KEAP1-driven co-mutations in lung adenocarcinoma unresponsive to immunotherapy despite high tumor mutational burden.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have demonstrated significant overall survival (OS) benefit in lung adenocarcinoma (LUAD). Nevertheless, a remarkable interpatient heterogeneity characterizes immunotherapy efficacy, regardless of programmed death-ligand 1 (PD-L1) expression and tumor mutational burden (TMB). KEAP1 mutations are associated with shorter survival in LUAD patients receiving chemotherapy. We hypothesized that the pattern of KEAP1 co-mutations and mutual exclusivity may identify LUAD patients unresponsive to immunotherapy. PATIENTS AND METHODS: KEAP1 mutational co-occurrences and somatic interactions were studied in the whole MSKCC LUAD dataset. The impact of coexisting alterations on survival outcomes in ICI-treated LUAD patients was verified in the randomized phase II/III POPLAR/OAK trials (blood-based sequencing, bNGS cohort, N = 253). Three tissue-based sequencing studies (Rome, MSKCC and DFCI) were used for independent validation (tNGS cohort, N = 289). Immunogenomic features were analyzed using The Cancer Genome Atlas (TCGA) LUAD study. RESULTS: On the basis of KEAP1 mutational co-occurrences, we identified four genes potentially associated with reduced efficacy of immunotherapy (KEAP1, PBRM1, SMARCA4 and STK11). Independent of the nature of co-occurring alterations, tumors with coexisting mutations (CoMut) had inferior survival as compared with single-mutant (SM) and wild-type (WT) tumors (bNGS cohort: CoMut versus SM log-rank P = 0.048, CoMut versus WT log-rank P < 0.001; tNGS cohort: CoMut versus SM log-rank P = 0.037, CoMut versus WT log-rank P = 0.006). The CoMut subset harbored higher TMB than the WT disease and the adverse significance of coexisting alterations was maintained in LUAD with high TMB. Significant immunogenomic differences were observed between the CoMut and WT groups in terms of core immune signatures, T-cell receptor repertoire, T helper cell signatures and immunomodulatory genes. CONCLUSIONS: This study indicates that coexisting alterations in a limited set of genes characterize a subset of LUAD unresponsive to immunotherapy and with high TMB. An immune-cold microenvironment may account for the clinical course of the disease.

Genotype-phenotype correlations in 5-fluorouracil metabolism: a candidate DPYD haplotype to improve toxicity prediction.

5-Fluorouracil is among the most widely used anticancer drug, but a fraction of treated patients develop severe toxicity, with potentially lethal injuries. The predictive power of the available pretreatment assays, used to identify patients at risk of severe toxicity, needs improvements. This study aimed to correlate a phenotypic marker of 5-fluorouracil metabolism (the individual degradation rate of 5-fluorouracil-5-FUDR) with 15 functional polymorphisms in the dihydropyrimidine dehydrogenase gene (DPYD). Single SNP (single-nucleotide polymorphism) analysis revealed that the SNPs rs1801160, rs1801265, rs2297595 and rs3918290 (splice site variant IVS14+1G>A) were significantly associated with a decreased value of 5-FUDR, and the rs3918290 causing the larger decrease. Multi-SNP analysis showed that a three-SNP haplotype (Hap7) involving rs1801160, rs1801265 and rs2297595 causes a marked decrease in 5-FUDR, comparable to that caused by the splice site variant rs3918290, which is the main pharmacogenetic marker associated with severe fluorouracil toxicity. The similar effect played by Hap7 and by the splice site variant rs3918290 upon individual 5-FUDR suggests that Hap7 could also represent a similar determinant of fluorouracil toxicity. Haplotype assessment could improve the predictive value of DPYD genetic markers aimed at the pre-emptive identification of patients at risk of severe 5-fluorouracil toxicity.The Pharmacogenomics Journal advance online publication, 28 July 2015; doi:10.1038/tpj.2015.56.

The importance of lymph node retrieval and lymph node ratio following preoperative chemoradiation of rectal cancer.

