PD-L1 and beyond: Immuno-oncology in cytopathology.

Over the past decade, immunotherapy has emerged as one of the most promising cancer treatments. Several monoclonal antibodies targeting the programmed death 1 (PD-1)/ programmed death ligand-1 (PD-L1) pathway have been integrated into standard-of-care treatments for a wide range of cancer types. Although all the available PD-L1 immunohistochemistry (IHC) assays have been developed on formalin-fixed histological specimens, a growing body of research has recently suggested the feasibility of PD-L1 testing on cytological samples. Although promising results have been reported, several important issues still need to be addressed. Among these are pre-analytical issues, cyto-hystological correlation, and inter-observer agreement. This review will briefly summarise the knowledge gaps and future directions of cytopathology in the immuno-oncology scenario.

Moving towards a local testing solution for undetermined thyroid fine-needle aspirates: validation of a novel custom DNA-based NGS panel.

AIMS: In thyroid cytopathology, the undetermined diagnostic categories still pose diagnostic challenges. Although next-generation sequencing (NGS) is a promising technique for the molecular testing of thyroid fine-needle aspiration (FNA) specimens, access to such technology can be difficult because of its prohibitive cost and lack of reimbursement in countries with universal health coverage. To overcome these issues, we developed and validated a novel custom NGS panel, Nexthyro, specifically designed to target 264 clinically relevant mutations involved in thyroid tumourigenesis. Moreover, in this study, we compared its analytical performance with that of our previous molecular testing strategy. METHODS: The panel, which includes 15 genes (BRAF, EIF1AX, GNAS, HRAS, IDH1, KRAS, NF2, NRAS, PIK3CA, PPM1D, PTEN, RET, DICER1, CHEK2, TERT promoter), was validated with a cell-line derived reference standard and 72 FNA archival samples previously tested with the 7-gene test. RESULTS: Nexthyro yielded 100% specificity and detected mutant alleles at levels as low as 2%. Moreover, in 5/72 (7%) FNAs, it detected more clinically relevant mutations in BRAF and RAS genes compared with the 7-gene test. Nexthyro also revealed better postsequencing metrics than the previously adopted commercial 'generic' NGS panel. CONCLUSION: Our comparative analysis indicates that Nexthyro is a reliable NGS panel. The study also implies that a custom-based solution for routine thyroid FNA is sustainable at the local level, allowing patients with undetermined thyroid nodules affordable access to NGS.

Thyroid fine-needle aspiration trends before, during, and after the lockdown: what we have learned so far from the COVID-19 pandemic.

PURPOSE: Nowadays, the clinical management of thyroid nodules needs to be multi-disciplinary. In particular, the crosstalk between endocrinologists and cytopathologists is key. When FNAs are properly requested by endocrinologists for nodules characterised by relevant clinical and ultrasound features, cytopathologists play a pivotal role in the diagnostic work-up. Conversely, improper FNA requests can lead to questionable diagnostic efficiency. Recently, recommendations to delay all non-urgent diagnostic procedures, such as thyroid FNAs, to contain the spread of COVID-19 infection, have made the interplay between endocrinologists and cytopathologists even more essential. The objective of this study was to assess the impact of COVID-19 pandemic on our practice by evaluating the total number of FNAs performed and the distribution of the Bethesda Categories before, during, and after the lockdown. METHODS: We analysed the FNA trends before (1st January 2019 to March 13th 2020), during (March 14th to May 15th), and after (May 16th to July 7th) the lockdown. RESULTS: Although the total number of weekly FNAs dropped from 62.1 to 23.1, our referring endocrinologists managed to prioritise patients with high-risk nodules. In fact, in the post-lockdown, the weekly proportion of benign diagnoses dropped on average by 12% and that of high-risk diagnoses increased by 6%. CONCLUSIONS: The lesson we have learned so far from this pandemic is that by applying safety protocols to avoid contagion and by increasing the threshold for FNA requests for thyroid nodules, we can continue to guarantee our services to high-risk patients even in times of a health crisis.

KRAS mutations testing in non-small cell lung cancer: the role of Liquid biopsy in the basal setting.

In advanced stage non-small cell lung cancer (NSCLC) patients, Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) testing may soon acquire a predictive significance to select patients for AMG510 treatment. Since tissue samples are not always available, liquid biopsy may represent a viable option for KRAS testing. Here, we review the last three years clinical practice performed on 194 plasma based liquid biopsies by next generation sequencing (NGS) SiRe® panel. In particular, 36 (18.6%) KRAS mutated cases were identified, with an overall median allelic frequency of 5.0% (ranging between 0.2% and 46.8%). No concomitant mutations were observed in the other NSCLC clinical relevant genes included in the SiRe® panel, such as epidermal growth factor receptor (EGFR) and v-Raf murine sarcoma viral oncogene homolog B (BRAF). Exon 2 p.G12C was the most common detected mutation (13/36, 36.1%). In conclusion, our data update and confirm that SiRe® NGS panel represents a robust analytical tool to assess KRAS mutational status on circulating tumor DNA. Further investigation is required to design more cost-effective diagnostic algorithms to harmonize clinical relevant biomarker testing on tissue and blood in advanced stage NSCLC clinical practice.

