of 6.5 years for the occurrence of breast cancer. All statistical tests were two-sided. RESULTS: During 57 516 person-years of follow-up, 315 women developed primary invasive or in situ breast cancer. Multivariate analyses that adjusted for age, obesity, and other covariates revealed that the risk of breast cancer for women in the highest quartile of BMD for all three skeletal sites was 2.7 (95% confidence interval [CI] = 1.4 to 5.3) times greater than that for women in the lowest quartile at all three skeletal sites. The magnitude of increased risk associated with high BMD differed by the stage of disease at diagnosis and was greater for more advanced tumors (relative risk [RR] for TNM [i.e., tumor-lymph node-metastasis] stage II or higher tumors = 5.6; 95% CI = 1.2 to 27.4) than for early-stage disease (RR for in situ/TNM stage I tumors = 2.2; 95% CI = 1.0 to 4.8). CONCLUSIONS: Elderly women with high BMD have an increased risk of breast cancer, especially advanced cancer, compared with women with low BMD. These findings suggest an association between osteoporosis and invasive breast cancer, two of the most prevalent conditions affecting an older woman's health.

Impaired spermatogenesis in men with congenital absence of the vas deferens.

It is generally assumed that men with congenital bilateral absence of the vas deferens (CBAVD) have azoospermia because of obstruction and that sperm production is normal. This study examines spermatogenesis in men with CBAVD to assess the validity of this assumption. We identified all men with CBAVD who had undergone either a diagnostic or therapeutic fertility procedure. Procedures included diagnostic biopsy, testis fine needle aspiration (FNA) mapping, microscopic epididymal sperm aspiration (MESA), and testis sperm extraction (TESE). Among 33 CBAVD men, 18 underwent testis biopsy, 27 had MESA/TESE, and 10 had FNA mapping. On evaluation of these procedures, normal spermatogenesis was present in 29 men. Four men (12%) demonstrated impaired spermatogenesis. One patient had FNA testis cytology consistent with late maturation arrest, another demonstrated hypospermatogenesis on biopsy and low sperm yield by MESA, and two patients had pure Sertoli cell only histology on biopsy. Aetiologies for impaired spermatogenesis included varicocele and underlying genetic abnormalities. Although patients with CBAVD are assumed to have normal spermatogenesis and infertility due simply to obstruction, the potential for concomitant defects in sperm production exists. A clinical suspicion of testis failure should prompt further diagnostic evaluation of spermatogenesis prior to sperm retrieval. In addition, genetic counselling should be offered and testing for genetic lesions, including cystic fibrosis gene mutations and/or variants, Y chromosome microdeletions, and karyotype abnormalities, should be considered.

Loss of chromosome 16q in lobular carcinoma in situ.

Lobular carcinoma in situ (LCIS) and infiltrating lobular carcinoma may represent different forms of the same disease based on their frequent clinical association and similar histologic features. Patients with LCIS are at increased risk of multicentric and bilateral disease. Thus, LCIS may represent both a precursor to infiltrating lobular carcinoma and a marker of risk for breast cancer. To identify genomic alterations in LCIS, comparative genomic hybridization was performed on 17 cases without concurrent invasive carcinoma. Loss involving chromosome 16q was present in 88% of cases and was the sole detected alteration in 29%. Gain involving 1q was second in frequency, occurring in 41% of tumors, and in all cases was associated with loss of 16q. Other recurrent changes were loss involving 17p (18%), 8p (12%), and 12q24 (12%). E-cadherin immunohistochemistry was performed on all LCIS cases to evaluate the correlation of loss involving 16q22, the site of the E-cadherin gene, and altered protein expression. Most cases with 16q22 loss showed altered E-cadherin expression (12 of 13). These results in LCIS are similar to changes reported in infiltrating lobular cancer, confirming a genetic relationship between them. HUM PATHOL 32:292-296.

Testicular fine-needle aspiration in infertile men: correlation of cytologic pattern with biopsy histology.

Open testicular biopsy is the standard method for histopathologic assessment of spermatogenesis. The need for testis biopsy has been questioned with the increased success of minimally invasive techniques such as fine-needle aspiration (FNA) mapping. This study examines whether FNA can provide cytologic information equivalent to histologic patterns by correlating diagnoses from testis FNA cytology with biopsy histology. Men (n = 87) who had undergone both diagnostic FNA mapping and open testis biopsy in the evaluation of infertility were identified. Biopsies were assessed by recognized histologic patterns of normal, hypospermatogenesis, early and late maturation arrest, and Sertoli cell only. FNA cytologic specimens were examined for adequacy and were classified similarly. Mixed patterns were also identified. The correlation between the two methods was 94%, with no differences among the different histologies. Discrepancies between cytology and histology were primarily the result of inadequate sampling and evidence of mixed patterns on FNA mapping. FNA cytology is a minimally invasive method of obtaining testicular tissue for diagnostic purposes. These data demonstrate that FNA cytology can evaluate accurately all classically defined histologic types, and may have the potential to replace testis biopsy in the assessment of spermatogenesis.

