Trop-2 and Nectin-4 immunohistochemical expression in metastatic colorectal cancer: searching for the right population for drugs' development.

BACKGROUND: Trop-2 and Nectin-4 are transmembrane proteins overexpressed in many tumours and targets of antibody-drug conjugates (ADC). In metastatic colorectal cancer (mCRC), the role of Trop-2 and Nectin-4 has been poorly investigated. METHODS: Tumour samples of patients randomised in the phase III TRIBE2 were assessed for Trop-2 and Nectin-4 expression. RESULTS: Three hundred eighty-six tumours were assessed for Trop-2 expression. 90 (23%), 115 (30%) and 181 (47%) were Trop-2 high, medium and low, respectively. Patients with low Trop-2 tumours achieved longer PFS (12 versus 9.9 months, p = 0.047) and OS (27.3 versus 21.3 months, p = 0.015) than those with high/medium Trop-2 tumours. These findings were confirmed in multivariate analysis (p = 0.022 and p = 0.023, respectively). A greater OS benefit from treatment intensification with FOLFOXIRI/bevacizumab was observed in patients with high/medium Trop-2 tumours (p-for-interaction = 0.041). Two hundred fifty-one tumours were assessed for Nectin-4 expression. Fourteen (5%), 67 (27%) and 170 (68%) were high, medium and low, respectively. No prognostic impact was observed based on Nectin-4 expression and no interaction effect was reported between Nectin-4 expression groups and treatment arm. CONCLUSIONS: In mCRC, expression levels of Trop-2 and Nectin-4 are heterogeneous, suggesting a target-driven development of anti-Trop2 and anti-Nectin-4 ADCs. Medium/high Trop-2 expression is associated with worse prognosis and higher benefit from chemotherapy intensification.

SMARCA4 as a support for the differential diagnosis of poorly differentiated lung carcinomas.

Among non-small cell lung cancers (NSCLCs), sarcomatoid carcinomas account for 3%. They are rare tumours with a poor prognosis, classified into three subgroups, namely pleomorphic carcinoma, pulmonary blastoma and carcinosarcoma. In the 5th edition of WHO Classification of Thoracic Tumours more space is given to SMARC4-deficient lung cancers. Although studies on SMARCA4-deficient lung tumours are limited, a small percentage of SMARCA4 loss is present within NSCLCs. This finding is clinically relevant, as the loss of the SMARCA4 gene is associated with a worse prognosis. In our study, we analysed the presence of the main catalytic subunit of the SMARCA4 gene, the BRG1 protein, in 60 sarcomatoid lung tumours. The results of our study show that 5.3% of sarcomatoid carcinomas have BRG1-loss in tumour cells, proving that a non-negligible amount of lung sarcomatoid carcinomas are SMARCA4-deficient. These data open the debate on the necessity of including the detection of SMARCA4 within a standardised immunohistochemical panel.

Limited Accuracy of Pan-Trk Immunohistochemistry Screening for NTRK Rearrangements in Follicular-Derived Thyroid Carcinoma.

Patients with advanced thyroid cancer harboring NTRK rearrangements can be treated with highly effective selective inhibitors. Immunohistochemistry (IHC) analysis, to detect Trk protein expression, represents an appealing screening strategy for NTRK rearrangements, but its efficacy has been poorly explored in thyroid cancer. The aim of this study is to investigate the diagnostic utility of Trk IHC in the identification of NTRK rearrangements. A series of 26 follicular-derived thyroid tumors, positive for NTRK rearrangements, and 28 NTRK fusion-negative controls were retrospectively analyzed by IHC using the pan-Trk monoclonal antibody (clone EPR17341) on the Ventana system. Area under the curve (AUC), sensitivity and specificity were calculated by ROC analysis. Trk expression was detected in 25 samples, including 22 out of the 26 NTRK-rearranged (84.6%) and three out of 28 NTRK-negative samples (10.7%). Four out of twenty-six NTRK-rearranged thyroid tumors were negative for Trk expression (15.4%), all carrying the ETV6/NTRK3 fusion. The AUC, sensitivity and specificity were 0.87, 0.85 and 0.89, respectively. A screening based on IHC analysis showed limited sensitivity and specificity in the identification of NTRK-rearranged tumors. Since falsely negative results could preclude the administration of effective targeted drugs, alternative detection strategies should be considered for thyroid cancer.

