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Background and aims: The role of overweight and obesity in the development of atrial fibrillation (AF) is well established; however, the differential effect on the occurrence and recurrence of AF remains uncertain. The aim of this review is to compare the effect of underweight and varying degrees of obesity on onset of AF and in recurrent post-ablation AF, and, when possible, in relation to sex. Methods: A systematic literature search was conducted in PubMed, Embase, and Cochrane Library from inception to January 31, 2023. Studies reporting frequency of newly-diagnosed AF and of recurrent post-ablation AF in different BMI categories, were included. 3400 records were screened and 50 met the inclusion criteria. Standardized data search and abstraction were performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) Statement. Data were extracted from the manuscripts and were analyzed using a random effect model. The outcome was the occurrence of AF in population studies and in patients undergoing ablation. Results: Data from 50 studies were collected, of which 27 for newly-diagnosed AF and 23 for recurrent post-ablation AF, for a total of 15,134,939 patients, of which 15,115,181 in studies on newly-diagnosed AF and 19,758 in studies on recurrent post-ablation AF. Compared to normal weight, the increase in AF was significant (p< 0.01) for overweight, obese, and morbidly obese patients for newly-diagnosed AF, and for obese and morbidly obese patients for recurrent post-ablation AF. Newly-diagnosed AF was more frequent in obese female than obese male patients. Conclusion: The effect of increased BMI was greater on the onset of AF, and obese women were more affected than men.

O1 Tumour heterogeneity: what does it mean? Dow-Mu Koh O2 Skeletal sequelae in adult survivors of childhood cancer Sue Creviston Kaste O3 Locoregional effects of breast cancer treatment Sarah J Vinnicombe O4 Imaging of cancer therapy-induced CNS toxicity Giovanni Morana, Andrea Rossi O5 Screening for lung cancer Christian J. Herold O6Risk stratification of lung nodules Theresa C. McLoud O7 PET imaging of pulmonary nodules Kirk A Frey O8 Transarterial tumour therapy Bernhard Gebauer O9 Interventional radiology in paediatric oncology Derek Roebuck O10 Image guided prostate interventions Jurgen J. Fütterer O11 Imaging cancer predisposition syndromes Alexander J. Towbin O12Chest and chest wall masses Thierry AG Huisman O13 Abdominal masses: good or bad? Anne MJB Smets O14 Hepatobiliary MR contrast: enhanced liver MRI for HCC diagnosis and management Giovanni Morana O15 Role of US elastography and multimodality fusion for managing patients with chronic liver disease and HCC Jeong Min Lee O16 Opportunities and challenges in imaging metastatic disease Hersh Chandarana O17 Diagnosis, treatment monitoring, and follow-up of lymphoma Marius E. Mayerhoefer, Markus Raderer, Alexander Haug O18 Managing high-risk and advanced prostate cancer Matthias Eiber O19 Immunotherapy: imaging challenges Bernhard Gebauer O20 RECIST and RECIST 1.1 Andrea Rockall O21 Challenges of RECIST in oncology imaging basics for the trainee and novice Aslam Sohaib O22 Lymphoma: PET for interim and end of treatment response assessment: a users’ guide to the Deauville Score Victoria S Warbey O23 Available resources Hebert Alberto Vargas O24 ICIS e-portal and the online learning community Dow-Mu Koh O25 Benign lesions that mimic pancreatic cancer Jay P Heiken O26 Staging and reporting pancreatic malignancies Isaac R Francis, Mahmoud, M Al-Hawary, Ravi K Kaza O27 Intraductal papillary mucinous neoplasm Giovanni Morana O28 Cystic pancreatic tumours Mirko D’Onofrio O29 Diffusion-weighted imaging of head and neck tumours Harriet C. Thoeny O30 Radiation injury in the head and neck Ann D King O31 PET/MR of paediatric brain tumours Giovanni Morana, Arnoldo Piccardo, Maria Luisa Garrè, Andrea Rossi O32 Structured reporting and beyond Hebert Alberto Vargas O33 Massachusetts General Hospital experience with structured reporting Theresa C. McLoud O34 The oncologist’s perspective: what the oncologist needs to know Nick Reed O35 Towards the cure of all children with cancer: global initiatives in pediatric oncology Carlos Rodriguez-Galindo O36 Multiparametric imaging of renal cancers Hersh Chandarana O37 Linking imaging features of renal disease and their impact on management strategies Hebert Alberto Vargas O38 Adrenals, retroperitoneum and peritoneum Isaac R Francis, Ashish P Wasnik O39 Lung and pleura Stefan Diederich O40 Advances in MRI Jurgen J. Fütterer O41 Advances in molecular imaging Wim J.G. Oyen O42 Incorporating advanced imaging, impact on treatment selection and patient outcome Cheng Lee Chaw, Nicholas van As S1 Combining ADC-histogram features improves performance of MR diffusion-weighted imaging for Lymph node characterisation in cervical cancer Igor Vieira, Frederik De Keyzer, Elleke Dresen, Sileny Han, Ignace Vergote, Philippe Moerman, Frederic Amant, Michel Koole, Vincent Vandecaveye S2 Whole-body diffusion-weighted MRI for surgical planning in patients with colorectal cancer and peritoneal metastases R Dresen, S De Vuysere, F De Keyzer, E Van Cutsem, A D’Hoore, A Wolthuis, V Vandecaveye S3 Role of apparent diffusion coefficient (ADC) diffusion-weighted MRI for predicting extra capsular extension of prostate cancer. P. Pricolo (paola.pricolo@ieo.it), S. Alessi, P. Summers, E. Tagliabue, G. Petralia S4 Generating evidence for clinical benefit of PET/CT – are management studies sufficient as surrogate for patient outcome? C. Pfannenberg, B. Gückel, SC Schüle, AC Müller, S. Kaufmann, N. Schwenzer, M. Reimold,C. la Fougere, K. Nikolaou, P. Martus S5 Heterogeneity of treatment response in skeletal metastases from breast cancer with 18F-fluoride and 18F-FDG PET GJ Cook, GK Azad, BP Taylor, M Siddique, J John, J Mansi, M Harries, V Goh S6 Accuracy of suspicious breast imaging—can we tell the patient? S Seth, R Burgul, A Seth S7 Measurement method of tumour volume changes during neoadjuvant chemotherapy affects ability to predict pathological response S Waugh, N Muhammad Gowdh, C Purdie, A Evans, E Crowe, A Thompson, S Vinnicombe S8 Diagnostic yield of CT IVU in haematuria screening F. Arfeen, T. Campion, E. Goldstraw S9 Percutaneous radiofrequency ablation of unresectable locally advanced pancreatic cancer: preliminary results D’Onofrio M, Ciaravino V, Crosara S, De Robertis R, Pozzi Mucelli R S10 Iodine maps from dual energy CT improve detection of metastases in staging examinations of melanoma patients M. Uhrig, D. Simons, H. Schlemmer S11Can contrast enhanced CT predict pelvic nodal status in malignant melanoma of the lower limb? Kate Downey S12 Current practice in the investigation for suspected Paraneoplastic Neurological Syndromes (PNS) and positive malignancy yield. S Murdoch, AS Al-adhami, S Viswanathan P1 Technical success and efficacy of Pulmonary Radiofrequency ablation: an analysis of 207 ablations S Smith, P Jennings, D Bowers, R Soomal P2 Lesion control and patient outcome: prospective analysis of radiofrequency abaltion in pulmonary colorectal cancer metastatic disease S Smith, P Jennings, D Bowers, R Soomal P3 Hepatocellular carcinoma in a post-TB patient: case of tropical infections and oncologic imaging challenges TM Mutala, AO Odhiambo, N Harish P4 Role of apparent diffusion coefficient (ADC) diffusion-weighted MRI for predicting extracapsular extension of prostate cancer P. Pricolo, S. Alessi, P. Summers, E. Tagliabue, G. Petralia P5 What a difference a decade makes; comparison of lung biopsies in Glasgow 2005 and 2015 M. Hall, M. Sproule, S. Sheridan P6 Solid pseudopapillary tumour of pancreas: imaging features of a rare neoplasm KY Thein, CH Tan, YL Thian, CM Ho P7 MDCT - pathological correlation in colon adenocarcinoma staging: preliminary experience S De Luca, C Carrera, V Blanchet, L Alarcón, E Eyheremnedy P8 Image guided biopsy of thoracic masses and reduction of pneumothorax risk: 25 years experience B K Choudhury, K Bujarbarua, G Barman P9 Tumour heterogeneity analysis of 18F-FDG-PET for characterisation of malignant peripheral nerve sheath tumours in neurofibromatosis-1 GJ Cook, E Lovat, M Siddique, V Goh, R Ferner, VS Warbey P10 Impact of introduction of vacuum assisted excision (VAE) on screen detected high risk breast lesions L Potti, B Kaye, A Beattie, K Dutton P11 Can we reduce prevalent recall rate in breast screening? AA Seth, F Constantinidis, H Dobson P12 How to reduce prevalent recall rate? Identifying mammographic lesions with low Positive Predictive Value (PPV) AA Seth (archana.seth@nhs.net), F Constantinidis, H Dobson P13 Behaviour of untreated pulmonary thrombus in oncology patients diagnosed with incidental pulmonary embolism on CT R. Bradley, G. Bozas, G. Avery, A. Stephens, A. Maraveyas P14 A one-stop lymphoma biopsy service – is it possible? S Bhuva, CA Johnson, M Subesinghe, N Taylor P15 Changes in the new TNM classification for lung cancer (8thedition, effective January 2017) LE Quint, RM Reddy, GP Kalemkerian P16 Cancer immunotherapy: a review of adequate imaging assessment G González Zapico, E Gainza Jauregui, R Álvarez Francisco, S Ibáñez Alonso, I Tavera Bahillo, L Múgica Álvarez P17 Succinate dehydrogenase mutations and their associated tumours O Francies, R Wheeler, L Childs, A Adams, A Sahdev P18 Initial experience in the usefulness of dual energy technique in the abdomen SE De Luca, ME Casalini Vañek, MD Pascuzzi, T Gillanders, PM Ramos, EP Eyheremendy P19 Recognising the serious complication of Richter’s transformation in CLL patients C Stove, M Digby P20 Body diffusion-weighted MRI in oncologic practice: truths, tricks and tips M. Nazar, M. Wirtz, MD. Pascuzzi, F. Troncoso, F. Saguier, EP. Eyheremendy P21 Methotrexate-induced leukoencephalopathy in paediatric ALL Patients D.J. Quint, L. Dang, M. Carlson, S. Leber, F. Silverstein P22 Pitfalls in oncology CT reporting. A pictorial review R Rueben, S Viswanathan P23 Imaging of perineural extension in head and neck tumours B Nazir, TH Teo, JB Khoo P24 MRI findings of molecular subtypes of breast cancer: a pictorial primer K Sharma, N Gupta, B Mathew, T Jeyakumar, K Harkins P25 When cancer can’t wait! A pictorial review of oncological emergencies K Sharma, B Mathew, N Gupta, T Jeyakumar, S Joshua P26 MRI of pancreatic neuroendocrine tumours: an approach to interpretation D Christodoulou, S Gourtsoyianni, A Jacques, N Griffin, V Goh P27 Gynaecological cancers in pregnancy: a review of imaging CA Johnson, J Lee P28 Suspected paraneoplastic neurological syndromes - review of published recommendations to date, with proposed guideline/flowchart JA Goodfellow, AS Al-adhami, S Viswanathan P29 Multi-parametric MRI of the pelvis for suspected local recurrence of prostate cancer after radical prostatectomy R Bradley P30 Utilisation of PI-RADS version 2 in multi-parametric MRI of the prostate; 12-months experience R Bradley P31 Radiological assessment of the post-chemotherapy liver A Yong, S Jenkins, G Joseph P32 Skeletal staging with MRI in breast cancer – what the radiologist needs to know S Bhuva, K Partington P33 Perineural spread of lympoma: an educational review of an unusual distribution of disease CA Johnson, S Bhuva, M Subesinghe, N Taylor P34 Visually isoattenuating pancreatic adenocarcinoma. Diagnostic imaging tools. C Carrera, A Zanfardini, S De Luca, L Alarcón, V Blanchet, EP Eyheremendy P35 Imaging of larynx cancer: when is CT, MRI or FDG PET/CT the best test? K Cavanagh, E Lau

