EGFR expression and activation are common in HER2 positive and triple-negative breast tumours.

EGFR has been associated with unfavourable prognosis in patients with triple-negative breast carcinomas, although little is known about EGFR activation in these tumours. In a series of breast carcinomas (archived formalin fixed tumours, n=100), we investigated EGFR phosphorylation status at Tyr992 (pEGFR-Y992) and Tyr1068 (pEGFR-Y1068) by immunohistochemistry, along with EGFR protein expression (extracellular domain), gene amplification status (fluorescent in situ hybridization) and conventional clinicopathologic parameters. EGFR protein was present in 21.9%, while phosphorylation at Y1068 and Y992 was observed in 27.8% and 50.5% of tumours, respectively. None of the tumours showed EGFR gene amplification, whereas 21.1% exhibited chromosome 7 polysomy. The above EGFR parameters were usually not simultaneously detected and were not associated with each other. High grade (p=0.003), lymph node positive (p=0.045), estrogen receptor (ER) negative (p<0.001) tumours often expressed EGFR protein. EGFR-Y992 and Y1068 phosphorylation was inversely associated with ER presence (p=0.023 and p=0.029, respectively) but positively with HER2 expression status (p<0.001 and p=0.002, respectively). The global positivity for any EGFR parameter did not significantly differ between triple-negative and HER2 positive tumours. In conclusion, EGFR phosphorylation is commonly encountered in breast carcinomas, although unrelated to EGFR protein presence and gene amplification. EGFR may appear activated even in cases where the extracellular domain of this protein is not observed with immunohistochemistry. These findings may be useful for further studies aiming at the assessment of EGFR parameters on this type of material.

Update on extranodal lymphomas. Conclusions of the Workshop held by the EAHP and the SH in Thessaloniki, Greece.

Classification and proper treatment of extranodal lymphoma is hindered by the diversity of lymphoma types and the relative rarity of many of these tumour types. In order to review controversial issues in extranodal lymphoma diagnosis, a joint Workshop of the European Haematopathology Association (EAHP) and the Society for Hematopathology (SH) was held, where 99 selected cases were reviewed and discussed. This Workshop summary is focused on the most controversial aspect of cutaneous B-cell lymphoma, other extranodal B-cell lymphomas, plasmablastic lymphoma and anaplastic large-cell lymphoma in extranodal sites, and makes practical recommendations about diagnosis and therapeutic approaches.

Comparative study of CD34, alpha-SMA and h-caldesmon expression in the stroma of gynaecomastia and male breast carcinoma.

AIMS: To address the fibroblastic/myofibroblastic nature of stroma in gynaecomastia and in male breast carcinoma, the expression of CD34, alpha-smooth muscle actin (SMA) and h-caldesmon in the stromal cells was investigated by immunohistochemistry. METHODS AND RESULTS: Representative archival paraffin blocks were collected from male patients with gynaecomastia (32 cases) and mammary carcinoma (24 cases) between 1984 and 2004 and CD34, alpha-SMA and h-caldesmon were assessed immunohistochemically using a streptavidin-biotin method. Thirty cases of gynaecomastia showed a CD34+, alpha-SMA- and h-caldesmon- immunophenotype with different CD34 staining intensity in the various histological subtypes. Positivity for alpha-SMA and negativity for CD34 and h-caldesmon was found in a case of florid gynaecomastia relating to reactive fibrosis due to previous surgical intervention. Acquisition of alpha-SMA expression by stromal fibroblasts but absence of CD34 staining was identified in 22 cases of male breast carcinoma. CONCLUSIONS: The immunophenotype of periductal connective tissue stroma in gynaecomastia appears to parallel the phenotype of normal breast stroma. In male breast carcinoma the stromal cell immunophenotype is similar to that of its female counterpart showing myofibroblastic differentiation. However alpha-SMA+ and CD34- are not specific to malignancy because such findings are also encountered in reactive fibrosis.

Relative expression of human telomerase catalytic subunit (hTERT) transcripts in astrocytic gliomas.