Role of Prophylactic Central Compartment Lymph Node Dissection on the Outcome Of Patients With Papillary Thyroid Carcinoma and Synchronous Ipsilateral Cervical Lymph Node Metastases.

OBJECTIVE: Prophylactic central compartment lymph node dissection (pCCND) results in a higher percentage of surgical-related complications. To date, no evidence of the impact of pCCND on the clinical outcome of papillary thyroid carcinoma (PTC) patients with synchronous ipsilateral cervical lymph node metastases has been reported. METHODS: We evaluated all consecutive patients affected by PTC and synchronous ipsilateral cervical, but without evidence of central compartment, lymph node metastases. We selected 54 consecutive patients (group A) treated by total thyroidectomy, ipsilateral cervical lymph node dissection, and pCCND and 115 patients (group B) matched for sex, age at diagnosis, number and dimension of the metastatic lateral cervical lymph nodes, without pCCND. Clinical outcome after a median of 5 years and surgical-related complications were assessed. RESULTS: The two groups were completely similar in terms of clinical features. Clinical outcomes showed a higher percentage of biochemical and indeterminate but not structural response in group B. Group B required significantly more radioiodine treatments, but no difference was shown in the need to repeat surgery for recurrences. Conversely, the prevalence of permanent hypoparathyroidism was significantly higher in group A (14.8%) than in group B (4.3%). CONCLUSION: In PTC patients with synchronous ipsilateral cervical lymph node metastases, in absence of clinically evident lymph node metastases of the central compartment, performing pCCND does not improve the 5-year outcome in terms of structural disease, despite a greater number of 131I treatments. However, pCCND is severely affected by a higher percentage of permanent hypoparathyroidism, even in the hands of expert surgeons. ABBREVIATIONS: IQR = interquartile range; pCCND = prophylactic central compartment lymph node dissection; PTC = papillary thyroid carcinoma; Tg = thyroglobulin; US = ultrasound.

Sodium-glucose co-transporter (SGLT)2 and SGLT1 renal expression in patients with type 2 diabetes.

AIM: To quantify the expression of sodium-glucose co-transporter (SGLT)2 and SGLT1, their cognate basolateral transporters, GLUT2 and GLUT1, and the transcriptional regulator of SGLTs in renal tissue obtained from people with T2DM and a group of well-matched people without diabetes. METHODS: We measured SGLT2 and SGLT1 expression in unaffected renal tissue from 19 people with T2DM and 20 people without diabetes, matched for age and estimated glomerular filtration rate (controls), undergoing unilateral nephrectomy. Expression of SGLT2 and SGLT1, as well as that of GLUT2 and GLUT1, was quantified using real-time and digital PCR; an affinity-purified antibody against human SGLT2 was used to localize SGLT2 by immunohistochemistry. RESULTS: SGLT2 expression was higher in control than T2DM tissue (median [interquartile range] target/ß-actin 1.62 [2.02] vs 0.67 [0.61]; P < .0001), and SGLT1 trended in the same direction (0.98 [1.19] vs 0.44 [0.48]; P = .08). Immunohistochemistry clearly localized SGLT2 to the tubular brush-border membranes, and was semi-quantitatively stronger in control than T2DM tissue (5.0 [1.0] vs 4.0 [1.0] score units; P = .043). GLUT2 (control vs T2DM: 1.00 [0.69] vs 0.49 [0.36]) and GLUT1 expression (control vs T2DM: 0.86 [0.73] vs 0.35 [0.30]; P = .0007 for both) were closely correlated with those of the respective SGLT partner. Hypoxia-inducible factor 1α, more abundant in control than T2DM tissue, might be a transcription factor involved in the modulation of SGLT2 expression. CONCLUSIONS: In whole renal tissue, expressions of SGLT2/GLUT2 and SGLT1/GLUT1 are coupled and slightly lower in typical people with T2DM as compared with well-matched people without diabetes.

