Pamela S. Larson

Loss of Heterozygosity or Allele Imbalance in Histologically Normal Breast Epithelium Is Distinct from Loss of Heterozygosity or Allele Imbalance in Co-Existing Carcinomas

To better understand early steps in human breast carcinogenesis, we examined allele imbalance or loss of heterozygosity (LOH), in co-existing normal-appearing breast epithelium and cancers. We microdissected a total of 173 histologically normal ducts or terminal ductolobular units (TDLUs) and malignant epithelial samples from 18 breast cancer cases, and examined their DNA for LOH at 21 microsatellite markers on 10 chromosome arms. Fourteen of 109 (13%) normal ducts/TDLUs, from 8 of 18 (44%) cases, contained LOH. The location of these 14 ducts/TDLUs appeared unrelated to distance from the cancer. LOH in normal-appearing epithelium involved only single markers, whereas LOH in cancers commonly encompassed all informative markers on a chromosome arm. In only 1 of 14 (7%) ducts/TDLUs with LOH, was the same LOH seen in the co-existing cancer. Global differences in LOH per arm in normal-appearing tissue were not demonstrated, but less LOH was seen at 11q and 17p than at 1q (P = 0.002), 16q (P = 0.01), and possibly 17q (P = 0.06). These results indicate that in a large fraction of women with breast cancer, histologically normal breast epithelium harbors occult aberrant clones. Individual clones rarely are precursors of co-existing cancers. However, they might constitute a reservoir from which proliferative lesions or second cancers develop once additional genetic abnormalities occur, they could contribute to intratumoral genetic heterogeneity, and they are consistent with a role for genetic instability early in tumorigenesis.

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. Copyright

Quantitative DNA Fingerprinting May Distinguish New Primary Breast Cancer From Disease Recurrence

PURPOSE Approximately 10% of women with breast cancer develop a second breast tumor, either a new primary or a recurrence. Differentiating between these entities using standard clinical and pathologic criteria remains challenging. Ambiguous cases arise, and misclassifications may occur. We investigated whether quantitative DNA fingerprinting, based on allele imbalance (AI) or loss of heterozygosity (LOH), could evaluate clonality and distinguish second primary breast cancer from recurrence. METHODS We developed a scoring system based on the AI/LOH fingerprints of 20 independent breast tumors and generated a decision rule to classify any breast tumor pair as related or unrelated. We validated this approach on eight related tumors (cancers and synchronous positive lymph nodes). Finally, we analyzed paired tumors from 13 women (bilateral cancers, primary tumors and contralateral positive axillary lymph nodes, or two ipsilateral tumors). Each pairs genetic classification was compared with their clinical diagnosis and outcome. RESULTS Each independent cancer had a unique fingerprint. Every tumor pairs relationship was quantifiable. Six of eight related tumor pairs were genetically classified correctly, two were indeterminate, and none were misclassified. Among the 13 women with two cancers, four of five clinically indeterminate pairs could be classified genetically. In three of 13 women, the pairs classification contradicted the clinical diagnosis. These women had bilateral cancers genetically classified as related and disease progression. This challenges the paradigm that bilateral cancers represent independent tumors. Overall, women with tumors genetically classified as related had poorer outcomes. CONCLUSION Quantitative AI/LOH fingerprinting is a potentially valuable tool to improve diagnosis and optimize treatment for the growing number of second breast malignancies.

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.

Allele Imbalance, or Loss of Heterozygosity, in Normal Breast Epithelium of Sporadic Breast Cancer Cases and BRCA1 Gene Mutation Carriers Is Increased Compared With Reduction Mammoplasty Tissues

PURPOSE Normal-appearing breast epithelium can contain genetic abnormalities, including allele imbalance (AI), also referred to as loss of heterozygosity. Whether abnormalities are associated with cancer or cancer risk is unknown. PATIENTS AND METHODS We performed a miniallelotype, using 20 microsatellites, on each of 460 histologically normal, microdissected breast terminal ducto-lobular units (TDLUs) from three groups of women: sporadic breast cancer patients (SP; n = 18), BRCA1 gene mutation carriers (BRCA1; n = 16), and controls undergoing reduction mammoplasty (RM; n = 18). We analyzed the results using Fishers exact tests, logistic regression, and generalized estimating equations. RESULTS AI was increased three-fold in SP and BRCA1 groups compared with RM. Both the number of TDLUs with AI increased (eight [5%] of 162 in the RM group compared with 24 [15%] of 162 in the SP and 22 [16%] of 136 in the BRCA1 groups; P = .0150), and the proportion of patients with AI increased (five [28%] of 18 in the RM group compared with 15 [83%] of 18 in the SP and 13 [81%] of 16 in the BRCA1 groups; P = .0007). The adjusted odds ratios (OR) for AI in TDLU increased in SP (OR = 15.5) and BRCA1 (OR = 13.7) patients compared with RM (P = .0025). This result was particularly evident on chromosome 17q (P = .0393), where more AI was seen in BRCA1 (OR = 12.4) than in SP (OR = 4.9) patients or RM controls. CONCLUSION Increased prevalence of AI in normal-appearing epithelium is associated with breast cancer and increased breast cancer risk. The increased prevalence may reflect dysregulation, even in normal-appearing epithelium, of genomic processes contributing to cancer development. The clinical significance of genetic alterations in the subset of controls remains to be determined.

Loss of heterozygosity in serial plasma DNA samples during follow-up of women with breast cancer

We evaluated the potential utility of occult circulating tumor DNA as a molecular marker of disease in subjects previously diagnosed with breast cancer. Using 24 microsatellite markers located at sites of frequent loss of heterozygosity (LOH) or allele imbalance in breast cancer, we analyzed DNA from 16 primary tumors (Stage IIA or more advanced) and 30 longitudinally collected plasma specimens. Clinical data at the time of plasma collection were obtained. All 16 tumors were characterized by an individual pattern of LOH. LOH was detected in 12 of 30 (40%) plasma samples, taken from 8 of 14 (57%) subjects. However, the number of LOH in plasma was small (n = 15), and the mean proportion of LOH was much lower than in the tumors (0.05 vs. 0.52). Although infrequent, 12 of 15 (80%) plasma LOH were concordant with abnormalities in the paired tumors, and the mean percent LOH was higher than in normal plasmas, suggesting that they were authentic tumor‐derived abnormalities. We found, despite this, no association, between plasma LOH and tumor stage or clinical status at time of blood collection (i.e., LOH was as common in subjects with no evident disease as in those with evident disease). In addition, detection of LOH was not consistent between serial samples from 5 of 11 subjects (45%), despite stable clinical conditions. No association with clinical outcome was evident, although the sample size was small. Microsatellite instability in plasma was infrequent, nonconcordant with paired tumor and inconsistent in serial samples. This pilot study suggests that identifying tumor‐specific LOH in the plasma of breast cancer subjects may not be useful for detecting occult metastases or for monitoring disease. Other detection techniques may be more promising, but circulating tumor DNA may not be a sufficiently accurate reflection of breast cancer clinical status or tumor activity.

In Utero Exposure to Diethylstilbestrol (DES) Does Not Increase Genomic Instability in Normal or Neoplastic Breast Epithelium

In 1992, the National Cancer Institute (NCI) established the Continuation of Follow‐Up of DES‐Exposed Cohorts to study the long‐term health effects of exposure to diethylstilbestrol (DES). Genetic effects on human breast tissue have not been examined. The authors investigated whether breast tissue of women exposed in utero to DES might exhibit the genetic abnormalities that characterize other DES‐associated tumors.

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 tumors 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. Int. J. Cancer (Pred. Oncol.) 89:105–110, 2000.