Molecular and biologic markers of premalignant lesions of human breast

There is currently great interest in the detection and characterization of putative precursor breast cancer lesions because of the possibility of chemoprevention. Knowledge of the biologic features of premalignant lesions, although limited, is rapidly evolving. Premalignant breast lesions have been examined for the presence of genetic alterations and for the expression of biomarkers such as the estrogen receptor (ER), Ki67, p53, and HER2/neu. Data obtained from genetic studies of precursor breast lesions clearly support the contention that genetic alterations begin quite early in selected subsets of histologically benign lesions. Although the results of biomarker expression profiles have been contradictory, most studies agree that precursor lesions significantly overexpress ER and that progressive alterations in ER expression accompany the transition of normal cells to hyperplastic lesions and carcinoma in situ. So far, the collected evidence indicates that precursor lesions in the breast demonstrate biomarker expression profiles and genetic abnormalities that are distinct from those of terminal ductal lobular units but share some of these features with invasive tumors. Future research in this field is urgently needed to identify specific biomarkers of prognostic and predictive value, which can help not only in the selection of patients for chemopreventive therapy but in monitoring the progression of high-risk lesions.     

Proliferative lesions of the breast: carcinoma in situ

Many breast carcinomas probably arise in a multi-step fashion through a series of intermediate lesions viz. ductal hyperplasia to atypical ductal hyperplasias to ductal carcinoma in situ (DCIS), and thence to invasive ductal cancer, each of which has a greater probability of becoming malignant than the one that preceded it. These precursor lesions have differing risk implications, hence treatment decisions vary with the risk. The heterogeneous lesions that come under the heading of hyperplasias and DCIS can cause problems for the histopathologists unless there is a conceptual understanding of the disease process. Since the surgical pathology report is the final word in cases of DCIS/LCIS, the pathologist becomes a vital partner in the decision making team.     

Expression of centrosomal tubulins associated with DNA ploidy in breast premalignant lesions and carcinoma

The centrosome plays an essential role in chromosomal segregation during cell division. Centrosome dysfunction might lead to aneuploidy and chromosomal instability. Invasive breast tumors with centrosome amplification often show aneuploidy.     

乳腺导管内癌分子分型应用研究

目的 采用免疫组织化学检测方法 对乳腺导管内癌进行分子分型.方法 收集50例乳腺导管内癌存档蜡块,用单克隆抗体CK5/6、CK8、CK18、34βE12、p63、S-100、SMA、CD10、CD117、EGFR、ER、PR和HER2进行免疫组织化学EnVision法染色,按照免疫表型分为5种类型:腺腔A型(ER+/PR+/HER2-)、腺腔B型(ER+/PR+/HER2+)、正常乳腺样型(ER-/PR-/HER2-且不表达基底/肌上皮标记及EGFR)、HER2过表达型(ER-/PR-/HER2+)和基底细胞样型(ER-/PR-/HER2-,且至少表达一种基底型角蛋白和(或)肌上皮标记物或EGFR).结果 腺腔A型16例(32%),腺腔B型19例(38%),HER2过表达型13例(26%),基底细胞样型2例(4%),无正常乳腺型.2例基底细胞样型,均表达CK5/6、CD117,例1同时表达SMA,例2表达CK8、CK18、34βE12、S-100,均为高级别导管内癌.结论 乳腺导管内癌可按免疫表型进行分子分型,部分导管内癌具有与基底细胞样癌相同的免疫表型,可能是基底细胞样癌的前驱病变,其诊断依赖于免疫组化检测.     

Morphometric analysis of basal cell layer in oral premalignant white lesions and squamous cell carcinoma.