Invasive anisakiasis by the parasite Anisakis pegreffii (Nematoda: Anisakidae): diagnosis by real-time PCR hydrolysis probe system and immunoblotting assay.

BACKGROUND: Anisakiasis is a fish-borne zoonosis caused by Anisakis spp. larvae. One challenging issue in the diagnosis of anisakiasis is the molecular detection of the etiological agent even at very low quantity, such as in gastric or intestinal biopsy and granulomas. Aims of this study were: 1) to identify three new cases of invasive anisakiasis, by a species-specific Real-time PCR probe assay; 2) to detect immune response of the patients against the pathogen. METHODS: Parasite DNA was extracted from parasites removed in the three patients. The identification of larvae removed at gastric and intestinal level from two patients was first obtained by sequence analysis of mtDNA cox2 and EF1 α-1 of nDNA genes. This was not possible in the third patient, because of the very low DNA quantity obtained from a single one histological section of a surgically removed granuloma. Real-time PCR species-specific hydrolysis probe system, based on mtDNA cox2 gene, was performed on parasites tissue of the three cases. IgE, IgG4 and IgG immune response against antigens A. pegreffii by Immunoblotting assay was also studied. RESULTS: According to the mtDNA cox2 and the EF1 α - 1 nDNA sequence analysis, the larvae from stomach and intestine of two patients were assigned to A. pegreffii. The Real-time PCR primers/probe system, showed a fluorescent signal at 510 nm for A. pegreffii, in all the three cases. In Immunoblotting assay, patient CC1 showed IgE, IgG4 reactivity against Ani s 13-like and Ani s 7-like; patient CC2 revealed only IgG reactivity against Ani s 13-like and Ani s 7-like; while, the third patient showed IgE and IgG reactivity against Ani s 13-like, Ani s 7-like and Ani s 1-like. CONCLUSION: The Real-time PCR assay, a more sensitive method than direct DNA sequencing for the accurate and rapid identification of etiological agent of human anisakiasis, was successfully assessed for the first time. The study also highlights the importance to use both molecular and immunological tools in the diagnosis of human anisakiasis, in order to increase our knowledge about the pathological findings and immune response related to the infection by zoonotic species of the genus Anisakis.

Sentinel Lymph Node Mapping in Lung Cancer: A Pilot Study for the Detection of Micrometastases in Stage I Non-Small Cell Lung Cancer.

Lymphadenectomy represents a fundamental step in the staging and treatment of non-small cell lung cancer (NSCLC). To date, the extension of lymphadenectomy in early-stage NSCLC is a debated topic due to its possible complications. The detection of sentinel lymph nodes (SLNs) is a strategy that can improve the selection of patients in which a more extended lymphadenectomy is necessary. This pilot study aimed to refine lymph nodal staging in early-stage NSCLC patients who underwent robotic lung resection through the application of innovative intraoperative sentinel lymph node (SLN) identification and the pathological evaluation using one-step nucleic acid amplification (OSNA). Clinical N0 NSCLC patients planning to undergo robotic lung resection were selected. The day before surgery, all patients underwent radionuclide computed tomography (CT)-guided marking of the primary lung lesion and subsequently Single Photon Emission Computed Tomography (SPECT) to identify tracer migration and, consequently, the area with higher radioactivity. On the day of surgery, the lymph nodal radioactivity was detected intraoperatively using a gamma camera. SLN was defined as the lymph node with the highest numerical value of radioactivity. The OSNA amplification, detecting the mRNA of CK19, was used for the detection of nodal metastases in the lymph nodes, including SLN. From March to July 2021, a total of 8 patients (3 female; 5 male), with a mean age of 66 years (range 48-77), were enrolled in the study. No complications relating to the CT-guided marking or preoperative SPECT were found. An average of 5.3 lymph nodal stations were examined (range 2-8). N2 positivity was found in 3 out of 8 patients (37.5%). Consequently, pathological examination of lymph nodes with OSNA resulted in three upstages from the clinical IB stage to pathological IIIA stage. Moreover, in 1 patient (18%) with nodal upstaging, a positive node was intraoperatively identified as SLN. Comparing this protocol to the usual practice, no difference was found in terms of the operating time, conversion rate, and complication rate. Our preliminary experience suggests that sentinel lymph node detection, in association with the accurate pathological staging of cN0 patients achieved using OSNA, is safe and effective in the identification of metastasis, which is usually undetected by standard diagnostic methods.