AIM: Preoperative chemoradiation (CRT) for rectal cancer decreases the number of examined lymph nodes (NELN) found in the resected specimen. However, the prognostic role of lymph node evaluation including overall numbers and the lymph node ratio (LNR) in patients having preoperative CRT have not yet been defined. The study has assessed the influence of CRT on the NELN and on lymph node number and LNR on the survival of patients with rectal cancer. METHOD: Between 2003 and 2011, 508 patients with nonmetastatic rectal cancer underwent mesorectal excision. Of these 123 (24.2%) received preoperative CRT. Univariate and multivariate analysis was performed to define the role of NELN and LNR as prognostic indicators of survival. RESULTS: Neoadjuvant CRT significantly reduced the NELN (P < 0.0001). Disease-free survival (DFS) and overall survival (OS) of patients with fewer or more than 12 nodes retrieved did not differ statistically. Node-negative patients with six or fewer lymph nodes were significantly associated with a poor DFS and OS on univariate analysis (P = 0.03 and P = 0.03). LNR significantly influenced the DFS and OS on multivariate analysis [DFS, P = 0.0473, hazard ratio (HR) 2.4980, 95% confidence interval (CI) 1.2631-9.4097; OS, P = 0.0419, HR 1.1820, 95% CI 1.1812-10,710]. CONCLUSION: The cut-off of 12 lymph nodes does not influence survival and should not be considered for cancer-specific prediction of patients having neoadjuvant CRT. In contrast LNR is an independent prognostic predictor of DFS and OS in such patients.

KRAS G12C mutation and risk of disease recurrence in stage I surgically resected lung adenocarcinoma.

KRAS G12C mutations are found in about 12-13% of LUAD samples and it is unclear whether they are associated with worse survival outcomes in resected, stage I LUAD. We assessed whether KRAS-G12C mutated tumours had worse DFS when compared to KRAS-nonG12C mutated tumours and to KRAS wild-type tumours in a cohort of resected, stage I LUAD (IRE cohort). We then leveraged on publicly available datasets (TCGA-LUAD, MSK-LUAD604) to further test the hypothesis in external cohorts. In the stage I IRE cohort we found a significant association between the KRAS-G12C mutation and worse DFS in multivariable analysis (HR: 2.47). In the TCGA-LUAD stage I cohort we did not find statistically significant associations between the KRAS-G12C mutation and DFS. In the MSK-LUAD604 stage I cohort we found that KRAS-G12C mutated tumours had worse RFS when compared to KRAS-nonG12C mutated tumours in univariable analysis (HR 3.5). In the pooled stage I cohort we found that KRAS-G12C mutated tumours had worse DFS when compared to KRAS-nonG12C mutated tumours (HR 2.6), to KRAS wild-type tumours (HR 1.6) and to any other tumours (HR 1.8); in multivariable analysis, the KRAS-G12C mutation was associated with worse DFS (HR 1.61). Our results suggest that patients with resected, stage I LUAD with a KRAS-G12C mutation may have inferior survival outcomes..

Cytopathological and chemico-physical analyses of smears of mucosa surrounding oral piercing.

OBJECTIVE: The aim of this comparative study was to analyze cytopathologically and chemico-physically the mucosa surrounding oral piercing to correlate results with adverse tissue signs. MATERIALS AND METHODS: The tongue superficial mucosa of 15 young subjects (control group) and the superficial mucosa surrounding oral piercing of 15 young subjects (test group, TG) were smeared on slides, Papanicolaou stained and analyzed under the optical microscope. Some smears were prepared for (back-scattered) scanning electron microscope (SEM) and X-ray microanalysis to study piercing fragments. RESULTS: Smears of TG displayed a variable extent of bacterial cytolysis of epithelial cells, fungi, hyperkeratosis, parakeratosis, granulocyte infiltration, calcium formations and bacterial flora; the four last statistically significant (P < 0.05). Foreign bodies surrounded by keratinocytes were detected under both light and SEM. X-ray microanalyses highlighted piercing alloy aggression, ion release and an inverse gradient of ion concentration inside keratinocytes. CONCLUSIONS: The pathological findings in smears correlated with adverse effects of oral piercing. Ion release may be related to direct toxic effects and belated reactions because of metal sensitization. A strict regulation of piercing is warranted.

