Gene expression in histologically normal epithelium from breast cancer patients and from cancer-free prophylactic mastectomy patients shares a similar profile.

INTRODUCTION: We hypothesised that gene expression in histologically normal (HN) epithelium (NlEpi) would differ between breast cancer patients and usual-risk controls undergoing reduction mammoplasty (RM), and that gene expression in NlEpi from cancer-free prophylactic mastectomy (PM) samples from high-risk women would resemble HN gene expression. METHODS: We analysed gene expression in 73 NlEpi samples microdissected from frozen tissue. In 42 samples, we used microarrays to compare gene expression between 18 RM patients and 18 age-matched HN (9 oestrogen receptor (ER)+, 9 ER-) and 6 PM patients. Data were analysed using a Bayesian approach (BADGE), and validated with quantitative real-time PCR (qPCR) in 31 independent NlEpi samples from 8 RM, 17 HN, and 6 PM patients. RESULTS: A total of 98 probe sets (86 genes) were differentially expressed between RM and HN samples. Performing hierarchical analysis with these 98 probe sets, PM and HN samples clustered together, away from RM samples. qPCR validation of independent samples was high (84%) and uniform in RM compared with HN patients, and lower (58%), but more heterogeneous, in RM compared with PM patients. The 86 genes were implicated in many processes including transcription and the MAPK pathway. CONCLUSION: Gene expression differs between the NlEpi of breast cancer cases and controls. The profile of cancer cases can be discerned in high-risk NlEpi from cancer-free breasts. This suggests that the profile is not an effect of the tumour, but may mark increased risk and reveal the earliest genomic changes of breast cancer.

Tamoxifen's protection against breast cancer recurrence is not reduced by concurrent use of the SSRI citalopram.

Tamoxifen remains an important adjuvant therapy to reduce the rate of breast cancer recurrence among patients with oestrogen-receptor-positive tumours. Cytochrome P-450 2D6 metabolizes tamoxifen to metabolites that more readily bind the oestrogen receptor. This enzyme also metabolizes selective serotonin reuptake inhibitors (SSRI), so these widely used drugs - when taken concurrently - may reduce tamoxifen's prevention of breast cancer recurrence. We studied citalopram use in 184 cases of breast cancer recurrence and 184 matched controls without recurrence after equivalent follow-up. Cases and controls were nested in a population of female residents of Northern Denmark with stages I-III oestrogen-receptor-positive breast cancer 1985-2001 and who took tamoxifen for 1, 2, or most often for 5 years. We ascertained prescription histories by linking participants' central personal registry numbers to prescription databases from the National Health Service. Seventeen cases (9%) and 21 controls (11%) received at least one prescription for the SSRI citalopram while taking tamoxifen (adjusted conditional odds ratio=0.85, 95% confidence interval=0.42, 1.7). We also observed no reduction of tamoxifen effectiveness among regular citalopram users (>or=30% overlap with tamoxifen use). These results suggest that concurrent use of citalopram does not reduce tamoxifen's prevention of breast cancer recurrence.

Quantitative analysis of allele imbalance supports atypical ductal hyperplasia lesions as direct breast cancer precursors.

It remains unclear whether hyperplastic breast lesions, especially with atypia, are cancer precursors or markers of increased cancer risk. Quantified comparisons of genomic alterations in coexisting lesions could address this question. Therefore, we examined allele imbalance (AI), also known as loss of heterozygosity (LOH), at 20 microsatellite markers on nine chromosome arms, in DNA from 106 samples microdissected from 17 randomly selected cancer-containing breast specimens: 13 simple (DH) and 45 atypical ductal hyperplastic (ADH) lesions, 30 in situ (DCIS) and 18 invasive ductal carcinomas (IDC). Data were analysed using regression models and generalized estimating equations. We found that AI increased as histology became more aberrant and varied with histology across the chromosome arms (p<0.0001). ADH had more AIs on 1q (p=0.03) and 16q (p=0.02) and fewer AIs on 17p (p=0.06) and 17q (p<0.0001) than on other arms. In cancers, AIs remained high on 1q and 16q, and became frequent on 17p and 17q. Concordance between AIs in ADHs and cancers exceeded the 50% expected if the lesions were separate clones in 16/20 (80%) ADHs (p=0.05), from 9/11 (82%) cases (p=0.03), and involved 41/51 (80%) evaluable markers (p=0.05). The occurrence of any AI in ADH predicted greater AI (p=0.009) and possibly lower grade (p=0.05) in coexisting cancers. Nevertheless, ADHs were not genetically identical to cancers or to each other. We found AIs discordant between ADHs and cancers (always on 1q and 16q), AIs unique to ADH (usually on 11q) and some genetic heterogeneity among coexisting ADHs. We conclude that ADH lesions are genetically advanced, with frequent alterations on 1q and 16q, and are often direct cancer precursors. Their global genetic characteristics predict features of cancers in the same breast. Nevertheless, the genetic heterogeneity detected suggests that hyperplasias and cancers may arise on a field at generalized increased cancer risk.

