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Primary myelofibrosis (PMF) is a rare myeloproliferative neoplasm characterized by clonal proliferation of mature myeloid lineages derived from stem cells (erythrocytes, leukocytes and magakaryocytes) with variable megakaryocyte atypia associated with reticulin and / or collagen bone marrow (BM) fibrosis, osteosclerosis, ineffective erythropoiesis, angiogenesis, extramedullary hematopoiesis and abnormal expression of cytokines. Allogeneic hemopoietic stem cell transplantation (alloHSCT) is currently the only curative approach for patients with myelofibrosis, and for this reason the number of allografts for these indications have been growing over the past years. Unfortunately relapse of myelofibrosis (MF) after an alloHSCT occurs in 10-40% of cases: patients usually present with a declining donor chimerism, and a reappearance of driver mutations if present; BM biopsy is usually consistent with typical megakaryocyte abnormalities and stromal fibrosis. Ultimately BM cells exhibit progressive loss of donor chimerism, and the relapse is therefore of recipient origin. Here we report two allografted MF patients who relapsed in donor cells.

Chronic lymphocytic leukemia can evolve to an aggressive lymphoma—in most of the cases diffuse large B cells lymphoma, rarely Hodgkin lymphoma—and this complication is defined Richter syndrome (RS). Immunogenotypic features that characterize RS include unmutated IgHV status with high prevalence of IgHV4-39/D6-13/J5 sequence; deletion of chromosome 17p or 11q; activation of oncogenes as NOTCH1 and c-MYC; inactivation of onco-suppressors as TP53 and CDKN2A; high expression of CD38 in lymph-nodes. The prognosis of this condition is very poor: patients experience a rapid clinical deterioration with frequent therapeutic failure since the current options include suboptimal strategies as standard chemo-immunotherapy followed by hematopoietic stem cells transplantation or enrollment in clinical trials which investigate the efficacy of target drugs. Understanding the biology of such a heterogeneous condition is crucial to personalize the treatment and improve patient's survival.

Background: Numerous studies showed that methylation analysis represents a newly developed urinary marker based on DNA methylation changes in a panel of genomic biomarkers and it could represent a valid tool in terms of the diagnosis and prediction of high-grade urothelial carcinoma recurrences. One of the limits of the use of this new molecular method during a follow-up is represented by the number of invalid tests in routine practice. Method: A total of 782 patients with a diagnosis of non-muscle-invasive high-grade carcinoma (NMIBC) was studied. The Bladder EpiCheck test (BE) was performed together with cytology in all cases within 1 year after the end of treatment. In 402 patients, the urinary samples were voided urine (UV), while, in 380 cases, the samples were collected after bladder washing (IU). For all the patients with invalid BE results, a second BE test was performed following the instructions for use that indicated the test should be repeated with a new urinary sample in the case of an invalid result. Results: Analyzing the two different groups (UV and IU), we found the invalid BE results seemed to be not related to urinary samples (p = 0.13 Fisher’s exact test), suggesting that the collection method was not relevant in order to reduce the number of invalid tests. Conclusions: In the follow-up for NMIBC, for patients for whom a BE test is planned, a combined approach of cytology and a methylation test is recommended in order to repeat the BE test with an invalid result only in those cases with a cytological diagnosis of atypical urothelial cells (AUC) suspicious for high-grade urothelial carcinoma (SHGUC) and high-grade urothelial carcinoma (HGUC).

