The evolving role of interventional cytopathology from thyroid FNA to NGS: Lessons learned at Federico II University of Naples.

Fine-needle aspiration (FNA) guided by ultrasound (US) has emerged as a highly precise diagnostic method for managing thyroid nodules, significantly diminishing unnecessary surgeries. The effectiveness of US-guided FNA is high when a single specialist performs the FNA procedure and the microscopy. This paradigm has paved the way for the evolution of interventional cytopathology, a specialist with a pivotal role in the preoperative diagnostic process, encompassing patient history review, clinical examination, FNA execution under US guidance, preparation, and microscopic interpretation of cytological samples. As the landscape of precision medicine unfolds, molecular testing assumes greater importance in thyroid cytopathology, particularly in refining the risk of malignancy for indeterminate nodules. The updated Bethesda classification system underscores the clinical significance of molecular tests, emphasizing their role in refining diagnostic accuracy. With this evolving landscape, interventional cytopathologists must adapt by acquiring expertise in molecular technologies and addressing ongoing challenges in workflow harmonization and optimization. This paper delves into our decade-long experience as interventional cytopathologists, focusing on recent endeavours to ensure adequate samples not only for microscopic diagnosis but also for molecular testing. Additionally, here we review the challenges of integrating next-generation sequencing (NGS) technology into clinical practice, highlighting the importance of integrating clinically meaningful molecular data into comprehensive molecular cytology reports.

KCL TEST: an open-source inspired asymptomatic SARS-CoV-2 surveillance programme in an academic institution.

Rapid and accessible testing was paramount in the management of the COVID-19 pandemic. Our university established KCL TEST: a SARS-CoV-2 asymptomatic testing programme that enabled sensitive and accessible PCR testing of SARS-CoV-2 RNA in saliva. Here, we describe our learnings and provide our blueprint for launching diagnostic laboratories, particularly in low-resource settings. Between December 2020 and July 2022, we performed 158277 PCRs for our staff, students, and their household contacts, free of charge. Our average turnaround time was 16 h and 37 min from user registration to result delivery. KCL TEST combined open-source automation and in-house non-commercial reagents, which allows for rapid implementation and repurposing. Importantly, our data parallel those of the UK Office for National Statistics, though we detected a lower positive rate and virtually no delta wave. Our observations strongly support regular asymptomatic community testing as an important measure for decreasing outbreaks and providing safe working spaces. Universities can therefore provide agile, resilient, and accurate testing that reflects the infection rate and trend of the general population. Our findings call for the early integration of academic institutions in pandemic preparedness, with capabilities to rapidly deploy highly skilled staff, as well as develop, test, and accommodate efficient low-cost pipelines.

Molecular testing for personalized therapy is underutilized in patients with borderline resectable and locally advanced pancreatic cancer - real world data from the NORPACT-2 study.

Background: International guidelines currently recommend the use of molecular testing in patients with advanced pancreatic cancer. The rate of actionable molecular alterations is low. The utility of molecular testing in patients with borderline resectable (BRPC) or locally advanced (LAPC) pancreatic cancer in real world clinical practice is unclear.Methods: 188 consecutive patients included in a prospective, population-based study (NORPACT-2) in patients with BRPC and LAPC (2018-2020) were reviewed. Molecular testing was performed at the discretion of the treating oncologist and was not recommended as a routine investigation by the national guidelines. All patients were considered fit to undergo primary chemotherapy and potential surgical resection. The frequency and the results of molecular testing (microsatellite instability (MSI) and/or KRAS status) were assessed.Results: Thirty patients (16%) underwent molecular testing. MSI tumour was detected in one (3.6%) of 28 tested patients. The patient received immunotherapy and subsequently underwent surgical resection. Histological assessment of the resected specimen revealed a complete response. KRAS wild type was detected in one (14.3%) of seven tested patient. Patients who initiated FOLFIRINOX as the primary chemotherapy regimen (p = 0.022), or were being treated at one of the eight hospital trusts (p = 0.001) were more likely to undergo molecular testing.Conclusions: Molecular testing was rarely performed in patients with BRPC or LAPC. Routine molecular testing for all patients with BRPC and LAPC should be considered to increase identification of targetable mutations and improve outcomes.

