Vacuolar Proton-Translocating ATPase May Take Part in the Drug Resistance Phenotype of Glioma Stem Cells.

The vacuolar proton-translocating ATPase (V-ATPase) is a transmembrane multi-protein complex fundamental in maintaining a normal intracellular pH. In the tumoral contest, its role is crucial since the metabolism underlying carcinogenesis is mainly based on anaerobic glycolytic reactions. Moreover, neoplastic cells use the V-ATPase to extrude chemotherapy drugs into the extra-cellular compartment as a drug resistance mechanism. In glioblastoma (GBM), the most malignant and incurable primary brain tumor, the expression of this pump is upregulated, making it a new possible therapeutic target. In this work, the bafilomycin A1-induced inhibition of V-ATPase in patient-derived glioma stem cell (GSC) lines was evaluated together with temozolomide, the first-line therapy against GBM. In contrast with previous published data, the proposed treatment did not overcome resistance to the standard therapy. In addition, our data showed that nanomolar dosages of bafilomycin A1 led to the blockage of the autophagy process and cellular necrosis, making the drug unusable in models which are more complex. Nevertheless, the increased expression of V-ATPase following bafilomycin A1 suggests a critical role of the proton pump in GBM stem components, encouraging the search for novel strategies to limit its activity in order to circumvent resistance to conventional therapy.

Exoscopic Microsurgery: A Change of Paradigm in Brain Tumor Surgery? Comparison with Standard Operative Microscope.

BACKGROUND: The exoscope is a high-definition telescope recently introduced in neurosurgery. In the past few years, several reports have described the advantages and disadvantages of such technology. No studies have compared results of surgery with standard microscope and exoscope in patients with glioblastoma multiforme (GBM). METHODS: Our retrospective study encompassed 177 patients operated on for GBM (WHO 2021) between February 2017 and August 2022. A total of 144 patients were operated on with a microscope only and the others with a 3D4K exoscope only. All clinical and radiological data were collected. Progression-free survival (PFS) and overall survival (OS) have been estimated in the two groups and compared by the Cox model adjusting for potential confounders (e.g., sex, age, Karnofsky performance status, gross total resection, MGMT methylated promoter, and operator's experience). RESULTS: IDH was mutated in 9 (5.2%) patients and MGMT was methylated in 76 (44.4%). Overall, 122 patients received a gross total resection, 14 patients received a subtotal resection, and 41 patients received a partial resection. During follow-up, 139 (73.5%) patients experienced tumor recurrence and 18.7% of them received a second surgery. After truncation to 12 months, the median PFS for patients operated on with the microscope was 8.82 months, while for patients operated on with the exoscope it was >12 months. Instead, the OS was comparable in the two groups. The multivariable Cox model showed that the use of microscope compared to the exoscope was associated with lower progression-free survival (hazard ratio = 3.55, 95%CI = 1.66-7.56, p = 0.001). CONCLUSIONS: The exoscope has proven efficacy in terms of surgical resection, which was not different to that of the microscope. Furthermore, patients operated on with the exoscope had a longer PFS. A comparable OS was observed between microscope and exoscope, but further prospective studies with longer follow-up are needed.

Perilesional resection technique of glioblastoma: intraoperative ultrasound and histological findings of the resection borders in a single center experience.

INTRODUCTION: The surgical goal in glioblastoma treatment is the maximal safe resection of the tumor. Currently the lack of consensus on surgical technique opens different approaches. This study describes the "perilesional technique" and its outcomes in terms of the extent of resection, progression free survival and overall survival. METHODS: Patients included (n = 40) received a diagnosis of glioblastoma and underwent surgery using the perilesional dissection technique at "San Gerardo Hospital"between 2018 and 2021. The tumor core was progressively isolated using a circumferential movement, healthy brain margins were protected with Cottonoid patties in a "shingles on the roof" fashion, then the tumorwas removed en bloc. Intraoperative ultrasound (iOUS) was used and at least 1 bioptic sample of "healthy" margin of the resection was collected and analyzed. The extent of resection was quantified. Extent of surgical resection (EOR) and progression free survival (PFS)were safety endpoints of the procedure. RESULTS: Thirty-four patients (85%) received a gross total resection(GTR) while 3 (7.5%) patients received a sub-total resection (STR), and 3 (7.5%) a partial resection (PR). The mean post-operative residual volume was 1.44 cm3 (range 0-15.9 cm3).During surgery, a total of 76 margins were collected: 51 (67.1%) were tumor free, 25 (32.9%) were infiltrated. The median PFS was 13.4 months, 15.3 in the GTR group and 9.6 months in the STR-PR group. CONCLUSIONS: Perilesional resection is an efficient technique which aims to bring the surgeon to a safe environment, carefully reaching the "healthy" brain before removing the tumoren bloc. This technique can achieve excellent tumor margins, extent of resection, and preservation of apatient's functions.

