p53 protein expression and nuclear DNA content in breast intraductal proliferations

Immunohistochemical analysis of the p53 gene protein and cytometric assessment of nuclear DNA were performed in a series of 51 cases of intraductal breast proliferation. The series included 22 cases of intraductal hyperplasia without atypia, 6 cases of intraductal hyperplasia with atypia, and 23 cases of pure intraductal carcinoma. Expression of p53 protein was detected in one case of intraductal hyperplasia without atypia (4·5 per cent), one case of intraductal hyperplasia with atypia (16·6 per cent) and six cases of intraductal carcinoma (26·0 per cent). No significant correlation was observed between p53 expression and histological subtype of intraductal carcinoma. Aneuploidy was demonstrated in two cases of intraductal hyperplasia with atypia (33·3 per cent) and in 18 cases of intraductal carcinoma (78·2 per cent). All cases of intraductal hyperplasia without atypia were euploid. No significant association was observed between p53 protein expression and ploidy in intraductal hyperplasia. The only case of intraductal hyperplasia without atypia positive for p53 was euploid, whereas the only p53-positive case of intraductal hyperplasia with atypia was aneuploid. Among the intraductal carcinomas, only the aneuploid cases showed positivity for p53, regardless of histological subtype. The results suggest that some of the changes observed in invasive breast carcinoma, such as p53 expression and aneuploidy, are already present in breast intraductal proliferation, especially in areas with atypia and in intraductal carcinoma. The expression of p53 in breast intraductal proliferation may reflect the acquisition of p53 gene mutations in cells unable adequately to repair DNA damage, with genomic instability which would lead to clonal expansion and putative evolution to invasive disease.     

Expression of p53 protein in benign epithelial hyperplasia,atypical ductal hyperplasia,non-invasive and invasive mammary carcinoma: an immunohistochemical study

To clarify whether p53 protein expression is involved in multistep carcinogenesis or the progression of mammary ductal carcinoma, we investigated p53 protein expression in 83 invasive ductal carcinomas (IDC), 10 IDC with a predominant intraductal component, 13 non-invasive ductal carcinoma (NIDC), 16 atypical ductal hyperplasia (ADH) and 39 benign epithelial hyperplasia (EH), using immunohistochemistry. Expression of p53 protein was detected in 24 (28.9%) cases of IDC, 5 (50%) cases of IDC with a predominant intraductal component and 1 (7.6%) case of NIDC. No expression was observed in either ADH or EH. In IDC, including cases with a predominant intraductal component, p53 protein expression was associated with a higher histological grade (P<0.0001) or mitotic index (P<0.0005). Although overexpression of c-erbB-2 protein has also shown a similar association with these prognostic indicators, expression of p53 protein correlated regardless of the status of c-erbB-2 overexpression. Completely coordinated expression of p53 protein was seen in both intraductal and invasive components. The intraductal component in IDC including cases with a predominant intraductal component which expresses p53 protein had significantly higher histological grade (P<0.0005) or more comedo-subtypes (P<0.0001). These results suggested that p53 protein expression occurs at a stage of NIDC with high histological grade or in comedo-subtypes. Its expression is maintained throughout invasion.     

乳腺增生病p53基因第5外显子突变及其蛋白表达

目的：探讨p53基因在乳腺癌发生早期的作用。方法：用免疫组化方法检测36例乳腺单纯性增生、31例不典型增生、14例原位癌和16例浸润癌中p53蛋白的表达，用PCR－SSCP检测了上述组织中p53基因第5外显子突变。结果：p53蛋白在单纯性增生、不典型增生、导管内癌、浸润癌中的表达率分别为0、22.6%(7/31)、42.8%(6/14)、50％（8／16），PCR－SSCP在各组中均未检测到该基因第5外显子突变。结论：乳腺癌发生早期阶段有p53基因的参与，但与第5外显子突变无明显关系。     

Comedonecrosis is an unfavorable marker in node-negative invasive breast carcinoma