Human telomerase catalytic subunit (hTERT) expression has been reported as a marker for malignancy in various tumor systems. The aim of the present study was to investigate the relative expression of hTERT (relhTERT) and its transcripts A+B+ (contained in the full-length product), Adel and Bdel in astrocytic gliomas (grades I-IV, n=38). relhTERT was assessed by duplex reverse transcription-PCR and the expression profile of Adel, Bdel and A+B+ transcripts by nested real time-PCR. relhTERT and A+B+ presence correlated well with each other ( P<0.001) and with histological grading [grades I-II (low) vs III-IV (high), P(relhTERT)=0.002 and P(A+B+)<0.001]. A+B+ was detected in one out of seven hTERT-positive low-grade tumors, while it was present in 96.3%, and predominantly expressed in 59.3% of high-grade tumors. Bdel predominance was observed only in three cases, irrespective of grading, while Bdel levels equal or close to those of A+B+ were found in 30.4% of grade IV tumors. In situ hybridization with specific Bplus and Bdel probes revealed positive signals for both mRNAs in association with relhTERT and respective variant profiles. In addition, this method was useful in assessing hTERT expression in cases where sampling errors for RT-PCR were unavoidable. Our findings show that except for differences in relhTERT, low- and high-grade astrocytic gliomas exhibit distinct hTERT variant profiles, most of which seem to be in line with the role attributed to hTERT regarding its contribution to the acquisition of malignant potential during astrocyte carcinogenesis. Low-grade tumors mainly express Adel and Bdel. High-grade tumors, especially grade IV, always express A+B+, mostly but not always in predominance over Adel and Bdel. In this same group, profiles with Bdel predominance or relatively equal A+B+/Bdel expression are also observed, and Adel is often missing. Whether these differences characterize tumors with different biological behavior remains to be elucidated.

Cyclin D1 overexpression in multiple myeloma. A morphologic, immunohistochemical, and in situ hybridization study of 71 paraffin-embedded bone marrow biopsy specimens.

Cyclin D1 expression was evaluated by immunohistochemical analysis and biotin-labeled in situ hybridization (ISH) in a series of 71 decalcified, paraffin-embedded bone marrow biopsy specimens from patients with multiple myeloma (MM). Cyclin D1 messenger RNA (mRNA) overexpression was detected by ISH in 23 (32%) of 71 cases, whereas cyclin D1 protein was identified by immunohistochemical analysis in 17 (24%) of 71 specimens. All cases that were positive by immunohistochemical analysis also were positive by ISH. Statistically significant associations were found between cyclin D1 overexpression and grade of plasma cell differentiation and between cyclin D1 overexpression and extent of bone marrow infiltration. Our findings demonstrate the following: (1) ISH for cyclin D1 mRNA is a sensitive method for the evaluation of cyclin D1 overexpression in paraffin-embedded bone marrow biopsy specimens with MM. (2) ISH is more sensitive than immunohistochemical analysis in the assessment of cyclin D1 expression. (3) Cyclin D1 overexpression in MM is correlated positively with higher histologic grade and stage.

In situ hybridization and reverse transcription-polymerase chain reaction for cyclin D1 mRNA in the diagnosis of mantle cell lymphoma in paraffin-embedded tissues.

Mantle cell lymphoma (MCL) is characterized by the chromosomal translocation t(11;14), which involves rearrangement of the bcl-1 proto-oncogene to the immunoglobulin heavy chain gene and results in overexpression of cyclin D1 mRNA. In this study, we evaluated the diagnostic relevance of three methods that may be helpful in the diagnosis of MCL: in situ hybridization (ISH) and a stringent reverse transcriptase-polymerase chain reaction (RT-PCR) protocol for cyclin D1 mRNA, and immunohistochemistry for cyclin D1 protein. The study group included 37 paraffin-embedded specimens (25 from lymph nodes and 12 from extranodal tissues) from 30 patients. MCL diagnosis was performed according to the Revised European-American Classification of Lymphoid Neoplasms. Twenty-nine patients with non-MCL lymphoproliferative disorders comprised the control group. Biotin-labeled ISH was performed in 28 cases of MCL, 24 (86%) of which were found to be positive. As shown by ISH in extranodal tissues, cyclin D1 mRNA was present not only in neoplastic lymphoid cells, but in other cell types as well. For this reason, RT-PCR results were considered reliable for MCL diagnosis only on informative material (from tissues that do not normally express cyclin D1); this method was evaluated as positive in 16 of 18 (89%) MCL cases. Cyclin D1 immunopositivity was present in 20 of 29 (69%) MCL cases. No members of the control group were found to express cyclin D1 mRNA by either ISH or RT-PCR under the stringent conditions used. In conclusion, stringent RT-PCR for cyclin D1 expression can be helpful in MCL diagnosis in paraffin-embedded material from lymph nodes. ISH is a sensitive method for cyclin D1 mRNA detection; its sensitivity is superior to that of cyclin D1 immunohistochemistry and similar to that of the stringent RT-PCR used. ISH is very specific as well, clearly more specific than RT-PCR, because it allows the correlation of molecular findings with morphology. This method can be applied on all types of paraffin-embedded tissues and provides an accurate tool for MCL diagnosis.