We have previously shown that the d16HER2 splice variant is linked to HER2-positive breast cancer (BC) tumorigenesis, progression and response to Trastuzumab. However, the mechanisms by which d16HER2 contributes to HER2-driven aggressiveness and targeted therapy susceptibility remain uncertain. Here, we report that the d16HER2-positive mammary tumor cell lines MI6 and MI7, derived from spontaneous lesions of d16HER2 transgenic (tg) mice and resembling the aggressive features of primary lesions, are enriched in the expression of Wnt, Notch and epithelial–mesenchymal transition pathways related genes compared with full-length wild-type (WT) HER2-positive cells (WTHER2_1 and WTHER2_2) derived from spontaneous tumors arising in WTHER2 tg mice. MI6 cells exhibited increased resistance to anoikis and significantly higher mammosphere-forming efficiency (MFE) and self-renewal capability than the WTHER2-positive counterpart. Furthermore, d16HER2-positive tumor cells expressed a higher fraction of CD29High/CD24+/SCA1Lowcells and displayed greater in vivo tumor engraftment in serial dilution conditions than WTHER2_1 cells. Accordingly, NOTCH inhibitors impaired mammosphere formation only in MI6 cells. A comparative analysis of stemness-related features driven by d16HER2 and WTHER2 in ad hoc engineered human BC cells (MCF7 and T47D) revealed a higher MFE and aldehyde dehydrogenase-positive staining in d16HER2- vs WTHER2-infected cells, sustaining consistent BC-initiating cell enrichment in the human setting. Moreover, marked CD44 expression was found in MCF7_d16 and T47D_d16 cells vs their WTHER2 and Mock counterparts. Clinically, BC cases from two distinct HER2-positive cohorts characterized by high levels of expression of the activated-d16HER2 metagene were significantly enriched in the Notch family and signal transducer genes vs those with low levels of the metagene.

