Joan Somja

18F-FDG Uptake Assessed by PET/CT in Abdominal Aortic Aneurysms Is Associated with Cellular and Molecular Alterations Prefacing Wall Deterioration and Rupture

Rupture of abdominal aortic aneurysms (AAAs) leads to a significant morbidity and mortality in aging populations, and its prediction would be most beneficial to public health. Spots positive for uptake of 18F-FDG detected by PET are found in 12% of AAA patients (PET+), who are most often symptomatic and at high rupture risk. Comparing the 18F-FDG–positive site with a negative site from the same aneurysm and with samples collected from AAA patients with no 18F-FDG uptake should allow the discrimination of biologic alterations that would help in identifying markers predictive of rupture. Methods: Biopsies of the AAA wall were obtained from patients with no 18F-FDG uptake (PET0, n = 10) and from PET+ patients (n = 8), both at the site positive for uptake and at a distant negative site of the aneurysmal wall. Samples were analyzed by immunohistochemistry, quantitative real-time polymerase chain reaction, and zymography. Results: The sites of the aneurysmal wall with a positive 18F-FDG uptake were characterized by a strikingly increased number of adventitial inflammatory cells, highly proliferative, and by a drastic reduction of smooth muscle cells (SMCs) in the media as compared with their negative counterpart and with the PET0 wall. The expression of a series of genes involved in the maintenance and remodeling of the wall was significantly modified in the negative sites of PET+, compared with the PET0 wall, suggesting a systemic alteration of the aneurysmal wall. Furthermore, a striking increase of several matrix metalloproteinases (MMPs), notably the MMP1 and MMP13 collagenases, was observed in the positive sites, mainly in the adventitia. Moreover, PET+ patients were characterized by a higher circulating C-reactive protein. Conclusion: Positive 18F-FDG uptake in the aneurysmal wall is associated with an active inflammatory process characterized by a dense infiltrate of proliferating leukocytes in the adventitia and an increased circulating C-reactive protein. Moreover, a loss of SMC in the media and alterations of the expression of genes involved in the remodeling of adventitia and collagen degradation potentially participate in the weakening of the aneurysmal wall preceding rupture.

Human papillomavirus DNA strongly correlates with a poorer prognosis in oral cavity carcinoma.

The prevalence of human papillomavirus (HPV) in a clinical series of 162 patients with oral squamous cell carcinoma (OSCC) was studied. Furthermore, we analyzed the correlation between the immunohistochemical expression of p16, p53, epidermal growth factor receptor (EGFR), and HPV status to predict survival in OSCC patients.

High incidence of high-risk HPV in benign and malignant lesions of the larynx

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. Paraffin-embedded 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.

Monoclonal antibodies against GARP/TGF-β1 complexes inhibit the immunosuppressive activity of human regulatory T cells in vivo

Monoclonal antibodies that inhibit human Treg function in vivo may be used therapeutically in cancer or infections. Immunotherapy according to GARP Regulatory T cells (Tregs) play a critical role in preventing autoimmunity but can be co-opted by cancer cells to block immune surveillance of tumors. Cuende et al. report that a membrane protein, GARP, which binds transforming growth factor–β1 (TGF-β1) on the cell surface of Tregs, is involved in Treg-mediated inhibition of immune responses. What’s more, the authors develop anti-GARP monoclonal antibodies that block TGF-β1 production by Tregs and inhibit the activity of these cells in a xenogeneic mouse model of graft-versus-host disease. Thus, blocking GARP, either alone or in combination with other checkpoint inhibitors, could add to our arsenal for cancer immunotherapy. Regulatory T cells (Tregs) are essential to prevent autoimmunity, but excessive Treg function contributes to cancer progression by inhibiting antitumor immune responses. Tregs exert contact-dependent inhibition of immune cells through the production of active transforming growth factor–β1 (TGF-β1). On the Treg cell surface, TGF-β1 is in an inactive form bound to membrane protein GARP and then activated by an unknown mechanism. We demonstrate that GARP is involved in this activation mechanism. Two anti-GARP monoclonal antibodies were generated that block the production of active TGF-β1 by human Tregs. These antibodies recognize a conformational epitope that requires amino acids GARP137–139 within GARP/TGF-β1 complexes. A variety of antibodies recognizing other GARP epitopes did not block active TGF-β1 production by Tregs. In a model of xenogeneic graft-versus-host disease in NSG mice, the blocking antibodies inhibited the immunosuppressive activity of human Tregs. These antibodies may serve as therapeutic tools to boost immune responses to infection or cancer via a mechanism of action distinct from that of currently available immunomodulatory antibodies. Used alone or in combination with tumor vaccines or antibodies targeting the CTLA4 or PD1/PD-L1 pathways, blocking anti-GARP antibodies may improve the efficiency of cancer immunotherapy.

NF-κB-induced KIAA1199 promotes survival through EGFR signalling

Constitutive activation of EGFR- and NF-κB-dependent pathways is a hallmark of cancer, yet signalling proteins that connect both oncogenic cascades are poorly characterized. Here we define KIAA1199 as a BCL-3- and p65-dependent gene in transformed keratinocytes. KIAA1199 expression is enhanced on human papillomavirus (HPV) infection and is aberrantly expressed in clinical cases of cervical (pre)neoplastic lesions. Mechanistically, KIAA1199 binds Plexin A2 and protects from Semaphorin 3A-mediated cell death by promoting EGFR stability and signalling. Moreover, KIAA1199 is an EGFR-binding protein and KIAA1199 deficiency impairs EGF-dependent Src, MEK1 and ERK1/2 phosphorylations. Therefore, EGFR stability and signalling to downstream kinases requires KIAA1199. As such, KIAA1199 promotes EGF-mediated epithelial–mesenchymal transition (EMT). Taken together, our data define KIAA1199 as an oncogenic protein induced by HPV infection and constitutive NF-κB activity that transmits pro-survival and invasive signals through EGFR signalling.

