Predicting locally advanced rectal cancer response to neoadjuvant therapy with 18F-FDG PET and MRI radiomics features.

PURPOSE: Pathological complete response (pCR) following neoadjuvant chemoradiotherapy or radiotherapy in locally advanced rectal cancer (LARC) is reached in approximately 15-30% of cases, therefore it would be useful to assess if pretreatment of 18F-FDG PET/CT and/or MRI texture features can reliably predict response to neoadjuvant therapy in LARC. METHODS: Fifty-two patients were dichotomized as responder (pR+) or non-responder (pR-) according to their pathological tumor regression grade (TRG) as follows: 22 as pR+ (nine with TRG = 1, 13 with TRG = 2) and 30 as pR- (16 with TRG = 3, 13 with TRG = 4 and 1 with TRG = 5). First-order parameters and 21 second-order texture parameters derived from the Gray-Level Co-Occurrence matrix were extracted from semi-automatically segmented tumors on T2w MRI, ADC maps, and PET/CT acquisitions. The role of each texture feature in predicting pR+ was assessed with monoparametric and multiparametric models. RESULTS: In the mono-parametric approach, PET homogeneity reached the maximum AUC (0.77; sensitivity = 72.7% and specificity = 76.7%), while PET glycolytic volume and ADC dissimilarity reached the highest sensitivity (both 90.9%). In the multiparametric analysis, a logistic regression model containing six second-order texture features (five from PET and one from T2w MRI) yields the highest predictivity in distinguish between pR+ and pR- patients (AUC = 0.86; sensitivity = 86%, and specificity = 83% at the Youden index). CONCLUSIONS: If preliminary results of this study are confirmed, pretreatment PET and MRI could be useful to personalize patient treatment, e.g., avoiding toxicity of neoadjuvant therapy in patients predicted pR-.

Long-term efficacy of microbiology-driven periodontal laser-assisted therapy.

Periodontitis represents a highly prevalent health problem, causing severe functional impairment, reduced quality of life and increased risk of systemic disorders, including respiratory, cardiovascular and osteoarticular diseases, diabetes and fertility problems. It is a typical example of a multifactorial disease, where a polymicrobial infection inducing chronic inflammation of periodontal tissues is favoured by environmental factors, life style and genetic background. Since periodontal pathogens can colonise poorly vascularised niches, antiseptics and antibiotics are typically associated with local treatments to manage the defects, with unstable outcomes especially in early-onset cases. Here, the results of a retrospective study are reported, evaluating the efficacy of a protocol (Periodontal Biological Laser-Assisted Therapy, Perioblast™) by which microbial profiling of periodontal pockets is used to determine the extent and duration of local neodymium-doped yttrium aluminium garnet (Nd:YAG) laser irradiation plus conventional treatment. The protocol was applied multicentrically on 2683 patients, and found to produce a significant and enduring improvement of all clinical and bacteriological parameters, even in aggressive cases. Microbiome sequencing of selected pockets revealed major population shifts after treatment, as well as strains potentially associated with periodontitis in the absence of known pathogens. This study, conducted for the first time on such a large series, clearly demonstrates long-term efficacy of microbiology-driven non-invasive treatment of periodontal disease.

Pathological non-response to chemotherapy in a neoadjuvant setting of breast cancer: an inter-institutional study.

To identify markers of non-response to neoadjuvant chemotherapy (NAC) that could be used in the adjuvant setting. Sixteen pathologists of the European Working Group for Breast Screening Pathology reviewed the core biopsies of breast cancers treated with NAC and recorded the clinico-pathological findings (histological type and grade; estrogen, progesterone receptors, and HER2 status; Ki67; mitotic count; tumor-infiltrating lymphocytes; necrosis) and data regarding the pathological response in corresponding surgical resection specimens. Analyses were carried out in a cohort of 490 cases by comparing the groups of patients showing pathological complete response (pCR) and partial response (pPR) with the group of non-responders (pathological non-response: pNR). Among other parameters, the lobular histotype and the absence of inflammation were significantly more common in pNR (p < 0.001). By ROC curve analyses, cut-off values of 9 mitosis/2 mm(2) and 18% of Ki67-positive cells best discriminated the pNR and pCR + pPR categories (p = 0.018 and < 0.001, respectively). By multivariable analysis, only the cut-off value of 9 mitosis discriminated the different response categories (p = 0.036) in the entire cohort. In the Luminal B/HER2- subgroup, a mitotic count <9, although not statistically significant, showed an OR of 2.7 of pNR. A lobular histotype and the absence of inflammation were independent predictors of pNR (p = 0.024 and <0.001, respectively). Classical morphological parameters, such as lobular histotype and inflammation, confirmed their predictive value in response to NAC, particularly in the Luminal B/HER2- subgroup, which is a challenging breast cancer subtype from a therapeutic point of view. Mitotic count could represent an additional marker but has a poor positive predictive value.

