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The Blessed Maria Lorenza Longo, founder of the hospital of Santa Maria del Popolo degli Incurabili and the Order of the Capuchin Poor Clares in Naples, Italy, died on 21 October 1539 and was recently beatified on 9 October 2021. The relic, a fully skeletonized cranium, underwent visual and radiological inspection. The biological profile supports the at¬tribution of female sex of the relic, whereas the age at death is estimated to be younger than that reported by historians. A paleopathological survey was conducted to evaluate the historical reports of poisoning or rheumatoid arthritis affecting Maria Longo. Given the limited skeletal data, it was not possible to confirm the presence of these claims. No obvious in¬dicators of dietary deficiencies were observed, and the tertiary syphilis hypothesized by textual sources was excluded. Postmortem alterations of the relic were clearly visible on the superior aspect of the cranium and testified to the worship of the relic.

Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences.Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology.All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification.Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational assays, and this could lead to better standardization of molecular cytopathology procedures. Cancer Cytopathol 2017;125:615-26. © 2017 American Cancer Society.

Fine-needle aspiration (FNA) cytology is accurate and cost-effective in the evaluation of thyroid nodules. Molecular techniques may contribute to risk stratification in indeterminate cases. Although next-generation sequencing (NGS) is a promising technique for the molecular testing of thyroid FNA specimens, thyroid-specific cancer gene panels are not commercially available. Conversely, the Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2), which includes the genes most frequently mutated in thyroid neoplasms, is commercially available and may represent an alternative to thyroid-specific panels. To the authors' knowledge to date, CHPv2 has performed well only on "ideal" cytological samples featuring abundant, high-quality DNA and satisfactory postsequencing metrics. The objective of the current study was to extend NGS to less-than-ideal samples, which represent a large percentage of routine clinical specimens.

AIMS: Patients with colorectal cancer harbouring KRAS mutations do not respond to antiepidermal growth factor receptor (anti-EGFR) therapy. Community screening for KRAS mutation selects patients for treatment. When a KRAS mutation is identified by direct sequencing, mutant and wild type alleles are seen on the sequencing electropherograms. KRAS mutant allele-specific imbalance (MASI) occurs when the mutant allele peak is higher than the wild type one. The aims of this study were to verify the rate and tissue distribution of KRAS MASI as well as its clinical relevance. METHODS: A total of 437 sequencing electropherograms showing KRAS exon 2 mutation was reviewed and in 30 cases next generation sequencing (NGS) was also carried out. Five primary tumours were extensively laser capture microdissected to investigated KRAS MASI tissue spatial distribution. KRAS MASI influence on the overall survival was evaluated in 58 patients. In vitro response to anti-EGFR therapy in relation to different G13D KRAS MASI status was also evaluated. RESULTS: On the overall, KRAS MASI occurred in 58/436 cases (12.8%), being more frequently associated with G13D mutation (p=0.05) and having a heterogeneous tissue distribution. KRAS MASI detection by Sanger Sequencing and NGS showed 94% (28/30) concordance. The longer overall survival of KRAS MASI negative patients did not reach statistical significance (p=0.08). In cell line model G13D KRAS MASI conferred resistance to cetuximab treatment. CONCLUSIONS: KRAS MASI is a significant event in colorectal cancer, specifically associated with G13D mutation, and featuring a heterogeneous spatial distribution, that may have a role to predict the response to EGFR inhibitors. The foreseen implementation of NGS in community KRAS testing may help to define KRAS MASI prognostic and predictive significance.