Primary central nervous system (CNS) extranodal marginal zone B-cell lymphoma (MZBL) is a rare low-grade non-Hodgkin lymphoma, characterised predominantly by small B cells, plasma cells, monocytoid cells and scattered large immunoblasts. Primary CNS MZBL is a slow-growing tumour that remains localised and is characterised by an excellent clinical prognosis. The present study describes the case of a 48-year-old HIV-negative female patient with a history of head trauma 1 year prior, who presented with worsening neurological symptoms and a magnetic resonance imaging finding of a ∼3-cm extra-axial mass within the left lateral ventricle. From histopathology and immunohistochemistry, the lesion was diagnosed as a CNS MZBL; as no other primary lesions were found, the base of the choroid plexuses of the left lateral ventricle was considered the primary site. To the best of our knowledge, the current case is the first study to report on primary CNS MZBL arising in this anatomical site and paves the way for further studies on the role of chronic inflammation (in the present case resulting from trauma) in the pathogenesis not only of primary CNS MZBL but also of lymphoma in general. Additionally, this report could serve as a starting point for studies analysing the role of meningothelial cells in the pathogenesis of primary CNS MZBL.

The cranial window (CW) technique provides a simple and low-cost method to assess tumor angiogenesis in the brain. The CW combined with histology using selective markers for tumor and endothelial cells can allow a sensitive monitoring of novel antiangiogenesis therapies in preclinical models. The CW was established in cyclosporine immunosuppressed rats that were stereotactically grafted with fluorescent U87MG glioblastoma cells. One to 3 weeks after grafting, brain vasculature was visualized in vivo and assessed by immunofluorescence microscopy using antibodies against endothelial and smooth-muscle cells and blood brain barrier. At 1-2 weeks after grafting, the CW reliably detected the hypertrophy of venous-venous anastomoses and cortical veins. These structures increased highly significantly their pregrafting diameter. Arterialized veins and hemorrhages were seen by three weeks after grafting. Immunofluorescence microscopy showed significant branching and dilation of microvessels, particularly those surrounded by tumor cells. Mechanistically, these changes lead to loss of vascular resistance, increased venous outflow, and opening of venous-venous anastomoses on the cortical surface. Data from the present study, namely, the hypertrophy of cortical venous-venous anastomoses, microvessel branching, and dilation of the microvessels surrounded by tumor cells, indicate the power of this in vivo model for the sensitive monitoring of early tumor angiogenesis.

KRAS mutation is reportedly associated with poor prognosis in patients with different cancer types. However, mutational data on hilar cholangiocarcinoma are few and controversial. The aim of this study was to evaluate the rate of KRAS mutations in a single-center homogeneous population resected for hilar cholangiocarcinoma and the subsequent impact on prognosis. KRAS mutation status was evaluated in 54 patients undergoing major hepatectomy combined with resection of the main biliary confluence and regional lymphadenectomy for hilar cholangiocarcinoma between 2001 and 2019. Among these 54 patients, 12 (22.2%) had a KRAS mutation. KRAS mutation was not related with pathologic characteristics of the tumor. Five-year overall survival (OS) in patients with KRAS mutation was significantly lower than that observed in patients with KRAS wild type (0 vs. 49.2%, respectively; p = 0.003). In the multivariable analysis; independent predictors of poor OS were KRAS mutation (HR = 5.384; p = 0.003) and lymph node metastases (HR = 2.805; p = 0.023). The results of our study suggested that KRAS mutation in hilar cholangiocarcinoma was not rarely observed. KRAS mutation was an independent strong predictor of poor OS. KRAS mutation analysis should be included in the routine pathologic evaluation of resected hilar cholangiocarcinoma in order to better stratify prognosis

