Jaime Ferrer

Establishment and characterization of a continuous human chondrosarcoma cell line, ch-2879: comparative histologic and genetic studies with its tumor of origin.

Chondrosarcomas are malignant cartilage-forming tumors that represent the second most common malignant solid tumor of bone. These biologically poorly understood neoplasms vary considerably in clinical presentation and biologic behavior. Chemotherapy and radiation therapy are generally ineffective. Here we describe the establishment and characterization of a new human chondrosarcoma cell line named ch-2879, and we compare the cell line with its tumor of origin. The cell line was established from a recurrent grade 3 chondrosarcoma of the chest wall and characterized by growth kinetics and morphologic studies. Immunocytochemistry and RT-PCR were performed to examine the expression of cartilage-specific phenotypes. Genetic characterization was performed using cytogenetics, fluorescence in situ hybridization, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control, amplification of MDM2, CDK4, and Cyclin D1, and mutations in the p53 gene. ch-2879 cells were subcultured for more than 80 passages. They expressed vimentin, HNK-1, HBA-71, Ki-67, cyclin D1, Fli-1, S-100, p21, p27, and p53 and were negative for cytokeratin, EMA, p14, p16, MDM2, Rb, and c-erb-b2 antigens. Cytogenetically the recurrent tumor showed a hyperhaploid karyotype with clonal numerical and structural abnormalities. The sole structural abnormality was a chromosome derivative of a t(1;21) translocation. The cell line at passage 3 showed two populations: the hyperhaploid and an exactly duplicated, hypotriploid population. After the 18th passage, only the hypotriploid population was present. The cells expressed collagen 2. Molecular comparison of the primary and recurrent tumor evidenced an in vivo molecular change consisting of a deletion of 9p21 genes in the recurrence, probably caused by a selection process. Because of its gene expression profile, including expression of genes implicated in chondrogenesis in uncoated plastic dishes, this cell line may prove useful for cellular and molecular studies as well as studies of chondrosarcoma characterization and treatment.

Anaplastic carcinoma of the thyroid with rhabdomyosarcomatous differentiation: a report of two cases

Anaplastic carcinoma of the thyroid gland (ACT) is a highly malignant tumor that is almost invariably associated with a fatal outcome. It demonstrates a variety of peculiar histological features, with squamoid, giant cell and spindle cell growth patterns. The spindle cell variant of ACT is usually indistinguishable from a true sarcoma and it can simulate fibrosarcoma, malignant fibrous histiocytoma (MFH), hemangiopericytoma and angiosarcoma or rhabdomyosarcoma. Although a rhabdomyosarcomatous appearance has sometimes been mentioned in the literature, true skeletal muscle differentiation has never been consistently proved. We report two cases of ACT with rhabdomyosarcomatous differentiation, as demonstrated by means of immunohistochemistry and electron microscopy. Both cases disclosed a very similar histological appearance, with a main population of small, pleomorphic, round-to-oval cells arranged in a storiform pattern, admixed with scattered pleomorphic giant cells, an image similar to that of the usual type of MFH. Stains for epithelial markers showed only few, scattered, weakly positive cells. Thyroglobulin and calcitonin were negative in tumor cells in both cases. On the contrary, positivity to vimentin was strong and generalized. Immunomarkers of muscular differentiation showed a consistent positivity. At the ultrastructural level, the cells disclosed the same spindle and pleomorphic morphology, with large, bizarre nuclei and cytoplasm with abundant mitochondria, rough endoplasmic reticulum, secretory granules and lipid droplets. There were also cells with wide cytoplasm filled with filamentous material, either of actin or myosin, as well as Z-band material. In conclusion, the cases reported here show a clear-cut rhabdomyosarcomatous differentiation of ACT, confirmed both immunohistochemically and ultrastructurally, a feature not previously reported in the literature. These findings may contribute to the broadening of the differentiation spectrum of this unusual neoplasm.