The organization and physiological functions of multicellular organisms depend also on the presence of connective tissues (1). Fibroblasts are versatile connective tissue cells and represent a heterogeneous cell population (2). They maintain the homeostasis of the extracellular matrix but can also acquire an immunoregulatory phenotype (2). Moreover, fibroblasts can form multicellular aggregates when activated in vivo to myofibroblasts, during wound repair and in fibrotic states (1). It is known that established human dermal fibroblasts, when forced to form multicellular aggregates named spheroids, are activated to produce massive amounts of COX-2, prostaglandins and proinflammatory cytokines: this process terminates in a programmed necrosis, designated as nemosis (2). In the present study we analysed spheroids of human primary fibroblasts from skin. To this aim we formed spheroids adapting the hanging-drops and agarose-coated U-bottom well plates methods. Immunohistochemical analysis of spheroids, collected at different times, detected the presence of vimentin, a myofibroblast marker. Moreover, hematoxylin and eosin staining showed very negligible areas of necrosis. Lactate dehydrogenase (LDH) release, associated with loss of membrane integrity, was estimated in conditioned media from fibroblasts grown as spheroids or monolayers. Marginal levels of LDH activity were detected in conditioned media from spheroids and monolayers, although after 96 h an increase of LDH release, more evident in monolayers media, was measured. Western blotting analysis of spheroid extracts showed the absence of COX-2 and the presence of a-smooth muscle actin, a marker of myofibroblast differentiation. Hence, very low levels of LDH activity associated with the absence of COX-2 demonstrate that human primary fibroblasts from skin, cultured as spheroids, don’t undergo nemosis. Furthermore, TUNEL staining of sectioned spheroids showed very few apoptotic cells, positive for DNA breaks. Our study highlights new aspects about fibroblasts biology and interactions., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Colorectal cancer (CRC) is one of the most frequent and deadly malignancies worldwide. Despite the progresses made in diagnosis and treatment, the identification of tumor markers is still a strong clinical need, because current treatments are efficacious only in a subgroup of patients. UbcH10 represents a potential candidate biomarker, whose expression levels could be employed to predict response or resistance to chemotherapy or targeted agents. UbcH10 mRNA and protein expression levels have been evaluated in a large group of CRC patients and correlated with clinico-pathological characteristics, including KRAS mutations. Moreover, the endogenous levels of UbcH10 and its role on cell growth have been evaluated in CRC cells. Finally, to investigate the impact of UbcH10 protein expression on the response to irinotecan, its active metabolite SN-38 and cetuximab treatment, UbcH10 silencing experiments were carried-out on two colon carcinoma cell lines, Caco-2, and DLD1. Overexpression of UbcH10 mRNA and protein was observed in the vast majority of patients analyzed. UbcH10 suppression decreased CRC cell growth rate (at least in part through deregulation of Cyclin B and ERK1) and sensitized them to pharmacological treatments with irinotecan, SN-38 and cetuximab (at least in part through a down-regulation of AKT). Taken together, these findings indicate that UbcH10 expression regulates CRC growth and could play an important role in the personalization of the therapy of CRC patients. © 2015 Wiley Periodicals, Inc.

The occurrence of head and neck palpable lesions is not exceptional in pediatric population, encompassing a wide range of pathological entities.1, 2 In particular, the non‐neoplastic conditions occurring in soft tissues usually are self‐limiting and do not require a surgical approach. In these cases, the minimal invasive fine‐needle aspiration (FNA) cytology may suffice to exclude malignancy in a pediatric patient, overcoming the need for sedation or general anesthesia.3-6 Focal (nodular) myositis (FM) is an infrequent mass‐forming inflammatory lesion presenting as a solitary lump in a given skeletal muscle.7 This condition has rarely been reported in children, as only 6% of cases occurred in patients with

