Variant alleles in factor V, prothrombin, plasminogen activator inhibitor-1, methylenetetrahydrofolate reductase and risk of thromboembolism in metastatic colorectal cancer patients treated with first-line chemotherapy plus bevacizumab.

Single-nucleotide polymorphisms (SNPs) related to hereditary thrombophilia were investigated as risk factors for thromboembolism in cancer patients. Their effect in metastatic colorectal cancer (mCRC) has never been explored so far. Our aim was to analyse the effect of coagulation factor V (FVL G1691A), prothrombin (PT G20210A), methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) and plasminogen activator inhibitor type 1 (PAI-1 5G/4G) allelic variants in this setting. Fifty-two patients treated with first-line chemotherapy plus bevacizumab who developed a thromboembolic event in their lifetime were initially genotyped. A contemporary cohort of 127 patients who did not experience any thromboembolic event was also analysed. DNA was extracted from peripheral blood and genotypes were determined by real-time PCR, using LightSNiP (TIB MOLBIOL) on LightCcler 480 (Roche). The association between thromboembolism and SNPs was investigated by univariable and multivariable analyses. All SNPs were in Hardy-Weinberg equilibrium (χ2 test P>0.20). FVL G1691A and PT G20210A were present only in heterozygosis in 4 (2.2%) and 7 (3.9%) patients, respectively; MTHFR C677T in homozygosis in 29 (16.2%), MTHFR A1298C in homozygosis in 13 (7.3%); PAI-1 5G/4G in 98 (54.7%) and 4G/4G in 41 (23%) patients. At univariable analysis, treatment duration was significantly associated with thromboembolism (P<0.001), whereas gender, age, obesity, platelets count and chemotherapy backbone were not. Similarly, FVL G1691A and PT G20210A as well as MTHFR C677T and PAI-1 4G allele were significantly associated, whereas MTHFR A1298C was not. At multivariable model including PT G20210A, MTHFR C677T and PAI-1 4G (age, obesity, treatment duration and chemotherapy backbone were included as adjustment factors), the three SNPs were significantlty associated with higher risk of thromboembolism (P=0.025, <0.0001 and P=0.033, respectively). Further validation studies are warranted in order to design a prospective trial of thromboprophylaxis in mCRC patients with high-risk genotypes.

HER2 loss in HER2-positive gastric or gastroesophageal cancer after trastuzumab therapy: Implication for further clinical research.

Mechanisms of acquired resistance to trastuzumab-based treatment in gastric cancer are largely unknown. In this study, we analyzed 22 pairs of tumor samples taken at baseline and post-progression in patients receiving chemotherapy and trastuzumab for advanced HER2-positive [immunohistochemistry (IHC) 3+ or 2+ with in-situ hybridization (ISH) amplification] gastric or gastroesophageal cancers. Strict clinical criteria for defining acquired trastuzumab resistance were adopted. Loss of HER2 positivity and loss of HER2 over-expression were defined as post-trastuzumab IHC score <3+ and absence of ISH amplification, and IHC "downscoring" from 2+/3+ to 0/1+, respectively. HER2 IHC was always performed, while ISH was missing in 3 post-progression samples. Patients with initial HER2 IHC score 3+ and 2+ were 14 (64%) and 8 (36%), respectively. Loss of HER2 positivity and HER2 over-expression was observed in 32 and 32% samples, respectively. The chance of HER2 loss was not associated with any of the baseline clinicopathological variables. The only exception was in patients with initial IHC score 2+ versus 3+, for both endpoints of HER2 positivity (80 vs. 14%; p = 0.008) and HER2 over-expression (63 vs. 14%; p = 0.025). As already shown in breast cancer, loss of HER2 may be observed also in gastric cancers patients treated with trastuzumab-based chemotherapy in the clinical practice. This phenomenon may be one of the biological reasons explaining the failure of anti-HER2 second-line strategies in initially HER2-positive disease.

Toward the molecular dissection of peritoneal pseudomyxoma.

BACKGROUND: Outcome of pseudomyxoma peritonei (PMP) after cytoreductive surgery (CRS) and hypertermic intraperitoneal chemotherapy (HIPEC) is heterogeneous even after adjusting for clinico-pathological prognostic variables. The identification of additional prognostic or even predictive biomarkers is an unmet clinical need. PATIENTS AND METHODS: Forty patients with mucinous appendiceal tumors and PMP were clinically eligible and had evaluable tumor samples obtained after CRS and HIPEC. We carried out next-generations sequencing (NGS) of 50 gene's hotspot regions contained in the Hotspot Cancer Panel v2 using the Ion Torrent Personal Genome Machine platform (Life Technologies). RESULTS: KRAS and GNAS mutations were found in 72% and 52%, and their allelic frequency was below 10% in 55% and 43% of samples, respectively. KRAS and GNAS mutations were associated with worse progression-free survival (PFS) at univariate analysis (P = 0.006 and 0.011, respectively). At multivariate analysis, only KRAS mutations were independently associated with PFS (P = 0.012); GNAS mutations were not-being significantly associated with other poor prognostic features such as incomplete cytoreduction or KRAS mutations. Validation of results was carried out in an independent bi-institutional cohort of 25 patients and the prognostic effect of KRAS mutations was again confirmed in the multivariate model (P = 0.029). NGS approach allowed the discovery of other potentially druggable mutations such as those in PI3K, AKT, LKB1, FGFR3 and PDGFRA. CONCLUSIONS: Given the homogeneity of this series and the sensitivity of NGS in this low-cellularity tumor, we demonstrated for the first time a poor prognostic role of KRAS mutations.

Italian law no. 1/2021 on the subject of vaccination against Covid-19 in people with mental disabilities within the nursing homes.