The size and shape of the cells in the basal cell layer of the oral epithelium in 100 specimens from oral mucosa were studied by using an interactive image analysis system (IBAS-1). Four groups of white lesions (traumatic keratosis, lichen planus, leucoplakia, and a "risk group") in addition to two control groups (normal mucosa and squamous cell carcinoma) were studied retrospectively. The results showed a progressive increase in the dimensions (area, perimeter, and maximum diameter) of the nuclei from normal mucosa through traumatic keratosis, lichen planus, leucoplakia and the "risk group" to carcinoma, with considerable differences. The nucleus in squamous cell carcinoma was twice as large as in normal mucosa. A substantial increase in the dimensions of both the cell and the nucleus was found in the "risk group." The nucleo:cytoplasmic ratio, contrary to what might have been anticipated in risk lesions, did not show considerable differences between the diagnostic groups. Furthermore, it was slightly decreased in the risk group compared with the normal mucosa. The shape factors (form PE and contour index) seemed to be less helpful in the identification of the "risk group." The size of the basal cell and its nucleus can be of diagnostic value for lesions with a high risk of malignant transformation.     

Differentiating nonhigh-grade duct carcinoma in situ from benign breast lesions

This study was undertaken to determine the discriminating cytological features between nonhigh-grade duct carcinoma in situ (NHGDCIS) and benign breast lesions and to determine any histological characteristics which would influence the cytological categorization. Smears of 12 each of histologically confirmed NHGDCIS and benign breast lesions were reviewed with regard to cellularity, cell discohesion, nuclear atypia, crowding of cells, tubule formation, necrosis, and presence of bare atypical nuclei and regular bare bipolar nuclei, and statistically analyzed. Architectural pattern, presence of necrosis, and the size of the lesion assessed at histological examination were compared with the initial cytological categorization. NHGDCIS lesions showed more cell discohesion (P = 0.04), bare atypical nuclei (P = 0.05), necrosis (P = 0.03), and sparse bare bipolar nuclei (P = 0.02) than benign lesions. These differences were statistically significant. Cellularity (P = 0.8), nuclear atypia (P = 0.06), crowding of cells (P = 0.1), and tubule formation did not show a significant difference. Six (out of six lesions) with a solid architectural pattern and six (of seven) with necrosis could be cytologically categorized as suspicious or malignant. Size of the lesion did not influence this. We conclude that cell discohesion, bare atypical and bare bipolar nuclei, and necrosis are discriminating features between NHGDCIS and benign breast lesions and NHGDCIS lesions with a solid architectural pattern and necrosis are more likely to be satisfactorily categorized cytologically.     

Cytogenetics of human malignant melanoma and premalignant lesions

Chromosome studies were done on direct preparations, early passage cultures, and/or cell lines derived from melanocytic lesions of 17 patients. There were 5 nevi (3 dysplastic); 1 early primary melanoma (radial growth phase); 1 advanced primary melanoma (vertical growth phase) with multiple metastases; and 10 metastatic lesions. The 5 nevi had normal karyotypes, while each of the tumors had a predominantly abnormal karyotype. The early melanoma was pseudodiploid, including a 6p;22 translocation. Ten of the 11 advanced melanomas had one or more aberrations involving chromosome #1, with 9 having deletions or translocations of Ip that involved the proximal segment 1p12→1p22 9 times in 8 lesions. Six advanced tumors had additional material involving 7q, including extra #7s (4 cases) and 7q (2 cases). Nine melanomas, including the early tumor, had alterations in chromosome #6. Three had additional copies of 6p (as iso6p or t6p); the others showed no consistent pattern. In one advanced tumor, the primary lesion and 5 metastases (removed seriatim over an 18-month period) had nearly identical karyotypes, indicating the clonal nature of the neoplasm. The nonrandom cytogenetic changes suggest that genes important in melanoma carcinogenesis are located on the proximal portion of 1p, on 7q, and on chromosome #6. More data on early lesions are needed to identify the relation of these various cytogenetic changes to the different stages of malignant melanoma development.     