DNA alterations in tumor scrapes vs. biopsies of squamous-cell carcinomas of the head and neck.

Genetic abnormalities in SCCHNs are frequent and may be useful for screening, follow-up and prognosis. A biopsy or resection generally is utilized to identify these alterations but analysis of scraped or exfoliated tumor cells has been proposed as simpler and more versatile. It is unknown how well genetic abnormalities in scrapes reflect those in the tumor. Therefore, we compared DNA alterations in tumor scrapes obtained prior to treatment with alterations in microdissected tumor biopsies. Eight primary squamous-cell carcinomas of the head and neck (SCCHNs) were examined at 14 loci to determine loss of heterozygosity (LOH) at sites on 3p, 9p, 11p, 11q and 17p and amplification of cyclin D1 (CCND1). All biopsies contained DNA alterations, but only 3/8 scrapes contained unequivocal abnormalities; 4/8 contained subtle alterations that could not have been definitively identified without comparison to the paired biopsies. Overall, 22 alterations were detected in the biopsies: 8/22 were found unequivocally in the scrapes; 7/22 were identifiable in scrapes only after the biopsy alterations were defined and 7/22 were absent from scrapes. One LOH in scrape, but not biopsy, DNA was found. Discrepancies between scrapes and tumors tended to increase if multiple tumor samples were examined. We conclude that DNA alterations can be detected in scrapes of SCCHNs but may inaccurately reflect the tumor's complex genetic abnormalities. This may be due to contamination of scrapes with normal cells or to genetic heterogeneity within the tumor not represented in the scrape. Although examining scrapes of SCCHNs is an attractive technique, its clinical utility may have limitations.

Lack of correlation between morphometric analysis and presence of microsatellite alterations in proliferative ductal lesions of the breast.

OBJECTIVE: To apply morphometric studies using an image analyzer to a previously reported group of proliferative duct lesions and to compare results to see if there was a correlation between nuclear size range and monoclonality. STUDY DESIGN: Fifteen proliferative lesions of the breast from 12 subjects who had no history of breast malignancy were retrieved from archival pathology specimens. Evidence of monoclonality was studied using a panel of polymerase chain reaction primers to examine microsatellite alterations on microdissected paraffin-embedded specimens. Variation in nuclear size was studied using an image analyzer. RESULTS: Of six proliferative breast lesions with demonstrated genetic instability, three showed cytologic evidence of uniformity in nuclear size, a cytologic feature of malignancy. One of the six, which showed microsatellite alterations at three loci, demonstrated a wide range of nuclear size variation. Of the eight proliferative lesions that showed no genetic instability, three showed very uniform nuclear size, and five showed significant variations in nuclear size. One lesion, which fell into the "uncertain but probably genetic instable" category, showed diverse nuclear size ranges. CONCLUSION: This study demonstrated that there is no correlation between monoclonality and monomorphic cell cytology of histologic proliferative breast lesions.

Loss of heterozygosity at 11q23 in squamous cell carcinoma of the head and neck is associated with recurrent disease.

Loss of heterozygosity (LOH) at chromosome 11q23 has been found in a variety of epithelial human neoplasms, suggesting that this region contains a tumor suppressor gene(s) important to tumorigenesis. We investigated whether LOH at 11q23 could be detected in squamous cell carcinoma of the head and neck (SCCHN), and whether loss at this site was associated with specific clinical parameters. Fifty-six matched blood and SCCHN tumor samples taken at the time of diagnosis were evaluated for LOH at three microsatellite markers at 11q23. Multiplex PCRs with [alpha-32P]dCTP labeling of the amplified DNA strands were performed. Clinical data were obtained from medical record review. LOH at 11q23 was found in 13 of 52 (25%) evaluable tumors. There was no association between LOH at 11q23 and amplification of the CCND1 (cyclin D1) oncogene or inactivation of the p53 gene, which had been determined previously. With a mean follow-up of 24 months, an association independent of tumor size or stage was found between LOH at 11q23 and recurrent disease (P = 0.04). Among subjects who received radiotherapy (RT) as a component of their treatment, LOH at 11q23 was associated with persistent or recurrent locoregional disease (P = 0.05). LOH at 11q23 occurs in a subset of SCCHN. It is associated with a higher likelihood of recurrent disease, perhaps related to resistance to RT. The specific gene(s) and mechanism(s) responsible remain to be identified. Until then, LOH at 11q23 might become a marker identifying patients likely to do poorly with conventional therapy.

