Clinical validation of a molecular assay for intra-operative detection of metastases in breast sentinel lymph nodes.

BACKGROUND: In breast cancer patients, the status of the sentinel lymph nodes (SLNs) has been shown to accurately reflect the presence of metastases in the axillary lymph nodes (ALNs). Intra-operative SLN evaluation by frozen section histology may miss positive cases, leading to a second surgery for complete ALN dissection. Permanent section histology itself has tissue sampling limitations and is partially dependent on pathologist expertise. METHODS: A prospective study (N=78) was conducted in our institution to validate a new intra-operative molecular assay, the GeneSearch breast lymph node (BLN) assay. This assay quantifies the expression of mammaglobin and cytokeratin-19 genes using quantitative RT-PCR technology to determine SLN status. Fresh SLN sections (2 mm thick) were analyzed alternatively by BLN assay or post-operative histology (haematoxylin-eosin and immunohistochemistry). The subject was considered positive when histology revealed a focus >0.2 mm. RESULTS: BLN assay results corroborated with histologic results in 75 out of 78 patients for an overall agreement of 96%, a sensitivity of 92%, and a specificity of 97%. The positive and negative predictive values of the BLN assay were of 86% (12/14) and 98% (63/64), respectively. Interestingly, a statistically significant correlation was observed between the metastases' histologic size and both assay markers' expression levels as represented by cycle time to positivity (rho > or = 0.71, all p<0.0001). CONCLUSIONS: The performance of the BLN assay in identifying nodal metastases >0.2 mm was similar to that of permanent section histology, with the added advantages of an objective and rapid output that could be used for intra-operative decision to remove additional ALN.

CXCR4 expression in vitreoretinal membranes.

BACKGROUND/AIM: Proliferative vitreoretinopathy (PVR) and macular pucker (MP) vitreoretinal membranes are caused by abnormal cell migration. By their role in chemotactism, chemokine receptors represent good candidates to sustain this process. The authors thus investigated the expression of one of them, CXCR4, in these pathologies. METHODS: Three PVR and four MP membranes were surgically removed and processed for immunochemical studies with antibodies for CXCR4, cytokeratins or smooth muscle actin. RESULTS: CXCR4 expression was found in all membranes. There was no relation between severity of PVR or MP and presence of CXCR4. In addition, there was no difference in CXCR4 expression between MP and PVR. CONCLUSION: CXCR4 is expressed in PVR and MP. Further experiments are needed to test if CXCR4 and other chemokine receptors are implicated in vitreoretinal membrane formation.

Interleukins-6 and -11 expression in primary breast cancer and subsequent development of bone metastases.

Breast cancers frequently metastasize to bone where they often cause extensive tumor-induced osteoclast-mediated osteolysis. Interleukin-6 (IL-6) and IL-11 are two cytokines exhibiting osteolytic properties through their potent stimulation of osteoclast formation. We investigated the expression of IL-6 and IL-11 in 99 invasive primary breast tumors by immunohistochemistry and in situ hybridization, respectively. We examined their potential as predictive factors for further development of bone metastases. 52/90 (57%) of tumor samples showed IL-6 cytoplasmic immunostaining. There was no significant association between IL-6 status and any of the classical prognostic factors. 15/89 (17%) of the tumor samples expressed IL-11 mRNA. A positive IL-11 mRNA status was associated with a low tumor grade (P=0.05). Tumors expressing IL-11 mRNA had a statistically significant (P=0.002) higher rate of bone metastases occurrence (12/15, 80%) than IL-11 negative tumors (27/74, 37%). Such association was not found for IL-6. Our findings demonstrate for the first time IL-11 gene expression in some primary invasive breast tumors and suggest the potential of this cytokine as possible biological predictive factor for the development of bone metastases.

In situ amplification of oestrogen receptor alpha mRNA in breast cancer cell lines and tumours.

