BL-MOL-AR Project, Preliminary Results about Liquid Biopsy: Molecular Approach Experience and Research Activity in Oncological Settings.

Background Liquid biopsy is mainly used to identify tumor cells in pulmonary neoplasms. It is more often used in research than in clinical practice. The BL-MOL-AR study aims to investigate the efficacy of next-generation sequencing (NGS) and clinical interpretation of the circulating free DNA (cfDNA) levels. This study reports the preliminary results from the first samples analyzed from patients affected by various neoplasms: lung, intestinal, mammary, gastric, biliary, and cutaneous. Methods The Biopsia Liquida-Molecolare-Arezzo study aims to enroll cancer patients affected by various malignancies, including pulmonary, intestinal, advanced urothelial, biliary, breast, cutaneous, and gastric malignancies. Thirty-nine patients were included in this preliminary report. At time zero, a liquid biopsy is executed, and two types of NGS panels are performed, comprising 17 genes in panel 1, which is already used in the routine tissue setting, and 52 genes in panel 2. From the 7th month after enrollment, 10 sequential liquid biopsies are performed up to the 17th month. The variant allele frequency (%) and cfDNA levels (ng/mL) are measured in every plasmatic sample. Results The NGS results obtained by different panels are similar even though the number of mutations is more concordant for lung pathologies. There are no significant differences in the actionability levels of the identified variants. Most of the molecular profiles of liquid biopsies reflect tissue data. Conclusions Preliminary data from this study confirm the need to clarify the limitations and potential of liquid biopsy beyond the lung setting. Overall, parameters related to cfDNA levels and variant allele frequency could provide important indications for prognosis and disease monitoring.

Radioimmune Imaging of α4ß7 Integrin and TNFα for Diagnostic and Therapeutic Applications in Inflammatory Bowel Disease.

Imaging using radiolabelled monoclonal antibodies can provide, non-invasively, molecular information which allows for the planning of the best treatment and for monitoring the therapeutic response in cancer, as well as in chronic inflammatory diseases. In the present study, our main goal was to evaluate if a pre-therapy scan with radiolabelled anti-α4ß7 integrin or radiolabelled anti-TNFα mAb could predict therapeutic outcome with unlabelled anti-α4ß7 integrin or anti-TNFα mAb. To this aim, we developed two radiopharmaceuticals to study the expression of therapeutic targets for inflammatory bowel diseases (IBD), to be used for therapy decision making. Both anti-α4ß7 integrin and anti-TNFα mAbs were successfully radiolabelled with technetium-99m with high labelling efficiency and stability. Dextran sulfate sodium (DSS)-induced colitis was used as a model for murine IBD and the bowel uptake of radiolabelled mAbs was evaluated ex vivo and in vivo by planar and SPECT/CT images. These studies allowed us to define best imaging strategy and to validate the specificity of mAb binding in vivo to their targets. Bowel uptake in four different regions was compared to immunohistochemistry (IHC) score (partial and global). Then, to evaluate the biomarker expression prior to therapy administration, in initial IBD, another group of DSS-treated mice was injected with radiolabelled mAb on day 2 of DSS administration (to quantify the presence of the target in the bowel) and then injected with a single therapeutic dose of unlabelled anti-α4ß7 integrin or anti-TNFα mAb. Good correlation was demonstrated between bowel uptake of radiolabelled mAb and immunohistochemistry (IHC) score, both in vivo and ex vivo. Mice treated with unlabelled α4ß7 integrin and anti-TNFα showed an inverse correlation between the bowel uptake of radiolabelled mAb and the histological score after therapy, proving that only mice with high α4ß7 integrin or TNFα expression will benefit of therapy with unlabelled mAb.

"Urethral-Sparing" Robotic Radical Prostatectomy: Critical Appraisal of the Safety of the Technique Based on the Histologic Characteristics of the Prostatic Urethra.