Breast carcinoma is usually accompanied by an invasive component with an intraductal component, and each component shows different morphological features. We evaluated whether the presence or absence of comedonecrosis is correlated with prognosis and biological features in node-negative invasive breast carcinoma. Ninety-four node-negative breast carcinomas with an intraductal component were classified into two types: comedo type (n = 36) showing comedonecrosis partly or extensively in the intraductal component, and non-comedo type (n = 58) showing either an absence or small foci of necrosis. The Kaplan-Meier method was used to calculate disease-free survival. Immunohistochemical examination for p53 and HER-2 was conducted on the comedo (n = 35) and non-comedo (n = 47) type tumor specimens. Disease-free survival was significantly shorter in the comedo type than in the non-comedo type (P = 0.019). The expression of p53 was observed in 16 (45.7%) of the 35 comedo type cases, but only in two (4.3%) of the 47 non-comedo type cases (P < 0.0001). HER-2 overexpression was observed in seven (20.0%) of the 35 comedo type cases, while none of the 47 non-comedo type cases overexpressed HER-2 (P < 0.0001). These results suggest that the presence of comedonecrosis may be predictive of an unfavorable prognosis with aggressive biological behavior in node-negative invasive breast carcinoma.     

Identification of chromosome 9 alterations and p53 accumulation in isolated carcinoma in situ of the urinary bladder versus carcinoma in situ associated with carcinoma

Carcinoma in situ (CIS) of the urinary bladder is a flat, aggressive lesion and may be the most common precursor of invasive bladder cancer. Although chromosome 9 alterations are among the earliest and most prevalent genetic alterations in bladder cancer, discrepancy exists about the frequency of chromosome 9 losses in CIS. We analyzed 22 patients with CIS of the bladder (15 patients with isolated CIS, 7 patients combined with synchronous pTa or pT1 carcinomas) for gains and losses of chromosome (peri)centromere loci 1q12, 7p11-q11, 9p11-q12, and 9p21 harboring the INK4A/ARF locus (p16(INK4A)/p14(ARF)) and INK4B (p15(INK4B)) by multiple-target fluorescence in situ hybridization, and for p53 protein accumulation by immunohistochemistry. In 15 of 20 (75%) CIS lesions analyzed p53 overexpression was detected, whereas aneusomy for chromosomes 1 and 7 was identified in 20 of 22 (91%) CIS. In 13 of 22 (60%) CIS cases analyzed, 12 of which were not associated with a synchronous pTa or pT1 carcinoma, no numerical losses for chromosome 9 (p11-q12 and 9p21) were detected as compared with chromosomes 1 and 7. Furthermore 6 of 12 (50%) patients showed a metachronous invasive carcinoma within 2 years. In the remaining nine biopsies CIS lesions (40%) were recognized that showed losses of chromosome 9p11-q12 and 9p21, six of these were associated with a synchronous pTa or pT1 carcinoma. Three of these carcinomas were pTa and exhibited loss of 9q12 as well as a homozygous deletion of 9p21. The others were invasive carcinomas in which CIS lesions were also recognized that showed no numerical loss of chromosome 9, but did show an accumulation of p53. In conclusion our data demonstrate that predominantly isolated CIS lesions contained cells with no specific loss of chromosome 9, as opposed to CIS lesions with synchronous carcinomas that showed evidence of chromosome 9 loss. Furthermore our data strengthen the proposition that p53 mutations (p53 overexpression) precede loss of chromosomes 9 and 9p21 in CIS as precursor for invasive bladder cancer, as opposed to noninvasive carcinomas where chromosome 9 (9p11-q12) losses are early and frequently combined with homozygous deletions of 9p21.     

Intraductal papillary-mucinous tumours represent a distinct group of pancreatic neoplasms: an investigation of tumour cell differentiation and K-ras,p53 and c-erbB-2 abnormalities in 26 patients