Validation and Implementation of a Somatic only Tumor Exome for Routine Clinical Application.

Next generation sequencing based genomic testing is standard of care for tumor workflows. However, its application across different institutions continues to be challenging given the diversity of needs and resource availability amongst different institutions globally. Moreover, the use of a variety of different panels including those from a few individual genes to those involving hundreds of genes results in a relatively skewed distribution of care for patients. It is imperative to obtain a higher level of standardization without having to be restricted to specific kits or require repeated validations which are generally expensive. We show the validation and clinical implementation of the DH-CancerSeq assay, a tumor only whole exome based sequencing assay with integrated informatics while providing similar input requirements, sensitivity and specificity to a previously validated targeted gene panel while also maintaining similar turnaround times for patient care.

Evaluation of Multiple RNA Extraction Protocols for Chikungunya Virus Screening in Aedes aegypti Mosquitoes.

Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat. The main urban vector of CHIKV is the Aedes aegypti mosquito, which is found throughout Brazil. Therefore, it is important to carry out laboratory tests to assist in the virus's diagnosis and surveillance. Most molecular biology methodologies use nucleic acid extraction as the first step and require quality RNA for their execution. In this context, four RNA extraction protocols were evaluated in Ae. aegypti experimentally infected with CHIKV. Six pools were tested in triplicates (n = 18), each containing 1, 5, 10, 20, 30, or 40 mosquitoes per pool (72 tests). Four commercial kits were compared: QIAamp®, Maxwell®, PureLink®, and PureLink® with TRIzol®. The QIAamp® and PureLink® with TRIzol® kits had greater sensitivity. Two negative correlations were observed: as the number of mosquitoes per pool increases, the Ct value decreases, with a higher viral load. Significant differences were found when comparing the purity and concentration of RNA. The QIAamp® protocol performed better when it came to lower Ct values and higher RNA purity and concentration. These results may provide help in CHIKV entomovirological surveillance planning.

The Role of Molecular Markers in Thyroid Cancer Diagnostics and Treatment.

Molecular testing for cytologically indeterminate thyroid nodules has demonstrated benefit by reducing the need for diagnostic thyroidectomies and reducing costs. Its use is currently recommended in practice guidelines from the American Thyroid Association and the American Association of Endocrine Surgeons when clinically appropriate. Moreover, there is growing evidence that molecular testing may provide prognostic information and can detect targetable genetic alterations which may expand treatment options for refractory advanced thyroid cancers.

Successive next-generation sequencing strategy for optimal fusion gene detection in non-small-cell lung cancer in clinical practice.

Metastatic non-small-cell lung cancer (NSCLC) displays various molecular alterations in the RAS-MAPK pathway. In particular, NSCLCs show high rates of targetable gene fusion in ALK, RET, ROS1, NRG1 and NTRK, or MET exon 14 skipping. Rapid and accurate detection of gene fusion in EGFR/KRAS/BRAF mutations is important for treatment selection especially for first-line indications. RNA-based next-generation sequencing (NGS) panels appear to be the most appropriate as all targets are multiplexed in a single run. While comprehensive NGS panels remain costly for daily practice, optimal sequencing strategies using targeted DNA/RNA panel approaches need to be validated. Here, we describe our lung cancer screening strategy using DNA and RNA targeted approaches in a real-life cohort of 589 NSCLC patients assessed for molecular testing. Gene fusions were analysed in 174 patients negative for oncogene driver mutations or ALK immunohistochemistry in a two-step strategy. Targetable alterations were identified in 28% of contributive samples. Non-smokers had a 63.7% probability to have a targetable alteration as compared to 21.5% for smokers. Overall survival was significantly higher (p=0.03) for patients who received a molecularly matched therapy. Our study shows the feasibility in routine testing of NSCLC DNA/RNA molecular screening for all samples in a cost- and time-controlled manner. The significant high fusion detection rate in patients with wild-type RAS-MAPK tumours highlights the importance of amending testing strategies in NSCLC.

