Francisco Giner

Right ventricular involvement in anterior myocardial infarction: a translational approach

AIMS The aim of the present study was to evaluate the involvement of the right ventricle (RV) in reperfused anterior ST-elevation myocardial infarction (STEMI). METHODS AND RESULTS Left anterior descending (LAD)-perfused area (using thioflavin-S staining after selective infusion in proximal LAD artery, %), infarct size (using triphenyltetrazolium chloride staining, %), and salvaged myocardium (% of LAD-perfused area) in the right and left ventricle (LV) were quantified in a 90-min LAD occlusion 3-day reperfusion model in swine (n = 8). Additionally, we studied, using cardiovascular magnetic resonance, 20 patients with a first STEMI due to proximal LAD occlusion treated with primary angioplasty. Area at risk (T2-weighted sequence, %), infarct size (late enhancement imaging, %), and salvaged myocardium (% of area at risk) in the right and LV were quantified. In swine, a large LAD-perfused area was detected both in the right and LV (30 +/- 5 vs. 62 +/- 15%, P< 0.001) but more salvaged myocardium (94 +/- 6 vs. 73 +/- 11%, P< 0.001) resulted in a smaller right ventricular infarct size (2 +/- 1 vs. 16 +/- 5%, P< 0.001). Similarly, in patients a large area at risk was detected both in the right and LV (34 +/- 13 vs. 43 +/- 12%, P = 0.02). More salvaged myocardium (94 +/- 10 vs. 33 +/- 26%, P < 0.001) resulted in a smaller infarct size (2 +/- 3 vs. 30 +/- 16%, P< 0.001) in the RV. CONCLUSION In reperfused extensive anterior STEMI, a large area of the RV is at risk but the resultant infarct size is small.

Histopathological characterization of small cell osteosarcoma with immunohistochemistry and molecular genetic support. A study of 10 cases.

Isabel Cano Marı́a Lozano Álvaro Rodrı́guez Alberto Mate Magdalena Adrados Marı́a del Mar López Ruben Carro Santiago Montes-Moreno Department of Hematology, Mancha-Centro Hospital, Ciudad Real, Lymphoma Group, Molecular Pathology Programme, Spanish National Cancer Centre, Madrid, and Department of Pathology, Mancha-Centro Hospital, Ciudad Real, Spain 1. Swerdlow SH, Campo E, Harris NL et al. WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues, 4th ed. Lyon, France: International Agency for Research on Cancer, 2008. 2. Nam-Cha S, Roncador G, Sanchez-Verde L, Montes-Moreno S et al. PD-1, a follicular T-cell marker useful for recognizing nodular lymphocyte-predominant Hodgkin lymphoma. Am. J. Surg. Pathol. 2008; 32; 1252–1257. 3. Rüdiger T, Gascoyne RD, Jaffe ES, de Jong D et al. Workshop on the relationship between nodular lymphocyte predominant Hodgkin’s lymphoma and T cell ⁄ histiocyte-rich B cell lymphoma. Ann. Oncol. 2002; 13(Suppl. 1); 44–51. 4. Greaves MF, Maia AT, Wiemels JL, Ford AM. Leukemia in twins: lessons in natural history. Blood 2003; 102; 2321–2333.

A Tissue Microarray Study of Osteosarcoma: Histopathologic and Immunohistochemical Validation of Xenotransplanted Tumors as Preclinical Models

BackgroundOsteosarcomas (OS) are aggressive neoplasms with a wide range of morphologic patterns. Materials and MethodsOS cases (primary and xenotransplanted) with paraffin blocks available were collected and included in tissue microarrays (TMAs). A morphologic evaluation including the different passages in mice was carried out according to the new WHO criteria. In addition, TMAs were analyzed with a wide panel of immunohistochemical (IHC) markers (osteonectin, osteocalcin,cytokeratin, S100, Sox-9, Ki-67, Bcl-2, p53, p16, survivin, CD99, and caveolin-1). ResultsA total of 61 cases were collected. The distribution of the cases according to the histopathologic pattern was: 38 osteogenic OS, 8 primary chondrogenic OS, 2 primary telangiectatic OS, 6 parosteal OS, 2 primary small cell OS, 2 primary poorly differentiated OS, 1 primary dedifferentiated OS, and 3 primary pleomorphic MFH-like OS. The tumor morphology in xenotransplants was similar to the primary or metastatic tumor of origin and was generally maintained over the passages. The IHC results were heterogeneous and osteonectin and osteocalcin were the most expressed in original tumor and xenografts. S100 and Sox-9 were expressed in chondrogenic areas. Caveolin and survivin showed significant IHC variation between the subsequent passages. p16 displayed heterogenic expression. p53 expression increased over the passages, and Ki-67 expression was not associated with a more undifferentiated pattern, but increased over the passages. ConclusionsAn accurate morphologic evaluation using TMAs in original tumor is essential for the OS diagnosis; hence there is no IHC marker that alone distinguishes the OS subtypes. Xenografts in OS allow the study of tumor progression in this type of aggressive neoplasm.