Lung adenocarcinoma and papillary thyroid carcinoma (PTC) share a number of microscopic and immunophenotypical features. Thus, patients presenting with thyroid and lung synchronous neoplasms may be difficult on fine-needle aspiration (FNA) samples to define the site of origin of the malignancy. In the case reported here, inherent to a 57-years-old man presenting with a right lung mass and a large (44 mm) thyroid nodule, an integrated cytological, immunocytochemical and molecular approach enabled to clarify the primary nature of the neoplasm. FNA cytology showed in both sites papillary structures and nuclear changes reminiscent of PTC. The lung origin of the neoplasm was suggested on cell-block immunocytochemistry showing thyroid transcription factor-1 positive and PAX8 and TGB negative neoplastic cells. Next generation sequencing performed on the Ion Torrent platforms by the Ion Ampliseq Colon and Lung Cancer panel showed a similar genomic profile in both neoplastic sites with a concurrent KRAS G12C mutation. An integrated approach on FNA biospecimen is safe, cost effective, and may be coupled effectively with modern ancillary molecular techniques that may be useful, besides their predictive value, as a adjunctive diagnostic tool when the synchronous occurrence of lesions featuring overlapping morphologies challenge the cytopathologist.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutations are reliably detected by referral laboratories, even if most lung cancer cytology specimens sent to such laboratories contain very few cells. However, EGFR mutations may be distributed heterogeneously within tumors, thereby raising concerns that mutations detected on cytology are not representative of the entire tumor and, thus, are less reliable in predicting response to tyrosine kinase inhibitor (TKI) treatment than mutations detected on histology. To address this issue, the authors reviewed their clinical practice archives and compared the outcome of TKI treatment among patients who were selected by cytology versus patients who were selected by histology. METHODS: From July 2010 to July 2012, 364 cytology samples and 318 histology samples were received. Exon 19 deletions and the L858R point mutation in exon 21, detected by fragment assay and TaqMan assay, respectively, were confirmed by direct sequencing; discrepancies were resolved by cloning polymerase chain reaction products. The response rate (RR) and progression-free survival (PFS) at 12 months (range, 3-34 months) were evaluable in 13 EGFRmutated patients who were selected for treatment by cytology and 13 patients who were selected by histology. RESULTS: The mutation rate was similar in histology samples (8.5%) and cytology samples (8.8%). The RR (54%) and PFS (9.2 months) were similar in histologically selected patients and cytologically selected patients (RR, 62%; PFS, 8.6 months; P5.88). The disease control rate (responsive plus stable disease) was 92% in histologically selected patients and 100% in cytologically selected patients. CONCLUSIONS: EGFR mutations detected on cytology specimens by a centralized laboratory can predict TKI treatment response equally well as mutations identified on histology samples.

La terapia mirata del carcinoma del polmone non a piccole cellule (NSCLC) con inibitori del dominio tirosin-chinasico (anti-TKI) del recettore del fattore di crescita epidermico (EGFR) è efficace solo nei pazienti con mutazioni di tale gene. Le mutazioni più frequenti sono rappresentate da delezioni nell'esone 19 e dalla mutazione puntiforme (L858R) nell'esone 21. A causa della scoperta in fase avanzara di tali neoplasie, spesso la diagnosi di carcinoma polmonare è esclusivamente citologica; il DNA estratto dai campioni citologici viene poi utilizzato per determinare lo stato mutazionale di EGFR. Tuttavia, la possibilità che le mutazioni siano distribuite nel tessuto polmonare eterogeneamente è tutt'ora discussa. E' possibile che i campioni citologici non siano del tutto rappresentativi dello stato mutazionale dell'intera neoplasia e quindi, il valore predittivo dell' analisi mutazionale effettuata esclusivamente su citologico è ancora dubbia. Questa Tesi Sperimentale esamina il ruolo del patologo nella gestione del materiale citologico da NSCLC, dalla valutazione pre-analitica del vetrino, alla supervisione delle fasi analitiche dei test molecolari e, infine, alla sintesi finale nel report diagnostico. È stata inoltre studiata la validità del campione citologico come substrato biologico adeguato nella predizione della risposta alla terapia anti-TKI.

Although the most important application of novel molecular markers is that on cytology, translation from the bench to the FNA is complex . To make DNA- and/or RNA-based testing cost-effective on cytology a close integration with morphology is needed . Thus, the informativeness of the sample for cytology needs to be preserved to keep the accuracy of microscopy high. To this end, each of the steps of traditional cytology, such as preparation of FNA material, search for morphological criteria, assignment to the correct diagnostic class, and suggestion of the appropriate post FNA options should not be altered by molecular analysis. Then, this latter can refine cytology. The final result is to effectively stratify into high-risk and low-risk categories, and the indeterminate cytology classes identified by the BSRTC . Thus, cytological specimens should be properly handled to provide both morphological and molecular information . Our method of preparation of FNA to harvest material sufficient for both tests was recently validated on a series of 128 routinely performed FNA . The rationale behind our sample collection method was to ensure first an adequate cytological diagnosis and, then, to exploit part of the diagnostic material for molecular testing . Thus, two passes from different areas of the lesion are performed. A representative air-dried Diff-Quik stained smear is prepared within few minutes and reviewed on site ] . In 44 cases, the cytological evidences were sufficient for morphological assessment and the third pass was directly collected in RNA or DNA buffer extraction. Conversely, in 84 cases the specimen was either deemed inadequate by the onsite evaluation or required an additional ethanol-fixed Papanicolaou-stained smear to better evaluate nuclear morphology. Thus, a third pass was dedicated to the preparation of an additional smears and only needle rinsing was collected for BRAF testing. Higher average of extracted DNA concentration was observed in the dedicated pass group (25.9 vs. 7.95 ng/ m l). However, the rate of successful exon 15 BRAF amplification was similar with (43/44; 97.7%) or without (79/84; 94%) the dedicated pass. Thus, our protocol is suitable for both tests. When necessary, BRAF testing may also be performed on the residual samples of thyroid nodules, without interfering with routine cytology. Similarly, as far as mRNA markers are concerned, we have shown that in most samples, qRT-PCR analysis does not interfere with cytology . In fact, in a recent study on UbcH10 expression, including 84 cases with a cytological diagnosis of either follicular neoplasm ( n = 57) or suspicious for malignancy ( n = 27), we found that most (73.8%) cases were adequate for both tests