The aim of this work was to develop a direct in situ reverse transcription polymerase chain reaction (in situ RT-PCR) assay for the detection of oestrogen receptor alpha (ERalpha) mRNA on in vitro cell lines and breast tumour cell smears. ERalpha mRNA amplification was performed on MCF-7 (ERalpha positive) and MDA-MB-231 (ERalpha negative) cell lines as well as on 51 cytological smears of breast tumour samples from patients. The in situ amplification of mRNA in cell lines and ex vivo breast tumours was successful. However, finding an equilibrium between optimal cell morphology and PCR performance varied with each tumour, leading to difficulty in standardisation for daily practice. Nonetheless, in situ RT-PCR is a useful tool for the detection of ERalpha mRNA in selected cases, both in vitro and ex vivo. J Clin PATHOL: Mol Pathol

Subcutaneous lymphoid hyperplasia arising at site of ethnic scarifications and mimicking subcutaneous panniculitis-like T-cell lymphoma.

The case of a 40-year-old black man, who developed a very unusual tumour-like lymphoid hyperplasia involving primarily the subcutaneous tissue, is reported. The lesion, which arose at a site of tribal scarifications, displayed a deceptive morphology that closely resembled subcutaneous panniculitis-like T-cell lymphoma (SPTCL). An accurate diagnosis could only be made following detailed immunohistochemical and molecular studies. Although SPTCL has been thought to represent a very specific clinicopathologic entity, the present case illustrates that its histological appearance can, however, be closely mimicked by reactive and benign conditions.

Evidence of human papilloma virus infection but lack of Epstein-Barr virus in lymphoepithelioma-like carcinoma of uterine cervix: report of two cases and review of the literature.

Lymphoepithelioma-like carcinoma (LELC) of uterine cervix is an uncommon variant of squamous cell carcinoma (SCC). We report here 2 new cases in which DNA sequences from human papilloma virus (HPV) types 16 and 18 were detected by polymerase chain reaction (PCR). To the best of our knowledge, HPV infection has not been previously described in similar cases occuring in European women. Moreover, Epstein-Barr virus (EBV), which is frequently associated with cervical LELC in Asian women, was absent in our 2 cases. These results suggest that HPVs but not EBV can play a role in the pathogenesis of LELC occuring in women originating from Western countries.

Estrogen receptor of primary breast cancers: evidence for intracellular proteolysis.

Iodinated oestradiol-labeled oestrogen receptor (ER) isoforms devoid of amino-terminal ABC domains represent about two-thirds of the whole receptor population detected in cytosol samples from human breast cancers. This high frequency could not be ascribed to the expression of truncated mRNAs, or to the proteolysis of the native ER peptide at the time of homogenization or assay, suggesting an intracellular proteolysis. Free amino-terminal and ligand-binding domains maintained together within oligomeric structure(s); increase of ionic strength separated them. The amino-terminal region was consistently detected in the cell nucleus by specific immunohistochemistry leading to the concept of a potential intranuclear association between ER cleavage products and/or other regulatory proteins.

Langerhans' cell histiocytosis arising at the site of basal cell carcinoma excision.

Langerhans' cell histiocytosis (LCH) may be associated with a wide range of neoplastic disorders. However, the combination of a LCH and a basal cell carcinoma (BCC) of the skin still represents a highly unusual condition. In this publication, we report the case of a 48-year-old woman who developed a localized LCH involving the area of a previously excised BCC. Although the exact pathogenesis of this peculiar association remains yet to be elucidated, the hypothesis of an exaggerated Langerhans' cell reaction in response to the underlying BCC appears to be attractive.

Sensitivity of HER-2/neu antibodies in archival tissue samples of invasive breast carcinomas. Correlation with oncogene amplification in 160 cases.