Electrical conditioning of adipose-derived stem cells in a multi-chamber culture platform.

In tissue engineering, several factors play key roles in providing adequate stimuli for cells differentiation, in particular biochemical and physical stimuli, which try to mimic the physiological microenvironments. Since electrical stimuli are important in the developing heart, we have developed an easy-to-use, cost-effective cell culture platform, able to provide controlled electrical stimulation aimed at investigating the influence of the electric field in the stem cell differentiation process. This bioreactor consists of an electrical stimulator and 12 independent, petri-like culture chambers and a 3-D computational model was used to characterize the distribution and the intensity of the electric field generated in the cell culture volume. We explored the effects of monophasic and biphasic square wave pulse stimulation on a mouse adipose-derived stem cell line (m17.ASC) comparing cell viability, proliferation, protein, and gene expression. Both monophasic (8 V, 2 ms, 1 Hz) and biphasic (+4 V, 1 ms and -4 V, 1 ms; 1 Hz) stimulation were compatible with cell survival and proliferation. Biphasic stimulation induced the expression of Connexin 43, which was found to localize also at the cell membrane, which is its recognized functional mediating intercellular electrical coupling. Electrically stimulated cells showed an induced transcriptional profile more closely related to that of neonatal cadiomyocytes, particularly for biphasic stimulation. The developed platform thus allowed to set-up precise conditions to drive adult stem cells toward a myocardial phenotype solely by physical stimuli, in the absence of exogenously added expensive bioactive molecules, and can thus represent a valuable tool for translational applications for heart tissue engineering and regeneration.

Primary breast cancer stem-like cells metastasise to bone, switch phenotype and acquire a bone tropism signature.

BACKGROUND: Bone metastases represent a common and severe complication in breast cancer, and the involvement of cancer stem cells (CSCs) in the promotion of bone metastasis is currently under discussion. Here, we used a human-in-mice model to study bone metastasis formation due to primary breast CSCs-like colonisation. METHODS: Primary CD44⁺CD24⁻ breast CSCs-like were transduced by a luciferase-lentiviral vector and injected through subcutaneous and intracardiac (IC) routes in non-obese/severe-combined immunodeficient (NOD/SCID) mice carrying subcutaneous human bone implants. The CSCs-like localisation was monitored by in vivo luciferase imaging. Bone metastatic CSCs-like were analysed through immunohistochemistry and flow cytometry, and gene expression analyses were performed by microarray techniques. RESULTS: Breast CSCs-like colonised the human-implanted bone, resulting in bone remodelling. Bone metastatic lesions were histologically apparent by tumour cell expression of epithelial markers and vimentin. The bone-isolated CSCs-like were CD44⁻CD24⁺ and showed tumorigenic abilities after injection in secondary mice. CD44⁻CD24⁺ CSCs-like displayed a distinct bone tropism signature that was enriched in genes that discriminate bone metastases of breast cancer from metastases at other organs. CONCLUSION: Breast CSCs-like promote bone metastasis and display a CSCs-like bone tropism signature. This signature has clinical prognostic relevance, because it efficiently discriminates osteotropic breast cancers from tumour metastases at other sites.

A gene trap vector system for identifying transcriptionally responsive genes.

We present a method for fast and efficient trapping of genes whose transcription is regulated by exogenous stimuli. We constructed a promoterless retroviral vector transducing a green fluorescent protein-nitroreductase (GFNR) fusion protein downstream from a splice acceptor site. Flow cytometric analysis of the infected population allows identification and sorting of cells in which the trap is integrated downstream from an active promoter. Conversely, the nitroreductase (NTR) moiety allows pharmacological selection against constitutive GFNR expression. Using hepatocyte growth factor (HGF) stimulation of liver cells combined with either positive or negative selection, we recovered cell populations carrying traps in induced or suppressed genes, respectively. Several distinct responsive clones were isolated, and regulated expression of the trapped gene was confirmed at the RNA level. Positive and negative selection can be calibrated to recover traps in genes showing different levels of basal expression or transcriptional regulation. The flexibility and efficiency of the GFNR-based trap screening procedure make it suitable for wide surveys of transcriptionally regulated genes.

