Unconventional strategy could be the future: From target to KRAS broad range treatment.

The RAS gene family comprises genes that regulate cell growth and differentiation. KRAS, a member of this family, is often mutated in different cancers, resulting in uncontrolled cell growth and tumor development. Recent clinical trial results on KRAS inhibition in NSCLC have defined the presence of a significant proportion of patients resistant to direct G12C inhibition. The presence of co-mutations and the occurrence of secondary resistance phenomena observed in preclinical and clinical settings partly justify these poor results. In addition, all other non-G12C mutations currently remain without specific strategies. Evidence of interactions between KRAS signaling and the TME suggests potential in vitro efficacy of immune checkpoint inhibitors. In this short paper, we have reviewed the most relevant data from recent conferences, with a focus on KRAS inhibitors resistance mechanisms and interactions with the peri-tumor immune system. Commentary.

Exploiting Tertiary Lymphoid Structures to Stimulate Antitumor Immunity and Improve Immunotherapy Efficacy.

Tumor-associated tertiary lymphoid structures (TLS) have been associated with favorable clinical outcomes and response to immune checkpoint inhibitors in many cancer types, including non-small cell lung cancer. Although the detailed cellular and molecular mechanisms underlying these clinical associations have not been fully elucidated, growing preclinical and clinical studies are helping to elucidate the mechanisms at the basis of TLS formation, composition, and regulation of immune responses. However, a major challenge remains how to exploit TLS to enhance naïve and treatment-mediated antitumor immune responses. Here, we discuss the current understanding of tumor-associated TLS, preclinical models that can be used to study them, and potential therapeutic interventions to boost TLS formation, with a particular focus on lung cancer research.

Flow cytometry-assisted quantification of cell cycle arrest in cancer cells treated with CDK4/6 inhibitors.

Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (i.e., palbociclib, abemaciclib, and ribociclib) are well known for their capacity to mediate cytostatic effects by promoting cell cycle arrest in the G1 phase, thus inhibiting cancer cell proliferation. Cytostatic effects induced by CDK4/6 inhibitors can be transient or lead to a permanent state of cell cycle arrest, commonly defined as cellular senescence. Induction of senescence is often associated to metabolic modifications and to the acquisition of a senescence-associated secretory phenotype (SASP) by cancer cells, which in turn can promote or limit antitumor immunity (and thus the efficacy of CDK4/6 inhibitors) depending on SASP components. Thus, although accumulating evidence suggests that anti-cancer effects of CDK4/6 inhibitors also depend on the promotion of antitumor immune responses, assessing cell cycle arrest and progression in cells treated with palbociclib remains a key approach for investigating the efficacy of CDK4/6 inhibitors. Here, we describe a method to assess cell cycle distribution simultaneously with active DNA replication by flow cytometry in cultured hormone receptor-positive breast cancer MCF7 cells.

Early changes in circulating tumor DNA (ctDNA) predict treatment response in metastatic KRAS-mutated colorectal cancer (mCRC) patients.

The detection of RAS mutations and co-mutations in liquid biopsy offers a novel paradigm for the dynamic management of metastatic colorectal cancer (mCRC) patients. Expanding the results of the prospective OMITERC (OMIcs application from solid to liquid biopsy for a personalized ThERapy of Cancer) project, we collected blood samples at specific time points from patients who received a first-line chemotherapy (CT) for KRAS-mutated mCRC. CTC quantification was performed by CellSearch® system. Libraries from cfDNA were prepared using the Oncomine™ Colon cfDNA Assay to detect tumour-derived DNA in cfDNA. The analysis involved >240 hotspots in 14 genes. Twenty patients with KRAS-mutated mCRC treated at the Medical Oncology Unit of Careggi University Hospital were prospectively enrolled. Nine patients had available data for longitudinal monitoring of cfDNA. After 6 weeks of first-line CT an increase of KRAS-mutated clone was reported in the only patient who did not obtain disease control, while all patients with decrease of KRAS clones obtained disease control. Overall, in patients with a short (<9 months) progression-free survival (PFS) we registered, at 6 weeks, an increase in cfDNA levels and in KRAS mutations or other co-mutations, i.e. PIK3CA, FBXW7, GNAS, and TP53. In selected cases, co-mutations were able to better anticipate radiological progressive disease (PD) than the increase of KRAS-mutated clones. In conclusion, our study confirms plasma ctDNA as a crucial tool for anticipating PD at an early time point and highlights the value of a comprehensive assessment of clonal dynamics to improve the management of patients with mCRC.