Validation of a Gene Expression Approach for the Cytological Diagnosis of Epithelioid and Biphasic Pleural Mesothelioma on a Consecutive Series.

Cytological diagnosis of pleural mesothelioma (PM) is controversial, even using ancillary markers (BAP1, MTAP and CDKN2A). Here, we aimed to prospectively validate a previously developed 117-gene expression panel for the differential cytological diagnosis of epithelioid, biphasic PM and mesothelial hyperplasia. Seventy-seven pleural effusions were classified using the 117-gene expression levels (NanoString system). Sixty-eight cases were also screened for ancillary markers. The performance of both gene panel and ancillary markers was evaluated using ROC metrics. A score using the top consistently deregulated genes between epithelioid and biphasic PM was built to subtype malignant effusions. The panel alone reached a diagnostic accuracy (0.89) comparable to the best marker combination (BAP1 plus MTAP: 0.88). Ancillary tests missed 8 PMs, 7 of which were correctly classified by the panel. The score built by averaging the expression levels of MSLN, CLDN15 and CFB showed an accuracy of 0.80 in subtyping epithelioid and biphasic effusions. The 117-gene panel is effective for PM cytological diagnosis of epithelioid and biphasic PM. This tool can be complementary to ancillary markers, reducing invasive procedures and allowing an earlier diagnosis. Finally, the possibility to subtype PM on effusions strengthens the panel's role in PM diagnosis and management.

Autopsy Study of Testicles in COVID-19: Upregulation of Immune-Related Genes and Downregulation of Testis-Specific Genes.

CONTEXT: Infection by SARS-CoV-2 may be associated with testicular dysfunction that could affect male fertility. OBJECTIVE: Testicles of fatal COVID-19 cases were investigated to detect virus in tissue and to evaluate histopathological and transcriptomic changes. METHODS: Three groups were compared: (a) uninfected controls (subjects dying of trauma or sudden cardiac death; n = 10); (b) subjects dying of COVID-19 (virus-negative in testes; n = 15); (c) subjects dying of COVID-19 (virus-positive in testes; n = 9). SARS-CoV-2 genome and nucleocapsid antigen were probed using RT-PCR, in situ hybridization, and immunohistochemistry (IHC). Infiltrating leukocytes were typed by IHC. mRNA transcripts of immune-related and testis-specific genes were quantified using the nCounter method. RESULTS: SARS-CoV-2 was detected in testis tissue of 9/24 (37%) COVID-19 cases accompanied by scattered T-cell and macrophage infiltrates. Size of testicles and counts of spermatogenic cells were not significantly different among groups. Analysis of mRNA transcripts showed that in virus-positive testes immune processes were activated (interferon-alpha and -gamma pathways). By contrast, transcription of 12 testis-specific genes was downregulated, independently of virus positivity in tissue. By IHC, expression of the luteinizing hormone/choriogonadotropin receptor was enhanced in virus-positive compared to virus-negative testicles, while expression of receptors for androgens and the follicle-stimulating hormone were not significantly different among groups. CONCLUSION: In lethal COVID-19 cases, infection of testicular cells is not uncommon. Viral infection associates with activation of interferon pathways and downregulation of testis-specific genes involved in spermatogenesis. Due to the exceedingly high numbers of infected people in the pandemic, the impact of virus on fertility should be further investigated.

Thyroid Metastasis from Primary Breast Cancer.