O1 The metabolomics approach to autism: identification of biomarkers for early detection of autism spectrum disorder A. K. Srivastava, Y. Wang, R. Huang, C. Skinner, T. Thompson, L. Pollard, T. Wood, F. Luo, R. Stevenson O2 Phenome-wide association study for smoking- and drinking-associated genes in 26,394 American women with African, Asian, European, and Hispanic descents R. Polimanti, J. Gelernter O3 Effects of prenatal environment, genotype and DNA methylation on birth weight and subsequent postnatal outcomes: findings from GUSTO, an Asian birth cohort X. Lin, I. Y. Lim, Y. Wu, A. L. Teh, L. Chen, I. M. Aris, S. E. Soh, M. T. Tint, J. L. MacIsaac, F. Yap, K. Kwek, S. M. Saw, M. S. Kobor, M. J. Meaney, K. M. Godfrey, Y. S. Chong, J. D. Holbrook, Y. S. Lee, P. D. Gluckman, N. Karnani, GUSTO study group O4 High-throughput identification of specific qt interval modulating enhancers at the SCN5A locus A. Kapoor, D. Lee, A. Chakravarti O5 Identification of extracellular matrix components inducing cancer cell migration in the supernatant of cultivated mesenchymal stem cells C. Maercker, F. Graf, M. Boutros O6 Single cell allele specific expression (ASE) IN T21 and common trisomies: a novel approach to understand DOWN syndrome and other aneuploidies G. Stamoulis, F. Santoni, P. Makrythanasis, A. Letourneau, M. Guipponi, N. Panousis, M. Garieri, P. Ribaux, E. Falconnet, C. Borel, S. E. Antonarakis O7 Role of microRNA in LCL to IPSC reprogramming S. Kumar, J. Curran, J. Blangero O8 Multiple enhancer variants disrupt gene regulatory network in Hirschsprung disease S. Chatterjee, A. Kapoor, J. Akiyama, D. Auer, C. Berrios, L. Pennacchio, A. Chakravarti O9 Metabolomic profiling for the diagnosis of neurometabolic disorders T. R. Donti, G. Cappuccio, M. Miller, P. Atwal, A. Kennedy, A. Cardon, C. Bacino, L. Emrick, J. Hertecant, F. Baumer, B. Porter, M. Bainbridge, P. Bonnen, B. Graham, R. Sutton, Q. Sun, S. Elsea O10 A novel causal methylation network approach to Alzheimer’s disease Z. Hu, P. Wang, Y. Zhu, J. Zhao, M. Xiong, David A Bennett O11 A microRNA signature identifies subtypes of triple-negative breast cancer and reveals MIR-342-3P as regulator of a lactate metabolic pathway A. Hidalgo-Miranda, S. Romero-Cordoba, S. Rodriguez-Cuevas, R. Rebollar-Vega, E. Tagliabue, M. Iorio, E. D’Ippolito, S. Baroni O12 Transcriptome analysis identifies genes, enhancer RNAs and repetitive elements that are recurrently deregulated across multiple cancer types B. Kaczkowski, Y. Tanaka, H. Kawaji, A. Sandelin, R. Andersson, M. Itoh, T. Lassmann, the FANTOM5 consortium, Y. Hayashizaki, P. Carninci, A. R. R. Forrest O13 Elevated mutation and widespread loss of constraint at regulatory and architectural binding sites across 11 tumour types C. A. Semple O14 Exome sequencing provides evidence of pathogenicity for genes implicated in colorectal cancer E. A. Rosenthal, B. Shirts, L. Amendola, C. Gallego, M. Horike-Pyne, A. Burt, P. Robertson, P. Beyers, C. Nefcy, D. Veenstra, F. Hisama, R. Bennett, M. Dorschner, D. Nickerson, J. Smith, K. Patterson, D. Crosslin, R. Nassir, N. Zubair, T. Harrison, U. Peters, G. Jarvik, NHLBI GO Exome Sequencing Project O15 The tandem duplicator phenotype as a distinct genomic configuration in cancer F. Menghi, K. Inaki, X. Woo, P. Kumar, K. Grzeda, A. Malhotra, H. Kim, D. Ucar, P. Shreckengast, K. Karuturi, J. Keck, J. Chuang, E. T. Liu O16 Modeling genetic interactions associated with molecular subtypes of breast cancer B. Ji, A. Tyler, G. Ananda, G. Carter O17 Recurrent somatic mutation in the MYC associated factor X in brain tumors H. Nikbakht, M. Montagne, M. Zeinieh, A. Harutyunyan, M. Mcconechy, N. Jabado, P. Lavigne, J. Majewski O18 Predictive biomarkers to metastatic pancreatic cancer treatment J. B. Goldstein, M. Overman, G. Varadhachary, R. Shroff, R. Wolff, M. Javle, A. Futreal, D. Fogelman O19 DDIT4 gene expression as a prognostic marker in several malignant tumors L. Bravo, W. Fajardo, H. Gomez, C. Castaneda, C. Rolfo, J. A. Pinto O20 Spatial organization of the genome and genomic alterations in human cancers K. C. Akdemir, L. Chin, A. Futreal, ICGC PCAWG Structural Alterations Group O21 Landscape of targeted therapies in solid tumors S. Patterson, C. Statz, S. Mockus O22 Genomic analysis reveals novel drivers and progression pathways in skin basal cell carcinoma S. N. Nikolaev, X. I. Bonilla, L. Parmentier, B. King, F. Bezrukov, G. Kaya, V. Zoete, V. Seplyarskiy, H. Sharpe, T. McKee, A. Letourneau, P. Ribaux, K. Popadin, N. Basset-Seguin, R. Ben Chaabene, F. Santoni, M. Andrianova, M. Guipponi, M. Garieri, C. Verdan, K. Grosdemange, O. Sumara, M. Eilers, I. Aifantis, O. Michielin, F. de Sauvage, S. Antonarakis O23 Identification of differential biomarkers of hepatocellular carcinoma and cholangiocarcinoma via transcriptome microarray meta-analysis S. Likhitrattanapisal O24 Clinical validity and actionability of multigene tests for hereditary cancers in a large multi-center study S. Lincoln, A. Kurian, A. Desmond, S. Yang, Y. Kobayashi, J. Ford, L. Ellisen O25 Correlation with tumor ploidy status is essential for correct determination of genome-wide copy number changes by SNP array T. L. Peters, K. R. Alvarez, E. F. Hollingsworth, D. H. Lopez-Terrada O26 Nanochannel based next-generation mapping for interrogation of clinically relevant structural variation A. Hastie, Z. Dzakula, A. W. Pang, E. T. Lam, T. Anantharaman, M. Saghbini, H. Cao, BioNano Genomics O27 Mutation spectrum in a pulmonary arterial hypertension (PAH) cohort and identification of associated truncating mutations in TBX4 C. Gonzaga-Jauregui, L. Ma, A. King, E. Berman Rosenzweig, U. Krishnan, J. G. Reid, J. D. Overton, F. Dewey, W. K. Chung O28 NORTH CAROLINA macular dystrophy (MCDR1): mutations found affecting PRDM13 K. Small, A. DeLuca, F. Cremers, R. A. Lewis, V. Puech, B. Bakall, R. Silva-Garcia, K. Rohrschneider, M. Leys, F. S. Shaya, E. Stone O29 PhenoDB and genematcher, solving unsolved whole exome sequencing data N. L. Sobreira, F. Schiettecatte, H. Ling, E. Pugh, D. Witmer, K. Hetrick, P. Zhang, K. Doheny, D. Valle, A. Hamosh O30 Baylor-Johns Hopkins Center for Mendelian genomics: a four year review S. N. Jhangiani, Z. Coban Akdemir, M. N. Bainbridge, W. Charng, W. Wiszniewski, T. Gambin, E. Karaca, Y. Bayram, M. K. Eldomery, J. Posey, H. Doddapaneni, J. Hu, V. R. Sutton, D. M. Muzny, E. A. Boerwinkle, D. Valle, J. R. Lupski, R. A. Gibbs O31 Using read overlap assembly to accurately identify structural genetic differences in an ashkenazi jewish trio S. Shekar, W. Salerno, A. English, A. Mangubat, J. Bruestle O32 Legal interoperability: a sine qua non for international data sharing A. Thorogood, B. M. Knoppers, Global Alliance for Genomics and Health - Regulatory and Ethics Working Group O33 High throughput screening platform of competent sineups: that can enhance translation activities of therapeutic target H. Takahashi, K. R. Nitta, A. Kozhuharova, A. M. Suzuki, H. Sharma, D. Cotella, C. Santoro, S. Zucchelli, S. Gustincich, P. Carninci O34 The undiagnosed diseases network international (UDNI): clinical and laboratory research to meet patient needs J. J. Mulvihill, G. Baynam, W. Gahl, S. C. Groft, K. Kosaki, P. Lasko, B. Melegh, D. Taruscio O36 Performance of computational algorithms in pathogenicity predictions for activating variants in oncogenes versus loss of function mutations in tumor suppressor genes R. Ghosh, S. Plon O37 Identification and electronic health record incorporation of clinically actionable pharmacogenomic variants using prospective targeted sequencing S. Scherer, X. Qin, R. Sanghvi, K. Walker, T. Chiang, D. Muzny, L. Wang, J. Black, E. Boerwinkle, R. Weinshilboum, R. Gibbs O38 Melanoma reprogramming state correlates with response to CTLA-4 blockade in metastatic melanoma T. Karpinets, T. Calderone, K. Wani, X. Yu, C. Creasy, C. Haymaker, M. Forget, V. Nanda, J. Roszik, J. Wargo, L. Haydu, X. Song, A. Lazar, J. Gershenwald, M. Davies, C. Bernatchez, J. Zhang, A. Futreal, S. Woodman O39 Data-driven refinement of complex disease classification from integration of heterogeneous functional genomics data in GeneWeaver E. J. Chesler, T. Reynolds, J. A. Bubier, C. Phillips, M. A. Langston, E. J. Baker O40 A general statistic framework for genome-based disease risk prediction M. Xiong, L. Ma, N. Lin, C. Amos O41 Integrative large-scale causal network analysis of imaging and genomic data and its application in schizophrenia studies N. Lin, P. Wang, Y. Zhu, J. Zhao, V. Calhoun, M. Xiong O42 Big data and NGS data analysis: the cloud to the rescue O. Dobretsberger, M. Egger, F. Leimgruber O43 Cpipe: a convergent clinical exome pipeline specialised for targeted sequencing S. Sadedin, A. Oshlack, Melbourne Genomics Health Alliance O44 A Bayesian classification of biomedical images using feature extraction from deep neural networks implemented on lung cancer data V. A. A. Antonio, N. Ono, Clark Kendrick C. Go O45 MAV-SEQ: an interactive platform for the Management, Analysis, and Visualization of sequence data Z. Ahmed, M. Bolisetty, S. Zeeshan, E. Anguiano, D. Ucar O47 Allele specific enhancer in EPAS1 intronic regions may contribute to high altitude adaptation of Tibetans C. Zeng, J. Shao O48 Nanochannel based next-generation mapping for structural variation detection and comparison in trios and populations H. Cao, A. Hastie, A. W. Pang, E. T. Lam, T. Liang, K. Pham, M. Saghbini, Z. Dzakula O49 Archaic introgression in indigenous populations of Malaysia revealed by whole genome sequencing Y. Chee-Wei, L. Dongsheng, W. Lai-Ping, D. Lian, R. O. Twee Hee, Y. Yunus, F. Aghakhanian, S. S. Mokhtar, C. V. Lok-Yung, J. Bhak, M. Phipps, X. Shuhua, T. Yik-Ying, V. Kumar, H. Boon-Peng O50 Breast and ovarian cancer prevention: is it time for population-based mutation screening of high risk genes? I. Campbell, M.