Interobserver agreement among expert pathologists on through-the-needle microforceps biopsy samples for evaluation of pancreatic cystic lesions.

BACKGROUND AND AIMS: The recent development of microforceps for EUS through-the-needle biopsy (TTNB) sampling of the wall of pancreatic cystic lesions (PCLs) allows the collection of histologic specimens never handled and evaluated before by pathologists. We aimed to estimate the interobserver agreement among pathologists in evaluating such samples. METHODS: TTNB specimen slides from 40 PCLs with worrisome features were retrieved and independently evaluated for specimen adequacy, presence of lining epithelium, grade of epithelial dysplasia, presence of ovarian type stroma, and specific diagnosis by 6 expert pathologists from 6 different tertiary care centers. The Gwet's AC1 was used to assess interobserver agreement. RESULTS: An almost perfect agreement was observed for specimen adequacy (AC1, .82; 95% confidence interval [CI], .79-.98), presence of lesional epithelium (AC1, .90; 95% CI, .86-.92), epithelial dysplasia (AC1, .97; 95% CI, .95-.99), and ovarian-like stroma (AC1, .90; 95% CI, .86-.93). When considering all diagnoses separately, a moderate to substantial agreement was observed (AC1, .62; 95% CI, .57-.67), similarly to mucinous cysts versus serous adenoma versus other diagnoses (AC1, .65; 95% CI, .59-.70) and for mucinous cysts versus all other diagnoses (AC1,.74; 95% CI, .68-.84). The agreement for diagnosis of mucinous cystic neoplasm versus intraductal mucinous papillary neoplasm was almost perfect (AC1, .88; 95% CI, .81-.95). CONCLUSIONS: Interobserver agreement between expert pathologists in the evaluation of TTNB samples from PCLs with worrisome features was close to perfection for all evaluated parameters, except definitive diagnosis. When mucinous cystic lesions were compared versus all other diagnoses, the agreement became substantial, thus indicating that TTNB specimens can provide important information for PCL management decisions.

Liver Perfusate Natural Killer Cells From Deceased Brain Donors and Association With Acute Cellular Rejection After Liver Transplantation: A Time-to-Rejection Analysis.

BACKGROUND: The ability to predict which recipients will successfully complete their posttransplant clinical course, which is crucial for liver transplant (LT) programs. The assessment of natural killer (NK) cell subset determined by flow cytometry from a monocentric series of consecutive liver perfusates could help identify risk factors portending adverse LT outcomes. METHODS: Liver perfusates were collected during the back-table surgical time after the procurement procedures for donors after brain death. Lymphocytic concentrations and phenotypes were matched with donors after brain death characteristics and indications, timing, surgical techniques, outcomes, and biopsy-proven acute cellular rejections (ACRs) in 46 adult recipients who underwent LT between 2010 and 2014 at our institute. Cox regression models were used to study relevant risk factors in order to estimate hazard ratios for episodes of rejection after LT. RESULTS: Percentage of NK cells was significantly associated with donor age (P = 0.05) and the percentage of NK T cellular subset (P = 0.001). The length of follow-up after LT was 41.0 ± 20.9 months, and 11 (23.9%) recipients experienced biopsy-proven ACR. At time-to-rejection proportional regression analysis, a cutoff value of 33.7% was optimal, with a sensitivity of 1, specificity of 0.57, and positive and negative predictive values of 0.42 and 1, respectively. The liver perfusate NK cell subset was strongly associated with biopsy-proven ACR (hazard ratio, 10.7; P = 0.02). CONCLUSIONS: Liver perfusate cytofluorimetric phenotyping may contribute as a targeted preoperative tool to predict the risk of ACR, and as clinical test in translational studies that aim to improve donor allograft procurement and transplant outcomes.

Endoscopic ultrasound-guided fine needle tissue acquisition biopsy samples do not allow a reliable proliferation assessment of gastrointestinal stromal tumours.

