Prevention of prostate cancer metastasis by a CRISPR-delivering nanoplatform for interleukin-30 genome editing.

Prostate cancer (PC) is a leading cause of cancer-related deaths in men worldwide. Interleukin-30 (IL-30) is a PC progression driver, and its suppression would be strategic for fighting metastatic disease. Biocompatible lipid nanoparticles (NPs) were loaded with CRISPR-Cas9gRNA to delete the human IL30 (hIL30) gene and functionalized with anti-PSCA-Abs (Cas9hIL30-PSCA NPs). Efficiency of the NPs in targeting IL-30 and the metastatic potential of PC cells was examined in vivo in xenograft models of lung metastasis, and in vitro by using two organ-on-chip (2-OC)-containing 3D spheroids of IL30+ PC-endothelial cell co-cultures in circuit with either lung-mimicking spheroids or bone marrow (BM)-niche-mimicking scaffolds. Cas9hIL30-PSCA NPs demonstrated circulation stability, genome editing efficiency, without off-target effects and organ toxicity. Intravenous injection of three doses/13 days, or five doses/20 days, of NPs in mice bearing circulating PC cells and tumor microemboli substantially hindered lung metastasization. Cas9hIL30-PSCA NPs inhibited PC cell proliferation and expression of IL-30 and metastasis drivers, such as CXCR2, CXCR4, IGF1, L1CAM, METAP2, MMP2, and TNFSF10, whereas CDH1 was upregulated. PC-Lung and PC-BM 2-OCs revealed that Cas9hIL30-PSCA NPs suppressed PC cell release of CXCL2/GROß, which was associated with intra-metastatic myeloid cell infiltrates, and of DKK1, OPG, and IL-6, which boosted endothelial network formation and cancer cell migration. Development of a patient-tailored nanoplatform for selective CRISPR-mediated IL-30 gene deletion is a clinically valuable tool against PC progression.

Effect of 5-Aminolevulinic Acid (5-ALA) in "ALADENT" Gel Formulation and Photodynamic Therapy (PDT) against Human Oral and Pancreatic Cancers.

Oral squamous-cell and pancreatic carcinomas are aggressive cancers with a poor outcome. Photodynamic therapy (PDT) consists of the use of photosensitizer-induced cell and tissue damage that is activated by exposure to visible light. PDT selectively acts on cancer cells, which have an accumulation of photosensitizer superior to that of the normal surrounding tissues. 5-aminolevulinic acid (5-ALA) induces the production of protoporphyrin IX (PpIX), an endogenous photosensitizer activated in PDT. This study aimed to test the effect of a new gel containing 5% v/v 5-ALA (ALAD-PDT) on human oral CAL-27 and pancreatic CAPAN-2 cancer cell lines. The cell lines were incubated in low concentrations of ALAD-PDT (0.05%, 0.10%, 0.20%, 0.40%, 0.75%, 1.0%) for 4 h or 8 h, and then irradiated for 7 min with 630 nm RED light. The cytotoxic effects of ALAD-PDT were measured using the MTS assay. Apoptosis, cell cycle, and ROS assays were performed using flow cytometry. PpIX accumulation was measured using a spectrofluorometer after 10 min and 24 and 48 h of treatment. The viability was extremely reduced at all concentrations, at 4 h for CAPAN-2 and at 8 h for CAL-27. ALAD-PDT induced marked apoptosis rates in both oral and pancreatic cancer cells. Elevated ROS production and appreciable levels of PpIX were detected in both cell lines. The use of ALA-PDT as a topical or intralesional therapy would permit the use of very low doses to achieve effective results and minimize side effects. ALAD-PDT has the potential to play a significant role in complex oral and pancreatic anticancer therapies.

Human papillomavirus independent status on pathologic response and outcomes in locally advanced cervical cancer managed with chemoradiotherapy followed by surgery.