Timeliness of EGFR/ALK Inhibitor Treatment and Survival in Lung Cancer: A Population-Based Analysis.

BACKGROUND: ALK or EGFR inhibitor is an ideal frontline treatment for patients with advanced non-small cell lung cancer (NSCLC) harboring targetable alteration in ALK or EGFR. However, in the real-world setting, frontline treatment may be delayed or not ideal. For such patients, the benefit of initiating ALK or EGFR inhibitor at a later timepoint remains uninvestigated. METHODS: We utilized a nationwide electronic health record-derived, deidentified database collected from diverse oncology practices across the United States to investigate the timeliness of preferred targeted therapy (PTT). Individualized data obtained from patients with stage IV NSCLC at diagnosis treated with PTT from 2018 to 2023 were analyzed. RESULTS: Data from 3250 patients were analyzed: 2640 patients (81%) with EGFR mutation and 610 patients (19%) with ALK rearrangement. The median time to PTT was 7 weeks from diagnosis with 26.4% of patients started PTT within 1 month. Landmark analyses using timepoints ranging from 1 to 12 months after diagnosis showed that at all timepoints, patients who had started on PTT had a significantly better survival than those who had not. In a multivariable analysis, time to PTT ≤ 1 month from diagnosis was an independent predictor of survival: HR 0.74 (95% CI: 0.62-0.89), P = .002. Time to PTT was significantly associated with age, smoking status and genomic class. CONCLUSIONS: In this population-based analysis, an initiation of PTT occurring as late as at least 1 year from diagnosis still resulted in a significant survival benefit, though the magnitude of benefit appeared decreased as time passed.

Detangling the Threads of Hairy Cell Leukemia, Beyond the Morphology and Into the Molecular.

Hairy cell leukemia (HCL) makes up 2% of leukemias in the United States and encompasses great molecular heterogeneity. The standard treatment paradigm involves purine nucleoside analogues in the upfront setting with high complete response rate to initial therapy but frequent relapses. There is an increasing role for BRAF inhibitors, with or without rituximab, in refractory and even in untreated patients. The response to purine analogues in HCL variant cases, otherwise classified as splenic lymphoma with prominent nucleolus in the 5th WHO edition classification, is less robust. Several antibodies, small molecular inhibitors, and combination regimens have been explored in HCL but data is frequently limited by case reports or small case series. Here we review available treatment options including their efficacy and safety profiles. We also explore investigational agents and potential future targets. The goal is to present a comprehensive therapeutic review of this rare disease entity and outline the ever increasing and novel therapeutic management options which interrupt key pathways in the pathogenesis of this malignancy.

Molecular Testing in Lung Cancer: Recommendations and Update.

Adoption of molecular testing in lung cancer is increasing. Molecular testing for staging and prediction of response for targeted therapy remain the main indications, and although utilization of blood-based testing for tumor is growing, the use of the diagnostic cytology and tissue specimens is equally important. The pathologist needs to optimize reflex testing, incorporate stage-based algorithms, and understand types of tests for timely and complete assessment in the majority of cases. When tissue is limited, testing should capture the most frequent alterations to maximize the yield of what are largely mutually exclusive alterations, avoiding the need for repeat biopsy.

A Prospective Study of Publicly Funded Molecular Testing of Indeterminate Thyroid Nodules: Canada's Experience.

