Loss of heterozygosity and microsatellite instability in breast hyperplasia. No obligate correlation of these genetic alterations with subsequent malignancy.

Loss of heterozygosity and microsatellite instability have been often reported in breast cancer and seldom in proliferative breast disease (PBD). DNA samples from microdissected PBD lesions, including papillomas (25 lesions), from 8 women were analyzed by polymerase chain reaction for loss of heterozygosity and microsatellite instability at 10 loci including INT-2 oncogene locus, D17S796 (the p53 gene region), and D17S579 (in the region of the BRCA-1 gene). In a patient, five loci with microsatellite instability and two loci with loss of heterozygosity were identified in one papilloma with florid hyperplasia and atypia, and 10 other PBD lesions were negative for genetic alteration (GA) and atypia. Three loci with microsatellite instability were identified in another PBD lesion without atypia, whereas another lesion from this second patient had minimal atypia without GAs. These two patients have been well for more than 20 years. No other patient, including a woman developing cancer, had GAs. We detected GAs in PBD (25% of women, 8% of lesions). Incomplete correlation between GAs and anatomic atypia was suggested. It seems evident that several GAs in PBD lesions may not indicate clinically meaningful premalignancy for remaining breast.     

Value of scoring system in classification of proliferative breast disease on fine needle aspiration cytology

Fine-needle aspiration (FNA) cytology is successful in identification of benign and malignant breast lesions, but its role in proliferative breast lesions which increase cancer risk is poorly defined. We have analyzed the cytomorphologic features of proliferative breast lesions in conjunction with cytologic scoring system proposed by Masood et al and with histopathology. Sixty two patients (14 cases of fibroadenoma, 15 cases of fibroadenoma with atypia, 11 cases of proliferative breast disease (PBD), 8 cases of PBD with atypia and 14 cases of carcinoma) diagnosed on routine FNA were subjected to scoring following Masood's criteria. All cases with the cytologic diagnosis of fibroadenoma were confirmed on histology. Of 11 cases of PBD on FNA, 10 were PBD without atypia on histology. One case, which showed atypical hyperplasia on histology, was missed by both the scoring system and cytomorphology and one case was over-diagnosed as PBD with atypia by the scoring system. FNA cytology correctly identified all the carcinoma cases, while the scoring system under-diagnosed 2 cases as PBD with atypia. Hence, in cases not suspected to be atypical or confirmed to be cancer on routine cytology, scoring added no information over and above cytomorphology and was not useful. All fourteen cases of fibroadenoma with atypia suspected on routine cytology were fibroadenoma on histology. Scoring system correctly placed 11/14 of these cases as PBD without atypia. Similarly 3/8 cases thought to be PBD with atypia were correctly placed as PBD without atypia by scoring. Only 2/8 cases thought to be PBD with atypia on cytology were confirmed to have atypical hyperplasia on histology. Scoring improved the diagnostic yield to 2/5. Hence, in cases of fibroadenoma or PBD, suspected on FNAC to have cytological atypia, Masood scoring gives additional information by eliminating benign cases and improving diagnostic yield. Application of scoring in a step-wise manner, on atypical aspirates, can help in selection of cases suitable for biopsy.     

Correlation of cytologic findings and chromosomal instability detected by fluorescence in situ hybridization in breast fine-needle aspiration specimens from women at high risk for breast cancer.