The continuing role of breast fine-needle aspiration biopsy after the introduction of the IAC Yokohama System For Reporting Breast Fine Needle Aspiration Biopsy Cytopathology.

BACKGROUND: Ultrasound-guided fine-needle aspiration biopsy (FNAB) (US-guided FNAB) is a rapid and cost-effective procedure for the diagnosis of breast lesions. Our Institution has a long tradition in breast FNAB performed by cytopathologists; recently we adopted both US guidance and a five-tiered classification system similar to that proposed by the International Academy of Cytology (IAC). The aim of this study was to demonstrate the continuing role of US-guided FNAB in the diagnosis of breast lesions, despite the growing adoption of core-needle biopsy (CNB). METHODS: The laboratory information database system was searched to obtain the breast FNAB diagnostic reports recorded from 2010 to 2017 and classified using a five-tiered Classification System; each entry was matched with the available histology. RESULTS: A total of 4624 breast FNAB samples were retrieved. Of these, 1745/4624 cases (37.7%) had histological follow-ups. The risk of malignancy (ROM) was 4.9% for benign, 20.7% for atypical, 78.7% for suspicious of malignancy, and 98.8% for malignant. When the atypical category was evaluated as a negative index, the positive predictive value was 93.73%, and the negative predictive value was 90.78%, reaching an overall diagnostic accuracy of 92.82%. CONCLUSIONS: The IAC Yokohama System for Reporting Breast FNAB Cytopathology clearly identifies different diagnostic categories with increasing ROM. Most of the FNAB samples were classified as benign or malignant (65.3%), warranting prompt management for these patients. Moreover, the inclusion of the atypical category as a low-risk indeterminate category avoided overtreatment of benign lesions. Thus, despite the well-established merits of CNB, US-guided FNAB still represents a cost-effective and rapid nonoperative diagnostic approach.

Cytology meets next generation sequencing and liquid biopsy: A case of lung adenocarcinoma presenting as metastasis to the phalanx.

Carcinomas do rarely metastasize to the finger. Non-small-cell lung cancer is most frequently involved. To date, only a few cases have been reported, on histology. Here we describe the cytological features of a metastatic lung adenocarcinoma case to the phalanx of the right forefinger as initial manifestation of the neoplastic disease in a 69-years-old woman. The cytological microscopic findings, complemented by a wide array of ancillary techniques, were confirmed by histology. Neoplastic cells were positive for cytokeratin 7 (CK7) and thyroid transcription factor 1 (TTF-1), harboring, in tissue and in bloodstream samples, an uncommon epidermal growth factor receptor (EGFR) exon 20 p.S768_D760DUP, detected by next generation sequencing (NGS) and liquid biopsy. Cytology corroborated by immunocytochemistry, NGS, and liquid biopsy is a modern approach to provide diagnostic and therapeutic information even in rare and challenging cases.

Rapid On-site Molecular Evaluation in thyroid cytopathology: A same-day cytological and molecular diagnosis.

BACKGROUND: Thyroid fine-needle aspirates (FNAs) with undetermined morphology can be outsourced to centralized laboratories for comprehensive molecular profiling. When a local, rapid screening rules out easily detectable BRAF and NRAS mutations outsourcing is minimized, leading to cost savings. The fully automated Idylla technology, that does not require trained staff, is an emerging option. However, Idylla platform has only been validated to process formalin fixed paraffin embedded (FFPE) sections. Here we investigate whether also the FNA needle rinse could be genotyped by the same cytopathologist who performs the FNA, a procedure that can be termed rapid on site molecular evaluation (ROME). METHODS: To validate this approach, the Idylla BRAF and NRAS Test was performed on the rinses from 25 simulated (bench-top) FNAs, in a first part of the study. Genotyping data were compared with those obtained on matched histological FFPE blocks. The second part of the study was carried out on 25 prospectively collected routine FNAs to assess the performance of the Idylla BRAF and NRAS assay against a gold standard real time polymerase chain reaction method. RESULTS: Idylla NRAS-BRAF Mutation Test was performed on needle rinse as well as histological FFPE blocks. A sensitivity of 88.9%, a specificity of 100.0% were obtained comparing the Idylla NRAS-BRAF Mutation Test on needle rinse to the reference method. CONCLUSIONS: The FNA needle rinse can be directly genotyped. This obviates the need of cell block preparation, making possible a rapid combined morphological and molecular evaluation. Since DNA extraction is no longer necessary, the cytopathologist can perform ROME him/herself.

Evaluation of BRAF, RAS, RET/PTC, and PAX8/PPARg alterations in different Bethesda diagnostic categories: A multicentric prospective study on the validity of the 7-gene panel test in 1172 thyroid FNAs deriving from different hospitals in South Italy.