The tyrosine kinase receptors Ron and Sea control "scattering" and morphogenesis of liver progenitor cells in vitro.

The mammalian RON and the avian sea genes encode tyrosine kinase receptors of poorly characterized biological functions. We recently identified macrophage-stimulating protein as the ligand for Ron; no ligand has yet been found for Sea. In this work we investigated the biological response to macrophage-stimulating protein in mouse liver progenitor cells expressing Ron. These cells were also transfected with a chimeric cDNA encoding the cytoplasmic domain of Sea, fused to the extracellular domain of Trk (nerve growth factor receptor). In the presence of nanomolar concentrations of the respective ligands, both receptors induced cell "scattering", extracellular matrix invasion, and DNA synthesis. When liver progenitor cells were grown in a tri-dimensional type-I collagen matrix, ligand-induced stimulation of either Ron or Sea induced sprouting of branched cell cords, evolving into ductular-like tubules. The motogenic, mitogenic, and morphogenic responses were also elicited by triggering the structurally related hepatocyte growth factor receptor (Met) but not epidermal growth factor or platelet-derived growth factor receptors. These data show that Ron, Sea, and Met belong to a receptor subfamily that elicits a distinctive biological response in epithelial cells.

Biochemical and immunological properties of the human carcinoma-associated CAR-3 epitope defined by the monoclonal antibody AR-3.

The monoclonal antibody AR-3 reacts with an epitope (CAR-3) carried on a high-molecular-weight glycoprotein associated with carcinomas of the pancreas, stomach, colon, uterus, and ovary. This study reports the partial purification and characterization of CAR-3-bearing molecule. The antigen was quantified by a double determinant immunoradiometric assay. CAR-3 antigen was purified by a three-step procedure, consisting of perchloric acid extraction, molecular sieving on Sepharose CL-4B, and affinity chromatography on AR-3 antibodies coupled to Sepharose 4B. Following this procedure CAR-3 antigen was purified about 400-fold with a 36% yield. Treatment of the CAR-3 antigen with 16 mM metaperiodate or with 1 N NaOH resulted in complete loss of activity. Antigenicity survived enzymatic treatments known to destroy proteins. The epitope was found to be carried on a molecule with a molecular weight of greater than 400,000 with a density of 1.45 g/ml, metabolically labeled with [35S]sulfate, [3H]glucosamine, and [35S]methionine. It is concluded that CAR-3 epitope is expressed on a carbohydrate moiety linked to a sulfo-mucin-like molecule via an O-glycosidic bond. Cross-competition experiments showed that CAR-3 epitope is strictly related or in close topographic proximity to Lewis(a) and Lewis(b) antigens. Cross-double determinant immunoradiometric assay experiments indicated that the same mucin carrying CAR-3 bears also CA 19-9, CA 125, and BW 494 epitopes.

Osteopontin is an autocrine mediator of hepatocyte growth factor-induced invasive growth.

In epithelial cells, hepatocyte growth factor (HGF) activates a genetic program involving cell-cell dissociation ("scattering"), growth and invasiveness. The full program is not elicited by other growth factors like epidermal growth factor, and is aberrantly activated during cancer progression to the invasive-metastatic phenotype. To identify genes involved in the onset of invasive growth, we explored by cDNA microarrays the in vitro transcriptional response to HGF of mouse embryo liver cells. We identified osteopontin (OPN), a secreted matrix protein, as a major HGF transcriptional target. The wave of OPN induction is maximal at 6 h, in concomitance with the initiation of scattering, and is specific, because no other matrix protein among those explored by the microarray is affected. Interestingly, HGF, but not epidermal growth factor, promotes cell adhesion to OPN via the CD44 receptor. Scattering is significantly impaired by antibodies against OPN and CD44; conversely, constitutive OPN overexpression dramatically increases the motile and invasive responses to HGF, leading to disruption of the ordered morphogenetic program triggered by this ligand.

Structural and functional domains critical for constitutive activation of the HGF-receptor (Met).