CONTEXT: Indeterminate thyroid nodules (ITNs) lead to diagnostic surgeries in many countries. Use of molecular testing (MT) is endorsed by several guidelines, but costs are limitative, especially in public healthcare systems like in Canada. OBJECTIVES: Primary objective: evaluate the clinical value of Thyroseq® v3 (TSv3) using benign call rate (BCR) in a real-world practice. Secondary objective: assess cost-effectiveness of MT. DESIGN: This is a multicentric prospective study. SETTING: This study was conducted in 5 academic centers in Quebec, Canada. PATIENTS OR OTHER PARTICIPANTS: 500 consecutive patients with Bethesda III (on 2 consecutive cytopathologies) or IV and TIRADS 3 or 4 nodules measuring 1 to 4 cm were included. INTERVENTION: MT was performed between November 2021 and November 2022. Patients with a positive TSv3 were referred to surgery. Patients with a negative TSv3 were planned for follow-up by ultrasonography for a minimum of 2 years. MAIN OUTCOME MEASURE: The BCR, corresponding to the proportion of ITNs with negative TSv3 results, was assessed. RESULTS: 500 patients underwent TSv3 testing, with a BCR of 72.6% (95% CI: 68.5-76.5; p<0.001). 99.7% of patients with a negative result avoided surgery. The positive predictive value of TSv3 was 68.2% (95% CI: 58.5-76.9). The cost-benefit analysis identified that the implementation of MT would yield cost savings of $6.1 million over the next 10 years. CONCLUSIONS: Use of MT (TSv3) in a well-selected population with ITNs led to a BCR of 72.6%. It is cost-effective and prevents unnecessary surgeries in a public healthcare setting.

Molecular Pathology of Lung Cancer.

The identification of targetable genomic alterations in lung cancer is required as standard of care to guide optimal therapy selection. With a constantly evolving landscape of ancillary molecular and biomarker testing in lung cancer, pathologists need to be aware of what specimens to test, how the testing should be performed, and which targets to test for to provide the clinically relevant genomic information necessary to treat these patients. Several guideline statements on the topic are currently available to help pathologists and laboratory personnel best use the small specimens obtained from patients with lung cancer for ancillary molecular testing.

Brief Report on Global Clinician Practices in the First-Line Management of Metastatic Non-Small Cell Lung Cancer.

INTRODUCTION: In the dynamic landscape of metastatic NSCLC (mNSCLC) management, marked by several frontline options and the integration of next generation sequencing (NGS) for informed decision-making, barriers persist despite advancements. This includes challenges in clinical trial recruitment. To gain global insights into clinicians' practices, we conducted a survey on their testing and management approaches for patients with mNSCLC. METHODS: The survey, conducted from July 12 to August 20, 2023, utilized multiple-choice questions and qualitative research questions, employing the Likert Scale for comprehensive insights. RESULTS: A total of 127 individuals responded, with 72% affiliated with academic health systems, and 55% practicing in the USA. Regarding testing practices, 93% consistently ordered NGS for non-squamous histology, while 54% did so for squamous cell histology. Concurrent tissue and liquid biopsies were routinely ordered by 28%, while 39% reported ordering both testing platforms concurrently for select cases only. Respondents cited logistical barriers, such as insufficient tissue and lack of infrastructure, as the most common hindrance to molecular testing (76%), followed by reimbursement challenges (56%) and concerns about delayed turnaround time (50%). While most respondents were confident in interpreting NGS results, 22% lacked confidence. Concerning treatment decisions, 72% preferred awaiting molecular testing results before initiating systemic therapy. Less than 50% routinely referred patients for clinical trials in the frontline setting for mNSCLC. For patients with disease expressing high PD-L1 levels, most oncologists preferred pembrolizumab monotherapy. For disease with low PD-L1 expression, a platinum doublet chemotherapy regimen combined with pembrolizumab was favored. In disease cases with negative PD-L1 expression, a platinum doublet chemotherapy regimen with pembrolizumab was preferred. Key factors influencing oncologists' preferred immune checkpoint inhibitor (ICI) included experience with one ICI over another, preferred status per national guidelines, availability of trial data with a significant follow-up period, and consideration of drug cost. CONCLUSION: Although this study demonstrates an improved awareness and adoption of ordering NGS for the management of mNSCLC, it underscores the persistence of various barriers that must be addressed to improve upon the quality of care for patients diagnosed with mNSCLC.

Metastatic Epithelioid Hemangioendothelioma of the Bone: A Case Report and Literature Review.