The effect of a triclosan dentifrice on mucositis in subjects with dental implants: a six-month clinical study.

OBJECTIVE: The objective of the present clinical study was to assess the effect of the use of a dentifrice containing triclosan on peri-implant mucositis in subjects that had been restored with dental implants. METHODS: The trial was designed as a double-blind, randomized, two-treatment, parallel-group clinical study. Sixty male and female subjects, aged 30-70 years, were recruited. All subjects had lost teeth due to periodontal disease, and had been restored with a minimum of two implants at least one year prior to the start of the trial. Subjects were randomly assigned to two treatment groups. The subjects in the test group (Test) brushed their teeth and implant-supported restorations with a dentifrice containing triclosan, while the control subjects brushed with a sodium fluoride dentifrice. Only subjects with a minimum of one implant site showing clinical signs of peri-implant mucositis, i.e., bleeding after probing, were enrolled in the study. Clinical examinations were performed at baseline, and after three and six months. The following parameters were scored: Probing pocket depth (PPD), bleeding on probing (BoP), and plaque. The change from baseline within each treatment group at three months and six months was evaluated for all parameters using ANOVA and ANCOVA. RESULTS: Subjects with peri-implant mucositis who used a dentifrice containing 0.3% triclosan, as an adjunct to mechanical tooth brushing, exhibited significantly fewer clinical signs of inflammation than subjects who used a regular fluoride dentifrice at six months. The BoP scores were reduced from 53.8% to 29.1% in the Test group, whereas in the same interval there was an increase from 52.3% to 58.8% in the Control group. Furthermore, the individual mean PPD, as well as the frequency of sites with 5 mm and > or = 6 mm deep pockets, were reduced significantly more in the Test than in the Control group. CONCLUSION: The regular use of a dentifrice containing triclosan may reduce the clinical signs of inflammation in the mucosa adjacent to dental implants.

The ambitious role of anti angiogenesis molecules: Turning a cold tumor into a hot one.

In renal cancer emerging treatment options are becoming available and there is a strong need to combine therapies to reformulate and adjourn clinical practice. We here highlight and discuss the need to take advantage of the common immune targets to design combined strategies to increase clinical responses.

Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage.

BACKGROUND AND PURPOSE: In circulatory shock, melanocortins have life-saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists. EXPERIMENTAL APPROACH: Severe haemorrhagic shock was produced in rats under general anaesthesia. Rats were then treated with either the non-selective agonist [Nle4, D-Phe7]-melanocyte-stimulating hormone (NDP--MSH) or with the selective MC4 agonists RO27-3225 and PG-931. Cardiovascular and respiratory functions were continuously monitored for 2 h; survival rate was recorded up to 24 h. Free radicals in blood were measured using electron spin resonance spectrometry; tissue damage was evaluated histologically 25 min or 24 h after treatment. KEY RESULTS: All shocked rats treated with saline died within 30-35 min. Treatment with NDP--MSH, RO27-3225 and PG-931 produced a dose-dependent (13-108 nmol kg-1 i.v.) restoration of cardiovascular and respiratory functions, and improved survival. The three melanocortin agonists also markedly reduced circulating free radicals relative to saline-treated shocked rats. All these effects were prevented by i.p. pretreatment with the selective MC4 receptor antagonist HS024. Moreover, treatment with RO27-3225 prevented morphological and immunocytochemical changes in heart, lung, liver, and kidney, at both early (25 min) and late (24 h) intervals. CONCLUSIONS AND IMPLICATIONS: Stimulation of MC4 receptors reversed haemorrhagic shock, reduced multiple organ damage and improved survival. Our findings suggest that selective MC4 receptor agonists could have a protective role against multiple organ failure following circulatory shock.

