The influence of polysomy 17 on HER2 gene and protein expression in adenocarcinoma of the breast: a fluorescent in situ hybridization, immunohistochemical, and isotopic mRNA in situ hybridization study

Breast carcinomas with amplification of HER2 on chromosome 17 are associated with HER2 protein overexpression, adversely affecting prognosis and predicting response to Herceptin therapy. Chromosome 17 polysomy is encountered in assessing HER2 gene status, and its impact on HER2 gene and protein expression remains unclear. This impact was investigated in breast carcinomas identified by fluorescence in situ hybridization (FISH) to have a gain of chromosome 17 (CEP17+; n = 56), using a dual probe assay, which detects HER2 gene copy number and enumerates chromosome 17 (HER2/CEP17; Vysis). Cases were immunostained for HER2 protein (CB-11, Ventana), and scored blinded to FISH. A subgroup was evaluated by isotopic in situ hybridization for HER2 mRNA expression. Controls included ten HER2 amplified and ten nonamplified tumors, eusomic for chromosome 17. Immunohistochemistry (IHC) for HER2 protein was negative (0 or 1+) in 69% (39 of 56), 2+ in 27% (15 of 56), and 3+ in 3% (2 of 56) of CEP17+ cases. The mean CEP17 copy number among the three groups was similar (3.1, 3.0, and 3.1 for IHC 0/1+, 2+, and 3+, respectively). Isotopic in situ hybridization for HER2 mRNA performed on 26 CEP17+ cases (16 IHC 0-1+, 10 IHC 2+ or 3+) showed no increased HER2 mRNA expression (normalized to beta-actin mRNA). The mRNA expression and the IHC staining of the HER2-amplified and nonamplified controls was concordant with their FISH status. These results suggest that chromosome 17 polysomy in the absence of HER2 amplification does not have a significant biologic influence on HER2 gene expression in breast carcinoma.     

Detection of c-sis transcripts and PDGF-like products by in situ hybridization and immunohistochemical study

To clarify the relationship between oncogene c-sis expression and tumorigenesis in human osteosarcoma, in situ hybridization and immunohistochemical studies were performed to detect c-sis mRNA and PDGF-like protein partially consisting of c-sis product. Formalin fixed-paraffin embedded sections of eight cases of osteosarcoma of bone were examined. Three osteosarcomas highly expressed c-sis mRNA with a fine granular pattern in their cytoplasms. Five osteosarcomas, including three cases with c-sis expression, contained PDGF-like protein in their cytoplasms. These results suggested that c-sis oncogene and PDGF-like protein were closely related to tumorigenesis in human osteosarcoma. The DNA-mRNA in situ hybridization technique applied by the author is as efficient as the immunohistochemical method in cancer research.     

Melanocytic nevi with an atypical epithelioid cell component: clinical, histopathologic, and fluorescence in situ hybridization findings

Combined melanocytic nevi can contain a phenotypically distinct population of large atypical epithelioid cells in a background of smaller banal-appearing melanocytes. On the basis of the pattern of proliferation and degree of pigmentation, nevi with this pattern have been referred to as nevi with an atypical epithelioid cell component (N-AECC). When N-AECC display sheet-like or an expansile nodular growth pattern, notable cytologic atypia, and any level of mitotic activity, they can be difficult to distinguish from melanoma. The clinical history and appearance of these lesions may similarly raise concern for melanoma. In view of this diagnostic problem, we present 28 cases of N-AECC from our dermatopathology consultation and in-house practice. All 28 cases were found to be negative on the basis of fluorescence in situ hybridization (FISH) for imbalanced chromosomal aberrations commonly found in melanoma. The clinical outcomes showed a benign clinical course for all cases for which the outcome information was available. FISH analysis also revealed that, in 4 of 28 cases (14%), the AECC of the lesion demonstrated polyploidy localized to the large epithelioid cell component. This is likely more common among cases of N-AECC that have an atypical spitzoid epithelioid cell component, particularly those with obvious senescent nuclear changes. Care must be taken to avoid the pitfall of misinterpreting these FISH findings as changes consistent with melanoma. The use of ancillary testing methods including FISH may be beneficial in improving the diagnostic accuracy involved in making the distinction of N-AECC from melanoma. Further, we report a novel finding of polyploidy seen in certain cases of benign N-AECC.     

