Skin inflammatory response and efficacy of anti-epidermal growth factor receptor therapy in metastatic colorectal cancer (CUTACETUX).

Anti-epidermal growth factor receptor (EGFR) monoclonal antibody is a standard treatment of metastatic colorectal cancer (mCRC) and its most common adverse effect is a papulopustular acneiform rash. The aim of the CUTACETUX study was to characterize the skin inflammatory response associated with this rash and its relation to treatment efficacy. This prospective study included patients with mCRC treated with first-line chemotherapy plus cetuximab. Patients underwent skin biopsies before the initiation of cetuximab (D0) and before the third infusion (D28), one in a rash zone and one in an unaffected zone. Expression of Th17-related cytokines (IL-17A, IL-21, IL-22), antimicrobial peptides (S100A7 and BD-2), innate response-related cytokines (IL-1ß, IL-6, TNF-α and OSM), T-reg-related cytokines (IL-10 and TGF-ß), Th1-related cytokine (IFN-γ), Th2-related cytokine (IL-4), Thymic stromal lymphopoietin and keratinocyte-derived cytokines (IL-8, IL-23 and CCL20) were determined by RT-PCR. Twenty-seven patients were included. Levels of most of the cytokines increased at D28 in the rash zone compared to D0. No significant association was observed between variations of cytokines levels and treatment response in the rash zone and only the increase of IL-4 (p = .04) and IL-23 (p = .02) levels between D0 and D28 in the unaffected zone was significantly associated with treatment response. Increased levels of IL-8 (p = .02), BD-2 (p = .02), IL-1ß (p = .004) and OSM (p = .02) in the rash zone were associated with longer progression-free survival. Expression of Th2-related and keratinocyte-derived cytokines in the skin was associated with anti-EGFR efficacy. If this inflammatory signature can explain the rash, the exact mechanism by which these cytokines are involved in anti-EGFR tumor response remains to be studied.

Oncostatin M exerts a protective effect against excessive scarring by counteracting the inductive effect of TGFß1 on fibrosis markers.

Wound healing is a complex physiological process that repairs a skin lesion and produces fibrous tissue. In some cases, this process can lead to hypertrophic scars (HS) or keloid scars (KS), for which the pathophysiology remains poorly understood. Previous studies have reported the presence of oncostatin M (OSM) during the wound healing process; however, the role of OSM in pathological scarring remains to be precisely elucidated. This study aims to analyse the presence and involvement of OSM in the pathological scarring process. It was conducted with 18 patients, including 9 patients with hypertrophic scarring and 9 patients with keloid scarring. Histological tissue analysis of HS and KS showed minor differences in the organization of the extracellular matrix, the inflammatory infiltrate and the keratinocyte phenotype. Transcriptomic analysis showed increased expression levels of fibronectin, collagen I, TGFß1, ß-defensin-2 and S100A7 in both pathological samples. OSM expression levels were greater in HS than in KS and control skin. In vitro, OSM inhibited TGFß1-induced secretion of components of the extracellular matrix by normal and pathological fibroblasts. Overall, we suggest that OSM is involved in pathological wound healing processes by inhibiting the evolution of HS towards KS by controlling the fibrotic effect of TGFß1.

Oncostatin M is overexpressed in skin squamous-cell carcinoma and promotes tumor progression.

Cutaneous squamous cell carcinoma (cSCC) is the second most common keratinocyte malignancy and accounts for 20% of skin cancer deaths. Cancer is closely related to inflammation, but the contribution of the tumor microenvironment to cSCC development is poorly understood. We previously showed that oncostatin M (OSM), a cytokine belonging to the IL-6 family, promotes normal keratinocyte proliferation and migration, skin inflammation, and epidermal hyperplasia, both in vitro and in vivo. Here, we show that OSM is overexpressed in human cSCC and is associated with type 1 immune polarization. In vitro, OSM induced STAT-3 and ERK signaling, modified the expression of genes involved in cytokine signaling, proliferation, inhibition of apoptosis, and immune responses, and promoted proliferation and migration of malignant keratinocyte PDVC57 cells. PDVC57 cells grafted in the skin of mice led to rapid cSCC development, associated with OSM expression by tumor-infiltrating neutrophils. Finally, the absence of OSM (OSM-KO mice) led to a 30% reduction of tumor size and reduced M2 polarization in the tumor microenvironment. Globally, these results support a pro-tumoral role of OSM in cSCC development and suggest that a new therapeutic approach targeting this cytokine could be considered.