Intraductal papillary growth of mucin producting hypersecreting, columnar cells characterizes a group of rare pancreatic exocrine neoplasms which we propose to call intraductal papillary-mucinous tumors (IPMT). We analysed the histopathology of 26 IPMT in relation to gastro-enteropancreatic marker expression, genetic changes and biology. Four IPMT showing only mild dysplasia were considered to be adenomas. Nine tumours displayed moderate dysplasia and were regarded as borderline. Severe dysplasia-carcinoma in situ changes were found in 13 IPMT which were therefore classified as intraductal carcinomas. Six of these carcinomas were frankly invasive and two of these had lymph node metastases. The invasive component resembled mucinous noncystic carcinoma in all but one tumour which showed a ductal invasion pattern. Immunohistochemically, an intestinal marker type was found in most carcinomas, while gastric type differentiation prevailed among adenomas or borderline tumours. K-ras mutations (seven at codon 12 and one at codon 13) were found in 31% of IPMT (2 adenomas, 1 borderline, 5 carcinomas). Nuclear p53 overexpression was detected in 31% of IPMT (6 carcinomas and 2 borderline IPMT) and correlated with p53 mutations (one at exon 8 and the other at exon 5) in two carcinomas. p53 abnormalities were unrelated to K-ras mutation. c-erbB-2 overexpression was observed in 65% of IPMT, with various grades of dysplasia. Twenty-two of 24 patients are alive and well after a mean post-operative follow-up of 41 months. Only two patients, both with invasive cancer at the time of surgery, died of tumour disease. It is concluded that pancreatic IPMT encompass neoplasms which, in general, have a favorable prognosis, but are heterogeneous in regard to grade of dysplasia and marker expression. Adenoma, borderline tumour, intraductal carcinoma and invasive carcinoma can be differentiated. p53 changes but not K-ras mutation or c-erbB-2 overexpression are related to the grade of malignancy. Most IPMT differ in histological structure, marker expression and behaviour from ductal adenocarcinoma.     

In situ hybridization and immunohistochemistry of p53 tumor suppressor gene in human esophageal carcinoma.

We have studied expression of p53, a tumor suppressor gene, by using both immunohistochemistry and in situ hybridization in 20 cases of squamous cell carcinoma of the esophagus. Immunohistochemical analysis was performed by using monoclonal antibody pAb1801. Immunoreactive p53 was observed in the nuclei of the tumor cells in 17 cases. We used 35S-labeled anti-sense single-stranded synthetic oligonucleotide probe ON102, which hybridized with DNA sequence near the 5' end of p53, for in situ hybridization. In all the cases of invasive squamous cell carcinoma studied, no significant accumulation of p53 hybridization signals was observed in carcinoma cells. This result indicates that overexpression of p53 observed by immunohistochemical staining is not due to an increase in the steady-state level of p53 mRNA in frank carcinoma cells. In six cases of morphologically normal esophageal mucosa distant from carcinoma, accumulation of hybridization signals was observed in basal and parabasal cells of the mucosa. The mucosa of these cases was negative for p53 immunoreactivity except for one case showing sporadic positivity. Accumulation of hybridization signals was observed in foci of squamous dysplasia not associated with invasion in three cases.     

E-cadherin alterations in atypical lobular hyperplasia and lobular carcinoma in situ of the breast.

Tumor development from an early lesion through to invasive disease is not a clearly defined progression in the breast. Studies of invasive lobular carcinoma have reported mutations, loss of heterozygosity (LOH) and loss of protein expression in epithelial (E)-cadherin, a protein involved in cell adhesion. Our study examines in situ lobular neoplastic lesions without concurrent invasive carcinoma for E-cadherin gene alterations and protein expression, beta-catenin, alpha-catenin and p120-catenin protein expression, and LOH at the chromosome 16q locus, with the goal of determining the events occurring at the stage of lobular neoplasia. In all, 13 atypical lobular hyperplasia lesions and 13 lobular carcinoma in situ lesions from archived cases were examined. E-cadherin sequence alterations were evaluated using single strand conformation polymorphism and DNA sequencing, and PCR-based LOH analysis was carried out for the 16q locus. Using immunohistochemistry, we assessed protein expression. A total of 23 of 24 lesions evaluated by immunohistochemistry were negative for both E-cadherin and beta-catenin protein expression, and 21 of 23 lesions were negative for alpha-catenin. Cytoplasmic (rather than membrane) localization of p120-catenin was observed in 20 of 21 cases. Lobular carcinoma in situ cases were characterized by mutations; however, atypical lobular hyperplasia cases were not. LOH at 16q was an infrequent event. From our study, we conclude that an altered E-cadherin adhesion complex is an early event affecting atypical lobular hyperplasia as well as lobular carcinoma in situ and occurs prior to progression to invasive disease. However, the loss of protein expression is accompanied by E-cadherin DNA alterations in lobular carcinoma in situ but not in atypical lobular hyperplasia. These cases lacking both protein expression and gene alterations suggest that another mechanism is involved, possibly as early as at the hyperplastic stage, causing silencing of the E-cadherin complex.     