Cytologic evaluation of ductal lavage or random periareolar fine-needle aspiration (FNA) specimens has been proposed to improve risk stratification of women at high risk for breast cancer. However, cytologic assessment of morphologic changes is subjective. To assess the utility of fluorescence in situ hybridization (FISH) in the categorization of breast lesions, we prospectively evaluated 32 random periareolar FNA specimens from 27 women at high risk for breast cancer. Cytologic specimens were prepared using the thin preparation technique, and diagnoses were made on the basis of previously published criteria. Specimens were also evaluated by FISH for chromosomes 1, 8, 11, and 17. Monosomy was defined as the loss of one signal or both signals in >20% of cells, and polysomy was defined as the presence of > or = 3 signals in >6% of cells. Cytologic smears from seven invasive ductal carcinomas and nine benign breast specimens from women at low risk for breast cancer were included for comparison. In the high-risk group, cytologic findings were nonproliferative epithelium (NPE) in 16 cases and hyperplasia in 16 cases. Chromosomal aberrations were detected in 11 (69%) of 16 NPE cases, 14 (89%) of 16 hyperplasia cases, seven (100%) of seven carcinoma cases, and none of the low-risk cases. High-risk cases had significantly more monosomy of chromosomes 1, 11, and 17 and polysomy of chromosome 8 compared to low-risk cases and significantly less polysomy of chromosomes 1, 8, 11, and 17 compared to patients with cancer. There were no significant differences in monosomy or polysomy of individual chromosomes or a combination of chromosomes between the NPE and hyperplasia groups. This study shows that aberrations of chromosome number are common in high-risk women irrespective of cytologic findings. Studies evaluating the association between specific patterns of chromosomal polysomy and progression to malignancy may be warranted.     

Risk factors for breast cancer in women with proliferative breast disease

To assess the importance of various risk factors for breast cancer in women with benign proliferative breast lesions, we reevaluated 10,366 consecutive breast biopsies performed in women who had presented at three Nashville hospitals. The median duration of follow-up was 17 years for 3303 women, 1925 of whom had proliferative disease. This sample contained 84.4 per cent of the patients originally selected for follow-up. Women having proliferative disease without atypical hyperplasia had a risk of cancer that was 1.9 times the risk in women with nonproliferative lesions (95 per cent confidence interval, 1.2 to 2.9). The risk in women with atypical hyperplasia (atypia) was 5.3 times that in women with nonproliferative lesions (95 per cent confidence interval, 3.1 to 8.8). A family history of breast cancer had little effect on the risk in women with nonproliferative lesions. However, the risk in women with atypia and a family history of breast cancer was 11 times that in women who had nonproliferative lesions without a family history (95 per cent confidence interval, 5.5 to 24). Calcification elevated the cancer risk in patients with proliferative disease. Although cysts alone did not substantially elevate the risk, women with both cysts and a family history of breast cancer had a risk 2.7 times higher than that for women without either of these risk factors (95 per cent confidence interval, 1.5 to 4.6). This study demonstrates that the majority of women (70 per cent) who undergo breast biopsy for benign disease are not at increased risk of cancer. However, patients with a clinically meaningful elevation in cancer risk can be identified on the basis of atypical hyperplasia and a family history of breast cancer.     

Paired ductal lavage and fine-needle aspiration specimens from patients with breast carcinoma

Ductal lavage (DL) is a new procedure for sampling of the mammary epithelium, but experience with this technique remains limited. We compared the findings in paired DL and fine-needle aspiration (FNA) specimens obtained from patients with breast carcinoma. Four reviewers evaluated all DL samples. Two reviewers also examined the FNA material and compared cellular composition and morphologic findings in paired samples. DL and FNA samples from six patients were satisfactory for evaluation. Two DL samples showed marked atypia, one showed mild atypia, and two were benign; there was no agreement in one case (mild atypia vs. benign). Overall, the atypical cells in DL samples resembled those in the paired FNA material, but low degree of cytologic atypia and relative paucity of atypical cells limited their correct identification. The interpretation of DL samples is more challenging than that of FNA material, but similar criteria apply. To increase the sensitivity of DL, the number of epithelial cells required for a satisfactory sample should be higher than previously set.     