Predictive factors for response to regorafenib in recurrent glioblastoma, IDH-wildtype, are scarcely recognized. The objective of this study was to identify molecular predictive factors for response to regorafenib using a clinically available platform. We analyzed a prospective cohort of 30 patients harboring recurrent glioblastoma, IDH-wildtype, and treated with regorafenib. Next-generation sequencing (NGS) analysis was performed on DNA extracted from paraffin-embedded tissues using a clinically available platform. Moreover, MGMT methylation and EGFRvIII expression analyses were performed. Six-month progression-free survival (PFS) was 30% and median overall survival (OS) was 7.5 months, in line with literature data. NGS analysis revealed a mutation in the EGFR pathway in 18% of cases and a mutation in the mitogen-activated protein-kinase (MAPK) pathway in 18% of cases. In the remaining cases, no mutations were detected. Patients carrying MAPK pathway mutation had a poor response to regorafenib treatment, with a significantly shorter PFS and a nonsignificantly shorter OS compared to EGFR-mutated patients (for PFS, 2.5 vs 4.5 months, p = 0.0061; for OS, 7 vs 9 months, p = 0.1076). Multivariate analysis confirmed that MAPK pathway mutations independently predicted a shorter PFS after regorafenib treatment (p = 0.0188). The negative prognostic role of MAPK pathway alteration was reinforced when we combined EGFR-mutated with EGFRvIII-positive cases. Recurrent glioblastoma tumors with an alteration in MAPK pathway could belong to the mesenchymal subtype and respond poorly to regorafenib treatment, while EGFR-altered cases have a better response to regorafenib. We thus provide a molecular selection criterion easy to implement in the clinical practice.

Hodgkin lymphoma (HL) is a neoplastic disease in which the inflammatory microenvironment plays a pivotal role in the tumorigenesis. Neutrophilia is a typical finding in HL at diagnosis and, in particular, in association with lymphocytopenia, is a negative prognostic factor. As the immune checkpoint Programmed Death (PD)-L1/PD-1 has become an important therapeutic target, we were interested in the expression of PD-L1 in peripheral blood (PB) leukocytes using flow cytometry and RT-PCR in patients with HL and healthy controls. Granulocytes were the major PB cell fraction expressing PD-L1. PD-L1 expression on granulocytes was higher in patients with HL than in controls and correlated with lower T-cell numbers in PB. We analyzed for associations between PD-L1 expression in PB granulocytes at the time of diagnosis with patient characteristics and outcome in 126 patients with HL treated with standard chemotherapy adriamycin, bleomycin, vinblastine, and dacarbazine. Increased PD-L1 expression in PB associated with advanced disease, systemic symptoms, positive interim positron emission tomography, and inferior progression-free survival (PFS). PFS at 4 years was 81% (95% C.I., 71–87%) in patients with normal PD-L1 expression and 56% (95% C.I., 35–72%) in patients with higher-than-normal PD-L1 expression (p = 0.002). In conclusion, PD-L1 expression in PB could become a potentially actionable prognostic factor in HL.

Background: The management of non-muscle invasive bladder carcinoma (NMIBC) after transurethral resection of a bladder tumor consists of adjuvant intravesical therapy and strict and long surveillance with urine cytology and cystoscopy. The Bladder EpiCheck test (Nucleix Ltd) (BE) is a newly developed urinary markers based on DNA methylation changes in a panel of 15 genomic biomarkers, with a promising performance in term of non-invasive NMIBC detection. Methods: In this study we prospectively enrolled 151 consecutive patients with high grade NMIBC, treated with intravesical BCG and mitomycin C therapy and evaluated during the follow-up by voided urine cytology and white-light cystoscopy, according to the European Association of Urology Guidelines. The Bladder EpiCheck test was performed at the same time of urine cytology in voided specimen. In all cases with positive cytology the diagnosis was confirmed by histology and a diagnosis was made according to the 2017 tumor, node, metastasis (TNM) classification and graded using both the 1973 and the 2004 World Health Organization (WHO) classifications. Results: At three months of follow-up, we reported similar overall specificity rates for BE and urine cytology (85,1% vs 86,3%). In the group of patients with carcinoma in situ (CIS), we found the same specificity for BE and urine cytology (81,4%), while in the groups of patients with papillary high grade NMIBC, the specificity of BE was higher compared to cytology (96,3% vs 90,4%). The sensitivity of BE was always higher compared to cytology during all the follow-up both for papillary NMIBC and CIS. Conclusion: In the early follow-up of NMIBC the EpiCheck test might replace urinary cytology.