Genetically abnormal clones in histologically normal breast tissue.

Breast cancer is believed to develop as multiple genetic abnormalities accumulate, each conferring some growth advantage, but the timing and nature of the earliest steps in this progression are not yet elucidated. Proliferative breast lesions, associated with an increased risk of breast cancer although considered benign, recently were shown to contain clonal genetic abnormalities. Therefore, we hypothesized that clonal genetic abnormalities might be detectable before any phenotypic abnormalities are evident, ie, in histologically normal breast tissue. We examined DNA extracted from 95 normal-appearing breast ducts or terminal ductal-lobular units from 20 individuals at varying degrees of risk (those undergoing reduction mammoplasties, those with atypical hyperplastic proliferative lesions, and those already diagnosed with breast cancer). Using nine microsatellite markers, we sought evidence of genetic instability or of allelic imbalance (most likely representing loss of heterozygosity). We found genetically abnormal clones in 21/95 (22%) seemingly normal samples from 10/20 (50%) women from all three risk groups. In women under age 50, trends toward increased rates of abnormalities were noted with increased cancer risk. The abnormalities identified were more likely to be at sites of known or postulated tumor suppressor genes rather than at random or neutral loci. Our data indicate that genetic abnormalities potentially critical to breast tumorigenesis accumulate before pathological detection even of high-risk lesions and are detectable in tissue that is not only histologically benign but also completely normal.

Oral appliances in the treatment of snoring and sleep apnea.

Oral appliances have been developed that are effective in snoring patients and in patients with mild to moderate sleep apnea. This article reviews the types of appliances that are available, their possible modes of action, and their efficacy. In addition, the clinician is provided with guidelines on how to choose the appropriate patient for this therapy.

The efficacy of oral appliances in the treatment of persistent sleep apnea after uvulopalatopharyngoplasty.

Twenty-four patients who failed uvulopalatopharyngoplasty (UPPP) for obstructive sleep apnea (OSA) had an adjustable oral (Herbst) appliance made to treat the persistent apnea. Six patients discontinued the device prior to sleep evaluation. Eighteen patients had polysomnographic evaluations at baseline, post-UPPP, and with the Herbst appliance in place. The apnea-hypopnea index baseline (AHI) and arterial oxygen saturation (SaO2) nadir were 42.3+/-6.1 and 83.6+/-1.8%, respectively. There was no significant change in either parameter with surgery. With the oral appliance, the AHI fell to 15.3+/-4.4 (p < or = 0.01) and the SaO2 nadir increased to 87.9+/-1.2% (p < or = 0.05). Ten of the patients had control of the OSA with the Herbst appliance with a fall in the AHI to < 10. There were, in addition, two partial responders as defined by an AHI of <20 and a >50% fall in AHI compared with baseline and post-UPPP values. All but one of the responders and partial responders had complete resolution of subjective symptoms of daytime sleepiness with the appliance. An adjustable oral appliance appears to be an effective mode of therapy to control OSA after an unsuccessful UPPP.

Microsatellite alterations indicating monoclonality in atypical hyperplasias associated with breast cancer.

One model of breast tumorigenesis postulates a sequential evolution from normal to proliferative epithelium and eventually to neoplasia, but genetic data to support this progression have been limited. We wished to determine whether atypical hyperplasia (AH), a proliferative lesion conventionally classified and treated as benign, but associated with an increased risk of developing carcinoma, might show evidence of genetic abnormalities. Using the polymerase chain reaction (PCR), we examined DNA extracted from 12 separate AH lesions, from six breast cancer patients' paraffin-embedded tissue specimens, for alterations in microsatellite repeat sequences. Five of 12 AH lesions, from three of six patients, demonstrated alterations in microsatellite sequences in patterns indicating that the AH lesions are monoclonal or contain a substantial monoclonal component. We conclude that a subset of AH lesions from patients with breast cancer are characterized by monoclonal microsatellite alterations; therefore, they may already be neoplastic. This finding lends support to one postulated sequence of breast tumorigenesis and suggests that some type of genetic instability may play a role early in breast tumor development.