Molecular alterations differentiate microinvasive carcinoma from ductal carcinoma in situ and invasive breast carcinoma: retrospective analysis of a large single-center series

Microinvasive carcinoma (MIC) of the breast is a rare lesion. The clinicopathologic features and biologic behavior of MIC are unclear. Whether MIC is a distinct entity or an interim stage in the progression from ductal carcinoma in situ (DCIS) to invasive breast carcinoma (IBC) remains to be determined. A retrospective review of clinicopathologic features and analysis of the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), and Ki-67 in patients with MIC (90 cases), DCIS (268 cases) and IBC (1504 cases) was performed. Most MICs (93.3%) exhibited an intermediate to high nuclear grade, and this proportion was larger than that of DCIS (62.7%, P < 0.001) or IBC (85.4%, P = 0.036). The incidence of sentinel lymph node metastasis in MIC (12.5%) was higher than that of DCIS (1.6%, P < 0.001), but much lower than that of IBC (39.7%, P < 0.001). MICs had higher expression of HER-2 and lower expression of ER and PR compared to DCIS and IBC; and MIC was more likely to present with a HER-2+ subtype. Furthermore, DCIS exhibited greater HER-2 overexpression or gene amplification (P < 0.001) levels and lower proliferation index of Ki-67 (P < 0.001) compared to IBC. Our results suggest that the clinicopathologic and molecular phenotype of MIC are different from DCIS and IBC. Thus, MIC may be a distinct entity rather than an interim stage in the progression from DCIS to IBC. The prognosis of MIC and the biologic behavior of this uncommon subset need to be further explored.     

In situ production of sex steroids in human breast carcinoma

It is well known that sex steroids are closely involved in the growth of human breast carcinomas, and the great majority of breast carcinomas express sex steroid receptors. In particular, recent studies have demonstrated that estrogens and androgens are locally produced and act in breast carcinoma tissues without release into plasma. Blockade of intratumoral estrogen production potentially leads to an improvement in the prognosis of invasive breast carcinoma patients, and, therefore, it is important to obtain a better understanding of sex steroid-producing enzymes in breast carcinoma. In this review, we summarize recent studies on tissue concentration of sex steroids and expression of enzymes related to intratumoral production of estrogens [aromatase, steroid sulfatase (STS), and 17β-hydroxysteroid dehydrogenase type 1 (17βHSD1)], and androgens (17βHSD5 and 5α-reductase) in invasive and in situ (noninvasive) breast carcinomas, and discuss the significance of intratumoral production of sex steroids in breast carcinoma.     

Molecular alterations in columnar cell lesions of the breast.

Columnar cell lesions of the breast include a morphologic spectrum of simple columnar cell change, columnar cell hyperplasia, columnar cell hyperplasia with atypia and ductal carcinoma in situ of micropapillary/cribriform type. Invasive carcinomas of low grade are often seen in association with this spectrum. The biologic significance of these lesions that are commonly found on breast biopsies is unknown. Three cases of formalin-fixed, paraffin-embedded breast tissues, each displaying the entire spectrum of columnar cell lesions through ductal carcinoma in situ and including foci of invasive carcinoma were microdissected at multiple sites to evaluate neoplasia progression. Minute tissue targets were microdissected (4-8/case) from unstained 4-microm thick recut paraffin sections and included non-neoplastic breast and sites of columnar cell change, hyperplasia, atypia, ductal carcinoma in situ and invasive carcinoma. Allelic imbalance for a broad panel of microsatellite markers in proximity to known tumor suppressor genes was quantitated using automated polymerase chain reaction/gel electrophoresis. Genomic loci evaluated 1p, 3p, 5q, 9p, 9q, 10q, 17p, 17q, 19q, 22q. The presence, topographic relationship and time course of mutational damage was correlated with columnar morphologic features. Detailed allelic imbalance information was obtained from each microdissection tissue target producing a detailed fingerprint of mutational damage in each case. Allelic damage was targeted predominately at 9q, 10q, 17p and 17q. Simple columnar cell change was without mutational changes and only present in one case of columnar cell hyperplasia. The remainder of the cases all show progressive accumulation of allelic damage in columnar cell changes with atypia, ductal carcinoma in situ and invasive carcinoma. The fractional mutation percentage increased progressively from columnar cell hyperplasia through invasive carcinoma. Low level of allelic imbalance was demonstrable in columnar cell lesions by the microdissection approach. A gradient of progressive mutational change could be delineated in each case manifesting allelic loss damage. Allelic loss damage appeared to preferentially target loci at 9q, 10q, 17p and 17q. The findings are consonant with the hypothesis that a select group of atypical columnar cell lesions are morphologic precursors to invasive carcinoma. Integrated molecular pathology analysis used here can help define the significance of columnar cell lesions and its role in breast cancer tumorigenesis on an individual patient basis.     