Background Type 5 phosphodiesterase (PDE5) expression in the normal and pathological prostate is controversial. Objectives This study aimed at identifying the cell type/s, if any, expressing PDE5 in human healthy or pathological prostate sections in order to further validate the rationale of PDE5 inhibitor (PDE5i) treatment of benign prostatic hyperplasia (BPH) and their safety in the treatment of erectile dysfunction following prostate cancer (PCa) surgery. Materials and methods By immunohistochemical analysis, we studied PDE5 expression in tissue microarrays containing sections obtained from healthy, BPH, and PCa samples. Results Our results showed that PDE5 is barely expressed in the epithelial or stromal compartment of normal human prostates, but it is highly expressed in the stromal compartment of BPH sections. We also found that a low but significant number of PCa samples (22%) expressed PDE5 in the epithelial cancer cells but not in stromal cells and that such expression was not correlated with the tumor aggressiveness, according to their Gleason score. Discussion and conclusion PDE5 overexpression in the stromal compartment of BPH samples supports the rationale of PDE5 as a target in lower urinary tract symptoms of BPH. PDE5 expression in a significant percentage of PCa samples but the lack of correlation with the Gleason score suggests that this enzyme is not correlated with tumor aggressiveness; however, a role of PDE5 in the minimal residual disease of PCa cannot be excluded.

Background: Hyalinizing trabecular tumors (HTTs) are rare, essentially benign, follicular cell–derived thyroid neoplasms characterized by a trabecular growth pattern and nuclear pseudoinclusions. Their cytological findings are misleading, because these tumors are often misinterpreted on fine needle aspirate cytology as malignant lesions, such as papillary thyroid cancer and/or medullary thyroid cancer, leading to unnecessary total thyroidectomy. The aim of this study was to analyze the cytomorphological features and application of ancillary techniques in a series of HTTs. Methods: Of 26 histological cases of HTT collected from September 2001 to December 2018, 18 cases had concomitant cytopathology. Cytological cases were processed with liquid-based cytology (LBC). Immunocytochemistry for HBME-1 and galectine-3 as well as molecular testing for BRAFV600E mutation were performed on both LBC and histological specimens. Results: The 18 lesions with fine needle aspirate cytology ranged in size from 5 to 45 mm. Cytological diagnoses included: 1 benign lesion favoring goiter (5.5%), 4 atypia of undetermined significance (22.2%), 6 follicular neoplasms (33.3%), 5 suspicious for malignancy favoring papillary thyroid cancer (28%), and 2 malignant (11%). Hence, 89% HTT had a negative concordant immunopanel, and they were 100% wild-type BRAFV600E. Conclusion: The majority of our HTTs (83.3%) were diagnosed in the indeterminate Bethesda categories, suggesting that their cytomorphological features pose issues for reaching a conclusive cytological diagnosis. The ancillary test results in our series support the fact that HTT is a benign neoplasm.

The clinical progression of B cell chronic lymphocytic leukemia (CLL) is associated with immune cell dysfunction and a strong decrease of miR-181b-5p (miR-181b), promoting the death of CLL cells. Here we investigated whether the reduction of miR-181b impairs the immune response in CLL. We demonstrate that activated CD4+ T cells increase miR-181b expression in CLL through CD40– CD40L signaling, which enhances the maturation and activity of cytotoxic T cells and, consequently, the apoptotic response of CLL cells. The cytotoxic response is facilitated by a depletion of the anti-inflammatory cytokine interleukin 10, targeted by miR-181b. In vivo experiments in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice confirmed that miR-181b promotes the apoptotic death of CLL cells only when functional T cells are restored. Overall, our findings suggest that the reinstatement of miR-181b in CLL cells could be an exploitable adjuvant therapeutic option for the treatment of CLL.