The MET gene, encoding the tyrosine kinase receptor for Hepatocyte Growth Factor, is a potentially harmful oncogene overexpressed in a significant fraction of human cancers. To study the molecular mechanisms responsible for oncogenic activation, the biochemical and biological properties of a number of MET constructs were analysed. The native heterodimeric receptor (alpha beta), the beta chain alone, as well as a kinase defective mutant did not transform rodent fibroblasts upon transfection. The cytoplasmic domain, truncated immediately below the transmembrane region, acquired constitutive tyrosine kinase activity in vivo, produced foci of transformation, and was tumorigenic in nude mice. Removal of the first 39 amino acids of the juxtamembrane domain resulted in loss of constitutive activation in vivo and transforming potential, without impairment of the in vitro kinase activity. Replacement of the juxtamembrane domain with 5' TPR sequences restored constitutive kinase activation and transforming properties. Site-directed mutagenesis of either of the two tyrosine residues involved in the positive regulation of the catalytic activity upon phosphorylation (Y1234 or Y1235 in the kinase domain of the HGF receptor), strongly impaired TRP-MET transforming potential. These data show that: (1) the truncated cytoplasmic HGF receptor has constitutive kinase activity and is oncogenic; (2) the first 39 amino acids of the juxtamembrane domain and (3) the regulatory tyrosines in the catalytic domain are required to unleash its transforming potential.

Expression of the Met/HGF receptor in normal and neoplastic human tissues.

The MET oncogene encodes a transmembrane tyrosine kinase receptor. Recently, hepatocyte growth factor (HGF), a potent growth factor for hepatocytes involved in liver regeneration, has been proposed as a ligand. In this paper, the physiological role of the human Met/HGF receptor is investigated by studying its specific distribution in normal and neoplastic tissues. Northern blot analysis has shown that the MET gene is selectively expressed in several epithelial tissues. High levels of MET mRNA have been found in liver, gastrointestinal tract, thyroid and kidney. Western blot analysis has shown that the levels of the Met protein generally correspond to those of the mRNA. However, in the thyroid, where there is a high level of MET mRNA, the protein was barely detectable, suggesting translational or post-translational regulation. The protein was also detected in the brain. Normal or increased levels of MET mRNA and Met protein were consistently found in fresh samples of carcinomas as well as in epithelial tumor cell lines. In thyroid carcinomas of a specific histiotype the amount of Met protein, almost undetectable in the normal counterpart, was found to be increased more than 100-fold. The tissue distribution of the Met/HGF receptor indicates that this molecule is involved in growth control of epithelial cells other than hepatocytes and suggests that its increased expression may confer a growth advantage to neoplastic cells.

The tyrosine kinase receptor met and its ligand HGF are co-expressed and functionally active in HHV-8 positive primary effusion lymphoma.

Primary effusion lymphoma (PEL) harbors consistent infection by human herpesvirus-8, preferentially develops in immunodeficient patients and selectively localizes to the serous body cavities. Histogenetic analysis has suggested that PEL originates from post-germinal center, pre-terminally differentiated B cells sharing phenotypic features with plasma cells. Here we have investigated the expression status and functional integrity of the Met tyrosine kinase receptor and of its ligand hepatocyte growth factor (HGF). Thirteen PEL (nine cell lines and four primary specimens) were analyzed for Met and HGF expression and function by multiple assays. For comparison, a panel of 34 high grade B cell non-Hodgkin lymphomas (NHL) other than PEL was also investigated. Co-expression of Met and HGF was found in all PEL analyzed, whereas it was restricted to 1/34 B cell NHL other than PEL (P < 0.001; chi2 test). The Met protein expressed by PEL displays biochemical characteristics typical of Met expressed by other cell types and is capable of tyrosine autophosphorylation. By using a combination of immunological and biological assays, production and secretion of a functional HGF species was identified in all PEL cell lines analyzed. HGF stimulation of PEL cells rapidly induces Met tyrosine phosphorylation, demonstrating the functional integrity of the Met/HGF loop. Because of the well known mitogenic and motogenic properties of Met/HGF interactions, these data may bear implications for PEL growth and dissemination. Among B cell neoplasms, Met/HGF co-expression selectively clusters with PEL and, as demonstrated by previous studies, with multiple myeloma plasma cells, thus reinforcing the notion that PEL displays biologic similarities with tumors derived from late stages of B cell differentiation.

The Healthgrid White Paper.