Clinical, histopathologic, and immunohistochemical features of microglandular adenosis and transition into in situ and invasive carcinoma

Microglandular adenosis (MGA) of the breast is widely known as a benign lesion that can mimic invasive carcinoma. In situ and invasive carcinomas have been described as arising in MGA, but which cases of MGA will progress to carcinoma is unclear. Criteria for distinguishing uncomplicated MGA, MGA with atypia (AMGA), and carcinoma arising in MGA (MGACA) are not standardized. The primary objective of this study was to illustrate the clinical, histopathologic, and immunophenotypical characteristics of MGA, AMGA, and MGACA in an effort to provide criteria for distinguishing the 3 types. We retrospectively identified 108 cases seen at M.D. Anderson Cancer Center between 1983 and 2007 that had a diagnosis of MGA. Of the 108 cases, 65 cases had available material for review. Inclusion criteria were glands of MGA expressing S-100 protein and lacking myoepithelial layer (smooth muscle actin negative). Eleven out of 65 cases qualified to have an MGA component; myoepithelial layer was detected in the remaining 54 cases and were classified as adenosis. Out of the 11 MGA patients, there were 3 patients with uncomplicated MGA, 2 had AMGA, and 6 had MGACA. Staining indices for the cell cycle markers p53 and Ki-67 were used to compare the 3 tumor categories. Additional staining for other tumor markers [estrogen and progesterone receptors, HER2, epidermal growth factor receptor (EGFR), c-kit, CK5/6, and CK18] were performed. Patient demographics, tumor radiologic features, and clinical follow-up data were collected for all cases. Multiple invasive histologic components were identified in each of the MGACA cases. All invasive MGACAs had a duct-forming component. In addition, basal-like component was present in 2 cases, aciniclike in 2, matrix producing in 4, sarcomatoid in 1, and adenoid cystic in 1. All tumors had strong and diffuse CK8/18 and EGFR expression but no estrogen receptor, progesterone receptor, HER2 (ie, triple negative), or CK5/6 expression. C-kit was focally expressed in 2 of the MGACAs. Ki-67 and p53 labeling indices was < 3% in all MGAs, 5% to 10% in the AMGAs, and > 30% in MGACAs. In a follow-up ranging from 14 days to 8 years, none of the MGA cases recurred. One of the AMGA cases recurred as invasive carcinoma in a background of AMGA after 8 years following incomplete excision of the lesion. Three out of 6 MGACA cases (50%) required multiple consecutive resections ending up with mastectomy due to involved margins by invasive or in situ carcinoma. Two out of 6 MGACA cases (34%) developed metastasis and died of disease. Our data showed that Ki-67 and p53 expression, in conjunction with the morphologic features, could be a reliable marker to distinguish MGA from AMGA and MGACA. Although 11 tumors were only included in our study, 64% of the tumors were carcinomas arising in MGA. This high incidence of MGACA may not represent the actual frequency of MGAs progressing into carcinoma and is likely due to referral bias in our institution. Nonetheless, the high association of carcinoma with MGA necessitates complete excision of MGA to rule out invasion. Although all the MGACA cases were triple negative and express EGFR (basal-like features), all the cases in our study showed a luminal type of differentiation by CK8/18 expression, indicating that MGACA may not fit well into the current proposed molecular classification of breast cancer.     

Altered expression of amyloid precursors proteins after traumatic brain injury in rats: in situ hybridization and immunohistochemical study

The expression of alternatively spliced mRNAs for amyloid precursor protein (APP) isoforms and their translation products were examined in the rat cerebral cortex 1, 3, 6, and 12 h and 1, 3, and 7 days (n = 4-5 in each group) after fluid-percussion brain injury. In situ hybridization studies demonstrated that the expression of APP695 mRNA decreased in and around the damaged area of the cerebral cortex exposed to fluid-percussion injury 1 h after the insult. On the other hand, APP751/770 mRNAs were increased in the regions surrounding the damaged cortical areas 1 day after the injury. An increase of immunoreactive APP was detected in the regions around the damaged cortical areas 3 h after traumatic injury and maintained for the following 3 days. The APP immunoreactivity in the damaged cortices declined to the level of sham-operated animals by post-experimental day 7. Using an anti-amyloid beta (Abeta) protein (17-24) antibody, no deposits of immunoreactive Abeta (17-24) were observed in any of the samples examined in these experiments. These results suggest that the induction of Kunitz-type protease inhibitor (KPI) domain-containing APP mRNAs and the increased accumulation of APP are involved in the physiological and neuropathological responses of brains under various neurodegenerative conditions, including head trauma.     