Aims Bladder EpiCheck is one of several urinary tests studied to identify bladder tumours and analyses 15 methylation biomarkers determining bladder cancer presence on the basis of methylation profile. Methods 374 patients diagnosed with high-grade non-muscle invasive bladder cancer were treated and followed for 1 year with voided urine cytology and white-light cystoscopy and biopsies according to European Association of Urology Guidelines. 268 cases were diagnosed with high-grade papillary carcinoma, while 106 cases were carcinoma in situ. Bladder EpiCheck test was performed together with cytology in all cases. Results Comparing cytological categories of negative for high-grade urothelial carcinoma (NHGUC) and atypical urothelial cells (AUCs), we found that an EpiScore <60 correlates with NHGUC (p=0.0003, Fisher's exact test), while comparing AUC and suspicious for high-grade urothelial carcinoma (SHGUC) or SHGUC and high-grade urothelial carcinoma (HGUC) categories, an EpiScore ≥60 correlates with SHGUC and HGUC, respectively (p=0.0031 and p=0.0027, Fisher's exact test). In each TPS category, we found that sensitivity, specificity, Positive Predicitve Value (PPV) and Negative Predictive Value (NPV) of the Bladder EpiCheck test in HGUC category were higher than those observed in SHGUC group (sensitivity=98%, specificity=100%, NPV=85.7%, PPV=100% vs sensitivity=86.6%, specificity=52.3%, NPV=84.6%, PPV=56.5%). Conclusions Analysing methylation study results, we demonstrated that different TPS cytological categories also carry a distinct molecular signature. Moreover, our results confirm that cytological categories SHGUC and HGUC are different entities also from a molecular point of view and should continue to represent distinct groups in TPS.

Objective: To identify in which cases after cytological diagnosis, the Bladder EpiCheck test could represent an effective tool in non-muscle invasive bladder carcinoma or an useless expence. Materials and methods: 375 patients diagnosed with non-muscle invasive bladder cancer, 269 with high grade urothelial carcinoma and 106 with carcinoma in situ, were treated and followed for 1 year. The treatment was an intravesical instillation of Bacillus Calmette-Guerin in 305 patients and Mitomycin-C in 70 patients. During the follow-up patients were evaluated by voided urine cytology and white-light cystoscopy, according to the European Association of Urology Guidelines. Bladder EpiCheck test was performed together with cytology in all cases. Results: Analyzing Bladder Epicheck results for each category defined by the Paris System for Reporting Urinary Cytology, we found that the Episcore >60 correlates with histological diagnosis of high grade urothelial carcinoma (HGUC) in atypical urothelial cells and Suspicious for High Grade Urothelial Carcinoma (P = 0.0002 Odds Ratio 0.05926 95% Confidence Interval from 0.01127 to 0.3116 and P = 0.0009 Odds Ratio 0.03155 95% Confidence Interval from 0.001683 to 0.5914, Fisher's exact test, respectively), while in Negative for high grade urothelial carcinoma and HGUC patients Episcore is not helpful to identify cases with histological diagnosis of HGUC (P = 0.101 and P = 0.58 Fisher's exact test, respectively). Considering an Episcore ≥ 90 in the HGUC cytological group, this seems not to be correlated with a histological diagnosis of HGUC (P = 0.090 Fisher's exact test). Conclusions: Cytology and Bladder EpiCheck test in combination may have the potential to reduce cystoscopies in the follow-up of non-muscle invasive bladder cancer only for cytological diagnoses of atypical urothelial cells and Suspicious for High Grade Urothelial Carcinoma . Moreover, in patients with a cytological diagnosis of Negative for high grade urothelial car

Cytological specimens from thyroid nodules are increasingly being adopted as the first available material for cost effectively managing patients in the era of personalized medicine. Cytology aspirates not only play a central role in providing accurate diagnoses, but are also being collected for ancillary molecular testing. Molecular analysis, including the evaluation of somatic mutations and other genomic alterations, has accordingly become well integrated in the cytological workup of thyroid lesions. Appropriately handled thyroid cytology preparations provide well-preserved and adequately cellular material with improved DNA/RNA quantity. The recent publication of the 2nd edition of The Bethesda System for Reporting Thyroid Cytopathology and the American Thyroid Association guidelines confirm the relevant role of molecular testing in the management of the different subcategories of indeterminate thyroid lesions. This review discusses the role of molecular testing for indeterminate thyroid nodules, including the recent introduction of the noninvasive, encapsulated follicular variant of papillary thyroid carcinoma (FVPTC), known also as noninvasive follicular neoplasm with papillary-like nuclear features (NIFTP).