Relationship between classic histological pattern and sperm findings on fine needle aspiration map in infertile men.

Systematic testis fine needle aspiration (FNA) mapping has been proposed as an adjunctive or alternative diagnostic procedure to biopsy to determine the presence of spermatozoa within infertile testes. This study related testis histology to the global presence or absence of spermatozoa in the same testes determined by FNA cytology. Testis biopsies and FNA mapping were performed in 87 infertile, azoospermic men. A mean of 1.3 biopsies and 14 FNA sites were taken per patient. Biopsies were assessed by recognized histological patterns of normal, Sertoli cell-only, hypospermatogenesis, early and late maturation arrest, sclerosis as well as mixed patterns that included at least two of these histologies. FNA cytological specimens were assessed for sperm presence by an experienced cytologist. Overall, spermatozoa were found by FNA mapping in 52% of patients. A comparison of histology and FNA findings revealed that pure patterns of Sertoli cell-only and early maturation were associated with a very poor likelihood of sperm detection (4-8%). In contrast, patients with other pure pattern histologies or mixed patterns had high rates of FNA sperm detection (77-100%). Similar to reported testicular sperm extraction (TESE) findings, sperm detection with FNA shows wide variation depending on testis histology. Unlike most TESE reports, however, some histological patterns generally reflect a more global testicular dysfunction and poorer likelihood of sperm identification, suggesting the possibility that these phenotypes have a genetic origin. Systematic testis sampling with FNA offers additional geographical information about spermatogenesis that routine biopsies lack and can further guide couple decision-making in severe male factor infertility.

Diagnostic findings from testis fine needle aspiration mapping in obstructed and nonobstructed azoospermic men.

PURPOSE: Although helpful for defining extratesticular obstruction, the testis biopsy offers limited information on nonobstructive azoospermic testes. Guided by diagnostic biopsies, testis sperm extraction procedures fail in 25% to 50% of patients with nonobstructive azoospermia, largely because it is clinically difficult to know where sperm are located. To provide a more complete assessment of spermatogenesis in nonobstructive azoospermic patients and to simplify the confirmation of sperm production in men with obstruction, we use a systematic, fine needle aspiration "mapping" procedure. We summarize the diagnostic findings in a series of azoospermic men. MATERIALS AND METHODS: From 118 azoospermic infertile men (22 with obstructed and 96 with nonobstructed azoospermia) fine needle aspiration data were used to generate location specific, sperm frequency maps for obstructed and nonobstructive azoospermic testes to determine if "sperm rich" locations existed. RESULTS: Fine needle aspiration map analysis revealed that all aspiration locations from obstructed cases showed sperm. In men with nonobstructive azoospermia, sperm was identified in the right testis in 134 of 652 (20.5%) and in the left testis in 151 of 716 (21.1%) separate aspirations. Rates of sperm detection among various intratesticular sites were not statistically different. In 27.1% of cases the fine needle aspiration map found sperm in men with sperm negative biopsies. The likelihood of heterogeneity in fine needle aspiration sperm findings was 25% within individual nonobstructive azoospermic testes and 19.2% between testis sides. At post-procedure followup of 88 patients (74%), no clinical or surgical complications were observed. CONCLUSIONS: Testis fine needle aspiration mapping is a simple, reliable and informative diagnostic tool in the evaluation of azoospermic infertile men.

The prognostic value of proliferation indices: a study with in vivo bromodeoxyuridine and Ki-67.