Genetic relation of lobular carcinoma in situ, ductal carcinoma in situ, and associated invasive carcinoma of the breast.

AIMS: The mutual relation of lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS) of the breast, as accepted precursor lesions of invasive breast cancer, is controversial. Because they display genetic heterogeneity, it is not clear how genetically advanced these entities are and what causes the transition to an invasive carcinoma. METHODS: Six cases of LCIS, four of them with associated lobular invasive carcinoma, four cases of intermediately differentiated DCIS with an associated invasive lobular carcinoma, and nine cases of intermediately and poorly differentiated DCIS with associated ductal invasive carcinoma were investigated by means of comparative genomic hybridisation (CGH) after microdissection and immunohistochemical staining of E-cadherin. RESULTS: LCIS was characterised by a low average rate of copy number changes, no evidence of amplifications, and a high rate of gains and losses of chromosomal material at 1q and 16q, respectively. A high degree of genetic homology with well differentiated DCIS was obvious, as reported previously. The cases of intermediately differentiated DCIS with associated lobular invasive components and lobular differentiation revealed striking homologies, and a significant difference of E-cadherin expression. The comparison of preinvasive and invasive breast lesions, irrespective of differentiation within the same patient, revealed no specific alteration that might be associated with invasion. Genetic alterations seen in invasive carcinoma were not necessarily seen in the adjacent precursor lesions. CONCLUSIONS: These results provide strong evidence that invasive breast cancer is a disease with multiple cytogenetic subclones already present in preinvasive lesions. Moreover, specific CGH alterations associated with invasion were not observed. Furthermore, the close genetic association between well differentiated and a subgroup of intermediately differentiated DCIS and LCIS led to the hypothesis that LCIS and a subgroup of DCIS are different phenotypic forms of a common genotype.     

The basement membrane and lobular carcinoma in situ of the breast.

The basement membrane (BM) was examined by light microscopy in cases of lobular carcinoma in situ of the breast (LCIS), employing the following stains: haematoxylin-eosin, van Gieson-Hansen, PAS, colloidal-iron-PAS, and reticulin. The BM was non-intact in 12 out of 26 investigated cases of LCIS when reticulin staining was applied, but appeared non-intact in about 70 per cents with the other staining methods. On the other side control areas of the BM were intact in 23 out of 26 cases with reticulin stain, but in only about half of the cases with other stains. It is concluded that reticulin staining is the most suitable in evaluating the BM in LCIS, and that more or less pronounced defects of the BM in LCIS cannot be considered evidence of imminent invasion. Although the overall occurrence of invasive breast carcinoma was significantly increased in 9 cases with epithelial cell protrusion through the BM, this feature should be investigated further before it is allowed to affect therapeutic decisions.     

Molecular analysis of oral lichen planus. A premalignant lesion?

Oral lichen planus (OLP) is a common mucosal condition that is considered premalignant by some, although others argue that only lichenoid lesions with dysplasia are precancerous. To address the question of whether OLP without dysplasia is premalignant, we used microsatellite analysis to examine 33 cases of OLP for allelic loss at nine loci located on chromosomes 3p, 9p, and 17p. Loss of heterozygosity (LOH) on these three arms occurs frequently in oral tumors, and the presence of these alterations in premalignant lesions suggests that they may play an important role in tumor progression. Results were compared with those observed in oral dysplasias (10 mild, 11 moderate, 16 severe/carcinoma in situ), 22 oral squamous cell carcinomas, and 29 reactive lesions. LOH was present in 6% of OLP, 14% of reactive lesions, 40% of mild dysplasia, 46% of moderate dysplasia, 81% of severe dysplasia/carcinoma in situ, and 91% of squamous cell carcinomas. LOH was detected on only a single arm in OLP and reactive lesions but occurred on more than one chromosome in dysplasia and cancer, and the frequency of this multiple loss correlated significantly with increasing degrees of dysplasia and progression into squamous cell carcinoma (P = 0.0028). Although these findings do not support OLP as a lesion at risk for malignant transformation, such results need to be confirmed by use of other genetic markers as OLP may undergo malignant transformation through genetic pathways different from those of oral dysplasia.     