The effectiveness of video support in the teaching of manual skills related to initial periodontal therapy tested on phantoms.

INTRODUCTION: The teaching of manual skills and competencies is among the most time-consuming aspects of oral health-care education, especially when large groups of students are involved. Video has been repeatedly used as an educational tool with varying results. PURPOSE: The present study aimed to investigate the effectiveness of a computer-based video support system during practical training of manual skills and competencies related to periodontal treatment. MATERIALS AND METHODS: Eighty-four students were randomized into 9 groups: 5 experimental and 4 control groups. The control groups received instruction in the use of scaling and root planing instruments during a 7-hour seminar, and 2 hours of manual practice. The experimental groups received the same instruction, but in addition had access to a computer-based video support system, the Visual Training System (VTS), during practical training. During the 2-hour long practice session, all students practiced 21 different procedures, which were video recorded. The videos were later evaluated by an independent observer. RESULTS: On the whole, the students in the experimental group performed significantly better than their colleagues in the control group. Specifically, the groups that utilized the VTS video support performed significantly better in 9 of the 21 procedures tested. CONCLUSION: These results suggest that this computer-based video support can be an effective aid in the teaching of manual skills related to oral health care.

Gonadotropin-releasing hormone, inhibin, and activin in human placenta: evidence for a common cellular localization.

The human placenta produces several hypophysiotropic factors that participate in the control of local hormone secretion. In particular, previous reports showed that inhibin and activin modulate both GnRH release and hormonal effect in cultured human placental cells. The present study evaluated the possible correlations among the synthesis and cellular distribution of inhibin, activin, and GnRH in placental villi at term. mRNA coding for inhibin alpha- and beta A-subunits and GnRH were localized in human placenta at term by in situ hybridization using the corresponding cDNA probes. Autoradiograms revealed that some placental cells express inhibin alpha- and beta A-subunit and GnRH mRNAs. A common localization of the three hormonal mRNAs has been found in the inner part of the villi. Using affinity-purified polyclonal antisera, immunocytochemical studies confirmed that in placental villi at term, immunoreactive inhibin alpha- and beta A-subunits and GnRH had a distribution that was superimposable in several areas. Both the outer layer and the inner trophoblasts contained immunoreactive hormonal products. The present data show that some placental cells at term can produce and release inhibin, activin, and/or GnRH. This complement of hypophysiotropic factors may, thus, represent a local paracrine/autocrine control mechanism within the placenta.

Age-related distribution of endocrine cells in the human prostate: a quantitative study.

A morphometric analysis was performed to obtain quantitative data on age-related changes in prostatic endocrine cell (PrEC) density. Sixty prostates from subjects aged 14-74 years were studied with a semi-automatic image analysis system (ASM 68K, Leitz) applied to sections immunostained for chromogranin A-reactive cells. The highest density of PrECs (0.366 cells/mm of epithelial length) was found in the 25-54 year age group, which was significantly different from that found in prostates of the younger (0.311 cells/mm) and the older (0.261 cells/mm) age groups. The data probably reflect the higher incidence of incompletely developed glandular units in the younger group and the formation of new alveoli related to the usual glandular hyperplasia that occurs with increasing age in the older group.

[Nle4,D-Phe7]alpha-MSH improves functional recovery in rats subjected to diencephalic hemisection.

Rats subjected to diencephalic hemisection were s.c. treated with alpha-MSH (20 micrograms/rat daily) or with [Nle4,D-Phe7]alpha-MSH (10 micrograms/rat every other day) for two weeks starting on day 3 after lesion. Apomorphine-induced (1 mg/kg s.c.) rotational behavior was studied on days 7, 14 and 21 after lesion, and a sensorimotor test battery was carried out on days 3, 10, 17 and 24 after lesion. [Nle4,D-Phe7]alpha-MSH greatly reduced rotational behavior and significantly improved sensorimotor performance. Histological studies showed that treatment with alpha-MSH and, even more markedly, with [Nle4,D-Phe7]alpha-MSH reduced the size of the lesion and the pseudoinflammatory reaction, and caused a marked proliferation and hypertrophy of astroglia. Binding studies showed that no supersensitivity of striatal dopamine receptors developed on the lesioned side of alpha-MSH- and [Nle4,D-Phe7]alpha-MSH-treated rats. The present results seem to further support the trophic role of MSH peptides on nerve tissue.