Infusion of clinical-grade enriched regulatory T cells delays experimental xenogeneic graft-versus-host disease.

We investigated the ability of clinical‐grade enriched human regulatory T cells (Treg) to attenuate experimental xenogeneic graft‐versus‐host disease (GVHD) induced by peripheral blood mononuclear cells (PBMNCs; autologous to Treg) infusion in NSG mice, as well as verified their inability to induce xenogeneic GVHD when infused alone.

Organized proteomic heterogeneity in colorectal cancer liver metastases and implications for therapies

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)

Unique recurrence patterns of cervical intraepithelial neoplasia after excision of the squamocolumnar junction.

Recent studies have identified a putative cell of origin for cervical intraepithelial neoplasia (CIN) and cervical cancer at the squamocolumnar junction (SCJ) and suggest that these cells may not regenerate after excision (loop electrosurgical excision procedure). Our study addressed the impact of SCJ excision on the temporal dynamics, histologic and viral (human papillomavirus, HPV) characteristics of recurrent CIN. One hundred and thirty‐one consecutive patients treated by excision and attending follow‐up visits were enrolled. We compared recurrent and initial CIN with attention to excision margins, timing of recurrence, CIN grade, HPV types, p16 immunophenotype and SCJ immunophenotype. During the follow‐up period (up to 4 years), 16 (12.2%) recurrences were identified. Four (25%) were identified at the first follow‐up visit, closely resembled the initial CIN 2/3 in grade and HPV type and were typically SCJ marker positive [SCJ(+)], suggesting nonexcised (residual) disease. Twelve (75%) manifested after the first postoperative visit and all were in the ectocervix or in mature metaplastic epithelium. All of the 12 delayed recurrences were classified as CIN 1 and were SCJ (−). In total, 9 out of 11 SCJ (−) recurrences (82%) followed regressed spontaneously. Taken together, these results show that new lesions developing from any HPV infection are delayed and occur within the ectocervix or metaplastic epithelium. This markedly lower risk of CIN 2/3 after successful SCJ excision suggests that the removal of the SCJ could be a critical variable in reducing the risk of subsequent CIN 2/3 and cervical cancer.

Cervical (pre)neoplastic microenvironment promotes the emergence of tolerogenic dendritic cells via RANKL secretion.

The progression of genital human papillomavirus (HPV) infections into preneoplastic lesions suggests that infected/malignant cells are not adequately recognized by the immune system. In this study, we demonstrated that cervical/vulvar cancer cells secrete factor(s) that affect both the maturation and function of dendritic cells (DC) leading to a tolerogenic profile. Indeed, DC cocultured with cancer cell lines display both a partially mature phenotype after lipopolysaccharide (LPS) maturation and an altered secretory profile (IL-10high and IL-12p70low). In addition, tumor-converted DC acquire the ability to alter T-cell proliferation and to induce FoxP3+ suppressive T cells from naive CD4+ T cells. Among the immunosuppressive factors implicated in DC alterations in genital (pre)neoplastic microenvironment, we identified receptor activator of nuclear factor kappa-B ligand (RANKL), a TNF family member, as a potential candidate. For the first time, we showed that RANKL expression strongly increases during cervical progression. We also confirmed that RANKL is directly secreted by cancer cells and this expression is not related to HPV viral oncoprotein induction. Interestingly, the addition of osteoprotegerin (OPG) in coculture experiments reduces significantly the inhibition of DC maturation, the release of a tolerogenic cytokine profile (IL-12low IL-10high) and the induction of regulatory T (Treg) cells. Our findings suggest that the use of inhibitory molecules directed against RANKL in cervical/vulvar (pre)neoplastic lesions might prevent alterations of DC functionality and represent an attractive strategy to overcome immune tolerance in such cancers.

18F-FPRGD2 PET/CT Imaging of Integrin αvβ3 in Renal Carcinomas: Correlation with Histopathology

This study aimed to correlate 18F-FB-mini-PEG-E[c(RGDyK)](2) (18F-FPRGD2) uptake to integrin αvβ3 expression and angiogenesis in renal tumors. Methods: 18F-FPRGD2 PET/CT was performed on 27 patients before surgical resection (median 4 d) of a renal mass. The 18F-FPRGD2 uptake was compared with integrin αvβ3, CD31, CD105, and Ki-67 using immunohistochemistry; with placental growth factor and vascular endothelial growth factor receptors 1 and 2 using reverse transcription polymerase chain reaction; and with vascular endothelial growth factor A isoforms using enzyme-linked immunosorbent assay. Results: Overall, 18F-FPRGD2 uptake significantly correlated (P < 0.0001) with integrin αvβ3 expression in renal masses. However, it correlated only with integrin αvβ3-positive vessels in the group of papillary carcinomas whereas it correlated with integrin αvβ3 expression by tumor cells in the clear cell carcinoma group. Conclusion: 18F-FPRGD2 uptake reflects the expression of integrin αvβ3 in renal tumors but represents angiogenesis only when tumor cells do not express the integrin.