Breast cancer (BC), the most commonly diagnosed malignancy, frequently metastasizes to the bone, lungs, brain and liver at advanced stages, whereas the thyroid gland represents a rare target site for secondary disease. We examined the most recent literature about thyroid metastasis (TM) from BC after we encountered a peculiar case of a 71-year-old woman who developed sudden dysphagia, severe hypothyroidism and hypoparathyroidism due to TM 18 years after the diagnosis of her primary cancer. Based on published data, the prevalence of TM in BC ranges from 3% to 34%, with a median onset time of 48.2 months, although longer time intervals are not infrequent. TM negatively impacts the prognosis of these patients, however thyroid surgery can limit the local disease burden. Therefore, we suggest that clinicians involved in the follow-up care of BC patients should consider a differential diagnosis of secondary thyroid malignancy when incidental lesions are diagnosed during radiological evaluations or local symptoms affect the cervical region, even many years after the diagnosis of the primary cancer.

Outcomes of the Tall-Cell Variant of Papillary Thyroid Carcinoma in Patients with Different Ages: A 17-Year Mono-Institutional Experience.

The tall-cell variant of papillary thyroid carcinoma (TCPTC) is the most common aggressive variant of papillary thyroid carcinoma (PTC) and typically occurs in older patients. In this study, we analyzed retrospectively the largest mono-institutional series of PTCs with tall-cell features (989 patients) over a 17-year period, re-evaluating tumors based on age at presentation and outcomes in different age groups. We divided patients into three age groups following different criteria (the criterion from the American Joint Committee on Cancer Tumor Node Metastasis (AJCC TNM) guidelines, criterion for the statistical division into tertiles and adolescent/post-adolescent criterion) to analyze the clinicopathological characteristics in different age groups, especially in terms of recurrence-free survival (RFS) and distant recurrence-free survival (DRFS). We obtained three main results: 1. the population is distributed among the different age groups, and therefore, this type of cancer is not exclusively found among those of an older age; 2. in the RFS analysis, we can see a higher probability of local recurrence in the younger and older groups and, unexpectedly, a lower probability of local recurrence in the "median age" group; and 3. in the DRFS analysis, we can observe a higher probability of distant recurrence in older patients. From a molecular perspective, no significant differences in the mutational status of BRAF were detected according to different age groups, while mutations in the TERT promoter were exclusively present in older patients of all age groups, highlighting the potential prognostic implications of TERT promoter mutations in PTCs. In conclusion, the results of this series confirm that TC morphology alone in PTCs does not have the same negative prognostic significance in the younger population as in the older population. The reason for these different outcomes remains unclear and needs further studies.

New Immunohistochemical Markers for Pleural Mesothelioma Subtyping.

Pleural mesothelioma (PM) comprises three main subtypes: epithelioid, biphasic and sarcomatoid, which have different impacts on prognosis and treatment definition. However, PM subtyping can be complex given the inter- and intra-tumour morphological heterogeneity. We aim to use immunohistochemistry (IHC) to evaluate five markers (Mesothelin, Claudin-15, Complement Factor B, Plasminogen Activator Inhibitor 1 and p21-activated Kinase 4), whose encoding genes have been previously reported as deregulated among PM subtypes. Immunohistochemical expressions were determined in a case series of 73 PMs, and cut-offs for the epithelioid and non-epithelioid subtypes were selected. Further validation was performed on an independent cohort (30 PMs). For biphasic PM, the percentage of the epithelioid component was assessed, and IHC evaluation was also performed on the individual components separately. Mesothelin and Claudin-15 showed good sensitivity (79% and 84%) and specificity (84% and 73%) for the epithelioid subtype. CFB and PAK4 had inferior performance, with higher sensitivity (89% and 84%) but lower specificity (64% and 36%). In the biphasic group, all markers showed different expression when comparing epithelioid with sarcomatoid areas. Mesothelin, Claudin-15 and CFB can be useful in subtype discrimination. PAI1 and PAK4 can improve component distinction in biphasic PM.

Dissecting tumor lymphocyte infiltration to predict benefit from immune-checkpoint inhibitors in metastatic colorectal cancer: lessons from the AtezoT RIBE study.

