Clinical Relevance and Interplay between miRNAs in Influencing Glioblastoma Multiforme Prognosis.

Glioblastoma multiforme (GBM) is usually treated with surgery followed by adjuvant partial radiotherapy combined with temozolomide (TMZ) chemotherapy. Recent studies demonstrated a better survival and good response to TMZ in methylguanine-DNA methyltransferase (MGMT)-methylated GBM cases. However, approximately 20% of patients with MGMT-unmethylated GBM display an unexpectedly favorable outcome. Therefore, additional mechanisms related to the TMZ response need to be investigated. As such, we decided to investigate the clinical relevance of six miRNAs involved in brain tumorigenesis (miR-181c, miR-181d, miR-21, miR-195, miR-196b, miR-648) as additional markers of response and survival in patients receiving TMZ for GBM. We evaluated miRNA expression and the interplay between miRNAs in 112 IDH wt GBMs by applying commercial assays. Then, we correlated the miRNA expression with patients' clinical outcomes. Upon bivariate analyses, we found a significant association between the expression levels of the miRNAs analyzed, but, more interestingly, the OS curves show that the combination of low miR-648 and miR-181c or miR-181d expressions is associated with a worse prognosis than cases with other low-expression miRNA pairs. To conclude, we found how specific miRNA pairs can influence survival in GBM cases treated with TMZ.

Pathologist Computer-Aided Diagnostic Scoring of Tumor Cell Fraction: A Swiss National Study.

Tumor cell fraction (TCF) estimation is a common clinical task with well-established large interobserver variability. It thus provides an ideal test bed to evaluate potential impacts of employing a tumor cell fraction computer-aided diagnostic (TCFCAD) tool to support pathologists' evaluation. During a National Slide Seminar event, pathologists (n = 69) were asked to visually estimate TCF in 10 regions of interest (ROIs) from hematoxylin and eosin colorectal cancer images intentionally curated for diverse tissue compositions, cellularity, and stain intensities. Next, they re-evaluated the same ROIs while being provided a TCFCAD-created overlay highlighting predicted tumor vs nontumor cells, together with the corresponding TCF percentage. Participants also reported confidence levels in their assessments using a 5-tier scale, indicating no confidence to high confidence, respectively. The TCF ground truth (GT) was defined by manual cell-counting by experts. When assisted, interobserver variability significantly decreased, showing estimates converging to the GT. This improvement remained even when TCFCAD predictions deviated slightly from the GT. The standard deviation (SD) of the estimated TCF to the GT across ROIs was 9.9% vs 5.8% with TCFCAD (P < .0001). The intraclass correlation coefficient increased from 0.8 to 0.93 (95% CI, 0.65-0.93 vs 0.86-0.98), and pathologists stated feeling more confident when aided (3.67 ± 0.81 vs 4.17 ± 0.82 with the computer-aided diagnostic [CAD] tool). TCFCAD estimation support demonstrated improved scoring accuracy, interpathologist agreement, and scoring confidence. Interestingly, pathologists also expressed more willingness to use such a CAD tool at the end of the survey, highlighting the importance of training/education to increase adoption of CAD systems.

Identification of MGMT Downregulation Induced by miRNA in Glioblastoma and Possible Effect on Temozolomide Sensitivity.

Glioblastoma multiforme (GBM) remains one of the tumors with the worst prognosis. In recent years, a better overall survival (OS) has been described in cases subjected to Gross Total Resection (GTR) that were presenting hypermethylation of Methylguanine-DNA methyltransferase (MGMT) promoter. Recently, also the expression of specific miRNAs involved in MGMT silencing has been related to survival. In this study, we evaluate MGMT expression by immunohistochemistry (IHC), MGMT promoter methylation and miRNA expression in 112 GBMs and correlate the data to patients' clinical outcomes. Statistical analyses demonstrate a significant association between positive MGMT IHC and the expression of miR-181c, miR-195, miR-648 and miR-767.3p between unmethylated cases and the low expression of miR-181d and miR-648 and between methylated cases and the low expression of miR-196b. Addressing the concerns of clinical associations, a better OS has been described in presence of negative MGMT IHC, in methylated patients and in the cases with miR-21, miR-196b overexpression or miR-767.3 downregulation. In addition, a better progression-free survival (PFS) is associated with MGMT methylation and GTR but not with MGMT IHC and miRNA expression. In conclusion, our data reinforce the clinical relevance of miRNA expression as an additional marker to predict efficacy of chemoradiation in GBM.