BACKGROUND: Thyroid fine-needle aspiration (FNA) is a reliable and cost-effective diagnostic tool for establishing the nature of thyroid nodules, although up to 30% of FNAs are still classified as "indeterminate." Molecular testing of FNAs could improve preoperative diagnosis, thereby reducing unnecessary surgery. In this multicenter prospective study the authors investigated, using a 7-gene assay, the distribution and diagnostic impact of BRAF, RAS, RET/PTC, and PAX8/PPARg, the most frequent genomic alterations occurring during thyroid oncogenesis. METHODS: In total, of 1172 routine FNAs from 7 centers in southern Italy were classified according to the Bethesda System for Reporting Thyroid Cytopathology. Each specimen was tested, and molecular data were compared with available histology or cytologic follow-up. RESULTS: In particular, for atypia of undetermined significance/follicular lesion of undetermined significance cases, the 7-gene test confirmed the high positive predictive value of BRAFV600E and BRAF-like mutations (80%) and the moderate positive predictive value of RAS-like alterations (32.4%), suggesting different surgical management, depending on the type of mutation. The rate of mutation-positive FNAs was strictly related to the risk of malignancy of each diagnostic class, supporting the identification of prognostically relevant diagnostic categories. CONCLUSIONS: The 7-gene panel test improves the preoperative risk stratification of indeterminate thyroid FNAs, especially when considering the biologic significance of the different types of mutations. Moreover, the rate of mutation-positive FNAs is related to the risk of malignancy of each diagnostic class.

Microfluidic chip technology applied to fine-needle aspiration cytology samples for IGH clonality assessment.

BACKGROUND: Most cases of non-Hodgkin lymphoma (NHL) can be diagnosed using a combination of fine-needle cytology (FNC) and flow cytometry together with immunoglobulin light chain restriction and/or specific phenotypic profiles. However, 5%-15% of B-cell NHLs lack these specific diagnostic features. In such cases, the diagnosis of NHL may be supported by molecular clonality testing based on the immunoglobulin heavy chain (IGH) assay of clonality by polyacrylamide heteroduplex analysis or by automated capillary electrophoresis via GeneScan analysis. Chip-based microfluidic technology (MT), based on miniaturized parallel capillary electrophoresis structures, is a viable alternative to capillary electrophoresis analysis, being less costly and cumbersome. In this study, we evaluated the performance of MT platform in IGH clonality assessment in a series of lymph node FNC samples. METHODS: Thirty-five consecutive lymph node FNCs were evaluated. In all cases, the first and the second passes were used to prepare a conventional smear and to collect material for flow cytometry analysis; residual material was collected for molecular clonality assessment, and PCR products were analyzed both by MT and GeneScan platforms. RESULTS: Molecular clonality assessment by MT had a sensitivity of 84.2% and a specificity of 76.9%; GeneScan analysis had a sensitivity of 88.8% and a specificity of 92.8%. The overall agreement between the two platforms was 85.7% (30/35). CONCLUSIONS: MT analysis proved to be a viable technique for IGH clonality assessment on FNC samples. Should our data be confirmed in larger studies, the MT procedure may be suitable for routine diagnostic practice, even on cytological samples.

Different qualifiers of AUS/FLUS thyroid FNA have distinct BRAF, RAS, RET/PTC, and PAX8/PPARg alterations.

BACKGROUND: The Bethesda System for Reporting Thyroid Cytopathology category of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) includes fine-needle aspiration (FNA) specimens that cannot straightforwardly be classified as benign or malignant. To determine whether morphological subcategorization based on atypia qualifiers and molecular testing could improve malignancy risk stratification of AUS/FLUS patients, this study assessed the correlation between these qualifiers and the molecular alterations commonly harbored by thyroid neoplasms. METHODS: A total of 162 AUS/FLUS cases were subcategorized by atypia qualifiers (Hürthle cell changes, architectural atypia, and cytologic atypia [CyA]) and were tested for BRAF, N-H-KRAS, RET/PTC, and paired box 8 (PAX8)/peroxisome proliferator activated receptor γ (PPARg) mutations. RESULTS: CyA was observed more frequently in mutation-positive AUS/FLUS (14 of 37 [37.84%]) than mutation-negative AUS/FLUS (20 of 125 [16.00%]; P < .0084), and it specifically harbored the BRAFV600E point mutation. Malignancy was confirmed in the available follow-up. Conversely, although RAS was the most frequent mutation identified in AUS/FLUS FNA specimens (26 of 37 cases [70.27%]; P < .0001), it was distributed across various AUS/FLUS subcategories and was not significantly associated with a specific atypia qualifier or malignant outcome according to the available follow-up. Rearrangements of both RET/PTC (n = 1) and PAX8/PPARg (n = 3) were rarely retrieved in the FNA samples. CONCLUSIONS: BRAF and RAS mutations are associated with different AUS/FLUS qualifiers and hence have different risks of malignancy. Consequently, a hybrid molecular and morphological subcategorization system could improve the malignancy risk stratification of thyroid FNA samples diagnosed as AUS/FLUS. Cancer Cytopathol 2018;126:317-25. © 2018 American Cancer Society.