Morphometric analysis of differentiation in human breast carcinoma. Tumor heterogeneity

The assumption that tumor grading is best performed in the region having the least differentiation was tested in a series of mammary ductal carcinomas by quantitating the relative amounts of neoplastic cells that remain within ducts (intraductal tumor [IN]) or participate in glandular formations (differentiated infiltrating tumor [DI]). Field-to-field variation in these two elements displayed a linear correlation with their overall level in each tumor. Since having less than 10% DI + IN is a marker for recurrence among breast cancer patients, this marker was applied to find variant regions of potential biological importance. Such regions were common, but their distribution was random, and they varied so much within themselves that they were not significantly different from the remainder of the tumor. Furthermore, the existence of such poorly differentiated regions did not correlate with a poor prognosis. Grading should be performed on a sample that is representative of the entire tumor, rather than its least differentiated part.     

Morphometric analysis of differentiation in human breast carcinoma. Tumor grading

Subjective grading systems suffer from limited reproducibility and do not assign a sufficiently large proportion of patients to the most predictive grades (1 and 3). In 50 cases of stage 1 or stage 2 mammary ductal carcinoma having at least five-year follow-up, we quantitated the proportion of neoplastic cells showing glandular differentiation (DI) or remaining within ducts (IN). Patients who experienced recurrence had significantly less of both DI and IN, and the point at which 10% of neoplastic cells was composed of DI + IN appeared to be predictive for recurrence; ie, 24 recurrences appeared among the 33 patients who had tumors with DI + IN less than 10%, whereas none developed among the 14 patients having tumors with DI + IN greater than 10%. Only three patients could not be classified with this marker. Tumor stage and grade were independent variables. Reproducibility among three observers averaged 95%. The morphometric evaluation can be performed by a technician under the direction of a pathologist.     

Cytological and architectural heterogeneity in ductal carcinoma in situ of the breast.

AIM: The traditional architecture based classification system of ductal carcinoma in situ (DCIS) has been criticised on the grounds that individual lesions often show more than one pattern resulting in a large mixed category. New DCIS classification systems have emphasised the importance of cytological grade, which is reputed to be more uniformly expressed throughout a lesion. This study investigates the hypothesis that cytological heterogeneity is less common than architectural heterogeneity within DCIS lesions. METHODS: 121 cases of DCIS were graded as poorly, intermediately, or well differentiated according to a recently developed classification system that employs cytonuclear morphology as the major diagnostic criterion. Cases were categorised as pure when only one grade was present and as mixed if more than one grade was observed. Architecturally the cases were classified as solid, cribriform, micropapillary, or papillary and were described as pure if only one architectural pattern was present and as mixed if more than one pattern was seen. The incidence of cytological heterogeneity was compared with that of architectural heterogeneity. The presence of necrosis was assessed as an independent parameter and the relation to DCIS grade evaluated. RESULTS: Using the cytology based classification system 102 cases (84%) were classified as pure (65 poorly differentiated, 25 intermediately differentiated, and 12 well differentiated) and 19 cases (16%) as mixed. Extensive necrosis was observed in 61 (50%) cases and was closely correlated to DCIS grade. Architecturally 46 cases (38%) were classified as pure (38 solid, 5 cribriform, 2 micropapillary, and 1 papillary) and 75 (62%) as mixed. CONCLUSIONS: Cytological heterogeneity is much less common than architectural heterogeneity in DCIS lesions. The assessment of cytonuclear morphology is therefore likely to provide more consistent information about DCIS, particularly in small biopsy specimens where only part of the lesion may be available for examination.