BACKGROUND: Tumor immune cells influence the efficacy of immune-checkpoint inhibitors (ICIs) and many efforts aim at identifying features of tumor immune microenvironment able to predict benefit from ICIs in proficient mismatch repair (pMMR)/microsatellite stable (MSS) metastatic colorectal cancer (mCRC). METHODS: We characterized tumor immune cell infiltrate, by assessing tumor-infiltrating lymphocytes (TILs), Immunoscore, Immunoscore-IC, and programmed death ligand-1 (PD-L1) expression in tumor samples of patients with mCRC enrolled in the AtezoTRIBE study, a phase II randomized trial comparing FOLFOXIRI/bevacizumab/atezolizumab to FOLFOXIRI/bevacizumab, with the aim of evaluating the prognostic and predictive value of these features. RESULTS: Out of 218 patients enrolled, 181 (83%), 77 (35%), 157 (72%) and 162 (74%) specimens were successfully tested for TILs, Immunoscore, Immunoscore-IC and PD-L1 expression, respectively, and 69 (38%), 45 (58%), 50 (32%) and 21 (13%) tumors were classified as TILs-high, Immunoscore-high, Immunoscore-IC-high and PD-L1-high, respectively. A poor agreement was observed between TILs and Immunoscore or Immunoscore-IC (K of Cohen <0.20). In the pMMR population, longer progression-free survival (PFS) was reported for Immunoscore-high and Immunoscore-IC-high groups compared with Immunoscore-low (16.4 vs 12.2 months; HR: 0.55, 95% CI: 0.30 to 0.99; p=0.049) and Immunoscore-IC-low (14.8 vs 11.5 months; HR: 0.55, 95% CI: 0.35 to 0.85; p=0.007), respectively, with a significant interaction effect between treatment arms and Immunoscore-IC (p for interaction: 0.006) and a trend for Immunoscore (p for interaction: 0.13). No PFS difference was shown according to TILs and PD-L1 expression. Consistent results were reported in the overall population. CONCLUSIONS: The digital evaluation of tumor immune cell infiltrate by means of Immunoscore-IC or Immunoscore identifies the subset of patients with pMMR mCRC achieving more benefit from the addition of the anti-PD-L1 to the upfront treatment. Immunoscore-IC stands as the most promising predictor of benefit from ICIs.

Malignancies within rhinophyma: report of three new cases and review of the literature.

BACKGROUND: Rhinophyma, which represents the end stage of rosacea, is characterized by sebaceous hyperplasia, fibrosis follicular plugging, and telangiectasia. Although it is commonly labeled as an aesthetic problem, it may also determine airway obstruction and because of its nature to hide the growth of tumors. Due to the increasing number of reports of nonmelanoma skin types of cancer within rhinophyma, further concern about a higher incidence of malignancies in rhinophyma than in the skin of normal noses is reasonable. METHODS: We describe three male patients who developed malignancies (2 basal and 1 squamous cell carcinoma) associated with rhinophyma disease. The tumors developed over a mean of 23.3 years after primary diagnosis of rhinophyma. One case had a previous history of facial skin tumor. Surgical excision with clear margins allowed resolution in all three patients with a mean follow-up of 34.8 months. CONCLUSIONS: These three new cases and the review of 43 cases reported in the literature call attention to the clinical features of carcinomas arising in the context of rhinophyma, raising further concerns about the possible association between these two entities. The need for histologic examination of all surgically removed tissue in patients with rhinophyma is highlighted. Several macroscopic changes, including ulceration, drainage, and a rapid growth pattern, should alarm the physician and should be considered as suspicious of a malignant degeneration. Unexpected clinical modifications of a preexisting long-lasting silent rhinophyma could indicate the possibility of hidden malignancy rather than a rhinophyma itself. Although evidence of an association between the two entities remains inconclusive, half of the malignancies reported in our review were incidental findings associated with rhinophyma. Thus, since rhinophyma should not be considered solely a cosmetic problem, we recommend that all specimens be reviewed by a pathologist and if malignancy is diagnosed, re-excision with clear margins should be achieved when necessary with periodic follow-up.