-A. Young, P. James, Lifepool O53 Comprehensive coverage from low DNA input using novel NGS library preparation methods for WGS and WGBS C. Schumacher, S. Sandhu, T. Harkins, V. Makarov O54 Methods for large scale construction of robust PCR-free libraries for sequencing on Illumina HiSeqX platform H. DoddapaneniR. Glenn, Z. Momin, B. Dilrukshi, H. Chao, Q. Meng, B. Gudenkauf, R. Kshitij, J. Jayaseelan, C. Nessner, S. Lee, K. Blankenberg, L. Lewis, J. Hu, Y. Han, H. Dinh, S. Jireh, K. Walker, E. Boerwinkle, D. Muzny, R. Gibbs O55 Rapid capture methods for clinical sequencing J. Hu, K. Walker, C. Buhay, X. Liu, Q. Wang, R. Sanghvi, H. Doddapaneni, Y. Ding, N. Veeraraghavan, Y. Yang, E. Boerwinkle, A. L. Beaudet, C. M. Eng, D. M. Muzny, R. A. Gibbs O56 A diploid personal human genome model for better genomes from diverse sequence data K. C. C. Worley, Y. Liu, D. S. T. Hughes, S. C. Murali, R. A. Harris, A. C. English, X. Qin, O. A. Hampton, P. Larsen, C. Beck, Y. Han, M. Wang, H. Doddapaneni, C. L. Kovar, W. J. Salerno, A. Yoder, S. Richards, J. Rogers, J. R. Lupski, D. M. Muzny, R. A. Gibbs O57 Development of PacBio long range capture for detection of pathogenic structural variants Q. Meng, M. Bainbridge, M. Wang, H. Doddapaneni, Y. Han, D. Muzny, R. Gibbs O58 Rhesus macaques exhibit more non-synonymous variation but greater impact of purifying selection than humans R. A. Harris, M. Raveenedran, C. Xue, M. Dahdouli, L. Cox, G. Fan, B. Ferguson, J. Hovarth, Z. Johnson, S. Kanthaswamy, M. Kubisch, M. Platt, D. Smith, E. Vallender, R. Wiseman, X. Liu, J. Below, D. Muzny, R. Gibbs, F. Yu, J. Rogers O59 Assessing RNA structure disruption induced by single-nucleotide variation J. Lin, Y. Zhang, Z. Ouyang P1 A meta-analysis of genome-wide association studies of mitochondrial dna copy number A. Moore, Z. Wang, J. Hofmann, M. Purdue, R. Stolzenberg-Solomon, S. Weinstein, D. Albanes, C.-S. Liu, W.-L. Cheng, T.-T. Lin, Q. Lan, N. Rothman, S. Berndt P2 Missense polymorphic genetic combinations underlying down syndrome susceptibility E. S. Chen P4 The evaluation of alteration of ELAM-1 expression in the endometriosis patients H. Bahrami, A. Khoshzaban, S. Heidari Keshal P5 Obesity and the incidence of apolipoprotein E polymorphisms in an assorted population from Saudi Arabia population K. K. R. Alharbi P6 Genome-associated personalized antithrombotical therapy for patients with high risk of thrombosis and bleeding M. Zhalbinova, A. Akilzhanova, S. Rakhimova, M. Bekbosynova, S. Myrzakhmetova P7 Frequency of Xmn1 polymorphism among sickle cell carrier cases in UAE population M. Matar P8 Differentiating inflammatory bowel diseases by using genomic data: dimension of the problem and network organization N. Mili, R. Molinari, Y. Ma, S. Guerrier P9 Vulnerability of genetic variants to the risk of autism among Saudi children N. Elhawary, M. Tayeb, N. Bogari, N. Qotb P10 Chromatin profiles from ex vivo purified dopaminergic neurons establish a promising model to support studies of neurological function and dysfunction S. A. McClymont, P. W. Hook, L. A. Goff, A. McCallion P11 Utilization of a sensitized chemical mutagenesis screen to identify genetic modifiers of retinal dysplasia in homozygous Nr2e3rd7mice Y. Kong, J. R. Charette, W. L. Hicks, J. K. Naggert, L. Zhao, P. M. Nishina P12 Ion torrent next generation sequencing of recessive polycystic kidney disease in Saudi patients B. M. Edrees, M. Athar, F. A. Al-Allaf, M. M. Taher, W. Khan, A. Bouazzaoui, N. A. Harbi, R. Safar, H. Al-Edressi, A. Anazi, N. Altayeb, M. A. Ahmed, K. Alansary, Z. Abduljaleel P13 Digital expression profiling of Purkinje neurons and dendrites in different subcellular compartments A. Kratz, P. Beguin, S. Poulain, M. Kaneko, C. Takahiko, A. Matsunaga, S. Kato, A. M. Suzuki, N. Bertin, T. Lassmann, R. Vigot, P. Carninci, C. Plessy, T. Launey P14 The evolution of imperfection and imperfection of evolution: the functional and functionless fractions of the human genome D. Graur P16 Species-independent identification of known and novel recurrent genomic entities in multiple cancer patients J. Friis-Nielsen, J. M. Izarzugaza, S. Brunak P18 Discovery of active gene modules which are densely conserved across multiple cancer types reveal their prognostic power and mutually exclusive mutation patterns B. S. Soibam P19 Whole exome sequencing of dysplastic leukoplakia tissue indicates sequential accumulation of somatic mutations from oral precancer to cancer D. Das, N. Biswas, S. Das, S. Sarkar, A. Maitra, C. Panda, P. Majumder P21 Epigenetic mechanisms of carcinogensis by hereditary breast cancer genes J. J. Gruber, N. Jaeger, M. Snyder P22 RNA direct: a novel RNA enrichment strategy applied to transcripts associated with solid tumors K. Patel, S. Bowman, T. Davis, D. Kraushaar, A. Emerman, S. Russello, N. Henig, C. Hendrickson P23 RNA sequencing identifies gene mutations for neuroblastoma K. Zhang P24 Participation of SFRP1 in the modulation of TMPRSS2-ERG fusion gene in prostate cancer cell lines M. Rodriguez-Dorantes, C. D. Cruz-Hernandez, C. D. P. Garcia-Tobilla, S. Solorzano-Rosales P25 Targeted Methylation Sequencing of Prostate Cancer N. Jäger, J. Chen, R. Haile, M. Hitchins, J. D. Brooks, M. Snyder P26 Mutant TPMT alleles in children with acute lymphoblastic leukemia from México City and Yucatán, Mexico S. Jiménez-Morales, M. Ramírez, J. Nuñez, V. Bekker, Y. Leal, E. Jiménez, A. Medina, A. Hidalgo, J. Mejía P28 Genetic modifiers of Alström syndrome J. Naggert, G. B. Collin, K. DeMauro, R. Hanusek, P. M. Nishina P31 Association of genomic variants with the occurrence of angiotensin-converting-enzyme inhibitor (ACEI)-induced coughing among Filipinos E. M. Cutiongco De La Paz, R. Sy, J. Nevado, P. Reganit, L. Santos, J. D. Magno, F. E. Punzalan , D. Ona , E. Llanes, R. L. Santos-Cortes , R. Tiongco, J. Aherrera, L. Abrahan, P. Pagauitan-Alan; Philippine Cardiogenomics Study Group P32 The use of “humanized” mouse models to validate disease association of a de novo GARS variant and to test a novel gene therapy strategy for Charcot-Marie-Tooth disease type 2D K. H. Morelli, J. S. Domire, N. Pyne, S. Harper, R. Burgess P34 Molecular regulation of chondrogenic human induced pluripotent stem cells M. A. Gari, A. Dallol, H. Alsehli, A. Gari, M. Gari, A. Abuzenadah P35 Molecular profiling of hematologic malignancies: implementation of a variant assessment algorithm for next generation sequencing data analysis and clinical reporting M. Thomas, M. Sukhai, S. Garg, M. Misyura, T. Zhang, A. Schuh, T. Stockley, S. Kamel-Reid P36 Accessing genomic evidence for clinical variants at NCBI S. Sherry, C. Xiao, D. Slotta, K. Rodarmer, M. Feolo, M. Kimelman, G. Godynskiy, C. O’Sullivan, E. Yaschenko P37 NGS-SWIFT: a cloud-based variant analysis framework using control-accessed sequencing data from DBGAP/SRA C. Xiao, E. Yaschenko, S. Sherry P38 Computational assessment of drug induced hepatotoxicity through gene expression profiling C. Rangel-Escareño, H. Rueda-Zarate P40 Flowr: robust and efficient pipelines using a simple language-agnostic approach;ultraseq; fast modular pipeline for somatic variation calling using flowr S. Seth, S. Amin, X. Song, X. Mao, H. Sun, R. G. Verhaak, A. Futreal, J. Zhang P41 Applying “Big data” technologies to the rapid analysis of heterogenous large cohort data S. J. Whiite, T. Chiang, A. English, J. Farek, Z. Kahn, W. Salerno, N. Veeraraghavan, E. Boerwinkle, R. Gibbs P42 FANTOM5 web resource for the large-scale genome-wide transcription start site activity profiles of wide-range of mammalian cells T. Kasukawa, M. Lizio, J. Harshbarger, S. Hisashi, J. Severin, A. Imad, S. Sahin, T. C. Freeman, K. Baillie, A. Sandelin, P. Carninci, A. R. R. Forrest, H. Kawaji, The FANTOM Consortium P43 Rapid and scalable typing of structural variants for disease cohorts W. Salerno, A. English, S. N. Shekar, A. Mangubat, J. Bruestle, E. Boerwinkle, R. A. Gibbs P44 Polymorphism of glutathione S-transferases and sulphotransferases genes in an Arab population A. H. Salem, M. Ali, A. Ibrahim, M. Ibrahim P46 Genetic divergence of CYP3A5*3 pharmacogenomic marker for native and admixed Mexican populations J. C. Fernandez-Lopez, V. Bonifaz-Peña, C. Rangel-Escareño, A. Hidalgo-Miranda, A. V. Contreras P47 Whole exome sequence meta-analysis of 13 white blood cell, red blood cell, and platelet traits L. Polfus, CHARGE and NHLBI Exome Sequence Project Working Groups P48 Association of adipoq gene with type 2 diabetes and related phenotypes in african american men and women: The jackson heart study S. Davis, R. Xu, S. Gebeab, P Riestra, A Gaye, R. Khan, J. Wilson, A. Bidulescu P49 Common variants in casr gene are associated with serum calcium levels in koreans S. H. Jung, N. Vinayagamoorthy, S. H. Yim, Y. J. Chung P50 Inference of multiple-wave population admixture by modeling decay of linkage disequilibrium with multiple exponential functions Y. Zhou, S. Xu P51 A Bayesian framework for generalized linear mixed models in genome-wide association studies X. Wang, V. Philip, G. Carter P52 Targeted sequencing approach for the identification of the genetic causes of hereditary hearing impairment A. A. Abuzenadah, M. Gari, R. Turki, A. Dallol P53 Identification of enhancer sequences by ATAC-seq open chromatin profiling A. Uyar, A. Kaygun, S. Zaman, E. Marquez, J. George, D. Ucar P54 Direct enrichment for the rapid preparation of targeted NGS libraries C. L. Hendrickson, A. Emerman, D. Kraushaar, S. Bowman, N. Henig, T. Davis, S. Russello, K. Patel P56 Performance of the Agilent D5000 and High Sensitivity D5000 ScreenTape assays for the Agilent 4200 Tapestation System R. Nitsche, L. Prieto-Lafuente P57 ClinVar: a multi-source archive for variant interpretation M. Landrum, J. Lee, W. Rubinstein, D. Maglott P59 Association of functional variants and protein physical interactions of human MUTY homolog linked with familial adenomatous polyposis and colorectal cancer syndrome Z. Abduljaleel, W. Khan, F. A. Al-Allaf, M. Athar , M. M. Taher, N. Shahzad P60 Modification of the microbiom constitution in the gut using chicken IgY antibodies resulted in a reduction of acute graft-versus-host disease after experimental bone marrow transplantation A. Bouazzaoui, E. Huber, A. Dan, F. A. Al-Allaf, W. Herr, G. Sprotte, J. Köstler, A. Hiergeist, A. Gessner, R. Andreesen, E. Holler P61 Compound heterozygous mutation in the LDLRgene in Saudi patients suffering severe hypercholesterolemia F. Al-Allaf, A. Alashwal, Z. Abduljaleel, M. Taher, A. Bouazzaoui, H. Abalkhail, A. Al-Allaf, R. Bamardadh, M. Athar