Cytogenetic and molecular findings related to rhabdomyosarcoma. An analysis of seven cases

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood. Histologically, it is subdivided histologically into two main subtypes: alveolar (ARMS) and embryonal (ERMS). ARMS is characterized by t(2;13)(q35;q14) or its variant t(1;13)(p36;q14), which fuse PAX3 and PAX7, respectively, with FKHR to produce chimeric genes. ERMS is frequently associated with loss of heterozygosity of 11p15.5. We investigated seven RMS (three ARMS and four ERMS) by means of cytogenetic, fluorescence in situ hybridization, and molecular analyses, including the study of the main genes implicated in the G1- to S-phase cell cycle transition, and correlated these studies with pathologic findings and clinical outcome. All tumors showed clonal, numerical, and structural chromosomal abnormalities. Two ARMS had the t(2;13)(q35;q14) and the third a PAX7/FKHR fusion, a cryptic t(1;13)(p36;q14), undetected by cytogenetic techniques, but revealed by reverse transcriptase polymerase chain reaction. One ERMS showed a der(11)t(3;11)(p21;p15) as a sole structural anomaly. Gene amplification was seen in four tumors, as double minutes or in the form of homogeneously staining regions. Overexpression of MYCN oncogene was found in two ARMS; N-myc DNA probe detected oncogene amplification located on the double minutes of these cases. Analysis of the regulatory genes responsible for G1- to S-phase transition showed a homozygous deletion of the 9p21 locus genes in a spindle-cell ERMS.

Hepatoid adenocarcinoma of the urinary bladder

Abstractu2002A new case of hepatoid adenocarcinoma was diagnosed in fragments obtained at transurethral resection (TUR) from a 71-year-old man who had complained of haematuria. The tumour was composed of trabeculae and small solid nests of polygonal atypical cells simulating hepatocarcinoma, together with glandular areas of an otherwise typical adenocarcinoma. Immunohistochemistry showed cytoplasmic reactivity to AFP, AAT, albumin and CAM 5.2. Membrane reactivity was seen in EMA immunostaining, and there was also positivity to polyclonal CEA following a canalicular pattern. Immunoperoxidase studies of hepatocyte growth factor (HGF) and its receptor, c-met, were positive. Their expression may be related to the aggressive behaviour of this tumour.

Diffuse Type of Giant-Cell Tumor of Tendon Sheath: An Ultrastructural Study of Two Cases With Cytogenetic Support

Two cases of the diffuse type of giant-cell tumor of the tendon sheath (GCTTS) are described. Both tumors arose in the vicinity of large joints of the lower extremity, showing similar clinical and radiological features. Histologically, a proliferation of polygonal mononuclear cells was seen, together with osteoclastlike giant cells, foam cells, and siderophages. The tumors were poorly delineated, displaying an infiltrative pattern into the neighboring soft tissues. Immunohistochemically, strong expression of vimentin, neuron-specific enolase, A 1 -antitrypsin, and CD68 was found in both mono- and multinucleated tumor cells. At the ultrastructural level, mononuclear cells revealed a diverse morphology, displaying features of histiocytelike and fibroblastlike cells, with the former being more numerous. Scarce neurosecretorylike granules, made up of electrondense membrane-bound material, were found in the cytoplasm of the mononuclear cells. Cytogenetic analysis of one case shows the presence of a clonal population with 47 chromosomes and two different translocations, t(2;3) and der(8) t(8;12). Present findings provide further support regarding the neoplasic nature of this tumoral entity.

Clear cell syringoid carcinoma : An ultrastructural and immunohistochemical study

Syringoid carcinoma (syringoid eccrine carcinoma or eccrine epithelioma) is a rare cutaneous tumor with some controversy regarding its correct definition. It may also be difficult to differentiate from its benign counterpart (syringoma), other adnexal carcinomas, and cutaneous metastasis from adenocarcinomas. We present a case of a syringoid carcinoma of the clear cell variant complemented with an immunohistochemical and ultrastructural study, the latter revealing cytoplasmic accumulation of glycogen and presence of intercellular and intracellular lumina in clear tumor cells, as well as diverse hallmarks of malignancy (i.e., perineural invasion, tumor necrosis, and deep invasion). Clear tumor cells showed cytoplasmic and membranous immunoreactivity to epithelial membrane antigen, carcinoembryonic antigen, keratins, and S-100. Our ultrastructural and immunohistochemical results support the ductal differentiation of the glycogen-filled clear cell tumor population.

Non-canonical NF-κB pathway activation predicts outcome in borderline oestrogen receptor positive breast carcinoma.