Intestinal Ischemia: Unusual but Fearsome Complication of COVID-19 Infection.

The pathophysiology of gastrointestinal damage in coronavirus disease (COVID-19) is probably multifactorial. It is not clear whether the etiology of intestinal ischemia may be directly related to viral replication or may result from hyper-coagulability following SARS-CoV-2 infection.To confirm a pathogenic role of COVID-19, we retrospectively investigated the presence of SARS-CoV-2 virus in the ischemic bowel of five COVID-19 patients undergoing emergency surgery for intestinal ischemia in the period of March 2020-May 2021. Immunohistochemical positivity with weak intensity was observed in four out of five cases, but only one case was strongly positive both at immunohistochemistry and at molecular analysis. The histological alterations in the intestinal tissue samples showed similarity with the well-known alterations described in typical targetorgans of the virus (e.g., the lung). This observation suggests a similar mechanism of action of the virus. Further larger studies are, thus, required to confirm this preliminary finding. Clinicians should carefully monitor all COVID-19 patients for the possible presence of a SARS-CoV-2 intestinal infection, a potential cause of ischemia and bowel perforation.

Predictive Biomarkers in Thyroid Cancer.

In molecular pathology, predictive biomarkers identify which patients are likely to respond to targeted drugs. These therapeutic agents block specific molecules directly involved in cancer growth, dedifferentiation and progression. Until few years ago, the only targeted drugs available for advanced thyroid cancer included multi-tyrosine kinase inhibitors, mainly targeting the MAPK pathway and the angiogenic signaling. The administration of these drugs does not necessarily require a molecular characterization of tumors to assess the presence of predictive alterations. However, the availability of new selective targeted drugs for thyroid cancer patients is changing the diagnostic strategies for the molecular characterization of these tumors. The search for targetable alterations can be performed directly on tumor tissue by using a variety of methodologies, depending also on the number and type of alterations to test (i.e. single nucleotide variation or gene rearrangement). Herein, a comprehensive review of the currently available targeted treatments for thyroid cancer, related predictive markers and testing methodologies is provided.

Inflammatory Profiles of Tracheal Biopsies From SARS-CoV-2 Patients.

Purpose: An increasing number of laryngotracheal complications in mechanically ventilated COVID-19 patients has been reported in the last few months. Many etiopathogenetic hypotheses were proposed but no clear explanation of these complications was identified. In this paper we evaluated the possibility that the tracheal mucosa could be a high viral replication site that could weaken the epithelium itself. Methods: Subjects for the COVID-19 group and the control group were selected retrospectively according to specific criteria. Patients' basic and clinical data were recorded and analyzed. Tracheal samples of both groups were collected during surgical tracheostomies and then analyzed from a histological and genetic-transcriptional point of view. Results: Four COVID-19 patients were enrolled in this study and compared with four non-COVID-19 patients. No laryngotracheal complications were identified in both groups. The SARS-CoV-2 was detected in one out of four COVID-19 samples. A subepithelial inflammatory lymphomonocyte infiltrate was observed in all patients but two cases of the COVID-19 group showed vasculitis of small subepithelial vessels associated with foci of coagulative necrosis. Two gene sets (HALLMARK_INFLAMMATORY_RESPONSE and HALLMARK_ESTROGEN_RESPONSE_LATE) were significantly deregulated in COVID-19 patients compared to the control group. Conclusion: The altered inflammatory response of the COVID-19 patients could be another possible explanation of the increasing number of laryngotracheal complications.

Gene Expression Analysis of Biphasic Pleural Mesothelioma: New Potential Diagnostic and Prognostic Markers.