BACKGROUND: The prostatic urethra (PU) is conventionally resected during robot-assisted radical prostatectomy (RALP). Recent studies demonstrated the feasibility of the extended PU preservation (EPUP). AIMS: To describe the histologic features of the PU. METHODS: The PU was evaluated using cystoprostatectomy and RALP specimens. Cases of PU infiltration by prostate cancer or distortion by benign hyperplastic nodules were excluded. The thickness of the chorion and distance between the urothelium and prostate glands were measured. Prostate-specific antigen expression in the PU epithelium was evaluated with immunohistochemistry. Descriptive statistics were used. RESULTS: Six specimens of PU were examined. Histologically, the following layers of the PU were observed: (1) urothelium with basal membrane, (2) chorion, and (3) prostatic peri-urethral fibromuscular tissue. The chorion measures between 0.2 and 0.4 mm. There is not a distinct urethral muscle layer, but rather muscular fibers that originate near the prostatic stroma and are distributed around the PU. This muscular tissue appears to be mainly represented in the basal and apical urethra, but not in the middle urethra. The mean distance between the chorion and prostatic glands is 1.74 mm, with significant differences between base of the prostate, middle urethral portion, and apex (2.5 vs. 1.49 vs. 1.23 mm, respectively). PSA-expressing cells are abundant in the PU epithelium, coexisting with urothelial cells. CONCLUSIONS: The exiguity of thickness of the PU chorion, short distance from glandular tissue, and coexistence of PSA-expressing cells in the epithelium raise important concerns about the oncologic safety of EPUP.

Extensive Histopathological Characterization of Inflamed Bowel in the Dextran Sulfate Sodium Mouse Model with Emphasis on Clinically Relevant Biomarkers and Targets for Drug Development.

This study aims to develop a reliable and reproducible inflammatory bowel disease (IBD) murine model based on a careful spatial-temporal histological characterization. Secondary aims included extensive preclinical studies focused on the in situ expression of clinically relevant biomarkers and targets involved in IBD. C57BL/6 female mice were used to establish the IBD model. Colitis was induced by the oral administration of 2% Dextran Sulfate Sodium (DSS) for 5 days, followed by 2, 4 or 9 days of water. Histological analysis was performed by sectioning the whole colon into rings of 5 mm each. Immunohistochemical analyses were performed for molecular targets of interest for monitoring disease activity, treatment response and predicting outcome. Data reported here allowed us to develop an original scoring method useful as a tool for the histological assessment of preclinical models of DSS-induced IBD. Immunohistochemical data showed a significant increase in TNF-α, α4ß7, VEGFRII, GR-1, CD25, CD3 and IL-12p40 expression in DSS mice if compared to controls. No difference was observed for IL-17, IL-23R, IL-36R or F480. Knowledge of the spatial-temporal pattern distribution of the pathological lesions of a well-characterized disease model lays the foundation for the study of the tissue expression of meaningful predictive biomarkers, thereby improving translational success rates of preclinical studies for a personalized management of IBD patients.

Pathology of triple negative breast cancer.

Triple negative breast cancer (TNBC) is a subtype of breast tumor lacking hormone receptors expression and HER2 gene amplification and represents 24 % of newly diagnosed breast neoplasms. In this review, pathological aspects of triple-negative breast cancer are illustrated, with particular attention to the seminal studies that defined this subtype of breast cancer by a molecular point of view. This paper also focuses on practical issues raised in clinical routine by the introduction of genetic expression breast cancer profiling and the innovative prognostic and predictive impact on triple-negative breast cancer pathology. Moreover, histopathological aspects of triple-negative neoplasms are also mentioned, underlying the importance of histologic diagnosis of particular cancer subtypes with decisive impact on clinical outcome. Importantly, focus on new therapeutic frontier represented by immunotherapy is illustrated, with particular mention of immune checkpoint inhibitors introduction in TNBC therapy and their impact on future treatments.

Current management of central nervous system metastasis from cholangiocarcinoma: the neurosurgical perspective. Literature review.

Purpose: Central nervous system (CNS) metastasis from cholangiocarcinoma (CCA) are extremely rare and associated prognosis is poor. The involvement of the CNS by metastatic CCA may discourage any further treatment; however, data from the literature are discordant, due to recent reports of exceptionally long follow-up after surgical resection of a brain metastasis.Material and Methods: Electronic databases, such as PubMed/MEDLINE and Google Scholar, were analyzed for studies published up to October 2018 using the search term "cholangiocarcinoma and central nervous system metastasis or brain metastasis".Results: We found a total of 18 studies cited in the literature of the 30 year span analyzed, and we added a new case we treated at our Institution, reaching a series of 32 patients. Among these, 7 patients had leptomeningeal dissemination and 25 presented solid CNS metastasis. We analyzed the treatment options and the outcomes, addressing also histopathological insights on tumoral markers possibly involved in the mechanism of metastases of cholangiocarcinomasConclusions: According to the literature data, the outcome remains poor, particularly for those with leptomeningeal diffusion. Nevertheless, long term follow-up is reported in case of surgical resection of CNS metastasis, when there is a good control of the primary tumor. Actually, the majority of patients are often in advanced state of disease at diagnosis and not suitable for initial resective procedure; in these cases neo adjuvant and adjuvant therapies have provided a slight improvement of the outcome.