New digital confocal laser microscopy may boost real-time evaluation of endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) from solid pancreatic lesions: Data from an international multicenter study.

BACKGROUND: Pancreatic cancer is an aggressive malignancy and a leading cause of cancer death worldwide; its lethality is partly linked to the difficulty of early diagnosis. Modern devices for endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) were recently developed to improve targeting and sampling of small lesions, but innovative technologies for microscopic assessment are still lacking. Ex vivo fluorescence confocal laser microscopy (FCM) is a new digital tool for real-time microscopic assessment of fresh unfixed biological specimens, avoiding conventional histological slide preparation and potentially being highly appealing for EUS-FNB specimens. METHODS: This study evaluated the possible role of FCM for immediate evaluation of pancreatic specimens from EUS-FNB. It involved comparison of the interobserver agreement between the new method and standard histological analysis during international multicenter sharing of digital images. Digital images from 25 cases of EUS-FNB obtained with real-time FCM technology and 25 paired digital whole-slide images from permanent conventional paraffin sections were observed by 10 pathologists from different Institutions in Europe, Japan, and the United States, in a blinded manner. The study evaluated 500 observations regarding adequacy, morphological clues, diagnostic categories, and final diagnosis. FINDINGS: Statistical analysis showed substantial equivalence in the interobserver agreement among pathologists using the two techniques. There was also good inter-test agreement in determining sample adequacy and when assigning a diagnostic category. Among morphological features, nuclear enlargement was the most reproducible clue, with very good inter-test agreement. INTERPRETATION: Findings in this study are from international multicenter digital sharing and are published here for the first time. Considering the advantages of FCM digital diagnostics in terms of reduced time and unaltered sample maintenance, the ex vivo confocal laser microscopy may effectively improve traditional EUS-FNB diagnostics, with significant implications for planning modern diagnostic workflow for pancreatic tumors. FUNDING: This study was not supported by any funding source.

Thyroid Nodules with Indeterminate FNAC According to the Italian Classification System: Prevalence, Rate of Operation, and Impact on Risk of Malignancy. An Updated Systematic Review and Meta-analysis.

A thyroid nodule classified as indeterminate on fine-needle aspiration cytology (FNAC), hereafter referred to as an indeterminate thyroid nodule (ITN), represents a clinical dilemma. The Italian Consensus for the Classification and Reporting of Thyroid Cytology (ICCRTC) divides ITNs into low- and high-risk categories (i.e., TIR3A and TIR3B, respectively) to better manage patients. This study aimed to achieve high-evidence estimates of the prevalence, rate of operation, and risk of malignancy of ITNs, including TIR3A and TIR3B ITNs. This systematic review was conducted according to MOOSE to retrieve all original studies citing ICCRTC. The last search was performed in February 2022. The risk of bias of the included studies was assessed. Separate proportion meta-analyses were performed with a random-effect model using OpenMeta[Analyst]. The online search processed 271 studies, and 33 were finally considered. First, the cancer prevalence among ITNs was 32.4%. Second, the cancer prevalence among TIR3As was 12.4%, with heterogeneity (I2 90%) explained by a linear correlation between sample size and cancer rate (p = 0.009). Third, the cancer prevalence among TIR3Bs was 44.4%, with heterogeneity (I2 75%) explained by the inverse correlation between sample size and cancer rate (p = 0.031). Fourth, the prevalence of ITNs, TIR3A, and TIR3B among FNACs was 29.6%, 12.6%, and 12.9%, respectively, with sample size and TIR3B prevalence being inversely correlated (p = 0.04). Fifth, the operation rates of ITNs, TIR3A, and TIR3B were 54.3%, 48.3%, and 75.2%, respectively, and the sample size and TIR3A operation rate were inversely correlated (p = 0.010). These data strongly support the division of ITNs into low- and high-risk subcategories. Importantly for clinical practice, the cancer rate among ITNs is significantly influenced by the study sample size.

