Tumor heterogeneity is a major obstacle for developing effective anticancer treatments. Recent studies have pointed to large stochastic genetic heterogeneity within cancer lesions, where no pattern seems to exist that would enable a more structured targeted therapy approach. Because to date no similar information is available at the protein (phenotype) level, we employed matrix assisted laser desorption ionization (MALDI) image-guided proteomics and explored the heterogeneity of extracellular and membrane subproteome in a unique collection of eight fresh human colorectal carcinoma (CRC) liver metastases. Monitoring the spatial distribution of over 1,000 proteins, we found unexpectedly that all liver metastasis lesions displayed a reproducible, zonally delineated pattern of functional and therapeutic biomarker heterogeneity. The peritumoral region featured elevated lipid metabolism and protein synthesis, the rim of the metastasis displayed increased cellular growth, movement, and drug metabolism, whereas the center of the lesion was characterized by elevated carbohydrate metabolism and DNA-repair activity. From the aspect of therapeutic targeting, zonal expression of known and novel biomarkers was evident, reinforcing the need to select several targets in order to achieve optimal coverage of the lesion. Finally, we highlight two novel antigens, LTBP2 and TGFBI, whose expression is a consistent feature of CRC liver metastasis. We demonstrate their in vivo antibody-based targeting and highlight their potential usefulness for clinical applications. Conclusion: The proteome heterogeneity of human CRC liver metastases has a distinct, organized pattern. This particular hallmark can now be used as part of the strategy for developing rational therapies based on multiple sets of targetable antigens. (HEPATOLOGY 2014;59:924-934) See Editorial on Page 757 D espite its great promise, clinically used targeted cancer therapy is unfortunately showing only limited success at a very high cost. 1 This failure is partly explained by the high level of genetic heterogeneity of malignant lesions and the fact that carcinogenesis is an evolutionally driven process that recapitulates Darwin's theory of selection of the fittest. 2 In such an unfavorable context, targeted therapy protocols are reaching only a subpopulation of tumor cells, leading to a punctual increase in the selective pressure, fueling resistance to therapy, and thus failing to cure the patient. 3 The notion of tumor heterogeneity and the apparent random distribution of tumor cells harboring different mutations are supported by recent genetic studies. [4][5][6] However, the lack of information at the protein level limits the understanding of how genetic Abbreviations: CRC, colorectal cancer; MALDI, matrix assisted laser desorption ionization; MS, mass spectrometry. From the

The pathogenesis of nontraumatic osteonecrosis (ON) remains unclear. Some studies have suggested that nontraumatic ON is attributed to increased osteocytic apoptosis. To test this hypothesis, a controlled study must compare the apoptosis of osteocytes and osteoblasts in cases of ON and osteoarthritis (OA). To assess either the localized or diffuse patterns of this increased osteocytic and osteoblastic apoptosis, we evaluated both the proximal and distal regions of necrotic areas. Femoral heads resected for total hip prosthesis were included for this study. Of these, 10 were ON cases-three were induced by corticosteroids, three by alcohol abuse, one resulted from trauma, one resulted from hyperlipemia, and two were idiopathic-10 were osteoarthritis cases, and 1 from a patient suffering from a subcapital fracture. The TUNEL reaction was used to detect the apoptosis in osteoblasts and osteocytes. A semi-quantitative evaluation was conducted, at both distal and proximal areas relative to the lesions, specifically in the area surrounding the necrotic region in the osteonecrosis cases, in the eburnated bone in the osteoarthritis cases, and in the subchondral bone fracture. The apoptosis of osteoblasts and osteocytes was statistically more frequent in the regions close to the necrotic areas in the ON group. No difference was found in the unpaired areas. In the ON group, no difference was found in terms of the etiological factors. During ON, the apoptosis of osteocytes and osteoblasts is increased proximally to the necrotic regions in the patients presenting with osteoarthritis and subcapital fractures. This increase was found not only in the corticosteroid-induced ON cases but also in the idiopathic and alcohol abuse- and trauma-induced ON cases.