Interleukin-17A-induced production of acute serum amyloid A by keratinocytes contributes to psoriasis pathogenesis.

BACKGROUND: Acute-serum Amyloid A (A-SAA), one of the major acute-phase proteins, is mainly produced in the liver but extra-hepatic synthesis involving the skin has been reported. Its expression is regulated by the transcription factors NF-κB, C/EBPß, STAT3 activated by proinflammatory cytokines. OBJECTIVES: We investigated A-SAA synthesis by resting and cytokine-activated Normal Human Epidermal Keratinocytes (NHEK), and their inflammatory response to A-SAA stimulation. A-SAA expression was also studied in mouse skin and liver in a model mimicking psoriasis and in the skin and sera of psoriatic and atopic dermatitis (AD) patients. METHODS: NHEK were stimulated by A-SAA or the cytokines IL-1α, IL-17A, IL-22, OSM, TNF-α alone or in combination, previously reported to reproduce features of psoriasis. Murine skins were treated by imiquimod cream. Human skins and sera were obtained from patients with psoriasis and AD. A-SAA mRNA was quantified by RT qPCR. A-SAA proteins were dosed by ELISA or immunonephelemetry assay. RESULTS: IL-1α, TNF-α and mainly IL-17A induced A-SAA expression by NHEK. A-SAA induced its own production and the synthesis of hBD2 and CCL20, both ligands for CCR6, a chemokine receptor involved in the trafficking of Th17 lymphocytes. A-SAA expression was increased in skins and livers from imiquimod-treated mice and in patient skins with psoriasis, but not significantly in those with AD. Correlations between A-SAA and psoriasis severity and duration were observed. CONCLUSION: Keratinocytes could contribute to psoriasis pathogenesis via A-SAA production, maintaining a cutaneous inflammatory environment, activating innate immunity and Th17 lymphocyte recruitment.

Ultrastructural study of liver and lead tissue concentrations in young mallard ducks (Anas platyrhynchos) after ingestion of single lead shot.

Lead (Pb) represents a serious threat to wildlife and ecosystems. The aim of this study was to examine the subcellular effects of dietary Pb pellet ingestion on mallard (Anas platyrhynchos) livers. After ingestion of a single Pb shot (LS4 size class: 0.177 ± 0.03 g) in 41 mallard ducks (22 males and 19 females) versus 10 controls (5 males and 5 females), all 7-week old, a morphologic study was conducted by TEM (transmission electron microscopy) of liver at the subcellular level. The results in treated mallards showed at a magnification of 2500 X that hepatic parenchyma was altered as evidenced by intralysosomal electron-dense deposits, which are compatible with Pb deposits. Further, at a higher magnification (15,000 X) in both genders, deterioration of mitochondria was observed in which the crests and, to a lesser extent, outer membrane were lysed. While the rough endoplasmic reticulum was fragmented, intracytoplasmic electron-dense material compatible with Pb deposits was maximally visible, thereby underscoring the deeply destructive effect of this metal on the subcellular architecture of the liver. In addition, applying an optimized and validated method in a clean room using electrothermal atomic absorption spectrophotometer (ETAAS) with Zeeman background correction, the objective was to improve and refine certain indispensable measurements pertaining to Pb impregnation in tissues other than liver such as kidneys, bones, and feathers of mallards. Data demonstrated show that compared with controls, Pb accumulation increases significantly, not only in the liver (3-fold), but also in the bones and the feathers (14-fold). No significant difference was noted between males and females. Bearing in mind the marked subcellular toxicity attributed to Pb, this study reinforces present-day arguments advocating limitation of game consumption.

High-Fat Diet-Induced IL-17A Exacerbates Psoriasiform Dermatitis in a Mouse Model of Steatohepatitis.