Tissue microarrays analysis in chondrosarcomas: light microscopy, immunohistochemistry and xenograft study.

BackgroundChondrosarcoma (Chs) is the third most frequent primary malignant tumour of bone and can be primary or secondary, the latter results mainly from the malignant transformation of a benign pre-existing tumour.MethodsAll the cases diagnosed as Chs (primary tumours, recurrences and/or metastasis and xenotransplanted Chs) from the files of our Department were collected. Only cases with paraffin blocks available were selected (Total 32 cases). Six Tissue Microarrays (TMAs) were performed and all the cases and biopsies were distributed into the following groups: a) only paraffin block available from primary and/or metastatic tumours (3 TMAs), b) paraffin block available from primary and/or metastatic tumours as well as from the corresponding Nude mice xenotransplant (2 TMAs), c) only paraffin block available from xenotransplanted Chs (1 TMA). A reclassification of all the cases was performed; in addition, conventional hematoxylin-eosin as well as immunohistochemistry staining (S100, SOX-9, Ki-67, BCL-2, p53, p16, CK, CD99, Survivin and Caveolin) was analyzed in all the TMA.ResultsThe distribution of the cases according to the histopathological pattern and the location of tumours were as follows: fourteen Grade I Chs (all primaries), two primary Grade II Chs, ten Grade III Chs (all primaries), five dedifferentiated Chs (four primaries and one primary with metastasis), and two Chs from cell cultures (Ch grade III). One recurrent extraskeletal myxoid Chs was included as a control in the TMA. Although there was heterogeneity in immunohistochemistry results of the different material analyzed, S100, SOX-9, Caveolin and Survivin were more expressed. The number of passages in xenotransplants fluctuated between 1 and 13. Curiously, in Grade I Chs, these implanted tumours hardly grew, and the number of passages did not exceed one.ConclusionThe study of Chs by means of TMA techniques is very important because it will improve the assessment of different antibodies applied in the immunohistochemical assays. Xenotransplanted tumours in TMA improve knowledge concerning the variability in the morphological pattern shown by these tumours during the evolution in nudes.

Ezrin immunohistochemical expression in chondrosarcomas, osteosarcomas and Ewing sarcoma family of tumors

Dear Sir, Ezrin immunohistochemical (IHC) expression has been described in bone sarcomas, being present mainly in osteosarcomas [1–5]. Salas et al. [4] described the utility of ezrin protein expression as a tool to differentiate between conventional chondrosarcomas and chondroblastic osteosarcomas, demonstrating a specificity of 100%. In the present study, we describe ezrin IHC expression in 397 bone tumors, including 33 osteosarcomas (eight lowgrade, 20 high-grade and five chondroblastic), 23 conventional chondrosarcomas (ten grade I, eight grade II and five grade III) and 341 Ewing sarcoma family of tumors (ESFT) [236 conventional, 42 primitive neuroectodermal tumor (PNET) and 63 atypical]. The sections were immunostained with ezrin antibody (clone 3C12, dilution 1:250). The cytoplasmic/membranous immunoexpression was scored as negative ( 50%). For the statistical analysis, negative and poor ezrin tumors were considered as negative, and moderate and intense ezrin tumors were considered as positive. Chi-square or Fisher’s exact test was performed for statistical analysis. Ezrin immunoexpression was heterogeneous in the present series. Ezrin stained in a higher proportion of analyzed osteosarcomas cases (69%) than ESFT (40.7%) and chondrosarcomas (30%, p=0.023) (Figs. 1, 2, 3). Interestingly, chondroblastic osteosarcoma tumors did not reveal ezrin expression (Fig. 2), and some cases of low-grade chondrosarcoma revealed poor ezrin immunoexpression; both results are in contrast with the study of Salas et al. [4]. However, similar to the results obtained by Salas et al. [4], the protein immunoexpression in the present series is associated with the histological grade, being higher in morphologically high-grade tumors. Highgrade osteosarcomas and chondrosarcomas stained ezrin in a higher proportion (95% and 100%, respectively, p< 0.001) than low-grade osteosarcomas and chondrosarcomas. Within the ESFT, ezrin expression was higher in atypical ESFT (58.9%, p<0.001) and PNET (58.6%, p<0.001) than in conventional ESFT (30%). The immunostaining in chondrosarcoma and osteosarcoma was mainly cytoplasmic, but several cases in ESFT also displayed a membranous pattern similar to CD99 expression. In ESFT with strong ezrin expression, apoptotic cells did not express ezrin compared with non-apoptotic cells (Fig. 3). In conclusion, based upon present experience, I. Machado : S. Navarro : F. Giner :A. Llombart-Bosch (*) Pathology Department, University of Valencia, Avda Blasco Ibanez 17, 46010 Valencia, Spain e-mail: Antonio.llombart@uv.es