Proliferation indices are intended to help patients and clinicians make treatment decisions. We have previously demonstrated that a proliferation index based on in vivo labeling of S-phase cells with bromodeoxyuridine (BrdUrd) correlates with Ki-67 labeling index (LI). We now compare the prognostic value of these indices. With written consent, we gave 129 women with biopsy confirmed breast cancer 200 mg/M2 BrdUrd during 30 min immediately preceding surgery. We used IU-4 anti BrdUrd antibody to count the immunohistochemical labeling index (LI) of DNA-incorporated BrdUrd in 2,000 cells and MIB-1 to count Ki-67 (118 cases). Patients received standard surgical and adjuvant treatment. No patients were lost to follow-up and patients were followed a minimum of 2 (median 5.1) years. We compared survival and recurrence in tumors with high vs low labeling indices. We found that women in the low BrdUrd LI group had better disease free survival (92% vs 67% 5-yr DFS p = 0.001) and overall survival (94% vs 70% 5-yr OS, p = 0.0001) than those with a high LI. In comparison, a low Ki-67 index predicted better OS (87% vs 80% 5-yr OS, p = 0.020) and a trend for better DFS (84% vs 72% DFS p = 0.055). The apparent superiority of BrdUrd LI over Ki-67 LI is likely due to chance (p = 0.18). In multivariate survival analyses we found that BrdUrd LI proliferative index significantly improves prediction of DFS or OS even when node status, age or tumor size is in the model. We conclude that markers of proliferation are useful adjuncts in predicting patient prognosis.

Fine needle aspiration in the diagnosis of metastatic melanoma.

BACKGROUND: Fine needle aspiration is an accurate technique to diagnose metastatic melanoma. Few reports exist in the literature describing its usefulness in many patients with melanoma confirmed by open biopsy. OBJECTIVE: The purpose of this study was to determine the utility and predictive value of fine needle aspiration in patients with malignant melanoma who presented with lesions suspected to be metastatic. METHODS: We retrospectively reviewed 99 cases of fine needle aspiration and the corresponding histologic findings obtained by open biopsy in 82 patients. RESULTS: Of the 99 cases, 86 were positive for melanoma, 12 were negative, and one was indeterminate. The positive predictive value of fine needle aspiration was 99%. One patient had a false-positive diagnosis. CONCLUSION: Fine needle aspiration is a rapid, accurate, and minimally invasive procedure that is useful in the diagnosis of metastatic melanoma. Patients with a positive aspirate of palpable regional nodes can proceed directly to surgery, bypassing the need for an open biopsy.

Chromosomal alterations in ductal carcinomas in situ and their in situ recurrences.

BACKGROUND: Ductal carcinoma in situ (DCIS) recurs in the same breast following breast-conserving surgery in 5%-25% of patients, with the rate influenced by the presence or absence of involved surgical margins, tumor size and nuclear grade, and whether or not radiation therapy was performed. A recurrent lesion arising soon after excision of an initial DCIS may reflect residual disease, whereas in situ tumors arising after longer periods are sometimes considered to be second independent events. The purpose of this study was to determine the clonal relationship between initial DCIS lesions and their recurrences. METHODS: Comparative genomic hybridization (CGH) was used to compare chromosomal alterations in 18 initial DCIS lesions (presenting in the absence of invasive disease) and in their subsequent ipsilateral DCIS recurrences (detected from 16 months to 9.3 years later). RESULTS: Of the 18 tumor pairs, 17 showed a high concordance in their chromosomal alterations (median = 81%; range = 65%-100%), while one case showed no agreement between the paired samples (having two and 20 alterations, respectively). Morphologic characterization of the DCIS pairs showed clear similarities. The mean number of CGH changes was greater in the recurrent tumors than in the initial lesions (10.7 versus 8.8; P =.019). The most common changes in both the initial and the recurrent in situ lesions were gains involving chromosome 17q and losses involving chromosomes 8p and 17p. The degree of concordance was independent of the time interval before recurrence and of the presence of positive surgical margins. CONCLUSIONS: In this study, DCIS recurrences were clonally related to their primary lesions in most cases. This finding is consistent with treatment paradigms requiring wide surgical margins and/or postoperative radiation therapy.

Menstrual and reproductive factors for salivary gland cancer risk in women.

Several observations suggest that salivary gland cancer may be, in part, a hormonally dependent disease. We examined associations between hormonally mediated life events and salivary gland cancer risk in a population-based case-control study. Of 76 women diagnosed between 1989 and 1993, 63 (83%) were interviewed. Of 111 population controls, 83 (75%) were interviewed. Early menarche (OR = 4.1, 95% CI = 1.4-12.1) and nulliparity (OR = 2.6, 95% CI = 1.0-6.7) were associated with increased risk whereas late age at first full-term pregnancy (OR = 0.19, 95% CI = 0.3-1.2) and longer duration of oral contraceptive use (OR = 0.31, 95% CI = 0.10-1.0) were associated with diminished risk. These findings are consistent with a hormonal component in salivary gland cancer risk.

Transmucosal fine-needle aspiration diagnosis of intraoral and intrapharyngeal lesions.