Primary paraganglioma strictly confined to the liver and mimicking hepatocellular carcinoma: an immunohistochemical and in situ hybridization study

We describe a case of primary nonfunctioning paraganglioma that, unlike any other previously reported case, was strictly confined to the liver and must therefore have arisen on liver parenchyma. An asymptomatic 46-year-old man was referred to us for laparotomy and a right hemihepatectomy after a preoperative diagnosis of fibrolamellar hepatocellular carcinoma, based on a fine-needle biopsy. An 8-cm resiliently firm, pale gray nodule with a large central area of fibrosis and a thin fibrous capsule was resected. The polygonal eosinophilic tumor cells containing round nuclei lacking nucleoli were arranged in small nests set in a vascularly rich stroma. At immunohistochemistry neoplastic cells were strongly positive for chromogranin A, neuron-specific enolase, synaptophysin, and IGF-II protein; they were negative for keratin, S-100 protein, CD10, vimentin, and smooth muscle actin. In situ hybridization confirmed that, as in other sites, liver paraganglioma can express IGF-II gene. Conversely (and unlike hepatocellular carcinomas), the neoplastic cells did not express albumin mRNA, which was detected only in surrounding hepatocytes. The clinical course was benign and the patient is well and free of neoplastic disease 9 years after surgery. Knowledge of the entity should avoid possible confusion with hepatocellular carcinoma, especially of the fibrolamellar variety.     

Persistent (recurrent) Spitz nevi: a histopathologic, immunohistochemical, and molecular pathologic study of 22 cases

This report describes 22 Spitz nevi that seemed to have been clinically removed but persisted and clinically recurred at the biopsy site. These were evaluated in terms of histopathology, immunohistochemistry, and molecular pathology using comparative genomic hybridization (CGH) and fluorescent in situ hybridization. One of these 22 lesions was originally reported as an atypical melanocytic proliferation with some features of a Spitz nevus and was included in the study set at an early stage but was later recognized as melanoma after metastasis to regional lymph nodes 3 years after the local recurrence. We noted four histopathologic patterns in the recurrent lesions: 1) a predominantly intraepidermal pattern resembling "pseudomelanoma" as seen in recurrent "common" melanocytic nevi, 2) a compound, mostly nested pattern above or within a scar that was nearly identical to the originally biopsied Spitz nevus, 3) a nodular growth pattern that closely simulated invasive melanoma, and 4) a desmoplastic pattern resembling an intradermal desmoplastic Spitz nevus. Although the majority of recurrent lesions exhibited asymmetry and pagetoid spread, the dermal component usually had a low mitotic rate and retained architectural and cytologic maturation, which allowed distinction from invasive melanoma. Except for the metastasizing melanoma, the immunostaining pattern with S-100 and HMB-45 was identical to that previously reported for Spitz nevi. Ki67 revealed a very low proliferation rate in all cases, including the melanoma. CGH performed in 10 cases yielded results consistent with Spitz nevi in eight cases. The remaining two cases showed CGH profiles more typical of melanoma, and one of these was the above-referenced case of melanoma, proven by metastasis. Although ancillary molecular techniques such as CGH are of great help in distinguishing these from melanoma, until such techniques become widely available we advocate complete but conservative excision of any recurrent Spitz nevus.     

Primary lymphoepithelioma-like carcinoma of minor salivary gland: a case report with immunohistochemical and in situ hybridization studies

BACKGROUND: Lymphoepithelioma-like carcinomas (LEC) of salivary glands represent rare epithelial malignancies, with most cases affecting the parotid gland. To our knowledge, there was only one LEC arising from the minor salivary gland described in the English-language literature. METHODS: We report the second LEC of the minor salivary gland in the buccal area of a 50-year-old Taiwanese woman, who underwent surgical resection and adjuvant radiotherapy and remained alive and well after 120 months of follow-up. Histologically, irregular tumor nests of undifferentiated epithelial cells with syncytial cell boundaries were found embedded within rich lymphoplasmacytic stroma. The tumor cells also showed strong c-KIT expression and evidence of Epstein-Barr virus (EBV) infection. CONCLUSIONS: Our case suggests potential pathogenic implications of both c-KIT and EBV in LEC of the minor salivary gland that can be cured by the combination of surgery and radiotherapy and has a very favorable long-term prognosis.     