Background:NF-κB signalling appears deregulated in breast tumours. The purpose of this study was to determine whether the non-canonical NF-κB pathway, is activated in oestrogen receptor positive (ER+) breast cancer, to identify any correlation between its activity and the clinico-pathological phenotype and to explore whether NF-κB2 and RelB subunits and/or any of their target genes might be used as a predictive marker.Methods:Two independent cohorts of ER+ early breast cancer patients treated with adjuvant endocrine therapy were included in the study. Activation of RelB and NF-κB2 subunits was determined in a training set of 121 patients by measuring DNA-binding activities in nuclear extracts from fresh frozen specimens by an ELISA-based assay. Samples of 15 ER− breast cancer patients were also included in the study. In a large validation cohort of 207 patients, nuclear immunostaining of RelB and NF-κB2 on formalin-fixed paraffin-embedded specimens was performed. Statistical correlation within clinico-pathological factors, disease-free survival (DFS) and overall survival (OS) was evaluated. Publicly available gene expression and survival data have been interrogated aimed to identify target genes.Results:Activation of NF-κB2 and RelB was found in 53.7 and 49.2% of the 121 ER+ tumours analysed, with similar levels to ER− breast tumours analysed in parallel for comparisons. In the validation cohort, we obtained a similar proportion of cases with activation of NF-κB2 and RelB (59.9 and 32.4%), with a 39.6% of co-activation. Multiplexing immunofluorescence in breast cancer tissue confirmed an inverse spatial distribution of ER with NF-κB2 and RelB nuclear expression in tumour cells. Interestingly, NF-κB2 and RelB mRNA expression was inversely correlated with ER gene (ESR1) levels (P<0.001, both) and its activation was significantly associated with worse DFS (P=0.005 and P=0.035, respectively) in ER+ breast cancer. Moreover, the co-activation of both subunits showed a stronger association with early relapse (P=0.002) and OS (P=0.001). Finally, higher expression of the non-canonical NF-κB target gene myoglobin was associated with a poor outcome in ER+ breast cancer (DFS, P<0.05).Conclusions:The non-canonical NF-κB pathway activation is inversely associated with oestrogen receptor expression in ER+ breast cancer and predicts poor survival in this subgroup. The myoglobin gene expression has been identified as a possible surrogate marker of the non-canonical NF-κB pathway activation in these tumours.

An evaluation of the impact of technical bias on the concordance rate between primary and recurrent tumors in breast cancer

PURPOSEnWhether or not to biopsy the metastasis in recurrent breast cancer has become mired in controversy. Several studies have shown an important discordance of the immunohistochemical (IHC) determinations for ER, PR and HER2 between primary (PT) and recurrent tumors (RT). Yet it remains unknown within this what impact technical issues have. The aim of our study was to assess whether technical variability might have an impact on the concordance between PT and RT.nnnMETHODSnIHC determinations in paired biopsies from PT and RT were compared under routine vs study conditions. In the former, pathological analysis reproduced the conditions used in the routine of a University Pathology Department. In the latter, in a technical bias-minimizing manner, samples were re-assessed at the same timing and by two independent observers.nnnRESULTSn128 paired biopsies from 64 patients were analyzed under both conditions. Concordance under routine vs study conditions for ER was 66% vs 93.4% (p = 0.001), for PR 58.7% vs 80.3% (p = 0.064) and for HER2 86.8% vs 96.8% (p = 0.25). Kappa index under routine versus study conditions for ER was 0.27 vs 0.79 (p = 0.002), for PR 0.26 vs 0.39 (p = 0.47) and for HER2 0.67 vs 0.9 (p = 0.14).nnnCONCLUSIONSnAlthough discordance rate between PT and RT decreased under conditions minimizing technical issues, some discordant cases appeared not to be subjected to this confounding factor. Either for clinical practice or for future studies reassessment of PT in recurrent breast cancer should be encouraged.