Cyclin D1, retinoblastoma, p53, and Her2/neu protein expression in preinvasive breast pathologies: correlation with vascularity.

OBJECTIVES: Preinvasive breast pathologies show a degree of vascularization that correlates with risk of invasion. Recently, numerous oncogenes and tumor suppressor genes have been shown to regulate neovascularization. Therefore, we examined archival tissues of preinvasive breast pathologies by immunohistochemistry for alterations in the expression of four proteins, cyclin D1, retinoblastoma (Rb), p53, and Her2/neu, known to be important in breast tumorgenesis, and correlated these data with tissue vascularity. METHODS: Vascularity was determined by immunologic detection of von Willebrand factor. For carcinoma in situ (CIS) both stromal vascularity (MVD) and vascular cuffing (MCD) were determined. RESULTS: We found that cyclin D1 expression was increased in usual hyperplasia (11% of cases). Atypical hyperplasia, noncomedo CIS and comedo CIS were positive in 43, 49, and 57% of cases, respectively. Changes in Rb and p53 were rare in hyperplasia but occurred in 8 and 10% of CIS, respectively. Her2/neu protein was identified rarely in atypical hyperplasia and in both noncomedo and comedo ductal CIS. Neither Rb nor Her2/neu expression correlated with vascularity. p53 immunoreactivity correlated positively with both MCD and MVD. Cyclin D1 was negatively associated with MVD. CONCLUSION: These data suggest that p53 and cyclin D1 proteins may regulate the microvessel density of preinvasive breast pathologies.     

Clinical, histopathologic, and biologic features of pleomorphic lobular (ductal-lobular) carcinoma in situ of the breast: a report of 24 cases.

We reviewed 10 cases of pleomorphic lobular (ductal lobular) carcinoma in situ (PL/DLCIS) of the breast and compared them with 14 cases of pleomorphic lobular carcinoma in situ (PLCIS) found in association with invasive pleomorphic lobular carcinoma. The histologic features; immunohistochemical staining for estrogen receptors (ERs), p53, Ki67, E-cadherin, and gross cystic disease fluid protein-15 (GCDFP-15); and results of fluorescence in situ hybridization for HER-2/neu gene amplification were evaluated in all 24 cases. Histologically, PL/DLCIS cells were similar to those of PLCIS with invasion in that they were discohesive and medium to large in size with moderate to marked nuclear pleomorphism, small to prominent nucleoli, and moderate to abundant eosinophilic or vacuolated cytoplasm. In both groups, central necrosis was present in a small number of cases, and classic LCIS coexisted with the in situ lesion in less than half of the cases; in situ carcinomas were positive for ERs in 23 (100%) of 23 cases, p53 in 6 (25%) of 24 cases, and GCDFP-15 in 14 (74%) of 19 cases. The percentage of Ki67-positive tumor nuclei indicated moderate to high (more than 20%) proliferative activity in 8 (47%) of 17 cases. Immunostaining for E-cadherin was negative in all 24 cases. HER-2/neu gene amplification was observed in 1 (4%) of 23 cases. In cases with associated invasion, PLCIS had cytologic features and immunostaining patterns similar to those of the invasive pleomorphic component. Seven of the 10 patients who had PL/DLCIS without invasion underwent lumpectomy or simple mastectomy. Six of these patients had no evidence of disease in follow-up periods ranging from 4 to 32 months; the seventh patient developed recurrent disease 12 months after undergoing lumpectomy. We conclude that the cytologic features and biomarker expression profile of PL/DLCIS are similar to those of PLCIS with invasion but somewhat different from those of classic LCIS and ductal carcinoma in situ. Long-term follow-up studies are needed to further define the natural history of PL/DLCIS and its optimal management.     

The role of HER2/neu overexpression/amplification in the progression of ductal carcinoma in situ to invasive carcinoma of the breast.