Neuroprotective effect of gamma-hydroxybutyrate in transient global cerebral ischemia in the rat.

The effect of gamma-hydroxybutyrate on the histological and behavioral consequences of transient brain ischemia was studied in the four vessel occlusion rat model. In saline-treated animals, 30 min ischemia caused a massive loss of neurons in the hippocampal CA1 subfield (normal neurons: 14%, 5%, 23% and 30% on the 3rd, 10th, 15th and 65th day after ischemia, respectively). gamma-Hydroxybutyrate - 300 mg/kg intraperitoneally (i.p.) 30 min before or 10 min after arteries occlusion, followed by 100 mg/kg i.p. twice daily for the following 10 days - afforded a highly significant protection (normal neurons on the 3rd, 10th, 15th and 65th day after ischemia: 88% and 91%, 80% and 80%, 91% and 90%, 72% and 71% in rats receiving the first dose before or after arteries occlusion, respectively). The ischemia-induced sensory-motor impairment was significantly attenuated in rats receiving the first dose of gamma-hydroxybutyrate before arteries occlusion. Finally, the ischemia-induced impairment in spatial learning and memory, evaluated starting 27 days after the ischemic episode, was significantly attenuated by gamma-hydroxybutyrate, either injected first at 30 min before or 10 min after arteries occlusion. Lower doses of gamma-hydroxybutyrate had no significant effect. In conclusion, these results indicate that gamma-hydroxybutyrate provides significant protection against both histological and behavioral consequences of transient global cerebral ischemia in rats.

An intrauterine progesterone contraceptive system (52 mg) used in pre- and peri-menopausal patients with endometrial hyperplasia.

An intrauterine progesterone contraceptive system (IPCS) (52 mg) was inserted in 25 women with cystic endometrial hyperplasia. Among these women, 11 complained of heavy climacteric symptoms and also received an oestrogen replacement therapy consisting of conjugated oestrogens (0.625 mg/day) administered cyclically for 3 out of 4 wk. Prior to the therapy and after 6 mth of treatment, follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), oestrone (E1) and oestradiol (E2) plasma levels were measured and endometrial histology was evaluated. In the women receiving IPCS treatment alone, there were no significant changes in FSH, LH, PRL, E1 and E2 plasma levels. However, there were remarkable changes found in their endometrial histology. In the remaining women receiving both treatments there was a sharp decrease in FSH and LH plasma levels and an increase in E1 and E2 plasma levels, while the prolactin levels remained unchanged. With the exception of two of these women, the endometrial histology changed remarkably. The endometrial morphology of the two exceptions remained unchanged. All climacteric symptoms disappeared after the administration of both IPCS and the oestrogen replacement therapy. The remarkable changes which did occur in the endometrial histology resulted in a less active glandular epithelium and stromal decidual formation, thus proving a useful effect of treatment.

Leptomeningeal carcinomatosis presenting as progressive multineuritis: clinical, pathologic, and MRI study.

Two patients developed a persistent illness characterized clinically and electrophysiologically by asymmetric involvement of spinal roots, of cranial and peripheral nerves. In the first case the disease was not discovered clinically but only after autopsy. The primary neoplasm remained undetected at autopsy. There was profound infiltration of the leptomeninges by tumor cells with features of metastatic adenocarcinoma. In the second patient onset of neurological symptoms occurred 16 years after surgery for breast cancer, which may be reasonably considered the primary malignancy-CSF cytology was positive only in the second patient in whom Gd-DTPA MRI supported the diagnosis. Our cases demonstrate that diagnosis in leptomeningeal carcinomatosis may be a challenging clinical problem.