Between psychopathy and deviant socialization: A close look at the mafia men.

The Italian mafia organizations represent a subculture with values, beliefs and goals that are antithetical to and undermining of the predominant society. The conduct of individual members includes such extreme violence for material gain, it may at least superficially suggest a severe personality disorder. Since the first edition of the DSM and into the 21st century, various terms have been used, sometimes interchangeably, but over time inconsistently, to designate the mentality and practices of mafia members. Only recently has the psychology of mafia members become a focus of serious scientific study. Following broader national multicenter research, the present study aimed at investigating the possible differences in psychopathy between those mafia associates who had been convicted only of mafia association (Group A, bosses), and those who were also convicted of violent crimes (Group B, soldiers). The Psychopathy Checklist-Revised (PCL-R) was administered to n = 48 male inmates convicted of mafia association (Mage 45.0 years, SD 10.9, range 20-80 years); Group A consisted of n = 26 (54%) subjects, Group B n = 22 (46%). Most of the sample (73%) did not manifest psychopathy (PCL-R ≥ 25) nor Mann-Whitney U test disclosed significant differences in the total PCL-R scores between the study groups. We found significantly higher scores of PCR-R factor 1 (interpersonal / affective) in the members of the mafia association also convicted of violent crimes (PCL-R F1, group A: 5.8 ± 3.7; group B: 7.9 ± 3.5; p < 0.05), this difference appeared explainable on the basis of a higher component of affective psychopathy. These initial results add to the limited literature on mafia and psychopathy and seem to suggest the existence of a specific component of psychopathy in the subgroup of mafiosi with overtly violent conduct.

Cellular senescence in the response of HR+ breast cancer to radiotherapy and CDK4/6 inhibitors.

BACKGROUND: Preclinical evidence from us and others demonstrates that the anticancer effects of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors can be enhanced with focal radiation therapy (RT), but only when RT is delivered prior to (rather than after) CDK4/6 inhibition. Depending on tumor model, cellular senescence (an irreversible proliferative arrest that is associated with the secretion of numerous bioactive factors) has been attributed beneficial or detrimental effects on response to treatment. As both RT and CDK4/6 inhibitors elicit cellular senescence, we hypothesized that a differential accumulation of senescent cells in the tumor microenvironment could explain such an observation, i.e., the inferiority of CDK4/6 inhibition with palbociclib (P) followed by RT (P→RT) as compared to RT followed by palbociclib (RT→P). METHODS: The impact of cellular senescence on the interaction between RT and P was assessed by harnessing female INK-ATTAC mice, which express a dimerizable form of caspase 8 (CASP8) under the promoter of cyclin dependent kinase inhibitor 2A (Cdkn2a, coding for p16Ink4), as host for endogenous mammary tumors induced by the subcutaneous implantation of medroxyprogesterone acetate (MPA, M) pellets combined with the subsequent oral administration of 7,12-dimethylbenz[a]anthracene (DMBA, D). This endogenous mouse model of HR+ mammary carcinogenesis recapitulates key immunobiological aspects of human HR+ breast cancer. Mice bearing M/D-driven tumors were allocated to RT, P or their combination in the optional presence of the CASP8 dimerizer AP20187, and monitored for tumor growth, progression-free survival and overall survival. In parallel, induction of senescence in vitro, in cultured human mammary hormone receptor (HR)+ adenocarcinoma MCF7 cells, triple negative breast carcinoma MDA-MB-231 cells and mouse HR+ mammary carcinoma TS/A cells treated with RT, P or their combination, was determined by colorimetric assessment of senescence-associated ß-galactosidase activity after 3 or 7 days of treatment. RESULTS: In vivo depletion of p16Ink4-expressing (senescent) cells ameliorated the efficacy of P→RT (but not that of RT→P) in the M/D-driven model of HR+ mammary carcinogenesis. Accordingly, P→RT induced higher levels of cellular senescence than R→TP in cultured human and mouse breast cancer cell lines. CONCLUSIONS: Pending validation in other experimental systems, these findings suggest that a program of cellular senescence in malignant cells may explain (at least partially) the inferiority of P→RT versus RT→P in preclinical models of HR+ breast cancer.