Host-donor interactions in healing of human split-thickness skin grafts onto nude mice: in situ hybridization, immunohistochemical, and histochemical studies.

The behavior of host and donor cell lines in human split-thickness skin grafts onto nude mice was studied by in situ hybridization (ISH) using genomic DNAs as probes, and immunohistochemically with species-specific or cross-species specific antibodies, at different stages ranging from day 3 to more than 1 year following grafting. Changes in the graft vascular and interstitial extracellular matrix were also assessed using species-specific or cross-species specific antibodies to human or murine type I, III, and IV collagens. Finally, transplant reinnervation was investigated using antibodies to various nerve cytoplasmic antigens and the thiocholine method to demonstrate acetylcholinesterase. Using these methods we were able to show the following: (1) the graft epidermis that is not replaced by mouse keratinocytes is progressively colonized by recipient Langerhans cells (LCs); (2) revascularization of the grafts begins soon by inoculation of the graft vessels with the host microcirculatory bed, and mouse endothelial cells growing into preexisting human capillary tubes produce a new basement membrane, prior to the replacement of the original one; (3) within 3-5 days following grafting, mouse fibroblasts migrate into the graft dermis. The density of the human and murine fibroblast populations then progressively increases. Characterization of the interstitial collagens identifies both human and murine type I and III collagens. Production of type III collagens decreases during the progression of fibrogenesis while human type I collagen becomes the predominant matrix protein; (4) transplant reinnervation is deficient, and neurites growing into severed graft nerve trunks were never detected.     

混合型肝癌的临床病理特点及预后分析

目的 探讨混合型肝癌的临床病理特点、诊断和手术方案选择.方法 回顾性分析北京协和医院1990年1月至2010年10月收治的经病理学确诊的12例混合型肝癌临床资料,总结其临床病理特点、诊断及手术方案选择,根据随访结果应用SPSS 13.0软件Kaplan-Meier法绘制生存曲线.结果 12例患者术前均误诊为原发性肝细胞癌,术后经病理学证实为混合型肝癌.其中男性10例(83.3％),女性2例(16.7％),年龄40～59岁,平均(51士6.3)岁.11例(91.7％)患者有乙型肝炎病史,10例(83.3％)存在不同程度的肝硬化.16.7％ (2/12)的患者术前甲胎蛋白(AFP)阳性C＞25 ng/ml),33.3％(3/10)的患者CA19-9阳性(＞37 U/ml).在获得随访患者中,术后1、3、5年生存率分别为63.6％、27.2％和9.1％.结论 混合型肝癌罕见,临床表现缺乏特异性,明确诊断依靠术后病理学检查.根治性手术切除是改善患者预后的惟一有效手段,但由于肿瘤恶性程度高,总体预后较差.     

Adenoid cystic carcinoma of the trachea and main-stem bronchus. A clinical, histopathologic, and immunohistochemical study

Twelve cases of adenoid cystic carcinoma of the trachea and main-stem bronchus were histologically analyzed, and the results were examined with reference to the growth pattern of the tumor and the prognosis. The tumors were histologically classified into tubular, cribriform, and solid subtypes. Three histologic grades were established: grade I, tumors with tubular and cribriform subtypes but without solid subtype; grade II, tumors with tubular and cribriform subtypes in which the solid subtype comprised less than 20% of the area; grade III, tumors in which the solid subtype comprised more than 20% of the area. Three gross infiltrating types were established: type I, entirely intraluminal; type II, predominantly intraluminal; type III, predominantly extraluminal. In most cases histologic grade correlated with gross tumor type; that is, grades, I, II, and III were grossly types I, II, and III, respectively. The tumors infiltrating along the tracheobronchial wall were of the tubular or cribriform subtype, but not of the solid subtype. In two patients who died of distant metastasis, the histologic studies revealed the solid subtype. Immunohistochemical analysis demonstrated that the tubular subtype was the most differentiated form and the solid subtype, the most undifferentiated form. The histologic subtype of adenoid cystic carcinoma of the tracheobronchial tree was an important factor in the growth pattern of the tumor and the prognosis.     