Animal models for the study of inflammatory bowel diseases: a meta-analysis on modalities for imaging inflammatory lesions.

Inflammatory bowel diseases are lifelong disorders affecting the gastrointestinal tract characterized by intermittent disease flares and periods of remission with a progressive and destructive nature. Unfortunately, the exact etiology is still not completely known, therefore a causal therapy to cure the disease is not yet available. Current treatment options mainly encompass the use of non-specific anti-inflammatory agents and immunosuppressive drugs that cause significant side effects that often have a negative impact on patients' quality of life. As the majority of patients need a long-term follow-up it would be ideal to rely on a non-invasive technique with good compliance. Currently, the gold standard diagnostic tools for managing IBD are represented by invasive procedures such as colonoscopy and histopathology. Nevertheless, recent advances in imaging technology continue to improve the ability of imaging techniques to non-invasively monitor disease activity and treatment response in preclinical models of IBD. Novel and emerging imaging techniques not only allow direct visualization of intestinal inflammation, but also enable molecular imaging and targeting of specific alterations of the inflamed murine mucosa. Furthermore, molecular imaging advances allow us to increase our knowledge on the critical biological pathways involved in disease progression by characterizing in vivo processes at a cellular and molecular level and enabling significant improvements in the understanding of the etiology of IBD. This review presents a critical and updated overview on the imaging advances in animal models of IBD. Our aim is to highlight the potential beneficial impact and the range of applications that imaging techniques could offer for the improvement of the clinical monitoring and management of IBD patients: diagnosis, staging, determination of therapeutic targets, monitoring therapy and evaluation of the prognosis, personalized therapeutic approaches.

Hypertension in kidney transplantation is associated with an early renal nerve sprouting.

BACKGROUND.: Normalization of arterial pressure occurs in just a few patients with hypertensive chronic kidney disease undergoing kidney transplantation. Hypertension in kidney transplant recipients may be related to multiple factors. We aimed to assess whether hypertension in kidney-transplanted patients may be linked to reinnervation of renal arteries of the transplanted kidney. METHODS.: We investigated renal arteries innervation from native and transplanted kidneys in three patients 5 months, 2 years and 11 years after transplantation, respectively. Four transplanted kidneys from non-hypertensive patients on immunosuppressive treatment without evidence of hypertensive arteriolar damage were used as controls. RESULTS: . Evidence of nerve sprouting was observed as early as 5 months following transplantation, probably originated from ganglions of recipient patient located near the arterial anastomosis and was associated with mild hypertensive arteriolar damage. Regeneration of periadventitial nerves was already complete 2 years after transplantation. Nerve density tended to reach values observed in native kidney arteries and was associated with hypertension-related arteriolar lesions in transplanted kidneys. Control kidneys, albeit on an immunosuppressive regimen, presented only a modest regeneration of sympathetic nerves. CONCLUSIONS: . Our results suggest that the considerable increase in sympathetic nerves, as found in patients with severe arterial damage, may be correlated to hypertension rather than to immunosuppressive therapy, thus providing a morphological basis for hypertension recurrence despite renal denervation.

Co-expression of receptors of the HER family correlates with clinical outcome in non-small cell lung cancer (NSCLC).

HER family receptors play a critical role in lung carcinogenesis. There is a growing body of evidence showing that cooperation between them contributes to a more aggressive tumor phenotype and impacts on their response to targeted therapy. We explored immunohistochemical co-expression of HER family receptors (HER1, HER2, HER3, HER4) and its potential role as prognostic factor in resected non-small cell lung cancer (NSCLC). Expression of HER family receptors was assessed by immunohistochemistry on 125 surgically resected NSCLC. Kaplan-Meier estimates of overall survival (OS), disease-free survival (DFS), and time to recurrence were calculated for clinical variables and HER expression, using the Cox model for multivariate analysis. HER1 and HER3 expression was detected more frequently in squamous cell carcinoma (p = 0.002 and p = <0.001, respectively). HER4 was more often expressed in patients older than 60 years (p = 0.02) and in tumors of low histological grade (p = 0.04). Cases which expressed only HER1 had a worse DFS (p = 0.01) and OS (p = 0.01) compared to cases expressing HER1 and one or more of the other family members and to cases which did not express HER1 but one of the other HERs. By multivariate analysis, stage was an independent prognostic factor for DFS and OS. Furthermore, different patterns of co-expression of HER family receptors showed a statistically significant correlation with a shorter DFS (p = 0.03) and OS (p = 0.02). Our findings suggest that expression of HER1 only is correlated with worse DFS and OS. A better understanding of the functional relationships between these receptors may lead to a useful predictive indicator of response to targeted therapy.