Epithelioid hemangioendothelioma (EHE) is an extremely rare vascular tumor, which can pose a diagnostic dilemma. It affects women more than men and is mainly found in the liver, lung, and bone. To date, there are no known predisposing factors. Limited data are available on the management of EHE at metastatic stages. The only optimal treatments to prevent metastatic dissemination are surgical resection and amputation in addition to radiotherapy at early stages. The oncologist in this rare entity plays an important role in the guided and standardized management of this disease, especially for advanced stages. In this article, we report the case of a 74-year-old patient admitted with swelling on the outer aspect of the right calf associated with pain and total functional impairment of the limb. The diagnosis favored a high-risk vascular tumor resembling EHE, confirmed by bone (tibia) and soft tissue biopsy. The patient underwent staging investigations, revealing diffuse metastases to the liver, bones, and lungs. The objective of this article is to advocate for oncological intervention in this entity, particularly in the advanced stages of the disease. Despite its rarity, the advancement of clinical trials and therapeutic recommendations remains crucial for optimal treatment.

Pediatric-type low-grade gliomas in adolescents and young adults-challenges and emerging paradigms.

Pediatric-type low-grade glioma (PLGG) encompasses a heterogeneous group of WHO grade 1 or 2 tumors and is the most common central nervous system tumor found in children. PLGG extends beyond pediatrics, into adolescents and young adults (AYA, ages 15-40). PLGG represents 25% of all gliomas diagnosed in AYA with differences in tumor location and molecular alterations compared to children, resulting in improved outcome for AYAs. Long-term outcome is excellent, though patients may suffer significant morbidity depending on tumor location. There are differences in treatment practices with radiation used to treat PLGG in AYAs more often than in children. Most PLGG in AYA harbor an alteration in the RAS/MAPK pathway, with limited insight into response to targeted therapy in this age group. This review discusses the epidemiology, current therapeutic approaches, and challenges in the management of PLGG in AYA.

[Unusual progression of polycythemia vera]. / Ungewöhnliche Progression einer Polycythaemia vera.

The case of a male patient with newly diagnosed polycythemia vera showing rare and unusually rapid progression with phenotypic change towards chronic myelomonocytic leukemia is presented. The case report illustrates remarkably rapid disease progression including a structural change in usually indolent polycythemia vera and highlights the prognostic relevance of enhanced molecular genetic testing.

Recommendations on maximising the clinical value of tissue in the management of patients with intrahepatic cholangiocarcinoma.

Background & Aims: Patients with intrahepatic cholangiocarcinoma can now be managed with targeted therapies directed against specific molecular alterations. Consequently, tissue samples submitted to the pathology department must produce molecular information in addition to a diagnosis or, for resection specimens, staging information. The pathologist's role when evaluating these specimens has therefore changed to accommodate such personalised approaches. Methods: We developed recommendations and guidance for pathologists by conducting a systematic review of existing guidance to generate candidate statements followed by an international Delphi process. Fifty-nine pathologists from 28 countries in six continents rated statements mapped to all elements of the specimen pathway from receipt in the pathology department to authorisation of the final written report. A separate survey of 'end-users' of the report including surgeons, oncologists, and gastroenterologists was undertaken to evaluate what information should be included in the written report to enable appropriate patient management. Results: Forty-eight statements reached consensus for inclusion in the guidance including 10 statements about the content of the written report that also reached consensus by end-user participants. A reporting proforma to allow easy inclusion of the recommended data points was developed. Conclusions: These guiding principles and recommendations provide a framework to allow pathologists reporting on patients with intrahepatic cholangiocarcinoma to maximise the informational yield of specimens required for personalised patient management. Impact and Implications: Biopsy or resection lesional tissue from intrahepatic cholangiocarcinoma must yield information about the molecular abnormalities within the tumour that define suitability for personalised therapies in addition to a diagnosis and staging information. Here, we have developed international consensus guidance for pathologists that report such cases using a Delphi process that sought the views of both pathologists and 'end-users of pathology reports. The guide highlights the need to report cases in a way that preserves tissue for molecular testing and emphasises that reporting requires interpretation of histological characteristics within the broader clinical and radiological context. The guide will allow pathologists to report cases of intrahepatic cholangiocarcinoma in a uniform manner that maximises the value of the tissue received to facilitate optimal multidisciplinary patient management.