OBJECTIVE: While human papillomavirus (HPV) has been shown to play a significant role in cervical cancer carcinogenesis (HPV associated cases), a considerable percentage of cervical cancers occur independently of HPV status (HPV independent). METHODS: In this retrospective study of 254 locally advanced cervical cancer patients treated with chemoradiotherapy and radical surgery, HPV genotypes were determined using the Anyplex II HPV28 kit that uses multiplex, real time polymerase chain reaction technology. The primary endpoints of this study were to evaluate the complete response to chemoradiotherapy (pathologic complete response), the presence of microscopic (<3 mm, pathologic micro partial response, group 1) and macroscopic (>3 mm, pathologic macro partial response, group 2) residual carcinoma in the cervix, and the persistence of metastatic lymph nodes (group 3) in HPV independent cervical cancers. Secondary endpoints were evaluation of disease-free survival and overall survival. RESULTS: Of 254 patients studied, 21 cases (8.3%) of cervical cancer were determined to be HPV independent. The percentage of pathologic complete response was found to be higher in the HPV associated group compared with the HPV independent group (p<0.001). In the HPV associated cervical cancer group, 5 year disease free survival was found to be 80.8% versus 59.9% in the HPV independent group (p=0.014). Overall survival was also higher in the HPV associated group (87.9%) compared with the HPV independent patients (69.4%) (p=0.023). In the multivariate analysis, the International Federation of Gynecology and Obstetrics (FIGO) stage and HPV genotypes maintained their relevant impact on pathologic complete response to chemoradiotherapy: FIGO stages IIIC1 and IIIC2 were associated with a 13-fold increased risk for the presence of metastatic lymph nodes compared with group 1 (p<0.001). HPV independent cervical cancers showed the highest risk for the development of macroscopic/stable disease (p=0.007), and persistence of metastatic lymph nodes (p=0.004) versus group 1, respectively. CONCLUSIONS: This study showed that HPV status at diagnosis could be a relevant factor for clinical outcomes in locally advanced cervical cancer patients.

The Oncobiome in Gastroenteric and Genitourinary Cancers.

Early evidence suggests a strong association of microorganisms with several human cancers, and great efforts have been made to understand the pathophysiology underlying microbial carcinogenesis. Bacterial dysbiosis causes epithelial barrier failure, immune dysregulation and/or genotoxicity and, consequently, creates a tumor-permissive microenvironment. The majority of the bacteria in our body reside in the gastrointestinal tract, known as gut microbiota, which represents a complex and delicate ecosystem. Gut microbes can reach the pancreas, stomach and colon via the bloodstream. Oral bacterial translocations can also occur. In the stomach, pancreas and colon, low microbial diversity is associated with cancer, in particular with a bad prognosis. The urogenital tract also harbors unique microbiota, distinct from the gut microbiota, which might have a role in the urinary and female/male reproductive cancers' pathogenesis. In healthy women, the majority of bacteria reside in the vagina and cervix and unlike other mucosal sites, the vaginal microbiota exhibits low microbial diversity. Genital dysbiosis might have an active role in the development and/or progression of gynecological malignancies through mechanisms including modulation of oestrogen metabolism. Urinary dysbiosis may influence the pathogenesis of bladder cancer and prostate cancer in males. Modulation of the microbiome via pre, pro and postbiotics, fecal or vaginal microbiota transplantation and engineering bacteria might prove useful in improving cancer treatment response and quality of life. Elucidating the complex host-microbiome interactions will result in prevention and therapeutic efficacy interventions.

Peritoneal HPV-DNA test in cervical cancer (PIONEER study): A proof of concept.

The aim of this study was to investigate the prevalence of peritoneal human papillomavirus (HPV) infection in different clinical cervical cancer (CC) settings, and its association with potential clinical and/or histological factors. This is a single-center, prospective, observational study. Consecutive patients with newly diagnosed or recurrent/persistent CC, between March 2019 and April 2020, were included. A group of patients undergoing surgery for benign gynecological conditions was included as control group. All patients underwent HPV-DNA test in the cervix and in the peritoneal cavity simultaneously at time of surgery. Two-hundred seventy-two patients had cervical and peritoneal HPV test analyzed. Cervical and peritoneal HPV positivity (PHP) was found in 235 (88.0%) and 78 (28.7%) patients, respectively; the prevalence of PHP was 17.7% in early stage, 28.8% in locally advanced cervical cancer (LACC) and 46.6% in the metastatic/persistent/recurrent setting (P = .001). No control patient was found to have peritoneal HPV infection. Higher frequency of PHP was documented in patients with larger tumor size (P = .003), presence of cervical HPV 16/18 genotypes (P < .001), higher number of cervical high-risk (HR)-HPV per patient (P = .018) and peritoneal carcinomatosis (P < .001). Multivariate analysis demonstrated that lack of preoperative cervical conization in early stages (P = .030), while higher International Federation of Gynecology and Obstetrics (FIGO) stage (P = .021) and presence of cervical HPV 16/18 (P = .001) in LACC, was associated with PHP. This is a proof-of-concept study. A number of potential clinical implications, including prognosis, could be obtained by further studies.