Recent studies suggest that psoriasis may be more severe in patients with nonalcoholic fatty liver disease, particularly in those with the inflammatory stage of steatohepatitis [nonalcoholic steatohepatitis (NASH)]. Herein, we investigated the impact of diet-induced steatohepatitis on the severity of imiquimod-induced psoriasiform dermatitis. Mice fed with a high-fat diet developed steatohepatitis reminiscent of human NASH with ballooning hepatocytes and significant liver fibrosis. Mice with steatohepatitis also displayed moderate cutaneous inflammation characterized by erythema, dermal infiltrates of CD45(+) leukocytes, and a local production of IL-17A. Moreover, steatohepatitis was associated with an epidermal activation of caspase-1 and cutaneous overexpression of IL-1ß. Imiquimod-induced psoriasiform dermatitis was exacerbated in mice with steatohepatitis as compared to animals fed with a standard diet. Scale formation and acanthosis were aggravated, in correlation with increased IL-17A and IL-22 expression in inflamed skins. Finally, intradermal injection of IL-17A in standard diet-fed mice recapitulated the cutaneous pathology of mice with steatohepatitis. The results show that high-fat diet-induced steatohepatitis aggravates the inflammation in psoriasiform dermatitis, via the cutaneous production of IL-17A. In agreement with clinical data, this description of a novel extrahepatic manifestation of NASH should sensitize dermatologists to the screening and the management of fatty liver in psoriatic patients.

Expression of a gap junction protein, connexin43, in a large panel of human gliomas: new insights.

Precise diagnosis of low and high grades of brain tumors permits determining therapeutical strategies. So far, diagnosis and prognosis of gliomas were based on histological and genetic criteria which need being completed by a panel of molecular markers. Highly distributed in brain, gap junction proteins, connexins, could be considered as markers of glioma progression as previous studies indicated that expression of a connexin type, connexin43 (Cx43), is inversely correlated to tumor grading. However, this assumption was weakened by the low number of glioma samples used. Taking advantage of tissue microarray technique, we pursued this analysis by studying in situ expression of Cx43 on 85 samples (37 grade IV, 18 grade III, 24 grade II, and 6 grades II to III). Our analysis confirmed the global diminution of Cx43 expression in glioblastomas that was observed in previous studies. However, this analysis brought new insights such as the following ones. First, the high number of samples permitted to show that more than 60% of glioblastomas still express Cx43. Second, no gradual decrease in Cx43 expression was observed between grades II and III, but Cx43 appeared to be a marker distinguishing oligodendrocytic and astrocytic grade III tumors. Third, independently from tumor grade, a Cx43 nuclear staining was detected in areas where leukocytes are present. In conclusion, our study emphasizes the importance of in situ immunohistochemical approaches by giving more precise insights in the subcellular localization of Cx43. It also emphasizes the necessity to carry out such analysis on a wide range of samples to circumvent the high glioma heterogeneity.

[Metastatic lymph node collision of a prostatic adenocarcinoma and an urothelial carcinoma and review of the literature]. / Double métastase ganglionnaire d'un adénocarcinome prostatique et d'un carcinome urothélial et revue de la littérature.

INTRODUCTION: Tumor collision is the encounter of two tumors from two different topographical sites. Cases of metastatic lymph node collision are exceptional. We report the case of a metastatic lymph node collision of an urothelial carcinoma and a prostatic adenocarcinoma. OBSERVATION: A 61-year-old man was hospitalized for a right nephroureterectomy with peri-ureteral lymph node dissection. He was followed since 2004 for prostatic adenocarcinoma and treated with radical prostatectomy then radiation therapy 4 years later due to a new increase of PSA. In the follow-up, an urothelial carcinoma of the lower right ureter was discovered in 2014. Histological analysis of a peri-ureteral lymph node showed a double metastasis of urothelial and prostatic origin. The prostatic adenocarcinoma was composed of acinar and ductal subtypes. Immunohistochemical study including CK7, CK20, PSA, GATA3, P63 antibodies confirmed the distinct phenotype of the 2 tumors. DISCUSSION: Metastatic collision of urothelial carcinoma and prostatic adenocarcinoma has been reported in 4 cases only. Our review of literature shows that prostatic adenocarcinoma always precedes the urothelial carcinoma. Immunohistochemical study, when carried out for distinguishing both tumors, should include CK7, CK20 and PSA. GATA3, androgen receptor and P63 could be added in a second time.