Quantification of HER2 oncoprotein in fine-needle aspirates of the breast

Measurements of either HER2 gene overexpression or its gene-coded protein (p185) are clinically useful for predicting prognosis in breast cancer. The measurements are also useful for identifying metastatic breast cancer patients who may benefit from Herceptin treatment. Since fine needle aspiration (FNA) of the breast has become an increasingly popular technique for obtaining tissue specimens, we have developed a sensitive method to quantify p185 in the aspirate. For this procedure, p185 from the cell pellet of FNA is extracted with a buffer containing Triton X-100, and the p185 is measured with an enzyme immunoassay. Most of the malignant breast tumors (N=7) in this study were associated with elevated p185 concentrations (6/7, 319+/-222 U/mg), compared to the p185 concentrations in normal breast tissue (42.8+/-35 U/mg, N=47) or benign lesions (43.1+/-20.2 U/mg, N=22). Quantification of p185 in FNA may improve the assessment of breast cancer patients, revealing whether they are at high risk and may benefit from Herceptin treatment.     

散发性乳腺癌及乳腺不典型导管增生组织BRCA1基因启动子区甲基化分析

目的 了解散发性乳腺癌及癌旁增生组织、乳腺不典型导管增生组织BRCA1基因启动子区甲基化状态,探讨其与乳腺癌发生的关系.方法 采用甲基化特异性PCR(MSP)结合巢式PCR技术,研究23例散发性乳腺癌及其癌旁增生组织、6例乳腺不典型导管增生组织及5例健康成人女性外周血淋巴细胞中BRCA1基因启动子区甲基化状态.结果 5例健康成人女性外周血淋巴细胞均表现BRCA1基因启动子区甲基化阴性;23例原发性乳腺癌组织中,BRCA1基因启动子区CpG岛甲基化率为65.22%(15/23);癌旁增生组织检出CpG岛甲基化者11例,甲基化率为47.83%(11/23),且均为癌组织阳性患者;6例乳腺不典型导管增生组织中,BRCA1基因启动子区CpG岛甲基化阳性者2例,甲基化率为33.33%(2/6);统计学检验结果表明,乳腺癌、癌旁增生组织之间,BRCA1基因启动子区甲基化阳性率无显著差异.结论 BRCA1基因启动子区CpG 岛甲基化是散发性乳腺癌发生过程中的早期事件,可能在乳腺癌发生中和乳腺增生病癌变过程中起重要生物学作用.     

Effectiveness of fine-needle aspiration cytology of breast: analysis of 2,375 cases from northern Thailand

At the Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand, 2,375 cases of breast lesions were sampled by fine-needle aspiration (FNA) from 1994-1999. Cytologic diagnoses were: benign (48%), suspicious for malignancy (5%), malignant (15%), and unsatisfactory (32%). Comparison with histology was possible in 721 cases. The diagnoses obtained by FNA showed a sensitivity of 84.4%, specificity of 99.5%, positive predictive value of 99.8%, negative predictive value of 84.3%, false-negative rate of 16.7%, false-positive rate of 0.5%, and overall diagnostic accuracy of 91.3%. We conclude that, in experienced hands, FNA of breast masses is reliable for diagnosis. Assessment of samples at the time of aspiration can reduce the number of inadequate specimens to near zero. Correlation of FNA results with clinical and radiologic findings can identify false-negatives and false-positives, ensuring optimal patient management. Many centers now recommend needle core biopsy instead of FNA. For regions such as ours, the added cost of this test would make it unavailable to many patients, which could delay a diagnosis of breast cancer. We advocate keeping FNA as a first-line diagnostic procedure, at least in areas under economic restrictions, in order to maximize the availability of health care to women with breast disease.     

Computerized analysis of shadowing on breast ultrasound for improved lesion detection