Over the last four years, a community of researchers working on Grid and High Performance Computing technologies started discussing the barriers and opportunities that grid technologies must face and exploit for the development of health-related applications. This interest lead to the first Healthgrid conference, held in Lyon, France, on January 16th-17th, 2003, with the focus of creating increased awareness about the possibilities and advantages linked to the deployment of grid technologies in health, ultimately targeting the creation of a European/international grid infrastructure for health. The topics of this conference converged with the position of the eHealth division of the European Commission, whose mandate from the Lisbon Meeting was "To develop an intelligent environment that enables ubiquitous management of citizens' health status, and to assist health professionals in coping with some major challenges, risk management and the integration into clinical practice of advances in health knowledge." In this context "Health" involves not only clinical procedures but covers the whole range of information from molecular level (genetic and proteomic information) over cells and tissues, to the individual and finally the population level (social healthcare). Grid technology offers the opportunity to create a common working backbone for all different members of this large "health family" and will hopefully lead to an increased awareness and interoperability among disciplines. The first HealthGrid conference led to the creation of the Healthgrid association, a non-profit research association legally incorporated in France but formed from the broad community of European researchers and institutions sharing expertise in health grids. After the second Healthgrid conference, held in Clermont-Ferrand on January 29th-30th, 2004, the need for a "white paper" on the current status and prospective of health grids was raised. Over fifty experts from different areas of grid technologies, eHealth applications and the medical world were invited to contribute to the preparation of this document.

The monoclonal antibody-defined CAR-3 antigen is a serological marker associated with pancreatic carcinoma.

The monoclonal antibody-defined CAR-3 antigen is a new carcinoma associated marker which is expressed on a mucin-like molecule. Serum concentrations of CAR-3 were assayed in 181 patients with carcinomas of different organs, 20 patients with non-carcinomatous malignancies, 123 patients with inflammatory diseases and 150 healthy controls. Serum levels of CAR-3 were significantly increased in 51% of the patients with pancreatic carcinomas, in 60% of patients with biliary tract carcinomas and in about 15% of the patients with carcinomas of the digestive apparatus. Sera from patients with breast carcinomas were negative, as well as sera from patients with melanomas or sarcomas. CAR-3 values in samples from patients with chronic pancreatitis were constantly negative, as were samples from healthy donors. Significant concentrations of CAR-3 were detected in 20% of the sera from patients with acute pancreatitis and in 15% of the sera from patients with cirrhosis. Because of its high specificity for pancreatic carcinomas compared to chronic pancreatitis, CAR-3 seems a promising marker for distinguishing between neoplastic and chronic inflammatory diseases of the pancreas, whose differential diagnosis is difficult.

The EGFR family members sustain the neoplastic phenotype of ALK+ lung adenocarcinoma via EGR1.

In non-small cell lung cancer (NSCLC), receptor tyrosine kinases (RTKs) stand out among causal dominant oncogenes, and the ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nonetheless, long-term RTK inhibition leads invariably to acquired resistance, tumor recurrence and metastatic dissemination. In ALK+ cell lines, inhibition of ALK signaling was associated with coactivation of several RTKs, whose pharmacological suppression reverted the partial resistance to ALK blockade. Remarkably, ERBB2 signaling synergized with ALK and contributed to the neoplastic phenotype. Moreover, the engagement of wild-type epidermal growth factor receptor or MET receptors could sustain cell viability through early growth response 1 (EGR1) and/or Erk1/2; Akt activation and EGR1 overexpression prevented cell death induced by combined ALK/RTK inhibition. Membrane expression of ERBB2 in a subset of primary naive ALK+ NSCLC could be relevant in the clinical arena. Our data demonstrate that the neoplastic phenotype of ALK-driven NSCLC relays 'ab initio' on the concomitant activation of multiple RTK signals via autocrine/paracrine regulatory loops. These findings suggest that molecular and functional signatures are required in de novo lung cancer patients for the design of efficacious and multi-targeted 'patient-specific' therapies.

The GAB2 signaling scaffold promotes anchorage independence and drives a transcriptional response associated with metastatic progression of breast cancer.