BACKGROUND: Current prognostication of gastrointestinal stromal tumours is validated on/applies to resected tumours, mainly because surgery is recommended whenever possible. However, pre-treatment prognostication is increasingly warranted, considering the follow-up strategy recently admitted for expectedly low-risk tumours and the possible distinctive molecular features/spontaneous regression of some small cases. AIMS: To investigate whether endoscopic ultrasound-guided fine-needle tissue acquisition-biopsies reflect prognosticators of resected gastrointestinal stromal tumours, for possibly developing reliable pre-treatment prognostic criteria. METHODS: The applicability/reliability of mitotic index/5mm(2) and MIB1 proliferative index/1000 cells were tested in 35 endoscopic ultrasound-guided fine-needle tissue acquisition-biopsies diagnosed as gastrointestinal stromal tumour, subsequently resected without intervening therapy, consecutively collected in thirty months. Size and mitotic/proliferative indexes were compared with resection specimens. The feasibility of bioptic genotyping was also tested. RESULTS: 35 patients were studied (45.7% males; mean age 61.6 years, range 26-83 years). Mitotic/proliferative indexes were determinable in 68.6%/88.6% of biopsies, respectively; they were nevertheless underestimated, as happened with endoscopic ultrasound-determined tumour size. Bioptic genotyping revealed reliable. CONCLUSIONS: Endoscopic ultrasound-guided fine-needle tissue acquisition does not reliably reflect gastrointestinal stromal tumours' proliferation and size. Alternative parameters should be validated for a pre-surgical prognostic classification. Considering the emerging potentially prognostic genetic markers in gastrointestinal stromal tumours, the reliability of bioptic genotyping is a promising result.

Survivin protein as predictor of pathologic response in patients with locally advanced cervical cancer treated with chemoradiation followed by radical surgery.

We investigated the correlation of pathologic response and immunohistochemically assessed expression of survivin protein in 71 patients with locally advanced cervical cancer treated with chemoradiation (CT/RT) followed by radical surgery. The prognostic role of survivin expression was also evaluated. Immunohistochemical analysis of survivin expression was carried out using the polyclonal rabbit antisurvivin antibody. Cytoplasmic survivin immunoreaction was observed in 69 (97.2%) of 71 cases and nuclear staining in 7 (9.8%) of 71 women. Median cytoplasmic survivin expression was 160 (range, 0-280), and higher levels were observed in patients with residual disease (≥3 mm) in the cervix (survivin level, 160 versus 120; P = .016) and in women with metastatic lymph nodes (survivin levels, 160 versus 150; P = .032). No differences were documented in the distribution of patients with positive nuclear staining, according to clinicopathological variables. In multivariate analysis, cytoplasmic survivin expression emerged as an independent predictor of residual cervical disease and lymph node status after CT/RT. During a follow-up period of 83 months (range, 8-175 months), recurrences occurred in 24 (33.8%) women, and all patients died of disease. Women with high cytoplasmic survivin experienced shorter disease-free survival compared with patients with low levels (5-year disease-free survival, 80.8% versus 55.3%; P = .033). Only a trend was observed for greater overall survival in patients with high expression (5-year overall survival, 81.0% versus 55.3%; P = .069). No survival differences were documented for nuclear survivin status. The immunohistochemically assessed survivin cytoplasmic levels at diagnosis represent a reliable and easily assessable tool to predict response to CT/RT in patients with locally advanced cervical cancer.

Mutational status of KRAS, NRAS, and BRAF in primary clear cell ovarian carcinoma.

Ovarian clear cell carcinoma (OCCC) is a subtype of epithelial ovarian cancer with characteristic biological features and aggressive clinical behavior. OCCCs show a pattern of gene mutations different from other type I ovarian malignancies, notably a higher frequency of PIK3CA mutations. In low grade serous ovarian cancer, KRAS and BRAF mutations are frequent, but little data are available on the mutational status of these genes in OCCCs. To clarify this issue, we designed a clinicopathological study with the aim to establish the incidence of KRAS, NRAS, and BRAF hot spot mutations in OCCC. Between December 2006 and June 2012, 22 patients with a proven diagnosis of OCCC were admitted to our Institutions. In all cases, final diagnosis was established according to FIGO and WHO criteria. All women received complete surgical staging. The PyroMark Q24 system (Qiagen GmbH, Hilden, Germany) was used for pyrosequencing analysis of KRAS, NRAS, and BRAF hot spot regions on 2.5-µm sections of formalin-fixed paraffin-embedded tissue from primary OCCC. Pyrosequencing analysis of KRAS, NRAS, and BRAF hot spot regions revealed the presence of mutations only at codon 12 in exon 2 of KRAS in 3 of 22 (14 %) cases. We found no mutations in the hot spot regions of NRAF (exons 2, 3, 4) or BRAF (exon 15). The median age of women with a KRAS mutated OCCC was 74 years. These OCCC were unilateral FIGO stage IA lesions in two cases associated with foci of endometriosis. We conclude that in 14 % of OCCCs, a KRAS mutation occurs in codon 2 exon 2. NRAS and BRAF mutations were not found.