ABSTRACT: The Coronavirus Disease-19 (Covid-19) pandemic, in the last year, has resulted in a significant number of infections and deaths among nursing homes' residents. This phenomenon has set up the necessity to subject these patients, often suffering from mental disabilities to a vaccination against Covid-19. However, vaccination has long been the subject of public atten-tion, being regulated differently in many European countries. In Italy, the Ministry of Health has given priority, vaccination-wise, to health facilities' patients. The government has regulated through-law no. 1 of January 5, 2021, art. 5, the manifestation of consent to be Covid-19 vac-cinated in incapacitated subjects admitted to assisted health facilities. This rule arose from the need to protect fragile individuals as well as providing real dispositions for the involved health professionals. Nursing homes' elderly guests could be divided into four catego-ries: a) subjects capable to express their will (affected by physical problems); b) subjects who, due to varying degrees of incapacitation, have their own legal guardian, curator or support administrator, ap-pointed in accordance with the law; c) incapacitated subjects without legal representatives d) subjects who, pursuant to law no. 219/2017, have appointed their own trustee. This paper provides for a clear exemplification of all the possible scenarios identified by the Italian law no.1/2021.

Recent Judgement of the Italian Judiciary about medical assisted procreation (MAP): is informed consent valid after parents separation?

ABSTRACT: Law No 40/2004 regulates in Italy the matter of medically assisted procreation (MAP). Recently, the Tribunal of Capua Vetere expressed its position on the subject of informed consent in a case of MAP. In the specific case, a couple entered the preliminary stages of the PMA procedures, carrying out the fertilization of the ovum and the embryo production. Afterwards, the couple separated and the man denied consent to the continuation of the MAP. The woman, willing to proceed with the implantation, the woman made an urgent judicial appeal, obtaining the judge's permission to transfer the embryo to the uterus. This paper analyses the different bioethical positions on MAP's informed consent. In fact, on the one hand, the paper highlight what is set out in Law 219/2017 which provides for the possibility of the patient to revoke at any time the consent to the treatment given. On the other hand, it should be noted that Law 40/2004, willing to protect the embryo, establishes the irrevocability of the position of parental consent after fertilization. The judgment in question seems to favour this latter position, placing itself in the protection of the cryopreserved embryo and recall-ing the principle of entrustment following the fertilization of the egg. Nevertheless, the matter is controversial a consistent amount of legal developments are expected to arise in the next future.

Production and Easy One-Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzymatic Assay (ELISA).

Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (Culicoides spp.) and is caused by Bluetongue virus (BTV). BTV is the type species of the Orbivirus genus within the Reoviridae family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay.

The effects of osmotic shock on the organ cultured mammalian ocular lens.

Freshly isolated rabbit lenses were incubated in anosmolar culture media to study the effects of osmotic shock on lens transparency and protein synthesis. The rate of protein synthesis was measured using three markers, namely alpha-crystallin, a water soluble protein, vimentin, a cytoskeletal protein and MP-26, an intrinsic membrane protein. It was found that only when lenses were cultured in a hyperosmolar medium of 450 mOsM, was there a significant decrease in the rate of lens protein synthesis. The hyperosmolar medium inhibited the synthesis of all three marker proteins. Lens clarity also depended on the solute used to prepare the hyperosmolar medium. Our experimental data showed that there is no relation between decreased protein synthesis and the appearance of lens opacity in the organ cultured rabbit lens.

Iris-ciliary complex inhibits protein synthesis by organ cultured mammalian ocular lens cells.

Freshly isolated rabbit ocular lenses with or without adhering iris-ciliary complex were organ cultured. It was found that lenses with iris-ciliary complex showed decreased protein synthetic ability. Vimentin, a cytoskeletal protein and MP-26, a membrane associated protein, were used as markers for protein synthetic activity. Removal of the iris-ciliary complex at least partially restores the protein synthetic capability of the lens. The ocular lenses cultured with iris-ciliary complex for 48 hr showed marked equatorial opacities.

Analysis of a failed alumina THR ball head.

The favourable properties, on which is based the successful history of the use of alumina in orthopaedic surgery, are due to the tough control made by manufacturers on the whole process from raw materials purity to the quality controls. The minimum requirements of a medical grade alumina are stated from 1981 in ISO 6474 standard. Nevertheless, due to the voluntary character of standards, and lack of national and international laws, it was possible in the past to introduce into the market materials with characteristics not in compliance with the standard ISO 6474. A THR implanted on a fifty years old patient was revised three years after implantation, due to the failure of the alumina ball head. To understand the causes of such an early failure, the fragments of the retrieved ball head were submitted to a series of non destructive and destructive controls. The results show that the poor quality of the design and manufacture of the implant was the origin of the failure.

The role of specific macronutrient availability in the effect of food restriction on length of lactational diestrus in rats.

In lactating rats, food restriction for the first two weeks postpartum extends the period of lactational diestrus by about 1 week. In these studies we investigated whether this effect results from caloric restriction or the reduced availability of a specific macronutrient. In Experiment 1 lactating rats nursing litters of eight pups were assigned to one of four conditions: 1) ad lib. fed; 2) protein-restricted; 3) carbohydrate-restricted; and 4) fat-restricted. Animals in all the restricted conditions were given access to 50% of ad lib. intake of the appropriate nutrient for Days 1-14 postpartum and ad lib. access to the other two macronutrients. In Experiment 2, ad lib. supplementation from one macronutrient source was provided to lactating rats given restricted access to a composite diet. No differential effect of specific macronutrient deprivation or supplementation on length of lactational diestrus was observed in these studies. Thus, the results of both studies are consistent with the hypothesis that caloric restriction plays a primary role in inducing the prolongation of lactational diestrus in food-restricted rats.