Gain of MYCN Region in a Wilms Tumor-derived Xenotransplanted Cell Line

Wilms tumor is one of the most common pediatric malignant tumors of the kidney. Although the WT1 gene, located at 11p13, has been proven to be implicated in the development of Wilms tumor, other genes such as MYCN are also involved. The purpose of this study is to genetically characterize a Wilms tumor metastasis xenotransplanted in nude mice. Immunogenotype evolution of the xenografts material was monitored for 29 months using molecular techniques, fluorescent in situ hybridization and multiplex ligation-dependent probe amplification, in addition to immunohistochemistry in tissue microarrays. Genetic alterations present in the original tumor and retained in the xenotransplanted tumor were located in +1q, +3, +6, −7p, +7q, +8, −9p, +9q, +12. The multiplex ligation-dependent probe amplification detected a nondeleted status of genes located close to WT genes, except for a deletion of the EGFR gene (located at 7p11.2) and the GHRHR gene (located at 7p15), both flanking the WT5 gene. The MYCN gene (2p24 exon 3) and DDX1 gene (2p24 exons 2, 7, 15, and 24) were gained in passage 4 and the following passages. MYCN expression was positive from the beginning, without evidence of MYCN gain by fluorescent in situ hybridization. Histopathologic and growth rate changes were observed at those passages where low extra copy number of MYCN was present. In addition to other genetic abnormalities, the WT5 gene located at 7p13-14 is deleted and the MYCN gene gain began after 16 months in vivo evolution in athymic nude mice. MYCN is already used as a stratifying marker in neuroblastomas, and it may be also useful in implementing MYCN testing in prospective studies of Wilms tumors.

Uterine Glomeruloid Hemangioma in a Patient Without POEMS Syndrome

Cutaneous glomeruloid hemangioma is a hallmark of POEMS syndrome. These patients have elevated serum levels of vascular endothelial growth factor. The authors report an extracutaneous uterine glomeruloid hemangioma in an 82-year-old woman with a history of breast and endometrial carcinomas. Within the lumen of myometrial vessels, a lobular, glomeruloid proliferation of capillary-like CD31 and vascular endothelial growth factor receptor-1-positive endothelial cells was found. The capillary loops were lined by endothelial cells, most of them containing PAS-positive and immunoglobulin-positive eosinophilic hyaline globules (thanatosomes). This vascular proliferation was consistent with a glomeruloid hemangioma. Although an extracutaneous glomeruloid vascular proliferation has been found in the retroperitoneal adipose tissue in a patient with POEMS syndrome, this study reports what seems to be the first case of visceral glomeruloid hemangioma in a patient without POEMS syndrome. The authors hypothesize that the glomeruloid endothelial cell proliferation with vascular endothelial growth factor receptor-1 expression may be a paraneoplastic phenomenon.