Overexpression and amplification of the HER-2 oncogene in patients with breast cancer has correlated with early onset of metastasis, resistance to hormonal therapy and some forms of chemotherapy, and shortened survival. Therefore, evaluation of this putative prognostic or predictive factor seems critical. Because different antibodies are used for the detection of the 185-kd HER-2 oncoprotein, we studied the sensitivity of 3 frequently used antibodies. Immunohistochemistry results were correlated with gene amplification level as assessed by fluorescence in situ hybridization. Protein overexpression was found in 17.2% and 12.5% of cases using antibodies against the external (TAB250) and internal (CB11) domains of the protein, respectively, and in 38.0% of cases using a rabbit polyclonal antibody. Fluorescence in situ hybridization was successful in all 160 tumors, and amplification was found in 37 tumors (23.1%). The monoclonal antibody TAB250 had the lowest misclassification rate, 9.6% (sensitivity, 67%; specificity, 97.5%).

HIV-associated multinucleated giant cells in lymphoid tissue of the Waldeyer's ring: a detailed study.

Hyperplastic lymphoid tissues of the Waldeyer's ring in human immunodeficiency virus (HIV)-infected patients may occasionally contain multinucleated giant cells (MGCs). These cells, which are unrelated to any opportunistic infection, previously have been demonstrated to harbor significant amounts of HIV. Studies undertaken to characterize these MGCs have generated conflicting results: some reports suggested a macrophage origin, whereas others supported a dendritic cell lineage. This study was performed to determine the occurrence of MGCs in a series of adenoid/tonsil specimens from HIV-seropositive patients showing no histological evidence of opportunistic infection in order to further characterize the phenotype of these cells and to investigate the role of a viral infection in their pathogenesis. Adenoid/tonsil tissue specimens from 21 HIV-seropositive patients with no documented opportunistic infection were scrutinized for the presence of MGCs and evaluated immunohistochemically on paraffin sections by antibodies directed against various macrophage and DC antigens. These antigens included CD68, the macrophage marker 3A5, major histocompatibility complex Class II, S-100 protein, CD1a, and CD83. Additional immunostainings directed at CD21 and CD35 as well as at the HIV-associated p24 antigen were also performed. Finally, the presence of Epstein-Barr virus and human herpesvirus 8 viral sequences was investigated by in situ hybridization and by polymerase chain reaction analysis, respectively. MGCs were found in 14 patients (66.7%), regardless of gender, age, method of viral transmission, CD4 cell count, viral load, or ethnic group. These cells were mostly localized at the lymphoepithelium layer of the tonsillar crypts and, to a lesser extent, in the interfollicular areas of the underlying lymphoid tissue, which consistently exhibited features of follicular hyperplasia. Phenotypically, MGCs were found to be CD68+, 3A5+, major histocompatibility complex Class II+, S-100 protein+/-, CD1a-, CD21-, CD35-, and CD83-. Although the HIV-associated p24 protein was consistently present in the cytoplasm of these cells, no sign of Epstein-Barr virus or human herpesvirus 8 infection could be demonstrated. Consequently, our study didn't show any conclusive evidence to support that MGCs in hyperplastic lymphoid tissues of the Waldeyer's ring from HIV-seropositive patients originated from dendritic cells. The definite nature of these cells has yet to be elucidated, but it is plausible that they simply represent activated macrophages that are infected with HIV present in the oropharyngeal secretions during the circulation of their precursor through the lymphoepithelium area of adenoids and tonsils.

Adhesion molecule profiles in atopic dermatitis vs. allergic contact dermatitis: pharmacological modulation by cetirizine.