Prevalence, incidence and predictors of anal high-risk HPV infections and cytological abnormalities in HIV-infected individuals.

OBJECTIVES: Our purpose was to investigate prevalence, incidence and risk factors of anal high risk-HPV infections and cytological abnormalities in HIV-positive individuals. METHODS: A cohort of consecutively enrolled HIV-positive patients underwent, at baseline visit, a sexual behaviors questionnaire, anoscopy, HPV testing and cytological examination. Hybridization and multiplex-PCR were used for DNA detection and typing; HPV E6-E7 mRNA expression was analyzed in HR-HPV+ patients. Logistic regression was used to assess predictors of HR-HPV infection and anal dysplasia. RESULTS: 233 HIV-infected patients were enrolled (81% males, median age 44 years). HR-HPV was detected in 144 anal swabs and showed a positive association with CDC stage C and a negative association with a higher CD4 count and the use of a NNRTI-based antiretroviral regimen. HR-HPV DNA detection and anal warts at baseline were associated to cytological abnormalities; a detectable HIV-RNA independently predicted new onset anal dysplasia at follow-up (incidence 15.4 per 100 patients-year). Incidence of new HR-HPV infection was 44.2 per 100 patients-year. CONCLUSIONS: The relevance of screening for anal dysplasia in HIV+ patients is emphasized, especially in those with detectable plasma HIV-RNA, anal HR-HPV infection or compromised immunological status.

Porphyromonas gingivalis and the pathogenesis of rheumatoid arthritis: analysis of various compartments including the synovial tissue.

INTRODUCTION: We evaluated the presence of Porphyromonas gingivalis (Pg) DNA in the synovial tissue through synovial biopsy and in other compartments of rheumatoid arthritis (RA) patients in comparison with patients affected by other arthritides. Possible links with clinical, immunologic and genetic features were assessed. METHODS: Peripheral blood (PB), sub-gingival dental plaque, synovial fluid (SF) and synovial tissue samples were collected from 69 patients with active knee arthritis (32 with RA and 37 with other arthritides, of which 14 had undifferentiated peripheral inflammatory arthritis - UPIA). Demographic, clinical, laboratory and immunological data were recorded. The presence of Pg DNA was evaluated through PCR. The HLA-DR haplotype was assessed for 45 patients with RA and UPIA. RESULTS: No differences arose in the positivity for Pg DNA in the sub-gingival plaque, PB and SF samples between RA and the cohort of other arthritides. Full PB samples showed a higher positivity for Pg DNA than plasma samples (11.8% vs. 1.5%, P = 0.04). Patients with RA showed a higher positivity for Pg DNA in the synovial tissue compared to controls (33.3% vs. 5.9%, P <0.01). UPIA and RA patients carrying the HLA DRB1*04 allele showed a higher positivity for Pg DNA in the synovial tissue compared to patients negative for the allele (57.1% vs. 16.7%, P = 0.04). RA patients positive for Pg DNA in the sub-gingival plaque had a lower disease duration and a higher peripheral blood leucocyte and neutrophil count. The presence of Pg DNA did not influence disease activity, disease disability or positivity for autoantibodies. CONCLUSIONS: The presence of Pg DNA in the synovial tissue of RA patients suggests a pathogenic role of the bacterium. The higher positivity of Pg DNA in full peripheral blood and synovial tissue samples compared to plasma and synovial fluid suggests a possible intracellular localization of Pg, in particular in patients positive for HLA-DR4.