Biphasic is the second most common histotype of pleural mesothelioma (PM). It shares epithelioid and sarcomatoid features and is challenging to diagnose. The aim of this study was to identify biphasic PM markers to improve subtyping and prognosis definition. The expression levels of 117 cancer genes, evaluated using the nanoString system, were compared between the three major histotypes (epithelioid, sarcomatoid, and biphasic), and expression differences within biphasic PM were evaluated in relation to the percentage of epithelioid components. Biphasic PM overexpressed CTNNA1 and TIMP3 in comparison to sarcomatoid, and COL16A1 and SDC1 in comparison to epithelioid PM. CFB, MSLN, CLDN15, SERPINE1, and PAK4 were deregulated among all histotypes, leading to the hypothesis of a gradual expression from epithelioid to sarcomatoid PM. According to gene expression, biphasic PM samples were divided in two clusters with a significant difference in the epithelioid component. ADCY4, COL1A1, and COL4A2 were overexpressed in the biphasic group with a low percentage of epithelioid component. Survival analysis using TCGA data showed that high COL1A1 and COL4A2 expression levels correlate with poor survival in PM patients. Herein, we identified markers with the potential to improve diagnosis and prognostic stratification of biphasic PM, which is still an orphan tumor.

Down-regulation of miR-7-5p and miR-548ar-5p predicts malignancy in indeterminate thyroid nodules negative for BRAF and RAS mutations.

PURPOSE: The value of molecular markers in refining preoperative risk assessment of indeterminate thyroid nodules is being widely investigated. MicroRNAs (miRNA) are emerging as promising biomarkers for diagnostic and prognostic purposes. The aim of this study is to identify miRNAs specifically deregulated in mutation-negative indeterminate thyroid nodules. METHODS: Ninety-eight nodules preoperatively diagnosed as TIR 3A or TIR 3B with available histological diagnosis of follicular adenoma (FA), noninvasive follicular neoplasm with papillary-like nuclear features (NIFTP), and follicular variant papillary thyroid carcinoma (FV-PTC) have been retrospectively selected. Mutations in BRAF and RAS genes have been tested in all samples by real-time PCR; miRNAs were purified from cytology slides of 60 samples; expression analysis of 798 miRNAs was measured by the nCounter system. RESULTS: Point mutations in BRAF and RAS genes were detected in 32 out of 98 nodules (32.7%), the majority of which in FV-PTCs. Differential expression of miRNA in wild-type nodules highlighted that two miRNAs, namely miR-7-5p and miR-548ar-5p, were downregulated in FV-PTCs compared to FAs. The combined expression of these miRNAs, tested by ROC analysis, showed an area under the curve of 0.79. Sensitivity and negative predictive value were high both in wild-type (93% and 92%, respectively) and in mutated nodules (94% and 85%, respectively). CONCLUSION: The analysis of miR-7-5p and miR-548ar-5p expression in indeterminate thyroid nodules demonstrated a promising value in ruling out malignancy.

Clinical-Pathological Features and Treatment Outcome of Patients With Hobnail Variant Papillary Thyroid Carcinoma.

Papillary thyroid carcinoma (PTC) with hobnail areas above 30% is classified as hobnail variant (HVPTC). Although it is widely accepted that HVPTC has a worse outcome than classical PTC, it is unclear whether PTC with hobnail features below 30% is as aggressive as HVPTC. We gathered the largest mono-institutional series of PTC with hobnail areas and HVPTC to evaluate differences in terms of pathological features of aggressiveness, molecular profile, and treatment outcome. A total of 99 PTC with hobnail features above 5% were retrospectively selected; 34 of them met the criteria for HVPTC (0.4% of all PTC diagnosed at our institution). All tumors showed high rates of extra-thyroidal extension (40.4%), lymph node metastasis (68.1% of patients with lymphadenectomy), and vascular emboli (49.5%), with no differences according to the 30% cutoff. On the other hand, distant metastases were present in HVPTC only (9.4%). Also, advanced age, advanced disease stage, and TERT promoter mutation were associated with HVPTC. More than half of the patients with follow-up had structural or biochemical persistence after 1 year from surgery. Structural persistence was significantly more common in patients with HVPTC (37.5% vs. 8.7%), while no differences were observed considering structural and biochemical persistence together. The presence of hobnail features identifies locally aggressive tumors, and, consequently, it should be always acknowledged in the pathological report. However, tumors with more than 30% hobnail areas frequently present TERT promoter mutations, advanced disease stage, and structural persistence after radioiodine ablation.