Carcinoma in situ (CIS) is believed to be a precursor of muscle-invasive carcinomas that may arise from these flat high-grade, superficial urothelial lesions. CIS accounts for approximately 10% of all bladder tumors. Therapeutic options for urothelial CIS are limited, and in order to inhibit disease progression and recurrence, current guidelines recommend transurethral resection (TURBT) followed by intravesical administration of Bacillus of Calmette-Guerin (BCG). Approximately 30–40% of patients fail the BCG therapy with recurrence and progression of disease. In the present study, we examined the expression of PD-L1 both in neoplastic epithelial cells and in stromal inflammatory cells in patients with diagnosis of CIS primary responders and not responders to BCG therapy, in order to verify if the PD-L1 expression could identify patients resistant to BCG treatment. Moreover, we analyzed on the same cases the immunoreactivities of anti-PD-L1 MoAbs such as SP263, C23, and SP142. Our results have showed that PD-L1 expression in tumor cells and in immune cell compartment is higher in BCG-unresponsive group than in BCG responders, but only the PD-L1 22C3 expression in tumor cells seems to be associated with recurrence of disease (p = 0.035; OR 0.1204; CI 95% from 0.0147 to 1.023). Hence, our data suggest that the PD-L1 22C3 expression could help to identify CIS that fail the BCG therapy, supporting the hypothesis that enhanced levels of intratumoral PD-L1 22C3 expressed by the tumor cells may explain the failure of BCG immunotherapy.

Background: Programmed death-ligand 1 (PD-L1) expression is emerging as an important predictive biomarker in anti–PD-L1 cancer immunotherapy. Its role has been clearly defined in various human cancers and is linked to a poor prognosis and resistance to anticancer therapies. The role of PD-L1 in thyroid cancers has not been well defined in fine-needle aspiration cytology (FNAC). The authors examined the performance of PD-L1 immunostaining in liquid-based cytology (LBC) to determine whether it could be a biomarker of malignancy or aggressive disease. Methods: From January 2018 to March 2019, 236 thyroid lesions, which had been diagnosed by FNAC as indeterminate lesions, suspicious for malignancy (SFM), and malignant, were enrolled. PD-L1 immunostaining was performed on both LBC and corresponding histology samples. Results: The FNAC cohort included 50 benign negative controls, 42 samples of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS), 33 samples of follicular neoplasm/suspicious for follicular neoplasm (FN/SFN), 53 samples that were suspicious for malignancy (SFM), and 58 malignant samples. AUS/FLUS samples included 3 goiters, 32 follicular adenomas (FAs), 1 noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), 5 invasive follicular variants of papillary thyroid carcinoma (I-FVPTCs), and 1 follicular carcinoma; whereas FN/SFN samples included 24 FAs and 9 malignancies (4 I-FVPTCs, 1 NIFTP, 3 papillary thyroid carcinomas [PTCs], and 1 oncocytic follicular carcinoma). The 53 SFM samples were diagnosed on histopathology as 2 FAs, 5 NIFTPs, 15 I-FVPTCs, and 31 PTCs; whereas the 58 malignant specimens included 5 NIFTPs, 5 I-FVPTCs, and 48 PTCs. Increased plasma membrane and cytoplasmic PD-L1 expression was found in 79 cases (38.5%), including 61 PTCs (conventional and variants). Negative PD-L1 expression was found in NIFTPs and FAs. A BRAF V600E mutation was identified in 15% of PD-L1–positive