Quantification of beta-galactosidase activity as a marker of radiation-driven cellular senescence.

Cellular senescence is a permanent state of cell cycle arrest that can be triggered by different stressors, including cancer treatments (the so-called "therapy-induced senescence"), such as radiation therapy (RT). Although senescent cells do not proliferate, they remain metabolically active and play a critical role in tumor progression, metastasis, and response to therapy. Therefore, investigating the induction of cellular senescence upon RT treatment is a critical read out for investigating RT efficacy or combinatorial strategies in cancer research. Senescent cells are characterized by a plethora of markers, including an increased content and activity of lysosomes, which can be detected by the activity of the lysosomal enzyme senescence-associated ß-galactosidase. In this chapter, we present a protocol for the gold standard cytochemical method for quantification of the activity of the senescence-associated ß-galactosidase in irradiated murine breast cancer cells in vitro.

Challenges in the treatment of small cell lung cancer in the era of immunotherapy and molecular classification.

For many years the standard of care for small cell lung cancer (SCLC) has remained unchanged. Despite decades of active research, current treatment options are limited and the prognosis of patients with extended disease (ED) SCLC remains poor. The introduction of immune checkpoint inhibitors (ICIs) represents an exception and the only recent approval for ED-SCLC. However, the magnitude of benefit obtained with immunotherapy in SCLC is much more modest than that observed in other malignancies. Different pro-immunogenic or immunosuppressive features within the tumor microenvironment of SCLC may either modulate the sensitivity to immunotherapy or conversely dampen the efficacy of ICIs. Beside immunotherapy, a deeper understanding of the molecular biology of SCLC has led to the identification of new therapeutic targets for this lethal malignancy. Recent epigenetic and gene expression studies have resulted into a new molecular classification of four distinct subtypes of SCLC, defined by the relative expression of key transcription regulators and each characterized by specific therapeutic vulnerabilities. This review discusses the rationale for immunotherapy in SCLC and summarizes the main ICIs-trials in this tumor. We provide also an overview of new potential therapeutic opportunities and their integration with the new molecular classification of SCLC.

Profiling Players Involved in Overkill: An Analysis of 71 Cases in Central Italy.

"Overkill" is characterized by the finding of excessive wounds on the victim's body. Despite the large use of this term, it does not have a definition in the literature yet. Our study aimed to analyze the information related to the dynamics of overkill cases, collecting objective variables, and producing a profile of the players involved in this type of homicides. Data on 71 overkill cases from reports of the autopsies performed in the Section of Legal Medicine of the University of Rome Tor Vergata from 1 January 2000 to 31 December 2020 were collected. The victims and the perpetrators of overkill shared similar characteristics: they were usually male aged between 20 and 50 years, more likely in the 20-35 years range; victims however also showed another age peak in the range 50-55 years. The type of damage can be linked to the sole action of a cold weapon or the simultaneous use of multiple harmful tools with no significant differences. The most common motive proved to be a dispute for futile reasons; however, in the case of a single perpetrator of the crime, the economic and passionate motives are as frequent as the previous one. Implications of the findings and avenues for future research are discussed.

Cytofluorometric assessment of cell cycle progression in irradiated cells.