Detection of monoclonal microsatellite alterations in atypical breast hyperplasia.

Atypical hyperplastic (AH) breast lesions are currently classified and treated as benign proliferative disorders, but their presence is associated with a four- to fivefold increased risk of developing breast cancer. Currently, it is not known if an AH lesion is a marker of increased risk, or is itself a premalignant lesion. To investigate this question, we used a series of 15 microsatellite loci to analyze 15 separate AH lesions microdissected from the archived pathology specimens of subjects with no coincident or previous breast malignancy. We found that a significant subset (6/15, or 40%) of these AH lesions demonstrated evidence of monoclonal microsatellite alterations, both length variation and allele loss. These monoclonal alterations suggest that the AH lesion has already undergone genetic changes conferring a growth advantage. Thus, these AH lesions may actually be early neoplasms. We also noted that monoclonality characterized AH lesions in younger as compared with older women (44 vs. 59 yrs, P < 0.05) and that a subset of monoclonal lesions (4/6) demonstrated microsatellite alterations at more than one locus, suggesting that an undetermined type of genetic instability may play a role early in the development of abnormal breast proliferations. These findings contribute to our understanding of the pathogenesis of AH lesions and may have implications regarding their relationship to breast tumors.

Efficacy of a Herbst mandibular advancement device in obstructive sleep apnea.

Treatment options for obstructive sleep apnea (OSA) may involve potential side effects or discomfort; nasal continuous positive airway pressure (CPAP) may not be tolerated by 25% of patients. We therefore sought to determine the efficacy of mandibular advancement as a treatment for OSA, and to investigate whether clinical and radiographic parameters can predict the response to this treatment. Sixteen male and 3 female subjects with documented OSA who had failed or been unable to tolerate nasal CPAP underwent baseline polysomnography and cephalometry, and were then fitted with a removable Herbst appliance to achieve forward mandibular advancement during sleep. All subjects then underwent a second cephalometric evaluation and polysomnography with the appliance in place. Fourteen of 15 subjects demonstrated significant improvement in the degree of OSA, based on the apnea-hypopnia index (AHI) (34.7 +/- 5.3 to 12.9 +/- 2.4 events/h, p < 0.002). Comparison of pre- and posttreatment cephalometric values revealed no significant change in the posterior airway space (PAS) despite a reduction in mean AHI. There was a significant decrease in the mandible-hyoid distance (MP-H) with treatment for the group as a whole. When the study population was evaluated on the basis of a successful response to mandibular advancement (posttreatment AHI < 10), the baseline MP-H was found to be significantly shorter in the responders than in nonresponders. MP-H after mandibular advancement was likewise shorter in responders than in nonresponders. In addition, the soft palate length (PNS-P) showed a significantly greater shortening in responders after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome 11 translocation breakpoints at the PRAD1/cyclin D1 gene locus in centrocytic lymphoma.

Centrocytic lymphoma is a CD5-positive B-cell neoplasm. Rearrangements at the chromosome 11q13 bcl-1 breakpoint loci are present in the majority of these lymphomas, as a result of reciprocal translocation with the 14q32 immunoglobulin heavy chain joining genes. Recently, a gene lying approximately 110 kb telomeric of the bcl-1 major translocation cluster breakpoint locus, designated PRAD1, was proposed as a candidate bcl-1 oncogene. Accumulated evidence now indicates that this gene is the postulated bcl-1 oncogene. It encodes a protein with homology to cyclin family proteins designated cyclin D1 (CCND1). In order to determine whether 11q13 translocation breakpoints were present near the PRAD1 coding region in addition to the previously defined bcl-1 sites, we analyzed 27 centrocytic lymphomas by Southern blot using genomic and cDNA probes flanking the first exon of PRAD1. Five samples showed rearrangement at PRAD1 sites. In four of these, the breakpoints could be mapped from approximately one to 25 kb upstream of the first PRAD1 exon; each showed comigration of rearranged PRAD1 and immunoglobulin heavy-chain joining gene bands consistent with the t(11;14)(q13;q32). The fifth case was rearranged with PRAD1 probes only on BamHI-digested DNA, indicating either a point mutation or a polymorphism at this site. This sample also had rearrangement on multiple enzyme digests with the bcl-1 p94PS probe. None of 80 non-centrocytic B-cell neoplasms showed PRAD1 rearrangement. Thus, rearrangement at both bcl-1 and PRAD1 loci is strongly associated with centrocytic lymphoma, and provides a useful molecular marker for classifying this subtype of lymphoma. Furthermore, translocation-induced aberrant expression of the PRAD1 cyclin may lead to deregulated cell cycle control and play an important role in the pathogenesis of centrocytic lymphoma.