A splice variant of the human gene HER2, lacking exon-16 (ΔHER2) which encodes a small extracellular region, has been described. This altered receptor forms disulfide bond-stabilized homodimers. We report here that the ΔHER2 splice variant represents about 9% of the HER2 mRNA obtained from most of the 46 breast carcinoma samples with HER2 expression levels ranging from 3+ to 0 by HercepTest. Analysis of human cells transfected with ΔHER2 or wild-type (WT) cDNA revealed no growth of WT cells in nude mice, whereas clones expressing 10-fold less ΔHER2 were tumorigenic. Unlike WT transfectants, ΔHER2-expressing cells showed low sensitivity to two new therapeutic drugs targeting receptors of the HER family (ZD1839 and Trastuzumab), whereas an inhibitor of the HER2 tyrosine kinase domain (Emodin) blocked activation of both ΔHER2 and WT transfectants. Taken together, our findings indicate that the ΔHER2 transcript encodes the transforming form of the oncoprotein. It is plausible that malignant transformation arises when a critical threshold of ΔHER2 is reached in HER2-overexpressing tumors. Specific inhibitors of HER2 catalytic activity represent a promising approach to therapy of HER2-overexpressing tumors.

A splice variant of the human gene HER2, lacking exon-16 (ΔHER2) which encodes a small extracellular region, has been described. This altered receptor forms disulfide bond-stabilized homodimers. We report here that the ΔHER2 splice variant represents about 9% of the HER2 mRNA obtained from most of the 46 breast carcinoma samples with HER2 expression levels ranging from 3+ to 0 by HercepTest. Analysis of human cells transfected with ΔHER2 or wild-type (WT) cDNA revealed no growth of WT cells in nude mice, whereas clones expressing 10-fold less ΔHER2 were tumorigenic. Unlike WT transfectants, ΔHER2-expressing cells showed low sensitivity to two new therapeutic drugs targeting receptors of the HER family (ZD1839 and Trastuzumab), whereas an inhibitor of the HER2 tyrosine kinase domain (Emodin) blocked activation of both ΔHER2 and WT transfectants. Taken together, our findings indicate that the ΔHER2 transcript encodes the transforming form of the oncoprotein. It is plausible that malignant transformation arises when a critical threshold of ΔHER2 is reached in HER2-overexpressing tumors. Specific inhibitors of HER2 catalytic activity represent a promising approach to therapy of HER2-overexpressing tumors.

The clinical interest in HER-2/neu is related to trastuzumab, a drug used to treat patients with invasive breast carcinoma overexpressing the HER-2/neu protein. It is very important to correctly identify those patients who may benefit from trastuzumab by accurate assessment of the HER-2/neu status. Of the various methods available, the Dako Herceptest for immunohistochemical assay is considered the most reliable to reach this goal. The aim of this study was to investigate within a group of Italian laboratories the reproducibility of the results of HER-2/neu assessment by means of the Dako scoring system on slides stained with the Herceptest kit. This study was also conceived as the continuation of one of our previous studies, which was similar in its aims but different in the classification criteria adopted. Our results show that, whereas the intra-observer reproducibility was generally satisfactory, the interobserver reproducibility was not. Moreover, our findings confirm that the two extreme classes (0 and 3+) are more easy to identify than the other two and that the Herceptest does not allow to discriminate optimally between scoring classes 2+ and 3+. These findings are relevant in clinical practice where the treatment choice is based on categories defined by this assay, suggesting the need of adopting educational programs and/or new reference materials to improve the assay performance.