Radiation therapy (RT) is well known for its capacity to mediate cytostatic and cytotoxic effects upon the accumulation of unrepaired damage to macromolecules, notably DNA. The ability of ionizing radiation to prevent malignant cells from replicating and to cause their demise is indeed an integral component of the anticancer activity of RT. Neoplastic cells are generally more sensitive to the cytostatic and cytotoxic effects of RT than their healthy counterparts as they exhibit increased proliferative rate and limited capacity for DNA repair. This provides a rather comfortable therapeutic window for clinical RT usage, especially with the development of novel, technologically superior RT modalities that minimize the exposure of normal tissues. Thus, while accumulating evidence indicates that cancer control by RT also involves the activation of tumor-targeting immune responses, assessing cell cycle progression in irradiated cells remains a central approach for investigating radiosensitivity in preclinical tumor models. Here, we detail a simple, flow cytometry-assisted method to simultaneously assess cell cycle distribution and active DNA replication in cultured estrogen receptor (ER)+ breast cancer MCF7 cells. With minimal variations, the same technique can be straightforwardly implemented to a large panel of human and mouse cancer cell lines.

Quantification of cytosolic DNA species by immunofluorescence microscopy and automated image analysis.

When employed according to specific doses and fractionation schedules, radiation therapy (RT) elicits potent tumor-targeting immune responses that rely on the secretion of type I interferon (IFN) by irradiated cancer cells. Most often, this is initiated by the ability of RT to promote the cytosolic accumulation of double-stranded DNA (dsDNA) molecules, which are detected by cyclic GMP-AMP synthase (CGAS) to engage the stimulator of interferon response cGAMP interactor 1 (STING1)-dependent transactivation of type I IFN-coding genes via interferon regulatory factor 3 (IRF3). Here, we describe a simple protocol for the quantification of cytosolic dsDNA species by immunofluorescence microscopy coupled to automated image analysis, as enabled by precise sample processing conditions that permeabilize plasma-but not nuclear or inner mitochondrial-membranes. As compared to subcellular fractionation-based techniques, this approach is compatible with assessments in individual cells aimed at gauging inter-cellular heterogeneity, as well as subcellular tests including co-localization studies.

RT-PCR-assisted quantification of type I IFN responses in irradiated cancer cells.

It is now clear that radiation therapy (RT) can be delivered in doses and according to fractionation schedules that actively elicit immunostimulatory effects. While such effects are often sufficient to drive potent anticancer immunity culminating with systemic disease eradication, the immunostimulatory activity of RT stands out as a promising combinatorial partner for bona fide immunotherapeutics including immune checkpoint inhibitors (ICIs). Accumulating preclinical and clinical evidence indicates that the secretion of type I interferon (IFN) by irradiated cancer cells is a sine qua non for RT to initiate ICI-actionable tumor-targeting immune responses. Here, we detail a simple protocol to quantitatively assess type I IFN responses in irradiated mouse hormone receptor (HR)+ TS/A cells by RT-PCR. With minimal variations, the same technique can be straightforwardly adapted to quantify type I IFN-associated transcriptional responses in a variety of human and mouse cancer cells maintained in vitro.

A Rapid Fatal Outcome of Iliopsoas Hematoma: Clinical and Autopsy Findings.

A case of massive muscular bleeding of iliopsoas resulting in lethal exsanguination is presented. The intramuscular bleeding occurred spontaneously in an old man with heart failure, presented to the emergency department after the acute onset of shortness of breath, and treated with therapeutic doses of antiplatelets and heparin to prevent thrombosis. On the sixth day of recovery, pain in the left lumbar region develops while there was a decrease in hemoglobin level. Computed tomography (CT) scan revealed a 10 × 3 cm hematoma of the left iliac muscle. The treatment was immediately stopped, but within 6 hours, the death was confirmed. The autopsy revealed that the hematoma, and its increased size since the latest imaging assessment, was the leading cause of death. Particularly in older patients with comorbidity, even in those with clotting parameters in the therapeutic range, the potential for fatal result of iliopsoas muscle bleeding should be considered. Identifying potential patience with increased risk of this complication could be important, especially in pandemic time of COVID-19, when the use of anticoagulant therapy-both for treatment and for prevention of severe disease-has become massive and addressed also to people without previous and specific pathologies.