Sonography is being considered for the screening of women at high risk for breast cancer. We are developing computerized detection methods to aid in the localization of lesions on breast ultrasound images. The detection scheme presented here is based on the analysis of posterior acoustic shadowing, since posterior acoustic shadowing is observed for many malignant lesions. The method uses a nonlinear filtering technique based on the skewness of the gray level distribution within a kernel of image data. The database used in this study included 400 breast ultrasound cases (757 images) consisting of complicated cysts, solid benign lesions, and malignant lesions. At a false-positive rate of 0.25 false positives per image, a detection sensitivity of 80% by case (66% by image) was achieved for malignant lesions. The performance for the overall database (at 0.25 false positives per image) was less at 42% sensitivity by case (30% by image) due to the more limited presence of posterior acoustic shadowing for benign solid lesions and the presence of posterior acoustic enhancement for cysts. Our computerized method for the detection of lesion shadows alerts radiologists to lesions that exhibit posterior acoustic shadowing. While this is not a characterization method, its performance is best for lesions that exhibit posterior acoustic shadowing such as malignant and, to a lesser extent, benign solid lesions. This method, in combination with other computerized sonographic detection methods, may ultimately help facilitate the use of ultrasound for breast cancer screening.     

Cell blocks of breast FNAs frequently allow diagnosis of invasion or histological classification of proliferative changes

Two major limitations of breast fine needle aspiration (FNA) compared with core needle biopsies (CNB) are the inability to determine whether a cancer is invasive and to classify proliferative lesions. We studied 40 consecutive "rapid cell blocks" from breast FNAs with surgical pathology follow-up to test whether cell blocks can overcome these limitations. Of 25 carcinomas, invasion could be identified in the cell block sections in 11 (44%). One cystosarcoma phyllodes was suspected based on the cell block sections. Cell blocks from 12 of 14 benign breast FNAs showed sufficient cells to assign a histologic diagnosis of no hyperplasia (1 case, confirmed on follow-up) and usual hyperplasia (11 cases; confirmed in eight of 11 on follow-up). Specific histologic diagnoses included intraductal papilloma (2 cases), and in situ lobular neoplasia (2 cases). Cell blocks complement smears and monolayers and appear to overcome major limitations of breast FNA.     

Comparison of fine-needle aspiration cytology and core biopsy for diagnosis of breast cancer

Both fine-needle aspiration (FNA) cytology and core biopsy are useful in the diagnosis of breast cancer. In order to compare the sensitivities of these procedures, we reviewed 209 patients with breast cancer who had either FNA, core biopsy, or both, and also either mastectomy or lumpectomy. Sensitivities for FNA and core biopsies for diagnosing breast cancer were calculated and compared. Sensitivity for FNA or core biopsies interpreted as either atypical or malignant was 93.8% for FNA and 90.1% for core biopsy (P > 0.05). Sensitivity for FNA or core biopsies interpreted as malignant was 65.4% for FNA and 88.7% for core biopsy (P < 0.0001). Sensitivities of FNA interpreted as either atypical or malignant were 92.4% for FNA performed by pathologists and 100% for FNA by nonpathologists (P > 0.05). Sensitivities of FNA interpreted as malignant were 75.8% for FNA by pathologists and 20.0% for FNA by nonpathologists (P < 0.00001). Both FNA and core biopsies are sensitive procedures for the detection of breast cancer. There was no significant difference between sensitivity of FNA and core biopsies interpreted as either atypia or malignancy, although the sensitivity of core biopsies interpreted as unequivocal malignancy was greater than that of FNA. FNAs performed by pathologists were more sensitive than FNAs performed by nonpathologists in making an unequivocal diagnosis of breast cancer.     