Cytologic presentation of ovarian large cell carcinoma with rhabdoid features detected on peritoneal washing. Report of one case with cyto-histologic correlation and previously undescribed inactivating SMARCA-4 mutations.

The SMARCA subgroup of genes belongs to the SWI1/SNF1 family, responsible for chromatin remodeling and repair within the nucleosome. The SMARCA4 gene is located on chromosome 19p13 and encodes the BRG1 (BRAhMA) protein. We report the cytological and histological findings in one case of large cell SMARCA4 deficient ovarian carcinoma with positive peritoneal washing in a 69-year-old woman. The neoplastic cells were present as singly lying or perivascular clusters and showed medium or large size, round to oval hyperchromatic nuclei, and scarce to moderate cytoplasms. Molecular pathology investigations performed on the ovarian surgical sample found two previously undescribed mutations in the SMARCA4 gene and additional mutations in the CTNNB1 (Beta Catenin gene) and in PIK3CA. To our knowledge, this case probably represents the third cytologic report of this variant of ovarian carcinoma and the first one with molecular pathologic study.

Percutaneous biopsy of small renal mass: can diagnostic accuracy be affected by hospital volume?

INTRODUCTION: High diagnostic performance and low morbidity for renal tumor biopsy (RTB) have been described in highly experienced centers. Here we present the five-year experience of our institute in performing RTB. The protocol used, the safety profile and the diagnostic accuracy obtained were analyzed. MATERIAL AND METHODS: The study is a retrospective single-institution clinical data review of 84 consecutive RTB of small renal masses. Post-biopsy complications were reported using the Clavien-Dindo system. To measure the concordance between biopsy and nephrectomy specimens regarding histological subtype and International Society of Urological Pathology/World Health Organization (ISUP/WHO) renal cell carcinoma grade, the kappa coefficient of Cohen was used. RESULTS: Median (IQR) follow-up time was 44 (29-58) months. In total, 94% of RTB procedures were free of complications; when complications did occur, 80% were grade I and 20% were grade II. No cases of tumor seeding were observed. Combining the first and repeated biopsies the overall diagnostic rate was 85.8%. Overall, 79.1% of diagnostic RTB were malignant. In 42 surgically treated patients, the concordance between the histological results of biopsies and surgical specimens was very good for histological subtypes (k = 0.87) and moderate for tumor grade (k = 0.51). CONCLUSIONS: RTB resulted in a high safety profile. The overall diagnostic rate was 85% and an unnecessary intervention was avoided in 21% of patients. RTB showed a very good accuracy in determining the histological subtype of renal cancer while it was moderate for the tumor grade. These results are similar to those reported in larger series and support feasibility of this procedure in low-volume centers.

A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies.

BACKGROUND: A major perspective for the use of circulating tumor DNA (ctDNA) in the clinical setting of non-small cell lung cancer (NSCLC) is expected as predictive factor for resistance and response to EGFR TKI therapy and, especially, as a non-invasive alternative to tissue biopsy. However, ctDNA is both highly fragmented and mostly low concentrated in plasma and serum. On this basis, it is important to use a platform characterized by high sensitivity and linear performance in the low concentration range. This motivated us to evaluate the newly developed and commercially available SensiScreen® EGFR Liquid assay platform (PentaBase) with regard to sensitivity, linearity, repeatability and accuracy and finally to compare it to our already implemented methods. The validation was made in three independent European laboratories using two cohorts on a total of 68 unique liquid biopsies. RESULTS: Using artificial samples containing 1600 copies of WT DNA spiked with 50% - 0.1% of mutant copies across a seven-log dilution scale, we assessed the sensitivity, linearity, repeatability and accuracy for the p.T790M, p.L858R and exon 19 deletion assays of the SensiScreen® EGFR Liquid assay platform. The lowest value detectable ranged from 0.5% to 0.1% with R2≥0,97 indicating good linearity. High PCR efficiency was shown for all three assays. In 102 single PCRs each containing theoretical one copy of the mutant at initiating, assays showed repeatable positivity in 75.5% - 80.4% of reactions. At low ctDNA levels, as in plasma, the SensiScreen® EGFR Liquid assay platform showed better sensitivity than the Therascreen® EGFR platform (Qiagen) and equal performance to the ctEGFR Mutation Detection Kit (EntroGen) and the IOT® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific) with 100% concordance at the sequence level. CONCLUSION: For profiling clinical plasma samples, characterized by low ctDNA abundance, the SensiScreen® EGFR Liquid assay is able to identify down to 1 copy of mutant alleles and with its high sensitivity, linearity and accuracy it may be a competitive platform of choice.