OBJECTIVES: The effectiveness of fine-needle aspiration biopsy (FNAB) for the diagnosis of neck, thyroid, and salivary gland masses is well documented. Very few reports explore the potential of an intraoral FNAB approach for the diagnosis of submucosal lesions. We describe our technique and present case examples of pertinent differential diagnostic entities. We recommend an expanded role for FNAB of the oral cavity and oropharynx. STUDY DESIGN: Retrospective review. METHODS: A uniform technique was employed for transmucosal FNAB of 76 patients with intraoral masses. In applicable cases, cytology results were compared with traditional biopsy methods and permanent histopathologic specimens for accuracy. RESULTS: Our experience demonstrates the high sensitivity (93%) and specificity (86%) of intraoral FNAB when compared with biopsy by conventional means. FNAB provides distinct advantages for the cytologic diagnosis of submucosal lesions, which may be difficult to reach and adequately sample through conventional biopsy. FNAB of the tonsil and tonsillar fossa provides a safe and effective means of diagnosing both lymphoma and squamous cell cancer. Transmucosal FNAB via the mouth led to rapid diagnosis of a number of benign and malignant lesions. Applying this uniform FNAB technique, we had no significant complications. CONCLUSION: We recommend transmucosal FNAB as an effective means for highly accurate diagnosis of submucosal lesions of the oral cavity and oropharynx. CLINICAL RELEVANCE: Traditional biopsy techniques in the oral cavity may require anesthesia and may have diagnostic difficulties, particularly for submucosal lesions. Transmucosal FNAB overcomes these shortcomings by providing a minimally invasive means to rapid diagnosis of intraoral lesions.

Genetic alterations in ERBB2-amplified breast carcinomas.

Amplification of the ERBB2 oncogene has recently received attention as a target for antibody-based therapies and as a predictor of response to adjuvant chemotherapy. Modification of treatment strategies based on ERBB2 status has led to further interest in the genetic alterations that accompany ERBB2 gene amplification or overexpression. In this study, chromosome alterations that are associated with ERBB2 amplification were defined by comparative genomic hybridization (CGH). Additionally, fluorescence in situ hybridization (FISH) was used to validate gene amplification, and protein expression was detected immunohistochemically. ERBB2-amplified tumors as detected by FISH, immunohistochemistry (IHC), or CGH had twice as many CGH-defined chromosomal alterations (means of 11.8, 11.0, and 12.7, respectively) as the nonamplified tumors (means of 6.8, 7.0, and 5.6, respectively). ERBB2 positivity correlated with the total number of genetic events. A wide spectrum of copy number gains and losses was seen by CGH in all of the tumors. An increased number of losses of 18q and gains of 20q was found in ERBB2-positive tumors. Other common aberrations for all of the tumors were copy number gains of 1q (58%), 8q (52%), 20q (30%), and losses of 18q (39%), 13q (39%), and 3p (33%). A high degree of concordance was observed among the three methods in 33 primary breast cancers. The concurrence for ERBB2 detection between FISH and IHC was 90%, between FISH and CGH was 82%, and between IHC and CGH was 84%. This study shows that breast tumors showing erbB2 overexpression or gene amplification are genetically distinct from erbB2-negative tumors. These differences may relate to the mechanisms underlying altered response to adjuvant therapies and may define the responsiveness to erbB2-directed immunotherapy.

Propagation of genetically altered tumor cells derived from fine-needle aspirates of primary breast carcinoma.

Because primary breast tumors are diagnosed earlier in the clinic, procurement of sufficient amounts of tumor tissue for in-depth biological characterization is becoming increasingly difficult. We demonstrate here that relatively small numbers of tumor cells within samples of fine-needle aspirates (FNA) can be propagated in culture. Of 25 cases attempted, 12 were passageable, resulting in up to 10(7) viable cells. FNA-derived cultures were evaluated for anchorage-independence, c-erb-B2 overexpression, aneusomy, and pattern of allelic loss. In every case examined, the cultured cells closely resembled the original tumor tissue and displayed one or more tumor phenotypes. The incidence of erb-B2 overexpressing tumors was similar in passageable and nonpassageable cases (33% versus 31%, respectively). FNAs that are expanded from a wide range of clinical breast material could be useful for functional studies presently limited to rare established cell lines, such as aberrant signal transduction and gene regulation, and for testing potential anticancer vaccines and drugs.

Methaemoglobin formation after the use of EMLA cream in term neonates.