The identification of specific biomarkers obtained directly from human pathological lesions remains a major challenge, because the amount of tissue available is often very limited. We have developed a novel, comprehensive, and efficient method permitting the identification and absolute quantification of potentially accessible proteins in such precious samples. This protein subclass comprises cell membrane associated and extracellular proteins, which are reachable by systemically deliverable substances and hence especially suitable for diagnosis and targeted therapy applications. To isolate such proteins, we exploited the ability of chemically modified biotin to label ex vivo accessible proteins and the fact that most of these proteins are glycosylated. This approach consists of three successive steps involving first the linkage of potentially accessible proteins to biotin molecules followed by their purification. The remaining proteins are then subjected to glycopeptide isolation. Finally, the analysis of the nonglycosylated peptides and their involvement in an in silico method increased the confident identification of glycoproteins. The value of the technique was demonstrated on human breast cancer tissue samples originating from 5 individuals. Altogether, the method delivered quantitative data on more than 400 potentially accessible proteins (per sample and replicate). In comparison to biotinylation or glycoprotein analysis alone, the sequential method significantly increased the number (≥30% and ≥50% respectively) of potentially therapeutically and diagnostically valuable proteins. The sequential method led to the identification of 93 differentially modulated proteins, among which several were not reported to be associated with the breast cancer. One of these novel potential biomarkers was CD276, a cell membrane-associated glycoprotein. The immunohistochemistry analysis showed that CD276 is significantly differentially expressed in a series of breast cancer lesions. Due to the fact that our technology is applicable to any type of tissue biopsy, it bears the ability to accelerate the discovery of new relevant biomarkers in a broad spectrum of pathologies.

Abstract. The aim of this study was to determine the prevalence of human papillomavirus (HPV) in patients with laryngeal benign lesions (LBLs) and laryngeal squamous cell carcinomas (LSCCs) using a sensitive E6/E7 type-specific PCR. Paraffinembedded samples from LBL (n=39) and LSCC patients (n=67) were evaluated for the presence of HPV DNA by GP5 + /GP6 + consensus PCR and E6/E7 type-specific PCR for HPV types 6,11,16,18,31,33,35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68. In LSCCs, immunohistochemical staining of p16, p53 and EGFR was also assessed. The E6/E7 type-specific PCR showed that 44 out of 59 LSCC patients (i.e., 75%) had high-risk (hr) HPV types and that 27 out of 35 LBL patients (i.e., 77%) had hrHPV types. HPV-16 viral load was significantly higher in LSCC than in LBL patients (p<10 -6 ). The presence of hrHPV DNA did not correlate with the proportion of disease-free patients. Comparable levels of p16, p53 and EGFR expression were observed in the hrHPV + tumor group (100% p16 + , 56% p53 + and 97% EGFR + ) and in the HPV -or low-risk (lr) HPV + tumor group (92% p16 + , 66% p53 + and 100% EGFR + ). A very high prevalence of oncogenic HPV-16 was found in a series of benign and malignant laryngeal lesions. LSCC appears to be characterized by an active hrHPV infection. In LSCCs, the hrHPV + subgroup had a similar prognosis (in terms of risk of recurrence) as the HPV -subgroup.

The identification of diagnostic and prognostic biomarkers from early lesions, measurable in liquid biopsies remains a major challenge, particularly in oncology. Fresh human material of high quality is required for biomarker discovery but is often not available when it is totally required for clinical pathology investigation. Hence, all OMICs studies are done on residual and less clinically relevant biological samples. Here after, we present an innovative, simple, and non-destructive, procedure named EXPEL that uses rapid, pressure-assisted, interstitial fluid extrusion, preserving the specimen for full routine clinical pathology investigation. In the meantime, the technique allows a comprehensive OMICs analysis (proteins, metabolites, miRNAs and DNA). As proof of concept, we have applied EXPEL on freshly collected human colorectal cancer and liver metastases tissues. We demonstrate that the procedure efficiently allows the extraction, within a few minutes, of a wide variety of biomolecules holding diagnostic and prognostic potential while keeping both tissue morphology and antigenicity unaltered. Our method enables, for the first time, both clinicians and scientists to explore identical clinical material regardless of its origin and size, which has a major positive impact on translation to the clinic.