BACKGROUND: Experimental data suggest that there is an imbalance between Th1 and Th2 cells in atopic dermatitis (AD) skin compared to allergic contact dermatitis (ACD). This imbalance (Th2 and Th1 predominance, respectively) implies the production of different cytokines in these two conditions leading to different expression of adhesion molecules on skin endothelial cells. OBJECTIVE: The expression of VCAM-1 (IL-4/Th2-dependent) and ICAM-1 (INF-gamma/IL-1) on dermal vessels was compared in six patients with AD and six patients with ACD. The effect of cetirizine, a highly selective H1-receptor antagonist on the expressions was studied. METHODS: Six patients with AD were challenged with Dermatophagoides pteronyssimus (DPT patch tests applied to clinically normal skin) and six patients with ACD challenged in the same way with allergens of the European standard series. Skin biopsies at challenged sites were performed before and 6, 24 and 48 h after challenge. The experiment was carried out under double-blind cross-over conditions during a 4-day treatment with a placebo and cetirizine. RESULTS: In AD patients, the scores for both VCAM-1 and ICAM-1 were high before and after challenge. In ACD patients, the ICAM-1 score was high at each experimental time, but the VCAM-1 score, which was significantly lower before challenge, increased at 6, 24 and 48 h after challenge. The administration of cetirizine significantly reduced the VCAM-1 expression in AD patients at each experimental time. CONCLUSION: It is concluded that the increased VCAM-1 expression in AD patients compared to ACD may reflect greater IL-4 and/or IL-13 production in situ. The study also confirms the existence of a modulating effect of cetirizine in vivo on adhesion molecule expression.

Development of a calcifying fibrous pseudotumour within a lesion of Castleman disease, hyaline-vascular subtype.

A nine year old boy with localised Castleman disease of the hyaline-vascular subtype developed a calcifying fibrous pseudotumour. This pathological association does not appear to have been described before. In this case, the development of this very unusual soft tissue tumour-like process was thought to be related to a previous fine needle aspiration biopsy, which was performed because of lymphadenopathy localised to the right inguinal area. This case provides further evidence of the reactive nature of calcifying fibrous pseudotumour and also broadens the pathological spectrum of the stromal cell proliferation that occasionally supervenes within lesions of Castleman disease, hyaline-vascular type.

Natural killer-like T-cell lymphoma of the stomach.

We report the case of a 69-year-old white woman who developed a natural killer (NK)-like T-cell lymphoma involving primarily the stomach. The tumour consisted of large and pleomorphic lymphocytes infiltrating the gastric mucosa. Immunohistochemistry performed on paraffin sections showed the neoplastic cells to be CD3+, CD5-, CD8-, CD43+, CD45RO+, and CD57+. In addition, these cells also expressed HLA-DR, granzyme B, and, to a lesser extent, the CD30 activation marker. No pathologic features suggesting Helicobacter pylori, Epstein-Barr virus infection, or lymphocytic gastritis were found within adjacent normal mucosa. The patient had no previous history of coeliac disease, and her serology for H. pylori was negative. Since lymphomas are usually considered the neoplastic counterpart of normal lymphocytic subsets, it is possible that in this case the tumour cells originate from a distinct cytotoxic T-cell population normally present within the gastric mucosa. The pathogenesis of this highly unusual neoplasm, however, remains a mystery.

Tumor-infiltrating dendritic cells in adenocarcinomas of the breast: a study of 143 neoplasms with a correlation to usual prognostic factors and to clinical outcome.

Dendritic cells (DC) are the most potent antigen-presenting cells, and induce antigen-specific immune responses. Infiltration of tumors by DC is thought to reflect the interaction between the host immune system and tumor cells. Tumor-infiltrating DC (TIDC) are believed to evolve into tumor-antigen pulsed cells and then to migrate to local lymph nodes, where they activate anti-tumor immune responses. Indirect clinical evidence supporting this theory is provided by studies showing that high TIDC densities are associated with favorable prognosis in some tumor types. In the present study, we evaluated 143 primary breast adenocarcinomas for the presence of DC, using immunohistochemistry with the anti-S100 protein antibody. We analyzed the relationship between the degree of infiltration by S100+ TIDC and the usual prognostic factors and clinical outcome. The results show that 42% of breast adenocarcinomas contain S100 TIDC. The number of S100+ TIDC varies according to the grade of tumors as follows: GRIII > GRII > GRI. A relationship is also found between S100+ TIDC and tumor size, lymph-node involvement, estrogen/progesterone receptor status and age. However, the presence of S100+ TIDC, even at the highest density, was not correlated with metastasis-free survival or overall survival.

The immune response induced in vivo by dendritic cells is dependent on B7-1 or B7-2, but the inhibition of both signals does not lead to tolerance.