EMLA cream 5% (a eutectic mixture of lidocaine and prilocaine) is a topical anaesthetic that has become widely used to minimize pain from venipuncture in children. It has not, however, been recommended in neonates owing to the potential risk of methaemoglobinaemia induced by prilocaine. The aim of this study was to establish the safety of 1 g EMLA cream 5% used on intact skin in term neonates. Forty-seven neonates, aged 0-3 months, with a postconceptual age of > or = 37 weeks and a body weight between 2.8 and 5.7 kg, were included in a double-blind, randomized, placebo-controlled study. After baseline observations a total dose of 1.0 g EMLA/placebo was applied to two sites (0.5 g site(-1)) for 60-70 min. Venous methaemoglobin (metHb) levels were determined in each patient at baseline and at three randomly assigned times, 0.5-18 h after application. Following application of the cream, the mean metHb levels were 1.17% (range 0.50-2.53) in the EMLA group and 0.96% (range 0.50-1.53) in the placebo group. The metHb concentrations were significantly higher in the EMLA group in the intervals from 3.5 to 13 h after application than in the placebo group, but were well below potentially harmful levels. Based on these results, a 1-h application of 1 g EMLA cream is safe when used on the intact skin of term neonates below 3 months of age.

High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays.

Gene dosage variations occur in many diseases. In cancer, deletions and copy number increases contribute to alterations in the expression of tumour-suppressor genes and oncogenes, respectively. Developmental abnormalities, such as Down, Prader Willi, Angelman and Cri du Chat syndromes, result from gain or loss of one copy of a chromosome or chromosomal region. Thus, detection and mapping of copy number abnormalities provide an approach for associating aberrations with disease phenotype and for localizing critical genes. Comparative genomic hybridization (CGH) was developed for genome-wide analysis of DNA sequence copy number in a single experiment. In CGH, differentially labelled total genomic DNA from a 'test' and a 'reference' cell population are cohybridized to normal metaphase chromosomes, using blocking DNA to suppress signals from repetitive sequences. The resulting ratio of the fluorescence intensities at a location on the 'cytogenetic map', provided by the chromosomes, is approximately proportional to the ratio of the copy numbers of the corresponding DNA sequences in the test and reference genomes. CGH has been broadly applied to human and mouse malignancies. The use of metaphase chromosomes, however, limits detection of events involving small regions (of less than 20 Mb) of the genome, resolution of closely spaced aberrations and linking ratio changes to genomic/genetic markers. Therefore, more laborious locus-by-locus techniques have been required for higher resolution studies. Hybridization to an array of mapped sequences instead of metaphase chromosomes could overcome the limitations of conventional CGH (ref. 6) if adequate performance could be achieved. Copy number would be related to the test/reference fluorescence ratio on the array targets, and genomic resolution could be determined by the map distance between the targets, or by the length of the cloned DNA segments. We describe here our implementation of array CGH. We demonstrate its ability to measure copy number with high precision in the human genome, and to analyse clinical specimens by obtaining new information on chromosome 20 aberrations in breast cancer.

The functional relationship between in vivo bromodeoxyuridine labeling index and Ki-67 proliferation index in human breast cancer.

Proliferation indices are used, along with other parameters, to estimate the risk of recurrence of breast cancer for individual patients. Because it is unlikely one index will be practical for all patients, it is important to understand the relationship between various indices of proliferation. For this reason, we compared a proliferation index based on in vivo labeling of S-phase tumor cells with the thymidine analog bromodeoxyuridine (BrdUrd), to a proliferation index based on an estimate of the growth fraction with the MIB-1 antibody to the Ki-67 antigen. With informed consent, we gave 145 patients 200 mg/m2 BrdUrd intravenously just prior to surgical removal of breast cancer. On histology sections, we visually counted S-phase cells which had incorporated BrdUrd using the Br-3 antibody which is specific to DNA-incorporated BrdUrd, and we counted cells in the growth fraction using the MIB-1 antibody to the Ki-67 antigen. We found that both indices were positively correlated with tumor size, number of positive nodes, and tumor grade, and both were negatively correlated with age and estrogen-progesterone receptor positivity. Using a linear functional relationship model, we found that the best (i.e. the maximal) fit between the two indices (correlation coefficient 0.79; p < 0.0001) occurred when each index was square root transformed, as is appropriate when counts follow a Poisson distribution. When we used the median as a cutpoint for each index, the classification of 19 percent of data pairs changed depending upon which index was used. We also estimated that the Ki-67 intercept (1.02 +/- 0.25) was significantly greater than zero. We conclude that the BrdUrd index of DNA synthesis in S-phase correlates highly with the MIB-1 index of the growth fraction, and both indices correlate well with other parameters of tumor aggressiveness. Because this correlation is driven by concordance of the extremes of high and low counts, clinical comparison will be necessary to determine which is the better prognostic marker for human breast cancer.