Fine-needle aspiration cytology of mesenchymal tumors of the breast

This report describes the fine-needle aspiration (FNA) cytologic findings of 15 cases of sarcomas involving the breast out of a combined series of 2,064 breast FNA biopsies, including 580 malignancies, thereby accounting for 2.6% of all the malignant breast tumors. The series consisted of 14 women and one man with a mean age of 48.4 yr (range, 29-63). There were eight cases of cystosarcoma phyllodes, including one malignant cystosarcoma phyllodes. Three benign cystosarcoma phyllodes had a significant concomitant atypical epithelial hyperplasia, which lead to a misdiagnosis of carcinoma in two of the cases. The third case was correctly identified as recurrent cystosarcoma phyllodes. In retrospect, features suggestive for cystosarcoma phyllodes and unusual for breast carcinoma include increased numbers of naked nuclei and hypercellular stromal fragments. Sarcomatous patterns in our four metaplastic carcinomas included chondrosarcoma (two cases), malignant fibrous histiocytoma (MFH) (one case), and fibrosarcoma (one case). Two additional pure primary MFHs (both of which had electron microscopic confirmation) and one metastatic fibrosarcoma to the breast were encountered. Recognition of unusual cytologic patterns for breast carcinoma should suggest the possibility of a primary or metastatic sarcoma to the breast. Potential pitfalls for misdiagnosis include the presence of atypical epithelial hyperplasia in some cases of cystosarcoma phyllodes, along with occasional cases having patterns indistinguishable from a fibroadenoma. The pleomorphic and bizarre cellular features can suggest the diagnosis of metaplastic and pure sarcomas of the breast, although the potential exists for confusion with very poorly differentiated carcinoma. FNA diagnosis of sarcomatous lesions of the breast is essential in order to insure proper surgical treatment.     

HER-2/neu detection in fine-needle aspirates of breast cancer: fluorescence in situ hybridization and immunocytochemical analysis

We evaluated HER-2 receptor status by immunocytochemical and immunohistochemical analyses and fluorescence in situ hybridization (FISH) in 51 fine-needle aspiration (FNA) specimens together with the corresponding formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from surgically resected breast cancers. Three fixation methods were compared: ethanol, formalin, and CytoLyt-ThinPrep (Cytyc, Boxborough, MA). HER-2 was overexpressed and amplified in 8 (16%) of 51 FFPE specimens. Of the 8 cases, gene amplification was observed in 8 FNA specimens (100%) and overexpression in 2 (25%) ethanol-, 4 (50%) CytoLyt-, and 5 (63%) formalin-fixed FNA specimens. Strong pairwise kappa association between FISH results performed on FNA specimens and FFPE tissue samples (ethanol fixation, kappa = 0.848; ThinPrep, kappa = 0.918) and moderate (ThinPrep, kappa = 0.692; formalin fixation, kappa = 0.667) to poor (ethanol, kappa = 0.300) pairwise kappa agreement between tissue immunohistochemical and FNA immunocytochemical results was demonstrated. We conclude that HER-2 protein expression on cytologic preparations was insufficiently reliable for clinical use, whereas HER-2 gene amplification determined by FISH demonstrated strong and consistent correlation with HER-2 status of FFPE tissue samples.     

P53 mutations analysis in benign and malignant breast lesions: using needle rinses from fine-needle aspirations

The fine-needle aspiration (FNA) technique is a widely used method for diagnostic assessment of breast diseases. In the current study we investigated the feasibility of sampling material for genetic studies from the same FNA samples as would be used for breast cytology. After making smears for cytological examination, the needle was rinsed into phosphate-buffered saline (PBS) solution. The material gained was sufficient for a polymerase chain reaction (PCR)-based study. As the FNA samples reflect a broad range of breast diseases, it is possible to study genetic changes at various stages of the neoplastic process. We looked for mutations in the p53 tumor suppressor gene in 198 FNA needle rinses, 42 from carcinomas and 156 from cytologically benign lesions. In the malignant samples, 22% carried mutations in the p53 gene. We also looked for p53 mutations in matching tissue sections from tumors and found the FNA needle rinses to represent the tumor well. In addition, three mutations in cytologically benign lesions were found, but none of these 3 patients were diagnosed with malignant tumors in the time frame of the study. The clinical significance of p53 mutations in benign breast tissue remains to be determined.     