Synchronous occurrence of multiple malignant neoplasms in the uterus (adenocarcinoma of the endometrium, large B-cell lymphoma of the cervix).

Multiple primary malignancies of the uterus are extremely rare. We report a case of endometrial adenocarcinoma and cervical large B-cell lymphoma occurring simultaneously in a 64-year-old woman with uterine bleeding. Adenopathy, hepatosplenomegaly or bone marrow infiltration were not found. Both malignant neoplasms mentioned above were diagnosed incidentally on the specimen (total hysterectomy with bilateral salpingo-oophorectomy) removed for uterine leiomyomas.

Malignant fibrous histiocytoma of the spine causing spinal neural foramen widening.

A case with a clinical picture of a chronic low back pain radiating to both lumbar regions caused by malignant fibrous histiocytoma is reported. Radiological, surgical and histopathological findings and treatment of this rare case are discussed.

Biliary cystadenocarcinoma with two types of tumour cells.

We report a rare case of biliary cystadenocarcinoma that occurred in the left hepatic lobe of a 62-year-old man and measured 20 cm in its greatest dimension. The neoplastic epithelium consisted of two types of cells: (1) cells with clear cytoplasm containing abundant mucin, and (2) cells with eosinophilic cytoplasm, which in some areas formed nodules with hepatocytoid features (polygonal cell shape, large nuclei with prominent nucleoli, and pseudoglandular structures). Histochemical stains revealed the presence of cytoplasmic mucin in the hepatocytoid areas, whereas immunohistochemical stains clearly showed a biliary phenotype (diffuse positive staining for "biliary type" cytokeratins, rare foci of positive staining with antibody to human hepatocytes (HEP-PAR1), absence of staining for alpha-fetoprotein, and no evidence of canalicular pattern of staining with polyclonal antibody to carcinoembryonic antigen). These findings indicate that areas reminiscent of hepatocellular carcinoma may occur in biliary cystadenocarcinomas. Histochemical and immunohistochemical stains are useful in reaching a definitive diagnosis in such cases.

Telomerase activity in precancerous hepatic nodules.

BACKGROUND: Recent studies have demonstrated that telomerase, a reverse transcriptase linked to cellular "immortalization," is activated in a variety of malignant human tumors. This study was conducted to determine whether telomerase activity represents a marker of malignant transformation in precancerous (dysplastic) nodules arising in patients with cirrhosis. METHODS: Telomerase activity was evaluated in frozen tissue samples of 14 cirrhotic liver specimens and 30 large nodular lesions contained therein, including 13 large regenerative nodules/low grade dysplastic nodules, 10 high grade dysplastic nodules, and 7 hepatocellular carcinomas (HCCs). A modified telomeric repeat amplification protocol was used. RESULTS: There was a clear-cut difference in telomerase activity levels between HCC (positive or strongly positive) and cirrhotic liver samples (weakly positive or negative). The majority of large noncancerous nodules (86%) exhibited telomerase activity levels similar to HCCs. However, such activity was not limited to dysplastic lesions but also was detected in some large regenerative nodules. CONCLUSIONS: These findings suggest that telomerase activation is an early event in large nodule formation in cirrhosis, which may facilitate the action of other factors in the process of carcinogenesis. Telomerase activity in large hepatic nodules is not always indicative of malignant transformation.

Expression of Bcl-2 protein in human primary breast carcinomas and its correlation with multifocality, histopathological types and prognosis.

The Bcl-2 gene product prevents programmed cell death (apoptosis) and possibly promotes tumour development. This protein has mainly been demonstrated in the cytoplasm of various normal and neoplastic cells, including normal mammary epithelia and breast carcinomas. The aim of this retrospective study was to correlate the immunohistochemical expression of Bcl-2 protein with the multi-unifocality and the histology of the two main types of breast carcinoma. We used monoclonal antibody 124 to investigate Bcl-2 expression in paraffin sections of 62 primary breast carcinomas. Bcl-2 expression was associated mainly with this lobular carcinoma. High Bcl-2 protein positivity was found in this type, and was statistically significant in comparison to the level of Bcl-2 in ductal, NOS carcinomas (lobular versus ductal, NOS, P < 0.0001). In the entire group, including all histological types, Bcl-2 expression was higher in multifocal tumours (P = 0.005). Statistical significance (P < 0.03) was also found within the group of ductal, NOS cases, showing that Bcl-2 protein expression is associated with multifocality, irrespective of the histology of breast carcinomas. No definite association between Bcl-2 expression and prognosis was found. Our results suggest that Bcl-2 protein plays some role in the development of multifocality in breast carcinomas.