The association between expression of the 67 kDa laminin receptor (67LR) and tumor aggressiveness has been convincingly demonstrated although the exact function of this molecule in the metastatic process has remained unclear. In this study, we tested whether the laminin-1, upon interaction with 67LR, promotes tumor cell aggressiveness; the investigation was based on: (i) the previous demonstration that soluble 67LR, as well as a 20-amino-acid peptide corresponding to the 67LR laminin binding site, changes the conformation of laminin upon interaction with this adhesion molecule and (ii) the known relevance of microenvironment remodeling by the tumor, leading to structural modification of extracellular matrix components in tumor progression. MDAMB231 breast carcinoma cells plated on peptide G-treated laminin-1 exhibited a polygonal array of actin filament bundles compared with cells seeded on native laminin-1 which presented the actin bundles organized as multiple cables parallel to margins. Furthermore, in cells seeded on peptide G-treated laminin-1, 67LR was distinct from the α6 integrin subunit in filopodia protrusions in addition to colocalizing with this integrin in focal adhesion plaques as it occurs when cells are plated on native laminin-1. In addition to differences in tumor cell adhesion and migration found in cells exposed to peptide G-treated vs native laminin-1, breast carcinoma cells seeded on modified laminin-1 showed a 6-fold increase in invasion capability compared with cells seeded on unmodified laminin-1. Alterations in actin organization as well as adhesion, migration and especially invasion observed in MDAMB231 cells in the presence of peptide G-treated laminin-1 were even found in MDAMB231 cells that, after selection for 67LR high expression, were seeded on native laminin-1. As the 67LR shedding is proportional to its expression level, these findings indicate a role for 67LR in changing laminin structure.Expression analysis of 97 genes encoding proteins that mediate cell matrix interactions, revealed significant differences between cells exposed to modified vs unmodified laminin-1 in 19 genes, 17 of which — including those encoding α3 integrin, extracellular matrix protein 1, proteolytic enzymes (such as MT1-MMP, stromelysin-3 and cathepsin L) and their inhibitors — were up-modulated in cells treated with modified laminin-1. Zymogram analysis clearly indicated a significant increase in the activity of the gelatinolytic enzyme MMP-2 in the culture supernatant from cells exposed to modified laminin-1, without an increase in mRNA abundance as observed in microarray analysis. Invasiveness of tumor cells conditioned by modified laminin-1, evaluated as the capability to cross Matrigel basement, was significantly more inhibited by MMPinhibitor TIMP-2 than invasiveness induced by native laminin-1. Taken together, our findings indicate that the role of 67LR in tumor aggressiveness rests in its ability to modify laminin-1 thereby activating proteolytic enzymes that promote tumor cell invasion through extracellular matrix degradation.

Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system capable of initiating immune responses to antigens. It is also well documented that cancer patients often experience anergy against tumor antigens. In this study we selected the best protocol for inducing the production of antibodies against the HER2 oncoprotein using DCs to overcome anergy. Murine DCs were pulsed in vitro, using different protocols, with recombinant HER2 fused to a human Fc (in order to improve DC antigen uptake) and were used to vaccinate mice. The obtained results indicate that antigen-pulsed DCs can induce an antibody response and that adding CpG after antigen pulsing greatly increases anti-HER2 antibody production.

In spite of the large number of studies on technical problems affecting the interlaboratory reproducibility of IHC HER-2/neu determination, only little is known about factors limiting the intra- and interobserver reproducibility in the actual practice of HER-2/neu expression analysis. The aim of the present INQAT study was to evaluate the intra- and interobserver reproducibility of IHC HER-2 analysis among pathologists routinely working in Italian laboratories. Twenty immunostained slides were distributed to 12 pathologists who had to report, for each slide, the semiquantitative analysis of the percentage of immunopositive cells and the qualitative evaluation of the intensity of membrane staining. The intra- and interobserver reproducibility as well as the reproducibility between each laboratory and the reference values were quantified adopting an approach based on computation of the weighted kappa statistic (Kw). Additionally, in order to evaluate the contribution of each category to the overall agreement, the kappa category-specific statistics (Kcs) were estimated for both classification criteria by jointly considering all the participating laboratories. The intraobserver analyses showed a satisfactory level of reproducibility for both the percentage of positive cells (median Kw, 0.94; range: 0.80–0.96) and membrane staining (median Kw, 0.86; range: 0.78–0.96). Similarly, a fairly good level of reproducibility for the percentage of cells (median Kw, 0.89; range, 0.73–0.96) and the intensity of membrane staining (median Kw, 0.84; range, 0.72–0.92) were observed from comparisons with reference values. When all possible pairwise comparisons were performed, a satisfactory level of interobserver reproducibility was found for most laboratories. Kwvalues varied between 0.51 and 0.98 (median Kw, 0.80) and between 0.61 and 0.94 (median Kw, 0.81) for semiquantitative and qualitative measurements, respectively. Analysis of the contribution of the extreme categories to the overall agreement showed a substantial or almost perfect agreement for both classification criteria. Conversely, the contribution of intermediate categories appeared to be scarce or slight for the percentage of immunostained cells and slight or fair for the intensity of membrane staining. We conclude that, overall, the interobserver reproducibility in IHC analysis of HER-2/neu expression is satisfactory, although classification of the intermediate categories is problematic, both with regard to the percentage of immunostained cells and the intensity of membrane staining.

In this study we investigated the immunochemical and cytochemical reactivity of two monoclonal antibodies against the 16-amino acid tandem repeat of MUC4 to demonstrate a possible variation of the mucin core peptide expression related to lung cancer. The immunocytochemical anti-MUC4 reactivity was analyzed in four lung cancer cell lines (Calu-1, Calu-3, H460, SKMES) and in other tumor cell lines, as well as in frozen materials from 21 lung adenocarcinomas (ACs), including five bronchioloalveolar carcinomas (BACs), and 11 squamous cell lung carcinomas (SqCCs). A weak fluorescence anti-MUC4 positivity (range: 10.3–16.2) was observed only in acetone-fixed lung cancer cell lines Calu-1, Calu-3 and H460. These three lung cancer cell lines also showed a cytoplasmic immunoperoxidase reactivity. The immunostaining in lung cancer tissues showed a granular cytoplasmic reactivity: 15/21 (71%) and 17/21 (80%) ACs were positive with BC-LuC18.2 and BC-LuCF12, respectively. All BACs were positive. Moderate to strong reactivity was present in well-differentiated ACs. In the normal lung parenchyma counterparts weak reactivity was found only in bronchiolar cells. All SqCCs were negative. Anti-MUC4 reactivity was also observed in the alveolar mucus. In conclusion, our anti-MUC4 MAbs detect a secretion product present in mucus and this product is elaborated by lung cancer cells and overexpressed in well-differentiated lung ACs.

Nerve growth factor (NGF) is known to exert a mitogenic effect on human breast cancer cells through proto-TrkA activation. Reverse transcriptase-PCR analysis of proto-TrkA expression in human breast carcinoma specimens and cell lines revealed trkA transcript in 12 of 14 human breast carcinoma specimens and in all of four cell lines tested. While cytofluorimetric and Western blot analysis indicated proto-TrkA expression in three of the four cell lines, NGF stimulated growth in only two of the three positive cell lines. Inhibition of NGF-induced MAPK activation by an antibody directed against the extracellular domain of TrkA but not by an inhibitor of TrkA phosphorylation demonstrated the requirement of NGF binding but not of proto-TrkA kinase activity for MAPK activation, suggesting the recruitment of another kinase for transmission of the mitogenic signaling. Indeed, NGF induced tyrosine phosphorylation and stimulated kinase activity of p185(HER2), a kinase receptor of the HER family. A TrkA phosphorylation inhibitor did not affect this activation. Moreover, the two receptors were coprecipitated by antibodies directed against proto-TrkA and p185(HER2). Down-modulation of p185(HER2) expression in a breast carcinoma line transfected with a construct containing an anti-p185(HER2) antibody sequence and expressing proto-TrkA impaired NGF-induced MAPK activation and proliferation. Together these data show that in cells expressing low levels of TrkA such as breast carcinoma cells, NGF must recruit other overexpressed receptors such as p185(HER2) in order to generate a biological signal that can induce breast cancer cell growth.