HER2/neu overexpression/amplification is seen more frequently in ductal carcinoma in situ, particularly high-grade ductal carcinoma in situ (50-60%), than in invasive ductal carcinoma of the breast (25-30%). To date, however, the role of HER2/neu in the progression of in situ to invasive disease has not been clarified. Two hundred fifty-one breast tumors were retrieved from the pathology files at Mount Sinai Hospital. These included 91 cases of ductal carcinoma in situ, 136 cases of invasive ductal carcinomas with associated ductal carcinoma in situ, and 24 cases of pure invasive carcinomas. All cases were reviewed and stained with two monoclonal antibodies to HER2/neu (CB11 and TAB250). Immunohistochemical staining was recorded using a semiquantitative scoring system (1). Representative cases were also investigated using fluorescence in situ hybridization. HER2/neu protein overexpression (defined as immunohistochemical staining with score of >or=5) was seen in 34% of cases of pure ductal carcinoma in situ, 17% of invasive carcinomas with associated ductal carcinoma in situ, and 12.5% of pure invasive carcinomas (P =.01). Sixty percent of cases of high-grade ductal carcinoma in situ showed HER2/neu protein overexpression, versus 29% of high-grade invasive carcinomas with associated ductal carcinoma in situ and 22% of high-grade pure invasive ductal carcinomas (P =.02). The concordance between the immunohistochemical staining in the in situ and invasive components of individual tumors was 90%. Thirty-three cases were also evaluated by fluorescence in situ hybridization and showed concordance between the immunohistochemical results and the degree of gene amplification in 91% of cases, whereas 3 of 33 cases showed HER2/neu gene amplification (HER2/CEP17 = 2.3-3.7) by fluorescence in situ hybridization in the absence of positive immunohistochemical staining. One case showed HER2/neu gene amplification in the associated ductal carcinoma in situ (HER2/CEP17 ratio = 6.5), with no evidence of gene amplification in the invasive tumor (HER2/CEP17 ratio = 1.14). Multiple genetic events are required for the development of an invasive phenotype. The findings from this study suggest that the genetic event of HER2/neu gene amplification/protein overexpression may not play a key role in the progression of ductal carcinoma in situ to invasive carcinoma and that other molecular alterations may be more important in the initiation of invasion in ductal carcinoma of the breast.     

HER2 status in pure ductal carcinoma in situ and in the intraductal and invasive components of invasive ductal carcinoma determined by fluorescence in situ hybridization and immunohistochemistry

AIM: To determine the HER2 status of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of the breast. The increased prevalence of HER2 amplification and overexpression in DCIS is considered to be maintained in the intraductal component of IDC; however, HER2 amplification and overexpression are detected much less in IDC. METHODS AND RESULTS: Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were performed to detect HER2 in 270 IDCs with an intraductal component and in 50 pure DCIS samples; IHC was also performed in 116 metastatic nodes. HER2 was found to be amplified in 77 cases (28.5%) and overexpressed in 79 (29.3%) of the 270 IDCs. HER2 amplification was similar between intraductal and invasive components of the same tumour. The concordance for HER2 status between invasive and intraductal components of individual tumours was 98.5% and 99.3% by FISH and IHC, respectively. HER2 was amplified in 25 (50%) of the 50 pure DCIS samples. HER2 overexpression in metastatic nodes resembled the HER2 status in the primary tumour for 108 (93.1%) of 116 cases (kappa =0.831). CONCLUSION: Our study indicates that the intraductal component of IDC may differ biologically when compared with pure DCIS. HER2 appears to lack a critical role in the progression from DCIS to IDC and HER2 status is maintained in metastatic lesions.     