Correction to: Functional and clinical significance of ROR1 in lung adenocarcinoma.

An amendment to this paper has been published and can be accessed via the original article.

Functional and clinical significance of ROR1 in lung adenocarcinoma.

BACKGROUND: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is normally detectable in embryonic tissues and absent in adult tissues. ROR1 was shown to inhibit apoptosis, potentiate EGFR signaling and reported to be overexpressed and associated with poor prognosis in several tumor models. This study aimed to assess the expression of ROR1 in lung adenocarcinoma (AC) patients. METHODS: We analyzed ROR1 expression by quantitative real-time PCR (qRT-PCR) in 56 histologically confirmed lung AC, stage I to IV, in addition we evaluated its association with TTF-1 (thyroid transcription factor-1) expression and the main molecular alterations involved in lung cancerogenesis. RESULTS: ROR1 overexpression was observed in 28.6% of the entire cohort, using a cut-off of 1, or in 51.8% of the cases using the median value as threshold. Among patients without any genetic alteration, ROR1 overexpression was observed in 34.8% considering a cut-off of 1 and 52.2% considering the median value. The distribution of ROR1 was homogeneous among the different molecular categories: we found no association of ROR1 expression and the presence of gene mutations/rearrangements or the expression of TTF-1. CONCLUSIONS: ROR1 overexpression could constitute a potential therapeutic target because altered in a consistent number of lung AC, especially in cases without druggable genetic alterations. ROR1 expression is independent of classical lung cancer molecular alterations and not correlated, in a Caucasian cohort, to TTF-1 expression.

Prostatic Ductal Adenocarcinoma with Papillary Features in a Bladder Washing: A Possible Pitfall in Urinary Cytology - Report of One Case.

In this article, we report on a case of combined, acinar and ductal prostatic adenocarcinoma affecting the prostatic urethra, which, due to a low degree of cytologic atypia and an exclusive papillary architecture with visible fibrovascular core, was erroneously diagnosed as a low-grade urothelial carcinoma based on its peculiar cytologic presentation in a bladder washing sample.

Risk of Malignancy (ROM) of Thyroid FNA Diagnosed as Suspicious for Malignancy or Malignant: an Institutional Experience with Systematic Review and Meta-Analysis of Literature.

A universal recommendation does not exist for thyroid FNA suspicious for malignancy (SFM). In this context, the guidelines have estimated a risk of malignancy (ROM) from 50 to 80% and both total thyroidectomy and lobectomy may be indicated. This study aimed to (1) retrospectively evaluate the SFM (i.e., TIR4) in a single institution to estimate their cancer prevalence at histology, and (2) systematic review the literature to obtain more robust information. The study period was 2015-2018. As a major inclusion criterion, both cytology and histology had to be performed in our institution. Histological diagnosis was the gold standard. For the systematic review, the online databases of Google Scholar, PubMed/MEDLINE, and Scopus were searched for papers using the same classification for thyroid FNA. A proportion meta-analysis was performed to obtain the pooled histological cancer rate among TIR4 and TIR5 (random-effects model). In the institutional database, there were 271 nodules with both histology and FNA and the cancer rate of TIR4 was 88.9%. By systematic review, five studies were selected for the meta-analysis. The pooled cancer rate was 85% in TIR4 and 99% in TIR5 (I2 = 0%; no publication bias). In conclusion, these new findings should prompt the guidelines board to fully revise the estimated ROM of SFM category. Clinical thyroidologists and thyroid surgeons should be aware of these data and the patients with SFM should be informed of their ROM.