Introduction: The detection of rosette-like clusters (RLC) of follicular cells in thyroid carcinoma has been reported mostly in the columnar cell variant of papillary thyroid carcinoma (PTC). Despite the fact that diagnosing variants of PTC is no longer encouraged by The Bethesda System for Reporting Thyroid Cytopathology, the identification of cytomorphological features such as RLC linked with these tumours might help reduce possible misinterpretation in thyroid fine needle aspiration (FNA) cytology. We accordingly investigated the potential correlation of architectural patterns including RLC with PTC variants. Methods: We analysed 225 thyroid FNA cytology cases diagnosed as suspicious for malignancy (SFM) and positive for malignancy (M) over a 1-year time where all samples had corresponding histology. We also included 150 benign lesions from the same period. The presence of RLC vs similar appearing solid clusters, papillary structures and microfollicles were evaluated. We also performed immunocytochemistry and molecular testing for BRAFV600E. Results: We included 100 (44.4%) SFM favouring PTC and 125 (55.6%) M cases with cyto-histological correlation. On histology, all SFM and M cases showed malignancy including 140 (62.2%) classic PTC and 85 (37.8%) PTC variants. The cytomorphological patterns in all FNA samples included solid (74%), papillary (89%), microfollicular (70%), and pseudo-RLC morphology (25.7%). We identified only pseudo-RLC in 33 FNA specimens from PTC variant cases that included tall cell variant (42.4%), hobnail variant (21.2%) and miscellaneous variants (36.3%) of PTC. No definitive RLC were detected in our series. Immunocytochemistry and BRAFV600E were not specifically linked with an RLC pattern. Conclusions: These findings demonstrate that in our dataset the architectural pattern of RLC was not recognised within PTC variants. However, we did identify a pseudo-RLC pattern that was observed in association with tall cell variant and hobnail variant

Alterations in the transcriptional factor c-MYC could be involved in the anti-EGFR resistance in metastatic colorectal cancer (mCRC). The c-MYC expression was evaluated in 121 RAS and BRAF wild-type mCRC before treatment with anti-EGFR+Folfiri therapy and in 33 subsequent metastases collected during target therapy (TT) or in TT resistance phase. We analyzed the expression and the functional role of some c-MYC linked miRNAs (miR-31-3p, miR-143 and miR-145) in our patient group and in two CRC cell lines, also performing a c-MYC target PCR array. Patients with higher c-MYC expression (HME) showed a significant lower PFS and OS when compared to those with low c-MYC expression (LME). HME pattern was significantly more frequent in the metastases after TT and significantly associated to anti-EGFR molecular resistance alterations. We also found a significant correlation between the expression of the above-mentioned c-MYC linked miRNAs, c-MYC level and anti-EGFR resistance. Moreover, expression gene profiling pointed out the pivotal role of c-MYC in CRC-related cell-cycle, apoptosis, signal transduction and cell-growth pathways. c-MYC expression might distinguish patients with a lower PFS and OS in anti-EGFR treated mCRC. The individuation of some miRNAs involved in the c-MYC pathway regulation and the downstream c-MYC effector genes could provide a new possible target to overcome the anti-EGFR resistance in mCRC.

The question whether perivascular glioma cells invading the brain far from the tumor bulk may disrupt the blood–brain barrier (BBB) represents a crucial issue because under this condition tumor cells would be no more protected from the reach of chemotherapeutic drugs. A recent in vivo study that used human xenolines, demonstrated that single glioma cells migrating away from the tumor bulk are sufficient to breach the BBB. Here, we used brain xenografts of patient-derived glioma stem-like cells (GSCs) to show by immunostaining that in spite of massive perivascular invasion, BBB integrity was preserved in the majority of vessels located outside the tumor bulk. Interestingly, the tumor cells that invaded the brain for the longest distances traveled along vessels with retained BBB integrity. In surgical specimens of malignant glioma, the area of brain invasion showed several vessels with preserved BBB that were surrounded by tumor cells. On transmission electron microscopy, the cell inter-junctions and basal lamina of the brain endothelium were preserved even in conditions in which the tumor cells lay adjacently to blood vessels. In conclusion, BBB integrity associates with extensive perivascular invasion of glioma cells.