Prognostic Relevance of Histomolecular Classification of Diffuse Adult High-Grade Gliomas with Necrosis.

Diffuse adult high-grade gliomas (HGGs) with necrosis encompass anaplastic oligodendrogliomas (AOs) with necrosis (grade III), glioblastomas (GBM, grade IV) and glioblastomas with an oligodendroglial component (GBMO, grade IV). Here, we aimed to search for prognostic relevance of histological classification and molecular alterations of these tumors. About 210 patients were included (63 AO, 56 GBM and 91 GBMO). GBMO group was split into "anaplastic oligoastrocytoma (AOA) with necrosis grade IV/GBMO," restricted to tumors showing intermingled astrocytic and oligodendroglial component, and "GBM/GBMO" based on tumors presenting oligodendroglial foci and features of GBM. Genomic arrays, IDH1 R132H expression analyses and IDH direct sequencing were performed. 1p/19q co-deletion characterized AO, whereas no IDH1 R132H expression and intact 1p/19q characterized both GBM and GBM/GBMO. AOA with necrosis/GBMO mainly demonstrated IDH1 R132H expression and intact 1p/19q. Other IDH1 or IDH2 mutations were extremely rare. Both histological and molecular classifications were predictive of progression free survival (PFS) and overall survival (OS) (P < 10(-4) ). Diffuse adult HGGs with necrosis can be split into three histomolecular groups of prognostic relevance: 1p/19q co-deleted AO, IDH1 R132H-GBM and 1p/19q intact IDH1 R132H+ gliomas that might be classified as IDH1 R132H+ GBM. Because of histomolecular heterogeneity, we suggest to remove the name GBMO.

EGFR, KRAS, BRAF, and HER-2 molecular status in brain metastases from 77 NSCLC patients.

The aim of this study was to determine the frequency of EGFR, KRAS, BRAF, and HER-2 mutations in brain metastases from non-small cell lung carcinomas (BM-NSCLC). A total of 77 samples of BM-NSCLC were included and 19 samples of BM from breast, kidney, and colorectal tumors were also studied as controls. These samples were collected from patients followed between 2008 and 2011 at Poitiers and Nice University Hospitals in France. The frequencies of EGFR, KRAS, BRAF, and HER-2 mutations in BM-NSCLC were 2.6, 38.5, 0, and 0% respectively. The incidence of KRAS mutation was significantly higher in female and younger patients (P < 0.05). No mutations of the four genes were found in BM from breast or kidney. However, among six BM from colorectal tumors, we identified KRAS mutations in three cases and BRAF mutations in two other cases. This study is the largest analysis on genetic alterations in BM-NSCLC performed to date. Our results suggest a low frequency of EGFR mutations in BM-NSCLC whereas KRAS mutations are as frequent in BM-NSCLC as in primitive NSCLC. These results raise the question of the variability of the brain metastatic potential of NSCLC cells in relation to the mutation pattern.

Epidermal growth factor receptor (EGFR) and KRAS mutations during chemotherapy plus anti-EGFR monoclonal antibody treatment in metastatic colorectal cancer.

It is now well established that metastatic colorectal cancer patients without KRAS mutation (codon 12) benefit from treatment with an epidermal growth factor receptor monoclonal antibody (anti-EGFR mAb). Recently, EFGR and KRAS mutations have been shown to exist in patients who developed resistance to anti-EGFR mAb. We analyzed KRAS, BRAF V600E and EGFR S492R mutations in 37 post-anti-EGFR mAb tumor samples from 23 patients treated with chemotherapy plus anti-EGFR mAb. No EGFR S492R mutation was detected. A KRAS mutation was found after anti-EGFR mAb in only one tumor. Our results suggest that acquired EGFR S492R and KRAS mutations do not constitute the main mechanism of resistance to anti-EGFR mAb in combination with chemotherapy.

Involvement of IL-1 and oncostatin M in acanthosis associated with hypertensive leg ulcer.