Nuclear cytometric changes in breast carcinogenesis

Breast cancer is thought to originate through progressively aberrant precursor lesions, paralleled by increasing morphological changes. The aim of this study was to quantify nuclear features by image cytometry in invasive breast cancer and its early (hyperplasia) and late (ductal carcinoma in situ) precursor lesions, in order to objectively describe nuclear changes in the spectrum of proliferative intraductal and invasive breast lesions. Image cytometry was performed on tissue sections of 20 samples of normal breast tissue, 71 of usual ductal hyperplasia (UDH), nine of atypical ductal hyperplasia (ADH), and 11 of well-differentiated and 13 of poorly differentiated ductal carcinoma in situ (DCIS) lesions. The invasive breast carcinomas consisted of 19 well-differentiated and 24 poorly differentiated lesions. Through the spectrum from normal breast tissue to invasive carcinoma, progressive changes in many nuclear features were measured. Significant differences were found between nuclei of florid ductal hyperplasia compared with mild and moderate ductal hyperplastic lesions, suggesting that florid ductal hyperplasia may be a more advanced lesion than assumed and may contain cancer precursor cells. No differences were found between ADH and well-differentiated DCIS, suggesting that these lesions are closely related. Feature values of well-differentiated DCIS were comparable to values found in well-differentiated invasive carcinoma and the same applied to poorly differentiated DCIS and invasive lesions. These results support the hypothesis that breast cancer develops through different routes of progression, one leading to well-differentiated invasive cancer through well-differentiated DCIS, and one leading to poorly differentiated invasive cancer through poorly differentiated DCIS. In conclusion, image cytometry reveals progressive changes in nuclear morphological and subvisual chromatin distribution features in the spectrum from intraductal proliferations to invasive breast cancer. This provides evidence for a progression from usual to atypical ductal hyperplasia and then to invasive cancer, through different routes for well-differentiated and poorly differentiated lesions.     

Sources of diagnostic discrepancies in fine-needle aspiration of the breast

Fine-needle aspiration (FNA) is a valuable technique to use in the evaluation of breast lesions; however, inadequate and discrepant diagnoses do occur. To identify the source and nature of inaccuracies related to the method we studied 39 cases in which FNA posed diagnostic problems. These problems could be attributed to sampling errors (71.8%), to the criteria of adequacy we use at our institution (25.6%), and to interpretation (2.6%). The nature of the breast lesion (68%) was the most common cause of inadequate sampling, followed by the experience of the aspirator (32%).     

Flow cytometric analysis of DNA ploidy and S-phase fraction in breast cancer using cells obtained by ex vivo fine-needle aspiration: an optimal method for sample collection

A total of 203 primary invasive breast cancers were sampled by ex vivo fine-needle aspiration (FNA), directly yielding adequate single cell suspensions for flow cytometric DNA analysis in 194 (96%). Labor-intensive and time-consuming steps of mechanical and enzymatic cellular disaggregation required by the use of fresh, frozen, or paraffin-embedded tissue were avoided, thereby minimizing preparation time. Conservation of tumor tissue allowed for the sampling of very small breast cancers. DNA ploidy and S-phase fraction data were comparable to flow cytometric data reported in other breast cancer studies using various sampling methods. Ex vivo FNA is the easiest and fastest method for sampling breast cancers for flow cytometric DNA analysis.     

Fine-needle aspiration of breast lesions: role and accuracy in a review of 7,495 cases

In the past 10 years, 7,495 cytological breast fine-needle aspirations (FNAs) were performed (4,756 FNAs of solid nodes and 2,739 of cystic nodes). Of these, 2,099 cases underwent surgery; 650 (31%) had histologically proven carcinoma. Sensitivity was 83.9%, specificity was 99.5%, the predictive value for negative results was 93.2% and for positive results was 98.6%, and the accuracy was 94.6%. Inadequate (13.3%) and doubtful samples (8.1%) were excluded from calculation. False-negative results (82 cases) mainly resulted from sampling errors. False suspicious results (six cases) lessened with increasing experience in breast pathology and with the application of strict diagnostic criteria, but most likely they will never reach zero. Frozen-section diagnosis could be bypassed only in selected cases. Guidelines on the role of FNA in management of solid breast lesions are given. FNA deserves further evaluation in diagnosing early stages of breast carcinoma: sensitivity was 7.5% in 57 carcinomas in situ, 67.5% in 55 minimally invasive carcinoma, and 92.7% in 538 nonminimally invasive carcinomas.