Solid papillary thyroid carcinoma with Hashimoto's thyroiditis: description of a further case with challenging cytological features.

Solid papillary thyroid carcinoma (SV-PTC) is a rare variant which is mainly observed in young patients with a history of exposure to ionising radiations. Neoplasms belonging to such category generally carry RET-PTC (REarranged during Transfection- Papillary Thyroid Carcinoma) fusions and seem to have a slightly worse prognosis with respect to classical and follicular variants of papillary thyroid carcinoma (PTC), though consistent prognostic and survival data are scarce. SV-PTC should be differentiated from trabecular-insular poorly differentiated thyroid carcinomas, which occur in a different age group and carry a dismal prognosis. These latter tumours do not show the typical nuclear features of PTC and show tumour necrosis with an high mitotic activity. In this report a further case of SV-PTC is described which was associated to Hashimoto's thyroiditis, a finding never described in the cytological literature up to now for SV-PTC; this association created further differential diagnostic problems. The neoplasm displayed RET-PTC1 fusion.

The Impact of Surgery in IDH 1 Wild Type Glioblastoma in Relation With the MGMT Deregulation.

Object: The treatment of choice in glioblastoma (GBM) is the maximal surgical extent of resection (EOR) followed by adjuvant chemo-radiotherapy. Furthermore, methylguanine-DNA methyltransferase (MGMT) promoter methylation is associated with prolonged overall survival (OS) and progression free survival (PFS). The objective of the present study is correlate the biomolecular aspects in relation with EOR. Materials and methods: We analyzed a series of 116 patients with IDH-1 wild type GBM and different EOR (Gross Total Resection-GTR-, Partial Resection-PR- and Biopsy), treated with adjuvant chemo-radiotherapy. The MGMT status was analyzed in terms of promoter methylation and protein expression. Results: When GTR was possible, OS and PFS were significantly better compared to the other two groups (p = 0.001 and p = 0.035, respectively). MGMT methylation was significantly associated with better OS in the biopsy group (p = 0.022) and better OS and PFS in PR (p = 0.02 and p = 0.012, respectively), but not in the GTR group (p = 0.252 for OS, p = 0.256 for PFS) nor the PFS in the biopsy group (p = 0.259). MGMT protein expression levels do not show any association with OS and PFS, regardless of the type of surgery. Conclusions: Our study confirms the positive association of a safe maximal EOR with better OS and PFS, and indicates a positive prognostic value of MGMT methylation status only in case of the presence of residual tumor tissue. MGMT protein expression seems not to play a clinical role in relation with the type of surgery.

Medullary-like hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent primary hepatic cancer. Pathological features can define the biological behavior and prognosis. Medullary-like HCC is a very rare variant that has been described only twice in literature. In the present study, we report the case of a non-cirrhotic 72-year-old man, who presented two HCC lesions on routine screening for hepatitis C virus liver disease. Radiological imaging and biopsy showed two different subtypes: one classic HCC, which was treated with chemoembolization, and a second PET/CT-positive carcinoma with a PET/CT-positive metastatic coeliac lymph node, which was resected laparoscopically with a left lateral sectionectomy and extended lymphadenectomy. Histopathology revealed a medullary-like HCC; lymph node analysis confirmed the metastatic nature of the PET/CT-positive coeliac node and showed an incidental B-cell lymphoma in the hepatic pedicle lymph nodes. To the best of our knowledge this is the third case of medullary-like HCC described in the literature, and the first associated to a concomitant typical HCC.

Histologic Outcome of Indeterminate Thyroid Nodules Classified at Low or High Risk.