Coding sequence of the overexpressed transcript of the putative oncogene PRAD1/cyclin D1 in two primary human tumors.

PRAD1 (cyclin D1) is a recently identified member of the family of cyclin genes, believed to play roles in regulating transitions through the cell cycle. The PRAD1 gene, located at 11q13, has been implicated in the pathogenesis of a variety of tumors, including parathyroid adenomas, t(11;14) bearing B-lymphoid tumors (particularly centrocytic lymphomas) where it is highly likely to be the BCL1 oncogene, and possibly in breast carcinomas and squamous cell cancers of the head and neck as well. PRAD1's tumorigenic influence appears to be effected through overexpression of its normal-sized transcript, but it has not been established whether the transcript's coding sequence is normal or contains oncogenic mutations. We have sequenced the coding region of the overexpressed PRAD1 transcript from two primary tumors with clonal PRAD1 region rearrangements: a benign parathyroid adenoma and a malignant centrocytic lymphoma. Each sequence is identical to the normal PRAD1 cDNA sequence, and presumably encodes normal PRAD1 protein. Thus, PRAD1 likely functions as a direct-acting oncogene whose rearrangement in tumors leads to overexpression or deregulated expression of its normal protein product.

Characterization of chromosome 11 translocation breakpoints at the bcl-1 and PRAD1 loci in centrocytic lymphoma.

The chromosome 11q13 bcl-1 locus is rearranged in the majority of centrocytic lymphomas, a CD5-positive B-cell non-Hodgkins lymphoma, as a result of reciprocal translocation with the 14q32 immunoglobulin heavy chain genes. Although several 11q13 bcl-1 breakpoint sites have been characterized, a postulated bcl-1 oncogene was not identified. Recently, however, a gene encoding cyclin D1, designated PRAD1, was proposed as a candidate bcl-1 oncogene; accumulated evidence now indicates this gene is bcl-1. To further characterize 11q13 breakpoints in B-cell neoplasms, we analyzed 26 centrocytic lymphomas and 68 other B-cell cancers by Southern blot using a panel of breakpoint probes spanning 110 kilobases of the bcl-1 and PRAD1 loci. Nineteen centrocytic cases (73%) showed rearrangement, 15 at bcl-1 breakpoint sites and 5 at PRAD1 sites. One case was rearranged at both bcl-1 and PRAD1 loci. All but the latter case showed comigration of rearranged bcl-1 or PRAD1 bands and immunoglobulin heavy chain joining gene bands, consistent with the t(11;14). bcl-1 rearrangement was present in only one of 68 noncentrocytic B-cell neoplasms; none showed PRAD1 rearrangement. Thus, bcl-1 and PRAD1 rearrangement is strongly associated with centrocytic lymphoma, providing a useful molecular marker for classifying this subtype of lymphoma and suggesting an important role for PRAD1 cyclin D1 in the pathogenesis of this neoplasm.

PRAD1, a candidate BCL1 oncogene: mapping and expression in centrocytic lymphoma.

Rearrangement of the BCL1 (B-cell lymphoma 1) region on chromosome 11q13 appears to be highly characteristic of centrocytic lymphoma and also is found infrequently in other B-cell neoplasms. Rearrangement is thought to deregulate a nearby protooncogene, but transcribed sequences in the immediate vicinity of BCL1 breakpoints had not been identified. PRAD1, previously designated D11S287E, was identified on 11q13 as a chromosomal breakpoint region rearranged with the parathyroid hormone gene in a subset of parathyroid adenomas; this highly conserved putative oncogene, which encodes a novel cyclin, has been linked to BCL1 and implicated also in subsets of breast and squamous cell neoplasms with 11q13 amplification. We report pulsed-field gel electrophoresis data showing BCL1 and PRAD1 to be no more than 130 kilobases apart. PRAD1 mRNA is abundantly expressed in seven of seven centrocytic lymphomas (Kiel classification), in contrast to 13 closely related but noncentrocytic lymphomas. Three of the seven centrocytic lymphomas had detectable BCL1 DNA rearrangement. Also, two unusual cases of CLL with BCL1 rearrangement overexpressed PRAD1, in contrast to five CLL controls. Thus, PRAD1 is an excellent candidate "BCL1 oncogene." Its overexpression may be a key consequence of rearrangement of the BCL1 vicinity in B-cell neoplasms and a unifying pathogenetic feature in centrocytic lymphoma.