Fine-needle tissue acquisition from subepithelial lesions using a forward-viewing linear echoendoscope.

BACKGROUND AND STUDY AIMS: The overall diagnostic accuracy of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) for subepithelial lesions (SELs) is suboptimal. The aim of this study was to evaluate the diagnostic accuracy, defined as the proportion of correct diagnoses, obtained using the EUS-guided fine-needle tissue acquisition (FNTA) sampling technique performed with the newly developed forward-viewing EUS scope (FV-EUS). PATIENTS AND METHODS: This was a retrospective analysis of a prospectively collected database including all consecutive patients with SELs who underwent EUS-FNTA using the FV-EUS scope from 2007 to 2011 in a tertiary referral center. All procedures were performed by a single expert endoscopist. RESULTS: A total of 121 consecutive patients with SELs (13 in the esophagus, 96 in the stomach, 10 in the duodenum, 2 in the rectum) underwent sampling of the lesion using the FV-EUS scope. The procedure was technically feasible in all but one patient, and no complication related to EUS-FNTA occurred. Full histological assessment including immunostaining could be completed in 93.4% (113/121) of the patients. Considering neoplastic vs. non-neoplastic diseases, the sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio were 92.8% (95%CI 86.3-96.8), 100% (95%CI 69.0-100%), infinity, and 0.07 (95%CI 0.04-0.14), respectively. CONCLUSIONS: EUS-FNTA performed in conjunction with the FV-EUS scope for sampling SELs of the gastrointestinal tract was safe and provided a very high diagnostic accuracy. Studies comparing FV-EUS with standard curved linear echoendoscopes are needed to clarify whether these results are due to the sampling technique or the type of scope utilized.

Ectopic immature renal tissue: clues for diagnosis and management.

Ectopic immature renal tissue has rarely been reported in literature, associated or not with teratoma. Its finding could be matter of concern, owing to the occasional possibility that Wilms tumor may develop in this setting. We report a 1-year-old male patient who underwent surgery for a sacral subcutaneous small teratoma with a prevalent component of immature renal tissue. The lesion appeared completely excised and, in absence of features of malignancy, only follow-up was suggested. The patient was alive and well 15 months postoperatively. Whenever ectopic immature renal tissue is detected, a proper histological interpretation is mandatory, in order to plan a suitable treatment of the patient. From an extensive analysis of cases reported in literature we draw some practical suggestions for the diagnosis and treatment of this rare condition.

C-reactive protein but not soluble CD40 ligand and homocysteine is associated to common atherosclerotic risk factors in a cohort of coronary artery disease patients.

OBJECTIVES: One third to one half of the variation in vascular disease occurrence remains unexplained by traditional risk factors. Since atherosclerosis may, in part, be an inflammatory disease, circulating factors related to inflammation may be predictors of cardiovascular disease. The aim of this study was to evaluate the association between common atherosclerotic risk factors and markers of inflammation. DESIGN AND METHODS: Serum levels of soluble CD40 (sCD40L), high-sensitive C-reactive protein (hs-CRP) and homocysteine (Hcy) were measured in 251 patients selected from a series of 438 subjects affected by previous myocardial infarction, angina or other cardiovascular diseases. RESULTS: sCD40L levels were lower in patients with previous myocardial infarction while no association was observed between sCD40L and Hcy levels and other risk factors. Only hs-CRP levels positively correlated with increased number of risk factors. CONCLUSION: In a setting of patients affected with coronary artery disease no association between sCD40L and homocysteine levels and atherosclerotic risk factors was observed; only hs-CRP showed increased levels according to the number of risk factors. Future studies using larger cohorts will be needed to validate the clinical use of markers of inflammation in the prediction of cardiovascular events.