Primary mammary small-cell carcinoma: a molecular analysis of 2 cases

Primary small-cell carcinoma of the breast is an exceedingly rare variant of breast carcinoma whose genetic profile has not been previously investigated. We report the molecular features of 2 cases of small-cell carcinoma of the breast: 1 with an adjacent intraductal carcinoma, and 1 with prior pleomorphic lobular carcinoma in situ. Laser capture microdissection followed by loss of heterozygosity (LOH) analysis revealed identical molecular alterations at multiple chromosomal regions, including BRCA-1, BRCA-2, p53, and retinoblastoma gene loci, in 1 case of small-cell carcinoma and its adjacent intraductal component. Additionally, LOH in 1 or both small-cell carcinomas was detected at 3p, 4q31.2-qter, 8p21-24, 11q13 (MEN-1 locus), 11q23.3, 11q24.1-25, 16q24.1 (H-cadherin locus), and 17q25. The results of our molecular analysis suggest that genetic changes in mammary small-cell carcinoma resembled those seen in both invasive ductal carcinomas and pulmonary small-cell carcinoma. Second, mammary small-cell carcinoma is clonally related to ductal carcinoma in situ and might represent an example of divergent differentiation occurring in a multipotential neoplastic stem cell.     

Fine-needle aspiration cytology of the breast: Invasive vs. in situ carcinoma

The surgical management of invasive breast carcinoma differs from that of in situ disease. Invasive carcinoma necessitates axillary lymph node dissection, a procedure that has associated morbidity. We studied 80 cases (66 invasive, 14 in situ) of breast carcinoma that had a histological diagnosis and a preoperative fine-needle aspirate. All slides were reviewed, with 17 cytologic features assessed. We found that six of these features showed a statistically significant difference between the invasive and in situ cases. These were infiltration of fat or stroma by malignant cells (72% of invasive cases demonstrated this feature, but it was not present in any of the in situ cases, P = 0.0002), the presence of myoepithelial cells overlying clusters of tumor cells (seen in 86% of in situ tumors and 7% of invasive cases, P < 0.00001), calcification (present in 71% of in situ and 15% of the invasive group, P = 0.001), foamy macrophages (noted in 64% of in situ tumors and 16% of invasive carcinomas, P = 0.0007), intracytoplasmic vacuoles (seen in 50% of invasive cases and 21% of in situ lesions, P = 0.08), and tubules (present in 30% of invasive and 7% of in situ tumors, P = 0.10). We demonstrate that invasion can be suggested in fine-needle aspirates of carcinomas, provided that true infiltration of fibrofatty connective tissue by neoplastic cells is present. In situ disease has characteristic features, but the presence of invasion cannot be excluded, even in the presence of stromal or adipose tissue fragments without tumor infiltration.     

Potential value of hormone receptor assay in carcinoma in situ of breast

The estrogen receptor (ER) expression of invasive breast cancer has been extensively studied both biochemically and with specific monoclonal antibodies against ER. Relatively few studies have attempted to characterize ER pattern in breast carcinoma in situ (CIS) and in other premalignant lesions. In the current study, the authors investigated the pattern of ER expression in 62 cases of breast CIS, 30 of which had a component of invasive cancer, and 36 cases of atypical hyperplasia. Paraffin sections of formalin-fixed breast tissue underwent enzyme pretreatment to expose nuclear antigenic sites as previously described. Breast tissues then underwent estrogen immunocytochemical assay using specific monoclonal antibodies (Abbott Laboratory, Chicago, IL). The cases were evaluated for heterogeneity, intensity of staining, and percentage of ER-positive cells. An attempt was made to study the relation between the pattern of ER expression, nuclear pleomorphism, and type of CIS. The results of ER immunocytochemical assay showed positive nuclear staining for ER in 75% of the CIS, 73% of CIS with invasive cancer, and 100% of atypical hyperplasias. ER expression in CIS agreed with that in the invasive carcinoma in 29 of 30 cases. This study also suggests that comedocarcinoma has a higher incidence of negative ER expression than the other types of CIS, particularly when it is associated with significant nuclear pleomorphism. There was no significant difference in ER tumor heterogeneity between premalignant and malignant lesions.     

Immunohistochemical and genetic analysis of non-small cell and small cell gallbladder carcinoma and their precursor lesions.