Hypertensive leg ulcer (HLU) is an inflammatory disease characterized by intense pain, alteration of vascularization, and skin necrosis. The optimal treatment relies on surgical removal of necrotic tissues covered by a split-skin graft. We studied the histomorphology of the lesions and investigated the involvement of inflammatory cells and cytokines to further define the physiopathology of HLU. We report epidermis acanthosis and a preferential occlusion of the precapillary arterioles with infiltration of neutrophils, macrophages, and T lymphocytes in the dermis. OSM, IL-1ß, and IL-6 were overexpressed in the ulcer, whereas the Th17-derived cytokines were not. In vitro, the addition of IL-1ß and OSM promoted acanthosis and destructuring of reconstructed epidermis. Exogenous IL-1ß and OSM synergistically induced epidermal acanthosis in mice. These data show that OSM and IL-1ß are not only a biological characteristic signature of HLU, but these cytokines reflect a specific inflammatory state, directly involved in the pathogenesis. We suggest that anti-cytokine biotherapies could be an alternative strategy to surgery to treat HLU.

Comprehensive re-sequencing of adrenal aldosterone producing lesions reveal three somatic mutations near the KCNJ5 potassium channel selectivity filter.

BACKGROUND: Aldosterone producing lesions are a common cause of hypertension, but genetic alterations for tumorigenesis have been unclear. Recently, either of two recurrent somatic missense mutations (G151R or L168R) was found in the potassium channel KCNJ5 gene in aldosterone producing adenomas. These mutations alter the channel selectivity filter and result in Na(+) conductance and cell depolarization, stimulating aldosterone production and cell proliferation. Because a similar mutation occurs in a mendelian form of primary aldosteronism, these mutations appear to be sufficient for cell proliferation and aldosterone production. The prevalence and spectrum of KCNJ5 mutations in different entities of adrenocortical lesions remain to be defined. MATERIALS AND METHODS: The coding region and flanking intronic segments of KCNJ5 were subjected to Sanger DNA sequencing in 351 aldosterone producing lesions, from patients with primary aldosteronism and 130 other adrenocortical lesions. The specimens had been collected from 10 different worldwide referral centers. RESULTS: G151R or L168R somatic mutations were identified in 47% of aldosterone producing adenomas, each with similar frequency. A previously unreported somatic mutation near the selectivity filter, E145Q, was observed twice. Somatic G151R or L168R mutations were also found in 40% of aldosterone producing adenomas associated with marked hyperplasia, but not in specimens with merely unilateral hyperplasia. Mutations were absent in 130 non-aldosterone secreting lesions. KCNJ5 mutations were overrepresented in aldosterone producing adenomas from female compared to male patients (63 vs. 24%). Males with KCNJ5 mutations were significantly younger than those without (45 vs. 54, respectively; p<0.005) and their APAs with KCNJ5 mutations were larger than those without (27.1 mm vs. 17.1 mm; p<0.005). DISCUSSION: Either of two somatic KCNJ5 mutations are highly prevalent and specific for aldosterone producing lesions. These findings provide new insight into the pathogenesis of primary aldosteronism.

Malignant pheochromocytoma: new malignancy criteria.

PURPOSE: The pathological diagnosis of malignancy in pheochromocytomas remains a controversial issue. According to the WHO, malignancy is defined in the presence of metastasis. Multiparameter scoring systems such as PASS (Pheochromocytoma of Adrenal gland Scaled Score) have been used but remain controversial. The aim of this study was to search for new immunohistologic elements allowing determination of pheochromocytoma malignancy. METHODS: Among 53 patients operated for pheochromocytoma between 1993 and 2009, we selected pheochromocytomas with proven metastasis, seven cases in group 1 (G1) and paired two others groups: group 2 (G2), patients who had "benign" pheochromocytoma with PASS ≥4 and group 3 (G3), patients who had "benign" pheochromocytoma with PASS <4. We retrospectively analysed PASS criteria, size, weight, tumour necrosis, Ki-67 and pS100 staining. RESULTS: The size and weight of the lesion were directly and significantly correlated to malignancy in all three groups: respectively 9.7 cm and 292.0 g (G1), 6.2 cm and 83.8 g (G2) and 3.8 cm and 37.1 g (G3) (p < 0.005 for both). Tumour necrosis (TN) was present in all G1 (p < 0.005) and respectively at 0% and 37.5% in G2 and G3. Ki-67 is directly correlated to presence of TN (p < 0.005) and malignancy (G1 14.1%, G2 1.8%, G3 2.6%; p < 0.001). All G1 had a Ki-67 index >4%, although one G3 presented an 11% Ki-67 index. There was an inverse statistically significant correlation between the three groups in staining using pS100 (p < 0.01). CONCLUSIONS: Size and weight of the pheochromocytoma are directly related to PASS and malignancy. The presence of tumour necrosis, Ki-67 index >4% and pS100 absence impose a close histopathological evaluation and follow-up with regard to cases presenting a high risk of malignancy/recurrence.