Abstract PD3-6: ConvertHER: Evolution of genomic alterations from primary to metastatic breast cancer

Background: Changes in breast cancer receptor status over disease progression and treatment have been described to a point that could alter response to therapy. There is growing interest in delivering biomarker/genomically-based targeted therapies. We aimed to determine the concordance of genomic alterations between primary (P) and metastatic (M) breast cancer in a prospective collection study. Methods: Targeted capture and next-generation sequencing was performed on formalin-fixed paraffin-embedded (FFPE) samples, profiling 202 cancer relevant genes in 61 pairs (primary and corresponding recurrence/metastasis). Tumors were classified at baseline as [hormone receptor (HR)+/HER2-, HR+/HER2+, HER-/HER2+, and triple negative breast cancer (TNBC)]. We aligned data to human reference assembly hg19 using Burrows-Wheeler Aligner9s (BWA) and removed duplicated reads. We identified somatic mutations variants and called copy number alterations (CNA) using an algorithm which reports gain or loss status of each exon. Alterations potentially targetable with established or investigational therapeutics were considered actionable. Results: Of the 61 cases, 15% changed breast cancer subtype. Of 747 mutations detected in 156 genes, 309 (41%) were discordant. Median number of mutations were 10 (range 6-11) in P and 8 (range 6-10) in M. Most common mutations occurred at NOTCH2, PCLO, MAP3K1, MLL3, NOTCH4, CRIPAK, TP53, PIK3CACSMD1, and FLG. Mutations were less common in HR-/HER2+ tumors in both P and M. Mutation discordance was not different in cases of changed breast cancer subtype (P=.31). Of 986 CNA detected in 173 genes, 758 (77%) were discordant. There was an increased frequency of EGFR1, ERBB2, FGFR3, CRIPAK, MEN1 and WT1 amplifications in M. CNA were less common in HR-/HER2+ tumors in both P and M. CNA discordances were more common in cases of changed breast cancer subtype (P Conclusion: Deep targeted exome sequencing of cancer-related genes revealed potentially targeted alterations. We found 41% and 77% mutation and CNA discordance between P and M. CNA were more common when breast cancer subtype changed. Citation Format: Ana Maria Gonzalez-Angulo, Ana Lluch, Agda K Eterovic, Angel Guerrero, Xiaofeng Zheng, Ramon Perez, Shuying Liu, Jose I Chacon, Ken Chen, Silvia Antolin, Gordon B Mills, Jaime Ferrer, Octavio Burgues, Begona Bermejo, Elia Munoz, Rosalia Caballero, Eva Carrasco, Eduardo Martinez, Funda Meric-Bernstam. ConvertHER: Evolution of genomic alterations from primary to metastatic breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-6.

Abstract P4-07-04: A distinctive miRNA profile highlights breast cancer in very young women (BCVY) as a new molecular subgroup

Background: Although less frequent (1% of all breast cancer) than in older women, breast cancer (BC) in very young women (BCVY),(≤35 years old) often exhibits larger tumor sizes, high pathological grade, high proliferation percentage, nodal involvement, and greater HER2+ percentage, usually associated with a poorer prognosis. BC and BCVY might arise as different disease entities derived from different underlying mechanisms. MicroRNAs (miRNAs) are short RNA molecules with post-transcriptional regulatory function, usually silencing targeted mRNAs. Recently, miRNA profiling has arisen as major approaching study technique, demonstrating a widespread miRNA deregulation in various tumor types. In this study we offer a miRNA profile of a subset of BCVY patients and evaluate whether there are any miRNA deregulated pointing out some pathway to better understand the ongoing mechanism and characterize BCVY as a new molecular entity. Material and methods: We performed a comprehensive study of miRNA expression using miRNA Affymetrix 2.0 array. We extracted RNA from FFPE tumor tissue of both 44 BC patients ≤35years (BCVY) and 46 older than 45years in two age groups (45-65 and >65), we used normal breast tissue as control and evaluated the differences in expression of each age group. We tried to validate most interesting miRNA by qRT-PCR. We performed enrichment analysis of multiple miRNA target genes (DIANA mirpath) to search for putative pathways that could be deregulated by the miRNAs. Results: We obtained a differential and unique miRNA expression profile of 121 miRNAs (p-value Conclusion: We performed a comprehensive study of the miRNA expression on a total of 89 BC patients, detecting a differential molecular profile in BCVY patients, suggesting that there might be a different underlying mechanism and that BCVY could be identified as a different entity. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-04.