Inside the thymus, thymic epithelial cells and thymocytes show an interdependent relationship for their functional differentiation and development. As regards possible interdependency for their mutual survival, it is clear that lympho-epithelial adhesion can control the survival of developing thymocytes whereas the effects of lymphoid adhesion on epithelial cell survival have never been described. To address this issue, we performed co-cultures between normal human thymic epithelial cells (TEC) and a mature lymphoid T cell line (H9) or unfractionated thymocytes. TEC were induced to apoptosis by growth factor deprivation and the level of cell death was measured by flow cytometry. TEC stimulated by cell adhesion showed a significant reduced apoptosis when compared to the control and this phenomenon was associated with increased binding activity of NF-(kappa)B, as measured by gel shift analysis. The activation of NF-(kappa)B was necessary to promote survival, since its inhibition by acetyl salicylic acid prevented the promoting effect. The mAb-mediated crosslinking of (alpha)(3)(beta)(1) was considered as a potential inducer of TEC survival, since we have previously demonstrated that the engagement of this integrin was able to induce NF-(kappa)B activation in TEC. The crosslinking of (alpha)(3)(beta)(1), which clustered at the lympho-epithelial contact sites, partially reproduced the promoting activity of cell adhesion. These results highlight that lympho-epithelial adhesion can control the survival of thymic epithelial cells through an intracellular pathway which requires the activation of NF-(kappa)B and is triggered by integrins of the (beta)(1) family.

The high-affinity nerve growth factor receptor corresponds to the tyrosine protein kinase encoded by the proto-oncogene trkA. Different findings suggest that nerve growth factor (NGF) can be operative in the growth modulation of tumor cell lines possessing high-affinity binding sites for this molecule. Using as immunizing material the SKNBE neuroblastoma cell line transfected with proto-trkA we produced a monoclonal antibody (MAb) able to recognize the high-affinity nerve growth factor receptor. The selected MAb, designated MGR12, is directed against an epitope present on the extracellular domain of the receptor since it showed reactivity on living trkA-expressing cells and was able to immunoprecipitate the proto-trkA molecule. The MGR12 MAb is directed against a non-functional epitope since it neither inhibited NGF binding nor induced receptor internalization. This new reagent appears to be an appropriate tool for analyzing the expression of high-affinity nerve growth factor receptor in tumors of different origin and for elucidating its involvement in tumor progression.

c-erbB-2 is an oncoprotein which is overexpressed in up to 40% of primary breast cancers. c-erbB-2 overexpression is a bad prognostic factor in patients with lymph node-positive disease. Unfortunately, there has been no agreement to date on whether c-erbB-2 overexpression is of prognostic significance in patients with lymph node-negative disease. c-erbB-2 overexpression is correlated with the absence of estrogen receptor expression in a number of publications. Correlation between c-erbB-2 overexpression and hormone sensitivity in the clinical setting is less well established and is the focus of ongoing studies. Both preclinical and clinical studies support an association between c-erbB-2 receptor overexpression and resistance to alkylating agents. In contrast, the data for c-erbB-2 and anthracyclines should be viewed in a slightly different manner. Anthracyclines appear to have a greater therapeutic effect in c-erbB-2-positive disease which may be dose sensitive. In c-erbB-2-negative disease not only is the therapeutic effect reduced but there does not appear to be any improved response to higher doses of anthracyclines. The data for c-erbB-2 and the taxanes is still not clear enough to provide any definite conclusions. If there is a correlation it would at present appear to be between paclitaxel and response rates, but this needs to be confirmed in larger studies. Few studies have looked at changes in c-erbB-2 on therapy. Those that have seem to show no significant change on either tamoxifen or chemotherapy.

By immunizing a mouse with human metastatic breast tumor cells from patient effusions and infiltrated lymph nodes, a monoclonal antibody (MLuC2), which identifies a new carcinoma-associated marker, was raised. The reactivity of this reagent was studied by immunohistochemistry on live and fixed cells from tumor cell lines and on frozen sections from surgical specimens. Besides reacting with 73% of breast carcinomas, MLuC2 also reacted with 93% of non-small cell lung carcinoma (NSCLC) and with a few normal tissues. The MLuC2-recognized molecule (CaMLuC2), whose MW was 90 KDa according to immunoblotting experiments, was found to be detectable in the serum and could therefore be of particular interest for serological diagnostic applications. Since the CaMLuC2 epitope was not polyexpressed on the bearing molecule, we produced a new generation of MAbs in order to define epitopes coexpressed with CaMLuC2 on the same 90 KDa molecule, and which are therefore suitable to develop a double-determinant immunoradiometric assay (DDIRMA) for the detection of this marker in the sera of lung carcinoma patients. Different analyses by immunohistochemistry, binding inhibition tests and DDIRMA, proved that the two new reagents developed, MLuC8 and MLuC9, recognize the same or closely related epitopes, which are however different from CaMLuC2, but which are all present on the same molecule. Preliminary immunoradiometric tests performed on sera from lung cancer and control patients showed a good specificity but a low sensitivity. In fact, only 42% of the 28 tested sera samples from NSCLC patients scored positive despite the fact that more than 90% of the NSCLC expressed the relevant antigen

The 67-kDa laminin receptor (67LR) is an important tumor marker whose molecular structure has not yet been fully elucidated. To shed new light on this molecule, we raised a series of eight new monoclonal antibodies, designated MPLR1 to 8, directed against the 37-kDa recombinant laminin receptor precursor (37LRP). Cross-competition experiments demonstrated that the epitopes recognized by MPLR2, 4 and 5 partially overlap, since MPLR4 and 5 compete with labelled MPLR2 for the binding to recombinant 37LRP. These three antibodies belong to the IgG1 class, whereas the other ones are all IgM. Presumably due to the fact that they are directed against partially unfolded antigenic determinants expressed on the recombinant protein, MPLRs did not recognize the native protein. Indeed, they showed no reactivity at the membrane level in cytofluorimetric analysis and they did not work in immunoprecipitation experiments. In contrast, these reagents are valuable tools in immunoblotting, since they clearly identify a 67-kDa protein (the mature laminin receptor) in addition to the 37-kDa precursor form. MPLRs are thus a new powerful tool which could help in the characterization of the still enigmatic 67LR molecule.

Two monoclonal antibodies (mAbs), designated MLuC5 and MLuC6, were produced against a human small cell lung carcinoma cell line. They were found to exhibit a superimposable reactivity on different cell lines and on platelets. Moreover, they both immunoprecipitated a 67 kDa molecule from the membrane of the reference target cells. Immunodepletion and cross-inhibition tests indicated that the two mAbs recognize two epitopes closely localized on the same molecule. The MLuC5 mAb was further characterized for its reactivity on platelets. Immunoprecipitation and ELISA assays demonstrate that this mAb recognizes the 67 kDa high afinity laminin receptor. MLuC5 reactivity was evaluated by immunohistochemistry on a variety of normal and tumor tissues, in particular breast specimens including normal epithelium, dysplastic lesions, in situcarcinomas, invasive primary carcinomas and distant metastases. The laminin receptor was found to be strongly expressed in 50% of the infiltrating carcinomas, whereas in situcarcinomas and benign lesions, as well as the normal mammary epithelium, were only weakly and focally positive. In metastatic lesions MLuC5 reactivity was only found in 11% of the samples tested, independently of the site of origin of the lesion.