Epidermal growth factor receptor (EGFR) expression in prostatic adenocarcinoma after hormonal therapy: a fluorescence in situ hybridization and immunohistochemical analysis

BACKGROUND: The progression of normal prostatic epithelium to androgen-dependent cancer and, eventually, hormone-refractory prostate cancer is a complex process involving many different growth regulatory signals. Activation of epidermal growth factor receptor (EGFR) has been implicated in prostate cancer cell growth. METHODS: This study was undertaken to investigate both amplification of EGFR gene by fluorescence in situ hybridization (FISH) and over-expression of EGFR by immunohistochemical staining in prostate tissue from 71 patients treated by hormonal therapy. RESULTS: EGFR gene amplification was present in 1 of 71 tumors, and polysomy of chromosome 7 was present in 24 of 71 tumors. Immunohistochemically, EGFR expression was demonstrable in 57 of 71 tumors. Membranous immunostaining for EGFR was observed in >75% of tumor cells in 11% of cases, in 51-75% of tumor cells in 20% of cases, in 26-50% of tumor cells in 21% of cases, in 11-25% of tumor cells in 21% of cases, and in 1-10% of tumor cells in 7% of cases. No immunostaining for EGFR was seen in 20% of cases. There was no correlation between EGFR protein expression and gene amplification. There was also no correlation between EGFR expression and clinicopathological characteristics or clinical outcome. CONCLUSIONS: We found that EGFR gene expression was detectable in 35% of this large series of hormone-treated prostate cancer, and that EGFR protein is frequently expressed in tissue from these patients. EGFR over-expression may serve as a reasonable target for therapeutic intervention in this otherwise difficult to treat subset of prostate cancer.     

Temporal bone fractures: a histopathologic study

Trauma or injury to the skull, if of sufficient force, may produce a temporal bone fracture. However, terms such as basilar skull fracture, labyrinthine hemorrhage, or labyrinthine concussion are inadequate in that they do not describe the precise histopathologic lesion. We carried out a histopathologic study of the 14 ears in seven patients; all the patients suffered at least one temporal bone fracture. Our objective was to review the histopathologic findings of various fracture lines through the base of the skull and correlate these with the history and clinical findings.     

In situ hybridization: principles and applications

In situ hybridization is a molecular biological technique recently introduced in histology. Its principle consists of forming stable nucleic hybrids in tissues or cells. In this review, the main steps of the technique are discussed in regard to the preparation of probes and their labelling, the fixation of tissues and cells and their permeabilization in order to facilitate the penetration of labelled probes. Conditions of hybridization and requisite control reactions are also analysed. Quantification possible when radioactive probes are used is detailed. The three current applications of in situ hybridization, the localization of a gene on a chromosome, the demonstration of viral genomes in cells and the investigation of messenger RNAs coding for a determined protein, are illustrated with specific examples.     

Adenoid cystic carcinoma of the prostate: A case report with immunohistochemical and in situ hybridization staining for prostate-specific antigen

A 43-year-old man with urinary outlet obstruction was referred to our hospital. A digital rectal examination revealed an elastic hard prostate. The serum prostate-specific antigen (PSA), serum prostatic acid phosphate and gamma-seminoprotein levels were found to be within the normal range, and transrectal ultrasound sonography provided normal findings. The patient underwent a subcapsular prostatectomy under a diagnosis of benign prostatic hyperplasia. Histopathologically, the lesion was diagnosed as an adenoid cystic carcinoma of the prostate. Because a further examination revealed a pathologic extension into the urinary bladder, a radical cystoprostatectomy was performed. The expression of PSA protein and PSA mRNA was studied by means of immunohistochemistry and an in situ hybridization technique. The adenoid cystic carcinoma in the patient did not show any positive signs for PSA protein or PSA mRNA.     

Squamous cell carcinoma in situ of the breast: a light microscopic and immunohistochemical study of a previously undescribed lesion

Three cases of squamous carcinoma in situ of the breast, one with an invasive component are described in women aged 35, 51, and 59 years. Two cases were detected by screening mammography. In 1 case, the squamous ductal carcinoma in situ was extensive. All cases showed obvious squamous features on standard hematoxylin and eosin sections. The in situ component of the lesions and the squamous differentiation were supported by immunohistochemistry. In 2 cases, the neoplastic cells showed actin positivity indicating myoepithelial cell differentiation. One case showed trilineage differentiation into luminal, squamous, and myoepithelial cells. These cases illustrate a variant of duct carcinoma in situ that has not been described in the current literature and provide insights into the dual epithelial-myoepithelial differentiation of some breast neoplasms.