Acquisition of independence from anchorage to the extracellular matrix is a critical event for onset and progression of solid cancers. To identify and characterize new genes conferring anchorage independence, we transduced MCF10A human normal breast cells with a retroviral cDNA expression library and selected them by growth in suspension. Microarray analysis targeted on library-derived transcripts revealed robust and reproducible enrichment, after selection, of cDNAs encoding the scaffolding adaptor Gab2. Gab2 was confirmed to strongly promote anchorage-independent growth when overexpressed. Interestingly, downregulation by RNA interference of endogenous Gab2 in neoplastic cells did not affect their adherent growth, but abrogated their growth in soft agar. Gab2-driven anchorage independence was found to specifically involve activation of the Src-Stat3 signaling axis. A transcriptional 'signature' of 205 genes was obtained from GAB2-transduced, anchorage-independent MCF10A cells, and found to contain two main functional modules, controlling proliferation and cell adhesion/migration/invasion, respectively. Extensive validation on breast cancer data sets showed that the GAB2 signature provides a robust prognostic classifier for breast cancer metastatic relapse, largely independent from existing clinical and genomic indicators and from estrogen receptor status. This work highlights a pivotal role for GAB2 and its transcriptional targets in anchorage-independent growth and breast cancer metastatic progression.

Met-driven invasive growth involves transcriptional regulation of Arhgap12.

Invasive growth is a complex biological program triggered by hepatocyte growth factor (HGF) through its tyrosine kinase receptor encoded by the Met proto-oncogene. The program involves-besides proliferation-cell dissociation, motility and invasiveness, controlled by intracellular signals impinging on PI3K and on the small G-proteins of the Rac/Rho family. The mechanism(s) unbalancing Rac/Rho activation are still not completely clarified. Here, we describe a functional link between HGF and Arhgap12, a gene encoding a previously uncharacterized protein of the RhoGAP family. We identified Arhgap12 as a transcriptional target of HGF, through a novel gene trapping strategy. We found that Arhgap12 mRNA and protein are robustly suppressed by HGF treatment, but not by serum. Arhgap12 displayed GTPase activating protein (GAP) activity towards Rac1 and, upon overexpression, impaired cell scattering, invasion and adhesion to fibronectin in response to HGF. Consistently, Arhgap12 silencing by RNA interference selectively increased the scatter and adhesion responses. These data show that HGF-driven invasive growth involves transcriptional regulation of a Rac1-specific GAP.

Cancer and blood coagulation.

In human patients, blood coagulation disorders often associate with cancer, even in its early stages. Recently, in vitro and in vivo experimental models have shown that oncogene expression, or inactivation of tumour suppressor genes, upregulate genes that control blood coagulation. These studies suggest that activation of blood clotting, leading to peritumoral fibrin deposition, is instrumental in cancer development. Fibrin can indeed build up a provisional matrix, supporting the invasive growth of neoplastic tissues and blood vessels. Interference with blood coagulation can thus be considered as part of a multifaceted therapeutic approach to cancer.

Biochemical and immunological properties of the human carcinoma antigen CAR-5 defined by the monoclonal antibody BD-5.

The monoclonal antibody (MAb) BD-5 reacts with an epitope (CAR-5) expressed in 83% of the gastric carcinomas and in 51% of the ductal pancreatic carcinomas. This MAb reacts also with epithelial cells of colorectal mucosa, but does not react at all with normal adult gastric mucosa or normal adult pancreas. We report the biochemical and immunochemical characterization of CAR-5-bearing molecule. The epitope was found to be carried on a mucin of more than 400 kDa with a density of 1.45 g/ml, metabolically labelled with 35S-sulfate, 3H-glucosamine, 3H-mannose and 35S-methionine. Antigenicity survived metaperiodate oxidation and alkalinization, while it was fully destroyed by pronase or papain. Trypsin, although cleaving the molecule, did not affect its antigenic activity. CAR-5 epitope is thus carried on the protein moiety of a sulfo-mucin. On the basis of its biochemical properties, the antigen was purified by a 3-step procedure, consisting of perchloric acid extraction, molecular sieving on Sepharose CL-4B and affinity chromatography on wheat-germ agglutinin coupled to Sepharose 4B. Cross-competition experiments, together with the chemical properties displayed by the different epitopes, clearly indicate that CAR-5 is different from all previously characterized carcinoma-associated determinants. Cross-DDIRMA experiments performed with different "catcher" and "tracer" antibody combinations showed that CAR-5 epitope may be expressed on the same mucin bearing CA 19-9, MOv2, DU-PAN-2, Lewisa and Lewisb epitopes.