Comparison of real-time PCR and pp65 antigen assays for monitoring the development of Cytomegalovirus disease in recipients of solid organ and bone marrow transplants.

Cytomegalovirus (CMV) infection is a frequent complication in transplant recipients. This retrospective study compared real-time PCR (rt-PCR) and a pp65 antigen assay as tools for monitoring CMV infection in solid organ (SOT) and bone marrow (SCT) transplant patients. The study tested 2662 samples by rt-PCR, and 1284 specimens with a pp65 antigen assay. 24.3% of the rt-PCR samples and 4.1% of the pp65 antigen samples were positive. 793 specimens, from 230 patients, were tested with both assays. In 6.7% of samples, both tests were positive; in 72.7% both were negative; in the remaining 20.6% of cases, the results were discordant. CMV disease was diagnosed in 50 patients. Results from the two methods were poorly correlated (r=0.460). The sensitivity of rt-PCR (94%) was higher than that of the pp65 antigen assay (27%). Both assays showed high specificity (92% and 99%, respectively). ROC curve analysis, performed separately for SOT and SCT patients, confirmed that rt-PCR outperformed the pp65 assay in the detection of CMV. These findings provide evidence that rt-PCR is a reliable diagnostic tool, and that it can be more effective than pp65 based assays in monitoring CMV infection progression and in guiding therapy in immunocompromised patients.

RNA (E6 and E7) assays versus DNA (E6 and E7) assays for risk evaluation for women infected with human papillomavirus.

In the majority of cases, high-risk human papillomavirus (HR HPV) infections regress spontaneously, with only a small percentage progressing to high-grade lesions. Current screening methods are based on DNA detection. An alternative would be to monitor expression of the E6 and E7 viral oncogenes continuously expressed by malignant phenotypes. In the work reported in this paper, we compared the two methods for a group of women with high-risk HPV infections. Cervical specimens from 400 women, previously found to be HPV DNA positive, were analyzed for HPV DNA by a liquid hybridization assay and typed by multiplex PCR (for types 16, 18, 31, and 33). Identification of HR HPV E6 and E7 RNA transcripts was performed using real-time reverse transcription-PCR and nucleic acid sequence-based amplification assays. Results were compared with concurrent cytological data. HR HPVs were found in 61.2% of patients. The most common genotype was HPV type 16 (HPV-16) (47.1%), followed by HPV-18, HPV-31, and HPV-33. Nine percent of cases involved other genotypes. Among 223 HPV DNA-positive samples, only 118 were positive in the RNA test. Among HPV DNA-positive patients with normal cytology, we detected E6 and E7 RNA transcripts in two cases (18.2%). The rate of detection increased gradually with the grade of the observed lesions, rising from 20% for patients with atypical squamous cells of undetermined significance to 48.1% for women with low-grade squamous intraepithelial lesions and 86.3% for those with high-grade squamous intraepithelial lesions. These results suggest that testing for HPV E6 and E7 transcripts could be a useful tool for screening and patient management, providing more accurate predictions of risk than those obtained by DNA testing.

BK virus DNA detection by real-time polymerase chain reaction in clinical specimens.

The BK polyomavirus (BKV) is widespread in the general population. In transplant recipients, the patients' weakened immune response may encourage reactivation of latent infection, leading to BKV-related diseases. Rapid and quantitative detection might help to delineate viral reactivation patterns and could thus play an important role in their clinical management. In our study we developed an "in-house" quantitative real-time PCR to detect BKV DNA. The effectiveness of this assay was evaluated by a retrospective analysis of 118 plasma specimens from 22 bone marrow transplant (BMT) recipients and 107 samples from immunocompetent subjects. Eight (36.3%) of the 22 bone marrow transplant recipients tested positive for BKV. The viral load varied from specimen to specimen (10 to 10(5) copies/ml). BKV related disease like hemorrhagic cystitis (HC) was diagnosed in three patients. Specimens from the control group all tested negative. Our results showed the high sensitivity of the real-time PCR, allowing accurate and reproducible measuring of the viral load in order to identify patients at risk for BKV-related diseases. With due caution in interpreting threshold values, the real-time PCR could provide a rapid, sensitive and specific tool for detecting BKV and distinguishing latent and active infection.