The role of the ThyGeNEXT oncogene panel used in combination with the expanded miRNA panel ThyraMIRv2 in Indeterminate thyroid nodules: A large, blinded, real-world, observational study.

BACKGROUND: Molecular analysis of fine-needle aspiration biopsies (FNAB) improves the diagnostic accuracy of cytologically indeterminate thyroid nodules (ITNs). Recently, the use of MPTXv2 has been shown to further improve the accuracy of risk stratification of ITNs. METHODS: A total of 338 patient samples with atypia of undetermined significance (n = 260) or follicular neoplasm (n = 78) cytology diagnosis and corresponding surgical outcomes or clinical follow-up, collected between 2016 and 2020 were included. All samples underwent multiplatform testing (MPTXv1), which includes an oncogene panel (ThyGeNEXT®) plus a microRNA risk classifier (ThyraMIR®). A blinded, secondary analysis was performed to assess the added utility of MPTXv2 (ThyraMIR®v2). The average length of follow-up for the surveillance group (n = 248) was 30 months. RESULTS: Sensitivity at moderate threshold was 96% and specificity at positive threshold was 99% for MPTXv2. At 14% disease prevalence, the negative predictive value at the moderate threshold was 99% and the positive predictive value at the positive threshold was 89% for MPTXv2. MPTXv2 had fewer patients classified into the moderate-risk group than MPTXv1, which was statistically significant (p < .001). Using surgical resection, the gold standard for outcomes, MPTXv2 showed a statistically greater area under the curve (p = .028) than MPTXv1, demonstrating greater accuracy for MPTXv2. CONCLUSION: Both test versions demonstrated robust performance with low false-positive molecular results. Data suggest that incorporation of MPTXv1, and more recently MPTXv2, into clinical practice within our healthcare network resulted in improved accuracy of ITN risk stratification.

Clinicopathological and molecular genetic analysis of 13 cases of primary retroperitoneal Ewing sarcoma.

Retroperitoneal Ewing sarcomas (RES) are very rare and mostly described in case reports. The purpose of this study was to retrospectively analyze the clinicopathology, molecular characteristics, biological behavior, and therapeutic information of 13 cases of primary RES with immunohistochemical staining, fluorescence in situ hybridization, RT-PCR and NGS sequencing detection techniques. The thirteen patients included eight males and five females with a mean age of 34 years. Morphologically, the tumors were comprised of small round or epithelial-like cells with vacuolated cytoplasm (6/13,46 %) arranged in diffuse, nested (8/13,62 %) and perivascular (7/13,54 %) patterns. Unusual morphologic patterns, such as meningioma-like swirling structures and sieve-like structures were relatively novel findings. Immunohistochemical studies showed CD99 (12/13; 92 %), CD56 (11/13; 85 %), NKX2.2 (9/13; 69 %), PAX7 (10/11;91 %) and CD117(6/9;67 %) to be positive.12 cases (92 %) demonstrated EWSR1 rearrangement and 3 cases displayed EWSR1::FLI1 fusion by FISH. ERCC4 splice-site variant, a novel pathogenic variant, was discovered for the first time via RNA sequencing. With a median follow-up duration of 14 months (6 to 79 months), 8/13 (62 %) patients died, while 5/13(38 %) survived. Three cases recurred, and five patients developed metastasis to the liver (2 cases), lung (2 cases) and bone (1 case). RES is an aggressive, high-grade tumor, prone to multiple recurrences and metastases, with distinctive morphologic, immunohistochemical, and molecular genetic features. ERCC4 splicing mutation, which is a novel pathogenic variant discovered for the first time, with possible significance for understanding the disease, as well as the development of targeted drugs.