The revised Italian consensus for the classification and reporting of thyroid cytology (ICCRTC) was published in 2014. Very recently, a high reliability of ICCRTC in classifying low and high risk indeterminate nodules (Tir 3A and Tir 3B, respectively) was demonstrated. This finding prompted us to review our case series of thyroid indeterminate lesions to verify these data. Only lesions undergone FNAC from December 2014 to October 2017 with subsequent histology at our institutions were eligible for the study. All cytologic samples had originally been classified according to ICCRTC in the subcategories of indeterminate lesion, such as Tir 3A and Tir 3B by three cytopathologists and another one with more than 10 years experience, when necessary. Sixty-three indeterminate FNAC were diagnosed during the study period, of which 51 were subsequently surgically treated. Overall, 9 carcinomas (7 follicular and 2 papillary) and 42 benign lesions were found at histology. The cancer rate observed in the Tir 3A category (3/40, 7.5%) was significantly (p = 0.0015) lower than that found in Tir 3B (6/11, 54.5%). No significant differences were found in age and size of the sampled nodules between the two subcategories. We can confirm in our series that the Italian reporting system for thyroid cytology shows high reliability in discriminating low risk indeterminate lesions from those at high risk of malignancy.

Pure intraductal carcinoma of the parotid gland: Cytologic findings on FNA sample. Report of one case.

One case of intraductal carcinoma of the parotid gland in a 67-year-old male patient is here introduced. The patient, who had a one-year history of a parotid mass, had undergone ultrasound and MRI examination that disclosed a 13x4x3 mm well delimited nodular mass of the accessory lobe of his left parotid gland. Ultrasound-guided Fine Needle Aspiration (FNA) had been performed by the clinician. The obtained smears showed widespread cellular necrosis in which cellular clusters with moderate and focally severe atypias displayed papillary and cribriform architecture and were admixed with sheets of epithelial cells with less striking nuclear atypias, squamous, or apocrine metaplasia. Histopathological examination disclosed a pure intraductal carcinoma of the parotid gland with classical morphology, which was radically excised. The differential cytological diagnosis of pure intraductal carcinoma of salivary glands may be difficult and comprises mucoepidermoid carcinoma as well as "in situ" carcinomas developping in the context of sclerosing polycystic adenosis, mammary analogue secretory carcinoma (MASC) of the salivary glands and cystic variants of salivary adenocarcinoma NOS (formerly called cystadenocarcinomas).

Synovial sarcoma metastatic to the parotid gland: A possible pitfall in salivary gland fine needle aspiration cytology. Report of a case with immunocytochemical and fluorescence in situ hybridization findings.

We report one case of metastatic synovial sarcoma (SS) to the parotid gland in a 93-year-old male. The patient had undergone upper left pulmonary lobectomy with mediastinal lymphadenectomy for SS of the lung 5 years before. The cytopathologic presentation and the immunocytochemical findings on the FNA sample were suggestive of a spindle cell myoepithelioma, while a SYT rearrangement was identified by a FISH performed on a cytological smear of the lesion. The diagnosis was further confirmed also by positive immunocytochemical expression of TLE1 on a section from the obtained cell block. The cytologic and immunophenotypic findings are shortly discussed in view of the reported immunophenotypic inconsistency of SS and of its differential diagnosis with spindle cell myoepithelioma of the salivary glands.The importance of the recently described TLE1 staining and its close correlation to SYT rearrangement is briefly discussed.

Cytological and Immunocytochemical Features of Merkel Cell Carcinoma on Fine Needle Cytology Samples: A Study of 22 Cases.

Merkel cell carcinoma (MCC) is an uncommon neuroendocrine small cell tumor derived from the transformation of the homonymous cells in the basal layer of the epidermis. MCC has a generally aggressive course, with a high tendency for local recurrence, lymph node involvement, and distant metastasis. Fine needle cytology (FNC) and immunocytochemistry were used as diagnostic procedures for 22 cases of MCC presented at our institute. All cases of MCC were successfully diagnosed on FNC. Among all of the monoclonal antisera used (CD56, CK20, CK MNF116, neuron-specific enolase (NSE), synaptophysin, and chromogranin), NSE and CD56 showed the highest frequency of positivity. The accuracy of the cytological diagnosis was 100% compared to the corresponding previous or subsequent pathological diagnoses. FNC and immunocytochemistry represent excellent and accurate diagnostic methods to distinguish MCC from other small-cell malignant entities.