Phenotype of Hodgkin and Sternberg-Reed cells and expression of CD57 (LEU7) antigen.

Possible associations between the immunophenotype of Hodgkin (H) and Sternberg-Reed (S-R) cells, the expression of CD57 (Leu 7) antigen, and the presence of Epstein-Barr virus (EBV) were investigated in lymph node specimens from 50 cases of Hodgkin's disease (HD), including 26 cases of mixed cellularity and 24 cases of nodular sclerosis. Tissues were fixed in 10% neutral formalin, or/and B5 solution. H and S-R cells were CD30+, CD15+ (85% of the cases) and LCA (CD45). A proportion of neoplastic cells positive for either T-cell markers (CD3) or B-cell markers (CD20) was observed in 10% and 34% of the cases, respectively. Membrane positivity for CD57 antigen was found in H and S-R cells in 10 cases (8 cases of mixed cellularity, and 2 cases of nodular sclerosis). Such immunopositivity was only observed in B5-fixed sections. No staining for CD57 antigen was identified in H and S-R cells of any case with CD20 positive neoplastic cells. H and S-R cells of both CD57-positive and CD57-negative cases were further studied by immunohistochemistry for LMP1, by in-situ hybridization for EBER and by polymerase chain reaction (PCR) for EBV-DNA. No association was identified between the expression of CD57 antigen and the presence of EBV sequences, transcripts or proteins. Our findings do not support a B-cell origin for H and S-R cells in CD57-positive cases of Hodgkin's disease and suggest that these neoplastic cells may be related to natural killer (NK) or T-cells expressing CD57 antigen.

Immunohistochemical phenotyping and PCNA detection in gastrointestinal stromal tumors.

The immunohistochemical profile and the expression of proliferating cell nuclear antigen (PCNA) were studied in a series of 44 mesenchymal tumors of the gastrointestinal tract (GIT). On routinely hematoxylin-eosin stained sections 31 cases were classified as leiomyomas or leiomyomatoid tumors, 12 as leiomyosarcomas and 1 as a neurilemmoma. Immunohistochemical stains for smooth muscle antigen (SMA), S-100 protein, glial fibrillary acidic protein (GFAP), vimentin and desmin were performed with the peroxidase-antiperoxidase method on paraffin sections. The streptavidin-biotin method for PCNA immunostaining was applied using the monoclonal antibody PC 10. On the basis of immunohistochemical findings, 32 cases were identified as leiomyomatoid tumors or leiomyosarcomas (SMA positive, S-100 protein negative), 2 cases as nerve sheath tumors (SMA negative, S-100 protein and GFAP positive), whereas 8 cases presented a mixed phenotype (SMA positive and S-100 protein positive). Two cases were negative for both SMA and S-100 protein. All tumors showed positive immunostaining for vimentin and a negative one for desmin. There was a correlation between the histologic grade and proliferating score, displayed by PCNA expression in tumors of smooth muscle origin. The PCNA expression in tumors of mixed phenotype was intermediate to that seen in leiomyosarcomas (high expression) and in leiomyomas (low expression).

Changes of hepatitis B core antigen (HBcAg) in liver biopsies of patients with chronic active hepatitis B treated with interferon.

Data have shown that hepatitis B core antigen (HBcAg) is detected in both the hepatocyte nucleus and cytoplasm. Its expression is associated with chronic hepatitis and active viral replication. The intrahepatic distribution of HBcAg was studied in liver biopsies of 14 patients with chronic active hepatitis B (CAH-B) (5 were hepatitis B e antigen [HBeAg]+/anti-HBe--, 9 were HBeAg--/anti-HBe+) by an immunohistochemical method (PAP) before and after 6-month treatment with interferon (IFN), and our findings were analyzed according to the response of patients to treatment. Our findings showed that, at the end of treatment, nuclear HBcAg was decreased or absent in 4 of 5 and cytoplasmic HBcAg in 2 of 4 HBeAg+/anti-HBe--patients, irrespective of the response to treatment. Loss of cytoplasmic expression was related to the outcome of treatment in 5 of 9 HBeAg--/anti-HBe+ patients. Four patients expressed no HBcAg before or at the end of treatment. These findings possibly reflect a different pattern of viral core antigen expression as a result of IFN therapy in the two groups of patients.

Immunohistochemical detection of the c-myc oncogene product in normal, hyperplastic and carcinomatous endometrium.