High-risk gastrointestinal stromal tumour (GIST) and synovial sarcoma display similar angiogenic profiles: a nude mice xenograft study

Background Gastrointestinal stromal tumour (GIST) is the most common primary mesenchymal tumour of the gastrointestinal tract. Spindle cell monophasic synovial sarcoma (SS) can be morphologically similar. Angiogenesis is a major factor for tumour growth and metastasis. Our aim was to compare the angiogenic expression profiles of high-risk GIST and spindle cell monophasic SS by histological, immunohistochemical and molecular characterisation of the neovascularisation established between xenotransplanted tumours and the host during the initial phases of growth in nude mice. Methods The angiogenic profile of two xenotransplanted human soft-tissue tumours were evaluated in 15 passages in nude mice using tissue microarrays (TMA). Tumour pieces were also implanted subcutaneously on the backs of 14 athymic Balb-c nude mice. The animals were sacrificed at 24, 48, and 96 h; and 7, 14, 21, and 28 days after implantation to perform histological, immunohistochemical, and molecular studies (neovascularisation experiments). Results Morphological similarities were apparent in the early stages of neoplastic growth of these two soft-tissue tumours throughout the passages in nude mice and in the two neovascularisation experiments. Immunohistochemistry demonstrated overexpression of pro-angiogenic factors between 24 h and 96 h after xenotransplantation in both tumours. Additionally, neoplastic cells coexpressed chemokines (CXCL9, CXCL10, GRO, and CXCL12) and their receptors in both tumours. Molecular studies showed two expression profiles, revealing an early and a late phase in the angiogenic process. Conclusion This model could provide information on the early stages of the angiogenic process in monophasic spindle cell SS and high-risk GIST and offers an excellent way to study possible tumour response to antiangiogenic drugs.

The Role of Immunohistochemistry in Rhabdomyosarcoma Diagnosis Using Tissue Microarray Technology and a Xenograft Model

Rhabdomyosarcomas (RMS) may resemble other non-myogenic sarcomas and malignant rhabdoid tumor (MRT). Alveolar rhabdomyosarcoma (ARMS) often harbors a typical translocation, but embryonal rhabdomyosarcoma (ERMS) lacks any specific rearrangement. Histopathology is not always sufficient for an unequivocal diagnosis, necessitating ancillary studies, including immunohistochemistry (IHC). Sixteen genetically tested RMS and two MRT were xenografted and followed in successive passages. Tissue microarrays were constructed including samples from original and xenograft tumors. Desmin, myogenin, CK, EMA, INI1, LSD1, AP2β, fibrillin-2, HMGA2, nestin, and SIRT1 were tested using immunohistochemical staining. Desmin and myogenin were positive in all RMS, and the epithelial markers were negative in almost all RMS. New markers (LSD1, AP2β, HMGA2, Nestin, and SIRT1) were positive in all RMS and MRT. There were no differences in IHC expression between the three RMS subtypes tested except fibrillin-2, which was negative in ARMS. Applying new IHC markers can contribute to RMS diagnosis. Nevertheless, most markers are also expressed in MRT, and further studies are needed to confirm their value against this and other small round cell tumors.

The epithelial mesenchymal transition process in wilms tumor: a study based on a xenograft model.

BackgroundUntil now, only a few mouse-transplanted human tumors or experimental Wilms tumor (WT) cell lines have been described. The aim of this study was to show the biological behavior, including histology, immunohistochemistry (IHC), and molecular biology, of a WT including the original tumor and metastasis transferred into nude mice and followed for successive generations in xenografts. MethodsA WT metastasis was xenotransplanted into nude mice and the mice was monitored for 7 passages over a period of 29 months; the original neoplasm was comparatively studied. The morphology was evaluated by optical and electron microscopy. The protein expression was analyzed by immunohistochemistry in whole sections and in tissue microarray. The molecular studies were carried out by multiplex ligation-dependent probe amplification and polymerase chain reaction analysis. ResultsThe histology changed markedly between the fourth and fifth transfer. The tumor exhibited an increased epithelial component (>40%) together with a slowing in the growth rate (8 mo). An epithelial-mesenchymal transition seemed to take place in the fourth passage and increased thereafter. The genetic studies also showed a WT5 deletion and a MYCN gain in all the tumor samples in passage 4 and beyond, but did not show E-cadherin, &bgr;-catenin, and APC mutations. ConclusionsAn epithelial pattern was associated with slow tumor growth, whereas the predominance of mesenchymal spindle cells with striated muscle cell differentiation was related with a high growth rate. The in vivo reorganization of the tumor components (blastemal, epithelial, and mesenchymal) does not seem to be related with the Wnt and EMT pathways.