Gallbladder carcinomas can be highly lethal neoplasms. Relatively little is known about the genetic abnormalities that underlie these tumors, particularly with respect to their timing in neoplastic progression. The authors evaluated 5 noninvasive dysplasias and 33 invasive gallbladder carcinomas (6 small cell carcinomas, 27 non-small cell carcinomas, of which 16 were accompanied by an in situ carcinoma component) for expression of the protein products of the p16, p53, Dpc4, and pRB tumor suppressor genes by immunohistochemistry. Neoplasms were also evaluated for the presence of activating K-ras oncogene mutations. Seventy-five percent of non-small cell gallbladder carcinomas demonstrated loss of p16 expression, whereas 63% accumulated high levels of p53. Loss of Dpc4 and pRB expression was less frequent, seen in 19% and 4% of the neoplasms, respectively. Thirty percent of neoplasms harbored activating K-ras mutations. In contrast, 100% of the small cell carcinomas of the gallbladder demonstrated inactivation of the pRB/p16 pathway; 67% showed loss of pRB expression, and the other 33% lost p16 expression. Eighty-three percent of small cell carcinomas accumulated high levels of p53, whereas loss of Dpc4 expression and activating K-ras mutations were not found. Among 15 evaluable in situ components, 13 harbored the same alterations found in the invasive component. Inactivation of p16 and p53 occur in the majority of non-small cell gallbladder carcinomas. Dpc4 inactivation and K-ras mutations occur in a significant minority of cases. pRB loss is uncommon in non-small cell gallbladder carcinoma, but virtually all small cell carcinomas inactivate the p16/pRB pathway, usually by retinoblastoma protein loss. It is noteworthy that all of these alterations occur at the level of carcinoma in situ.     

Relationship between the extent of intraductal component and that of the invasive component in ductal carcinomas of the breast

To clarify the relationship between the extent of the intraductal component and the invasive component in cases of invasive ductal carcinoma (IDC) of the breast, we divided 87 such cases into two groups, Group I in which the intraductal component extended for less than 10 mm (36 cases) and Group II in which the intraductal component extended for 10 mm or more (51 cases). On histological slides, there was an association between the extent of the intraductal component and the pattern of invasion. The majority (80.6%) of Group I IDC cases showed an invasive component composed of one invasive nodule, whereas 62.7% of Group II IDC cases showed an invasive component composed of two or more such nodules. On gross and microscopical examination, Group II IDC showed a significantly larger mean tumor size than Group I IDC (23.4 +/- 8.9 mm vs. 18.3 +/- 6.6 mm, P less than 0.002 & 24.1 +/- 14.1 mm vs. 17.1 +/- 6.5 mm, P less than 0.002). A similar result was obtained by clinical examination (41.8 +/- 17.0 mm vs. 29.5 +/- 11.6 mm, P less than 0.0001). These results suggest that Group II ductal carcinomas may frequently develop multiple stromal invasion, resulting suddenly in a sizeable breast mass perceived by the patient.     

Protein overexpression and gene amplification of c-erbB-2 in breast carcinomas: a comparative study of immunohistochemistry and fluorescence in situ hybridization of formalin-fixed, paraffin-embedded tissues

We evaluated 173 consecutive breast carcinomas for c-erbB-2 using a combination of immunohistochemistry (IHC) with a commercial polyclonal antibody (Nitirei) and dual-color fluorescence in situ hybridization (FISH) using the c-erbB-2-specific probe and the chromosome 17 centromere-specific probe from Vysis (Downers Grove, IL) and compared the results with the histologic characteristics of intraductal spread, cancer invasion, and intratumoral heterogeneity. With correction for chromosome 17 copy number, c-erbB-2 amplification was observed in 26 tumors (13.5%): high-level amplification in 23 tumors, and low-level amplification in 3. The gene amplification was positively correlated with c-erbB-2 protein overexpression, defined as 2+ or 3+ immunostaining, on a case-by-case basis (P < .000001). All 3+ immunostaining tumors (19 tumors) showed high-level amplification, although gene amplification was found in only 5 of 27 2+ immunostaining tumors. Although the rates of overexpression and gene amplification did not differ in ductal carcinomas in situ and invasive carcinomas (P = .46 and .53, respectively), they were significantly higher in invasive carcinomas with intraductal spreading (P < .0001). Intratumoral heterogeneity of c-erbB-2 amplification was found in only 1 case; however, in 17 invasive carcinomas, intraductal components expressed c-erbB-2 more intensely than invasive components. We conclude that in breast carcinomas, c-erbB-2 overexpression occurs mostly in tumors with high-level gene amplification, and such overexpression appears to endow carcinoma cells with the capacity for intraductal spreading. The best method for detecting breast carcinomas with c-erbB-2 aberrations using archival tissues is to screen cases by IHC; however, follow-up FISH assays are indispensable for excluding false-positive results.     