Antithrombotic activity of a 2-kDa heparin fragment in an experimental model of carotid artery thrombosis in rats.

The antithrombotic activity of a 2-kDa heparin fragment was studied in a rat model of common carotid artery thrombosis that causes a completely occlusive thrombus with cessation of the blood flow within 10-15 min. The compound reduced thrombus formation in a dose-dependent manner, starting from an intravenous dose of 5 mg kg-1. A dose of 20 mg kg-1 completely prevented thrombus formation and apparently induced the almost complete lysis of the already formed occlusive thrombus. At none of the doses used did the compound cause increased bleeding or the formation of haematomas. The present results indicate that low molecular weight heparins, which have an established, highly beneficial effect in venous thromboembolism, are also highly effective in an animal model of arterial thrombosis.

Resistance to bacterial aggression involving exposed nonresorbable membranes in the oral cavity.

Bacterial colonies split implanted membranes that are exposed to oral biologic fluids as a consequence of dehiscence. The clinical and histologic behavior of 14 implanted polyurethane membranes was observed during the period of exposure to oral fluids for 2, 3, 4, and 6 weeks and without dehiscence (after 8 weeks). Statistical analysis indicated that the decrease in the number of neutrophils after 5 weeks, associated with the increase in the number of activated fibroblasts, cellular debris, giant cells, and aggression of bacteria, was statistically significant (from P < .05 and P < .01 for activated fibroblasts to P <.005 and P < .001 for neutrophilic cells). The increase in bacterial passage through the polyurethane membranes and in the number of giant cells and cellular debris after 8 weeks represents late dissolution of the membranes; the progressive increase of activated fibroblasts is significant because the longer the membrane resists, the better the cells can grow and give way to the process of tissue regeneration.

Immunohistochemical detection of cell-cycle associated markers on paraffin embedded and formalin fixed needle biopsies of prostate cancer: correlation of p120 protein expression with AgNOR, PCNA/cyclin, Ki-67/MIB1 proliferation-scores and Gleason gradings.

Paraffin embedded and formalin fixed needle biopsies of prostate cancer (PC) were used to immunocytochemically detect the p120 nucleolar protein in relation to the Gleason histological gradings (GHG), the labelling indices of proliferating nuclear immunocytochemical markers (PCNA/Cyclin, Ki-67/MIB1) and the argyrophilic nucleolar region (AgNOR) rate. The twenty-six cases of PC (6 from large histological samples and 20 from needle biopsies) were equally distributed into low (< or = 6) or high (> or = 7) GHG groups. The p120 nucleolar protein immunocytochemical reaction was randomly expressed in large histological sections but uniformly distributed without gaps in needle biopsy sections. Only on the latter were quantitative values of PCNA/Cyclin (23.2 in low and 45.3 in high GHG), Ki-67/MIB1 (13.8 in low and 43.3 in high GHG) and AgNOR (5.0 in low and 7.5 in high GHG) related to those of p120 nucleolar protein (0.8 in low and 3.8 in high GHG). The values of all these cell cycle markers increased from low to high GHG of PC, all four reaching high statistical significance between the two groups (ANOVA-two tailed p < 0.0001). The PCNA/Cyclin index showed a higher positivity than the Ki-67/MIB1 index in PC with low GHG but not in PC with high GHG. In conclusion, paraffin embedded and formalin fixed PC needle biopsies exhibit a higher diagnostic PCNA/Cyclin than Ki-67/MIB1 index for cases presenting differentiated features, whereas p120 nucleolar protein detection seems to be a suitable marker of poorer outcome of PC.

[Ovarian cancer and pregnancy. Description of a clinical case]. / Carcinoma ovarico e gravidanza. Descrizione di un caso clinico.

A case of conservatively treated ovarian cancer in pregnancy is presented. Careful diagnosis is proposed and above all personalisation of each individual case so as not to use the customary, easy radical surgery.