We investigated the effect of peptide G, a synthetic peptide derived from the sequence of the 37-kDa laminin receptor precursor, on the interaction of laminin in two tumor cell lines one of which produces laminin and one of which does not. Addition of peptide G to the culture medium induced a significant increase in the amount of endogenous laminin detectable on the cell membrane of both cell lines. Moreover, pretreatment of exogenous laminin with peptide G dramatically increased laminin binding on both cell lines. Kinetics analysis of membrane-bound labeled laminin revealed a 3-fold decrease in the kd of peptide G-treated laminin compared with untreated or unrelated or scrambled peptide-treated laminin. Moreover, the affinity constant of peptide G-treated laminin increased 2-fold, with a doubling of the number of laminin binding sites, as determined by Scatchard analysis. Expression of the VLA6 integrin receptor on the cell membrane increased after incubation with peptide G-treated laminin. However, the lower binding inhibition of peptide G-treated laminin after anti-VLA6 antibody or cation chelation treatment indicates that membrane molecules in addition to integrin receptors are involved in the recognition of peptide G-modified laminin. These "new" laminin-binding proteins also mediated cell adhesion to laminin, the first step in tumor invasion. Together, the data suggest that peptide G increases and stabilizes laminin binding on tumor cells, involving surface receptors that normally do not take part in this interaction. This might explain the abundant clinical and experimental data suggesting a key role for the 67-kDa laminin receptor in the interaction between cancer cells and the basement membrane glycoprotein laminin during tumor invasion and metastasis.

Abstract.: Lung cancer belongs to the group of malignant lesions that specifically select bone as secondary implantation site. The molecular bases for this property, defined as osteotropism, is still largely unknown. The recent demonstration that human breast cancer cells express and attach to bone sialoprotein (BSP), a sulfated phosphoprotein rich in bone and other mineralized tissues, could provide a clue to elucidating bone metastases formation. BSP contains the integrin binding peptide Arg-Gly-Asp (RGD), as well as non-RGD cell attachment domain. Using an immunoperoxidase technique and a specific polyclonal antibody directed against a BSP synthetic peptide, we examined the expression of BSP in 48 lung lesions including 25 squamous carcinoma, 21 adenocarcinoma, and 2 bronchioloalveolar cancers, as well as 38 human ovarian carcinoma that constitute a group of generally nonosteotropic cancers. BSP was not specifically detected in normal lung tissue with the exception of cartilage associated with bronchi. Most of the adenocarcinoma (74%) and all squamous carcinoma of the lung examined exhibited detectable levels of BSP. Staining was mainly cytoplasmic and membrane associated. The two bronchioloalveolar lung cancers examined did not show detectable amounts of BSP. When microcalcifications were observed in pulmonary malignant lesions, they were usually associated with cancer cells expressing BSP. Only 21% of the ovarian cancers examined contained malignant cells with 2+ or 3+ positivity for BSP. We further demonstrated that in 8 of 10 additional lung cancers, BSP was detected at the mRNA level. Our observation is the first demonstration that BSP is expressed in non-small cell lung carcinoma. Lung cancer cells are now the second type of osteotropic malignant cells described to express BSP. Added to the observation that BSP expression is not frequent in ovarian carcinoma, a low osteotropic cancer, our study supports our hypothesis that BSP could play a role in determining the affinity of cancer cells to bone.

Three MAbs, MLuC2, MLuC8 and MLuC9, directed against a molecule that is produced and secreted by carcinoma cells were studied with the aim of developing a double-determinant immunoradiometric assay (DDIRMA). We demonstrated by means of immunoblotting, immunodepletion and DDIRMA techniques, that MLuC9 reacted against the CEA molecule, whereas MLuC2 and MLuC8 reacted against a 90 Kd molecule related to CEA. The DDIRMA performed with the anti-CEA as a catcher MAb and the anti-90 Kd as a tracer MAb was found to be positive with the HT29 soluble extract, which suggests the existence of CEA/90 Kd dimeric molecules. The same reactivity was found when sera from patients with lung carcinomas were tested, which excludes that this molecule could be an artefact due to the cell solubilization procedures. The association between CEA and the 90 Kd molecule was further confirmed by immunodepletion experiments in which the immunoprecipitation with one MAb not only removed the recognized molecule, but also partially immunodepleted the material from the other.

The interactions between tumor cells and laminin or other components of the extracellular matrix have been shown to play an important role in tumor invasion and metastasis. However, the role of the monomeric 67-kDa laminin receptor (67LR) remains unclear. We analyzed the regulation of 67LR expression under different culture conditions with respect to the expression of other well characterized laminin receptors. In A431 cells treated with laminin for different time periods, the regulation of 67LR expression correlated with expression of the alpha6 integrin subunit but not with the expression of other laminin receptors. Moreover, cytokine treatment resulted in down-modulated expression of the alpha6 integrin subunit and the 67LR. Co-regulation of the expression of the two receptors was further suggested by the observation that specific down-modulation of the alpha6-chain by antisense oligonucleotides was accompanied by a proportional decrease in the cell surface expression of 67LR. Biochemical analyses indicated co-immunoprecipitation of 67LR and the alpha6 subunit with an anti-alpha6 but not an anti-beta1 monoclonal antibody. Co-regulation of 67LR and alpha6 subunit expression, together with the physical association between the two receptors, supports the hypothesis that 67LR is an auxiliary molecule involved in regulating or stabilizing the interaction of laminin with the alpha6beta4 integrin.

The interaction between laminin and the oncoprotein encoded by the c-erbB-2 oncogene was studied in vitro and in vivo in human breast carcinomas. In vitro analysis of breast carcinoma cell lines overexpressing p185HER2 revealed that laminin, but not fibronectin, induced tyrosine phosphorylation and down-modulation of oncoprotein membrane expression. Laminin also specifically inhibited growth of p185HER2-positive cell lines. No direct binding between the recombinant extracellular domain of p185HER2 and laminin was found. Induction of oncoprotein down-modulation by anti-integrin antibodies and coprecipitation of the oncoprotein with the beta 4 integrin subunit indicate that the interaction between p185HER2 and laminin occurs through integrin molecules. The relevance of this in vitro observation was verified in vivo by analysing the prognostic value of p185HER2 overexpression as a function of laminin production on archival paraffin-embedded sections of 887 primary breast tumours. The results revealed an association between p185HER2 overexpression and unfavourable prognosis in tumours negative for laminin production, whereas in laminin-producing tumours, the oncoprotein overexpression was not associated with tumour aggressiveness.

Methods such as immunohistochemistry that have enhanced the detection of carcinoma cells in bone marrow aspirates appear to be useful in identifying patients with aggressive tumours. To detect epithelial cells in bone marrow aspirates from breast carcinoma patients, we used a pool of five different monoclonal antibodies (MAbs), which recognise 100% of breast carcinomas, together with the alkaline phosphatase method on cytospun cells obtained from sternum and iliac crest. Primary tumours were also analysed for the expression of the c-erbB-1 and c-erbB-2 oncogene products, and of two differentiation-related markers and laminin receptors. Immunoreactive cells were detected in the bone marrow of 62 of the 197 patients tested (31%) without any correlation with clinical parameters such as tumour size or lymph node metastasis, whereas a significant (P < 0.01) correlation was found with enhanced monomeric laminin receptor expression in the primary tumour. In fact, this receptor was expressed in respectively 63% and 38% of primary tumours from patients with and without immunoreactive cells in the bone marrow aspirates. Thus, the presence of immunoreactive cells in bone marrow correlates with the expression in the primary tumour of a marker of the metastatic potential of the tumour, the 67 kDa laminin receptor.

Analysis of the fate of the p185HER2oncoprotein following activation by heregulin β1 revealed the induction of the tyrosine‐phosphorylation, down‐modulation, and polyubiquitination of p185HER2. Receptor ubiquitination was suppressed in cells treated with heregulin β1 in the presence of sodium azide, an inhibitor of ATP‐dependent reactions, or genistein, a tyrosine kinase protein inhibitor, indicating the requirement for kinase activity and ATP in p185HER2polyubiquitination. Ubiquitinated p185HER2was degradated by the 26S proteasome proteolytic pathway. Kinetics and inhibition experiments indicated that endocytosis of the receptor occurs downstream of the initiation of the degradation process.

Analysis of the fate of the p185 HER2oncoprotein following activation by heregulin β1 revealed the induction of the tyrosine-phosphorylation, down-modulation, and polyubiquitination of p185 HER2. Receptor ubiquitination was suppressed in cells treated with heregulin β1 in the presence of sodium azide, an inhibitor of ATP-dependent reactions, or genistein, a tyrosine kinase protein inhibitor, indicating the requirement for kinase activity and ATP in p185 HER2polyubiquitination. Ubiquitinated p185 HER2was degradated by the 26S proteasome proteolytic pathway. Kinetics and inhibition experiments indicated that endocytosis of the receptor occurs downstream of the initiation of the degradation process.