Molecular and immunohistochemical analysis of intraductal papillary neoplasms of the biliary tract

Intraductal papillary neoplasms (IPNs) of the biliary tract are uncommon lesions that may be solitary or may spread extensively along the biliary tree. Some biliary IPNs are histologically and radiologically similar to intraductal papillary mucinous tumors (IPMNs) of the pancreas and present a risk for progression to invasive cholangiocarcinoma. Unlike pancreatic IPMNs, little is known about their molecular pathogenesis. We studied 14 biliary IPNs (including 5 cases with associated invasive cholangiocarcinoma) for genetic alterations in the APC/beta-catenin pathway, K-ras oncogene mutations, p53/chromosome 17p alterations, and Dpc4/18q alterations. Immunohistochemistry was performed for beta-catenin, p53, and Dpc4, and microdissected tissue was analyzed using direct DNA sequencing for exon 1 of K-ras and exon 3 of beta-catenin and allelic loss assays on chromosomes 5q, 17p, and 18q. Activating mutations in codon 12 of the K-ras oncogene were present in 4 of 14 (29%) biliary IPNs. Of these 4 cases, 2 patients had associated invasive cholangiocarcinoma, and identical K-ras mutations were present in both the intraductal and invasive components. Allelic loss on chromosome 18q was present in 4 of 13 informative cases (31%); however, no loss of normal Dpc4 expression was detected by immunohistochemistry. Nuclear accumulation of beta-catenin protein was demonstrated in 3 of 12 cases (25%); however, there were no beta-catenin gene mutations, and allelic loss on 5q was present in only 1 of 10 informative cases (10%). Both immunohistochemistry for p53 and 17p allelic loss assays were negative. Biliary IPNs therefore demonstrate a K-ras gene mutation frequency that is lower than that previously reported for pancreatic IPMNs, but similar to that reported for hepatic cholangiocarcinomas. The presence of K-ras mutations in 2 purely intraductal neoplasms, and identical K-ras mutations in 2 cases with both intraductal and invasive components, suggests that these mutations arise early in tumorigenesis. Finally, the frequency of allelic loss on 18q suggests that a locus on 18q is involved in the molecular pathogenesis of biliary IPNs, but this locus is not DPC4.     

Impact of the presence and quantity of ductal carcinoma in situ component on the outcome of invasive breast cancer

Introduction: The role of ductal carcinoma in situ (DCIS) component on the outcome of invasive breast cancer is not yet completely clear. Our study aims to assess the impact of the presence and quantity of DCIS component on the outcome of patients operated for invasive breast cancer. Materials and methods: We collected retrospective data about patients operated at their breast for invasive cancer between 2007 and 2012, focusing on the presence of DCIS component. Then, we divided patients into four groups based on the quantity of DCIS component as follows: not found (group A), minimal (group B, <25%), extensive (group C, 25-75%), and prevalent (group D, >75%). We further defined “extensive intraductal component” (EIC) groups C and D together. Results: DCIS component was associated with young age, familial history of breast cancer and worse biological characteristics, including high grading, higher prevalence of Her2/Neu overexpression, hormone receptors negativity, comedo-like necrosis and multifocality/multicentricity. Despite the unfavorable prognostic factors, invasive cancers associated with EIC were frequently treated with radical surgery and resulted to have long disease-free survival and low local recurrence rate. In patients with DCIS component (groups B, C, and D) the extension of this component resulted indirectly correlated with local recurrence rate, tumor lymphovascular invasion, and lymphnode extracapsular invasion. The highest prevalence of local recurrences was found in group B, which tended to be less frequently treated with radical surgery than group D (P<0.05) and C (P=n.s.). Conclusions: Different clinical and tumor features among invasive breast cancer with and without DCIS component indicate that they are distinct entities probably originating by different pathways that deserve to be studied. Furthermore, the controversial results about the management of cancer with minimal intraductal component require further studies in order to reduce local recurrence.