The expression and the distribution of the c-myc oncogene product (p62) was studied by a 3-step immunoperoxidase technique using the monoclonal antibody myc 1-6 E10 in 22 cases of normal endometrium (11 proliferative and 11 secretory phase), 43 endometrial hyperplasias (24 adenomatous and 19 adenocystic) and 26 endometrial carcinomas. Increased expression of c-myc product appeared in endometrial carcinomas compared with respective non-neoplastic tissue (p < 0.001). The immunolocalization of the c-myc protein shows a consistent difference between the various histologic patterns of non-neoplastic and neoplastic endometrium. Nuclear staining of the c-myc product was demonstrated in epithelial cells of the proliferative phase and predominantly in poorly differentiated forms of endometrial carcinomas. On the other hand cytoplasmic staining was found predominantly in the secretory phase and in well differentiated carcinomatous endometrium. In hyperplastic endometrium an intermediate immunohistochemical pattern was observed. The results of the present study emphasize that c-myc product overexpression and localization plays an important role in initiation, differentiation and progression of endometrial carcinomas.

Epstein-Barr virus and primary lung lymphoma: a study utilizing the polymerase chain reaction.

Seven cases of primary lung lymphoma were analyzed for the presence of Epstein-Barr virus DNA sequences by the polymerase chain reaction. The series included: four cases of diffuse, small lymphocytic lymphoma; one case of diffuse, intermediate lymphocytic lymphoma; one case of diffuse, small cleaved cell lymphoma; and one case of large cell, immunoblastic lymphoma. The latter occurred in a patient with acquired immunodeficiency syndrome. A 200-bp sequence of the Epstein-Barr nuclear antigen 1 gene was used as a template for PCR amplification. The only tumor that contained Epstein-Barr virus sequences was the immunoblastic lymphoma. These findings support previous observations that small lymphocytic lymphomas of the lung are not related to Epstein-Barr virus infection. In contrast, some large cell lymphomas may represent Epstein-Barr-virus--associated lymphoproliferative disorders.

[Chondroma of the parotid gland. Clinical--histologic--immunohistochemical findings of a rare case]. / Chondrom der Glandula parotis. Klinische--Histologische--Immunohistochemische Befunde eines seltenen Falles.

A unique extraskeletal chondroma is reported which occupied the entire right parotid gland of a 32-year old man. The midsized tumour was hard and well circumscribed. Its histological pattern was typical of a chondroma with lobules of hyaline cartilage and several areas of calcification. The chondrocytes were positive for S-100 protein and negative for smooth muscle and myoepithelial markers. Epithelial neoplastic elements, as found in pleomorphic adenomas, were not detected in the present case, neither morphologically nor immunohistochemically.

Langerhans cells and lymphocyte subsets in human gastrointestinal carcinomas. An immunohistological study on frozen sections.

In an immunohistochemical study of 38 human gastric and 40 human colonic carcinomas Langerhans cells, suppressor and helper lymphocytes were identified on frozen sections by using anti-CD1, anti-CD8 and anti-CD4 monoclonal antibodies. Tumours were divided into those with few (< 3 per high power field) and those with many (> 3 per high power field) Langerhans cells as well as into those with high number of CD4 and CD8 cells (> 30 per high power field). No significant difference in the number of Langerhans cells regarding histologic types, degree of differentiation and metastatic/non-metastatic groups of either gastric or colonic carcinomas was found. On the contrary the numbers of Langerhans cells related significantly (p < 0.05) to density of T-cell and especially CD4 cell infiltrations of gastric and colonic carcinomas. This finding supports the role of Langerhans cells as antigen presenting cells and their involvement in T-cell activation against neoplastic cells of human gastrointestinal carcinomas.

Immunohistochemical detection of estrogen receptors on paraffin sections of normal, hyperplatic and carcinomatous endometrium.

The present study is the first dealing with the demonstration of estrogen receptors (ER) in up to 8-year-old paraffin blocks of endometrial curettage samples routinely fixed in 10% formalin. The Mab ER-ICA was used in a modified peroxidase-antiperoxidase method after pretreatment of paraffin sections with pronase. Eleven cases with proliferative, 11 cases with secretory endometrium, 20 cases with adenocystic, 21 with adenomatous hyperplasia and 27 endometrial adenocarcinomas were tested. The two main parameters, namely the percentage of ER-positive cells and the intensity of the immunostaining, were higher in the proliferative phase followed in a declining sequence by adenocystic hyperplasia, adenomatous hyperplasia, adenocarcinomas and the secretory phase of endometrium. Interestingly, the intensity of the immunostaining showed a positive relationship to the percentage of ER-positive cells (r = 0.93, p less than 0.001). It seems that the immunohistochemical demonstration of ER in paraffin sections of uterine specimens is an easy and reliable method for the mapping of the heterogeneous expression of ER and their comparative study with the well preserved histopathological features even in old archival paraffin-embedded material.