Targeting oncogene and non-oncogene addiction to inflame the tumour microenvironment.

Immune checkpoint inhibitors (ICIs) have revolutionized the clinical management of multiple tumours. However, only a few patients respond to ICIs, which has generated considerable interest in the identification of resistance mechanisms. One such mechanism reflects the ability of various oncogenic pathways, as well as stress response pathways required for the survival of transformed cells (a situation commonly referred to as 'non-oncogene addiction'), to support tumour progression not only by providing malignant cells with survival and/or proliferation advantages, but also by establishing immunologically 'cold' tumour microenvironments (TMEs). Thus, both oncogene and non-oncogene addiction stand out as promising targets to robustly inflame the TME and potentially enable superior responses to ICIs.

Health Management in Italian Prisons during COVID-19 Outbreak: A Focus on the Second and Third Wave.

The SARS-CoV-2 spread is a threatening and challenging issue for correctional systems worldwide because of many factors, particularly overcrowding and of the intrinsic characteristics of the population. The prevention measures adopted by the Italian Government were aimed to protect and preserve both inmates' and prison workers' health. The present study aimed to evaluate the efficacy of the adopted strategies. METHODS: Data regarding Italian prisons' occupation and prisoners' population from January 2019 to June 2021, as well as the cumulative weekly increase of confirmed cases and the number of doses of vaccine administered among the population of inmates, the prison workers, and Italian population from November 2020 to the end of June 2021, were collected. RESULTS: Prisons' occupation dropped from 120% to 106% after the beginning of the pandemics. The confirmed cases between inmates were consistently lower than among the Italian population and prison workers. A time-series chart showed a time lag of one week between the peaks of the different population. CONCLUSIONS: The containing strategies adopted by the Italian correctional system have proved their effectiveness in terms of the prevention and protection of both inmate and staff health.

From overkill to beheading: A case report of a schizophrenic matricide.

Initial evidence exists on a subtype of matricide committed by subjects suffering from severe mental disorders. Matricide perpetrators often undergo a forensic psychiatric evaluation during the subsequent criminal trial because of supposed legal insanity. The few studies on matricide by mentally disordered perpetrators suggested a possible association between such extreme form of violence and schizophrenia spectrum disorders, especially in case of active delusions or hallucinations. METHODS: we analyze a case of a young male with a recent diagnosis of psychotic disorder who committed matricide by inflicting multiple injuries and beheading. Data emerging from the forensic pathological analysis of the victim, as well as the forensic psychiatric analysis of the matricide perpetrator are discussed within an interdisciplinary perspective. RESULTS: the autopsy revealed multiple stab wounds in the regions of the upper limbs, abdomen, chest, and neck, the latter determining beheading. The forensic psychiatric evaluation yielded a diagnosis of schizophrenia and clarified the causal role in the homicidal behavioral of active psychotic symptoms. CONCLUSION: the specific type of delusional content, and perpetrator-victim relationship might contribute explaining a subtype of extremely violent homicide in recent onset schizophrenia.

Radiotherapy as a tool to elicit clinically actionable signalling pathways in cancer.

A variety of targeted anticancer agents have been successfully introduced into clinical practice, largely reflecting their ability to inhibit specific molecular alterations that are required for disease progression. However, not all malignant cells rely on such alterations to survive, proliferate, disseminate and/or evade anticancer immunity, implying that many tumours are intrinsically resistant to targeted therapies. Radiotherapy is well known for its ability to activate cytotoxic signalling pathways that ultimately promote the death of cancer cells, as well as numerous cytoprotective mechanisms that are elicited by cellular damage. Importantly, many cytoprotective mechanisms elicited by radiotherapy can be abrogated by targeted anticancer agents, suggesting that radiotherapy could be harnessed to enhance the clinical efficacy of these drugs. In this Review, we discuss preclinical and clinical data that introduce radiotherapy as a tool to elicit or amplify clinically actionable signalling pathways in patients with cancer.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.