Dendritic cells (DC) can be used as physiological adjuvant in vivo. Indeed, a single injection of DC, pulsed in vitro with antigen, induces activation of specific T and B lymphocytes in syngeneic mice. The unique capacity of DC to sensitize naive T lymphocytes correlates with elevated expression of MHC antigens as well as co-stimulatory molecules. The aim of this work was to evaluate the functional role of the individual CD28 ligands in the induction of primary humoral and cellular responses, and to characterize the nature of the immune response induced in the presence of selected co-stimulatory molecules. Our data show that the primary response is strictly B7 dependent, and that B7-1 and B7-2 mediate overlapping co-stimulatory functions, as either molecule alone is sufficient to initiate an immune reaction. Inhibition of B7-1 and B7-2, however, does not lead to tolerance as predicted by the two-signal hypothesis. Rather, recognition of antigen in the absence of B7 appears as a null event, since subsequent immunogenic stimulation results in a primary response. Blockade of B7-2 co-stimulatory molecules significantly inhibits antigen-specific IgG1 but not IgG2a production, suggesting that B7-2 may direct the development of Th2 cells. These data emphasize the critical role of the CD28/B7 pathway in the induction of the immune response by DC, which appear to be the initiating antigen-presenting cells in situ.

Dendritic cells fused with mastocytoma cells elicit therapeutic antitumor immunity.

Characterization of the spontaneous immune response that frequently occurs in tumor-bearing animals, as well as immunization using dendritic cells pulsed with tumor antigens, suggests that a limiting factor of the tumor-specific immune response may be a defect in the co-stimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach to improve the antigen-presenting capacity of tumor cells, which does not require a source of purified tumor-associated antigen. We fused P815 mastocytoma cells with bone marrow-derived dendritic cells. We obtained one hybrid that displayed the phenotypic and functional properties of dendritic cells and expressed mRNA coding for the tumor-associated antigen P815 A/B. Injections of irradiated hybrid cells prevented the growth of preimplanted mastocytoma and induced long-lasting tumor resistance.

Interleukin-12-activated natural killer cells recognize B7 costimulatory molecules on tumor cells and autologous dendritic cells.

Activation of natural killer (NK) cells in the presence of interleukin-12 (IL-12) augments the capacity of these effector cells to recognize B7-1- and B7-2-expressing target cells. These effector cells also efficiently lyse autologous B7-positive progenitor or organ-derived dendritic cells, suggesting a physiologic regulatory pathway between IL-12, NK cells, and B7-expressing antigen-presenting cells. Although IL-12-activated NK cells secreted higher levels of interferon-gamma, this cytokine did not play a role in synergistic effects of IL-12 and B7 on NK activation. The B7-counterreceptor was found to be selectively upregulated on IL-2/IL-12 as compared with IL-2-activated NK cells. CD28 is functionally involved in the recognition of B7 on target cells since IL-2/IL-12-activated NK cells derived from CD28 knockout mice were strongly reduced in their capacity to lyse syngeneic B7-positive tumor cells as well as antigen-presenting cells. However, recognition of B7 on allogeneic targets did not require the expression of CD28 on the IL-2/IL-12-activated NK cells. Hence, IL-12 triggers the expression of both CD28-dependent and CD28-independent mechanisms that allow NK cells to eliminate B7-positive target cells including autologous dendritic cells.

Nature of the T lymphocytes in lymphocyte predominance Hodgkin's disease.

Little is known about the function of the T lymphocytes in lymphocyte predominance Hodgkin's disease. We report here the case of a 37-year-old man with a diffuse LPHD, featuring a similar increase in T lymphocytes in both the peripheral blood and the tumor, thus allowing for their characterization by functional assays. These cells were CD4+CD45RO+ and produced high amounts of IL-2 and IFN-gamma, consistent with a TH1-type profile. This subset of T helper cells is involved in cellular immunity and could reflect a cytotoxic reaction directed against the neoplastic cells.