Altered expression of the gap junction protein connexin43 is associated with papillary thyroid carcinomas when compared with other noncancer pathologies of the thyroid.

BACKGROUND: Gap junctions are membrane structures composed of connexins (Cx) that allow diffusion of small molecules between cells. They are involved in tissue homeostasis, and various organ dysfunctions have been associated with gap junction defects. To verify their possible involvement in thyroid pathologies, the expression of connexin43 (Cx43), the major Cx in the human thyroid, was evaluated in a variety of diseases including cancer. METHODS: There were 122 samples from various thyroid pathologies that were collected to analyze the presence of Cx43 by immunofluorescence. Through confocal microscopy, different patterns of Cx43 localization were identified as normal (membrane) or abnormal (cytoplasmic or lack of detection). The analysis of Cx43 expression was further performed by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry in a subset of 25 papillary carcinomas and compared with nontumoral thyroid tissues. RESULTS: The presence of Cx43 was commonly altered in thyroid cancer, as abnormal Cx43 staining was detected in 94.1% of cancer, 47.4% of adenomas, 45.7% of multinodular goiter, 16.7% of Graves' disease, and 25% of thyroiditis. In papillary carcinoma samples, the deregulation of Cx43 expression was mostly the consequence of a decrease of Cx43 mRNA (68% of cases) when compared with normal tissue. When Cx43 mRNA was not downregulated (32% of cases), both loss of membrane staining and aberrant cytoplasmic distribution of the protein were observed. CONCLUSIONS: These results show that aberrations of Cx43 expression are associated with thyroid papillary carcinoma.

Immunohistochemical detection, regulation and antiproliferative function of G-protein-coupled receptor kinase 2 in thyroid carcinomas.

TSH, via its G-protein-coupled receptor, activates cell growth of both benign and malignant thyroid tumors. G-protein-coupled receptors (GR) kinase 2 (GRK2) has been reported to regulate the TSH receptor but its role in cancer is unknown. To determine a possible function for GRK2 in the growth process of thyroid cancers, we analysed its expression in normal and tumoral thyroid tissues and studied thyroid cancer cell line proliferation after GRK2 overexpression. Thirty one thyroid tissues, including 16 non-medullary thyroid cancers and 15 adjacent normal tissues, were analysed by immunohistochemistry. Five paired tissues were also studied by western blotting for the GRK2 enzymatic activity. Immunohistochemical staining showed an increase in GRK2 in thyroid cancers including papillary, follicular, and anaplastic types, compared with their adjacent normal tissues. Immunoblot analysis and GRK2 enzymatic activity measurement confirmed immunohistochemical study. TSH and TSH in association with insulin or IGF-I stimulated GRK2 protein accumulation in normal human thyroid cells in primary culture. The TSH effect on the GRK2 expression was mimicked by forskolin. After GRK2 overexpression in two poorly differentiated thyroid cell lines, all the clones showed a significant reduction in cell proliferation, ranging from 28 to 65% inhibition compared with vector alone after 96-h culture. In conclusion, thyroid mitogenic factor-stimulated GRK2 accumulation may explain, in part, high GRK2 levels in differentiated carcinoma, because TSH, insulin, or IGF-I is known to be involved in the thyroid cancer progression. Surprisingly, instead of stimulating, GRK2 reduced cell proliferation revealing a new role for this kinase in the growth of thyroid cancers.

Solitary intestinal fibromatosis associated with congenital ileal atresia.

We report for the first time an association between congenital solitary intestinal fibromatosis and intestinal atresia. The spindle cell proliferation showed a high apoptotic index contrasting with a low proliferation rate, suggesting that the tumor may have undergone focal and spontaneous regression, leading to intestinal atresia.