BACKGROUND: Fine needle cytology (FNC) of thyroid nodules is not always diagnostic. Most of FNCs undeterminated for malignancy belong to the cytological class of "follicular neoplasm/suspicious for follicular neoplasm" lesions (FN). In this group only 10-30% of cases are malignant and the most appropriate surgical management is still controversial. Here, this issue was addressed and the more reliable predictive criteria of malignancy were also evaluated. METHODS: We retrospectively evaluated 472 patients, surgically treated after a FN diagnosis in a tertiary care referral center. In patients affected by bilateral thyroid disease with a cytological diagnosis of FN, or when high-risk clinical features and familiarity for thyroid cancer were present, total thyroidectomy (TT) was performed. Conversely, hemithyroidectomy (HT) was preferred when the nodule was single and when the age was ≤ 45 years. Frozen section examination was not used, and if cancer was diagnosed by definitive pathology of the HT specimen, the remnant thyroid lobe was removed. Histological features, surgical complications, and long-term outcomes of the remnant lobe were reported. Clinical features predictivity was also evaluated. RESULTS: TT was performed in 154/472 pts (32.62%), while HT was carried out in 318/472 cases (67.37%). The overall malignancy rate (MR) was 18.85% (89/472 pts), respectively 16% (51/318pts) following HT, and 24.6% (38/154pts) following TT, with a statistically significant difference. Similarly, the rates of transient and definitive hypoparathyroidism and the mean hospital stay following TT were higher than after HT (and statistically significant). Age < 45years and female gender were more frequently associated to malignancy. The rate of complications following second surgery was comparable to that of primary HT. In the HT group incidence of unexpected contralateral papillary thyroid cancer (PTC) was 9.8% and, after 88.2 ± 30.42 months mean follow-up, completion surgery for benign pathology was carried out in 6.7% of cases. CONCLUSIONS: Our data show that histology following a cytological FN diagnosis is malignant only in a low percentage of cases (89/472, 18.85%). Following TT, a MR higher than in HT was observed. Even if some clinical features are cancer associated, malignancy cannot be reliably predicted before surgery. Thus, in solitary low-risk lesions, HT is still the standard of care. Its lower complication rates makes HT the safest procedure. In case of multiglandular disease TT may be recommended. Further investigation is warranted to achieve a better preoperative diagnostic accuracy in order to reduce the amount of surgical operations with diagnostic aim.

Aim: ALK, ROS1, NTRK and RET gene fusions and MET exon 14 skipping alterations represent novel predictive biomarkers for advanced non-small-cell lung cancer (NSCLC). Therefore, testing patients for these genetic variants is crucial for choosing the best selective treatment. Over the last couple of decades, next-generation sequencing (NGS) platforms have emerged as an extremely useful tool for detecting these variants. Materials & methods: In the present study, we report our NGS molecular records produced during a year of diagnostic activity. Results: Overall, our in-house developed NGS workflow successfully analyzed n = 116/131 (88.5%) NSCLC samples. Of these, eight (6.8%) and five (4.3%) out of 116 patients harbored ALK and RET gene rearrangements, respectively: one case harbored ROS1 gene fusion (0.7%). Conclusion: Our results highlight that an RNA-based NGS analysis can reliably detect gene fusion alterations, thereby playing a pivotal role in the management of NSCLC patients.

Noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) are low-risk thyroid lesions most often characterised by RAS-type mutations. The histological diagnosis may be challenging, and even immunohistochemistry and molecular approaches have not yet provided conclusive solutions. This study characterises a set of NIFTPs by Matrix-Assisted Laser Desorption/Ionisation (MALDI)–Mass Spectrometry Imaging (MSI) to highlight the proteomic signatures capable of overcoming histological challenges. Archived formalin-fixed paraffin-embedded samples from 10 NIFTPs (n = 6 RAS-mutated and n = 4 RAS-wild type) were trypsin-digested and analysed by MALDI–MSI, comparing their profiles to normal tissue and synchronous benign nodules. This allowed the definition of a four-peptide signature able to distinguish RAS-mutant from wild-type cases, the latter showing proteomic similarities to hyperplastic nodules. Moreover, among the differentially expressed signals, Peptidylprolyl Isomerase A (PPIA, 1505.8 m/z), which has already demonstrated a role in the development of cancer, was found overexpressed in NIFTP RAS-mutated nodules compared to wild-type lesions. These results underlined that high-throughput proteomic approaches may add a further level of biological comprehension for NIFTPs. In the future, thanks to the powerful single-cell detail achieved by new instruments, the complementary NGS–MALDI imaging sequence might be the correct methodological approach to confirm that the current NIFTP definition encompasses heterogeneous lesions that must be further characterised.

Background: The management of cutaneous melanoma has dramatically changed in recent years thanks to the development of tyrosine kinase and immune-checkpoint inhibitors (ICIs). Thus, multiple biomarker testing is becoming ever more important to identify patients potentially eligible for these treatments. One reliable approach for molecular evaluation of metastatic melanoma is fine-needle cytology (FNC). To examine the utility of this approach for PD-L1 assessment, we evaluated the cellular adequacy of residual cell block (CB) material from metastatic melanomas previously tested for BRAF and NRAS mutations. Methods: We retrieved from our internal archives a series of FNC samples of metastatic melanoma subjected to molecular testing on residual CB material or dedicated needle rinse, between January 2016 and July 2022. Briefly, RT-PCR was adopted to assess BRAF and NRAS status, and SP263 assay was employed to assess PD-L1 expression level. Results: Overall n=19 cases were selected. Of these, 11 (57,9%) cases revealed a BRAF exon 15 p.V600E mutation, one case (5,3%) revealed NRAS mutation, and seven cases (36,8%) showed no mutations. Regarding PD-L1 assessment, 16/19 (84,2%) cases were deemed adequate, containing at least 100 viable cells. Conclusions: We highlighted the feasibility of PD-L1 assessment on residual CB material from metastatic melanomas previously tested for BRAF and NRAS mutations. Moreover, we pointed out that FNC needle rinses may be an alternative sources of nucleic acids for molecular testing, preserving CB material for ICC evaluation.

AimsGene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated.MethodsCell lines harbouringEML4(13)–ALK(20) andSLC34A2(4)–ROS1(32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides.ResultsFour (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms.ConclusionsReference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches.

Background: The DIO2 Thr92Ala polymorphism (rs225014), which occurs in about 15-30% of Caucasian people, determines a less efficient type 2 deiodinase (D2) enzyme. The aim of this study was to determine the impact of DIO2 Thr92Ala polymorphism on the serum thyrotropin (TSH) levels in thyroidectomized patients with hypothyroidism and to evaluate whether TSH levels and aging could be related, at pituitary level, to D2 activity. Methods: This prospective study was performed on 145 thyroid cancer patients, treated with total thyroidectomy, and undergoing radioiodine treatment after 3 weeks of levothyroxine (LT4) withdrawal. A mouse model has been used to determine D2 protein and mRNA levels in pituitary during aging. Results: Genetic analysis identified DIO2 Thr92Ala polymorphism in 56% of participants: 64/145 (44%) patients were homozygous wild type (WT) (Thr/Thr), 64 (44%) heterozygous (Thr/Ala), and 17 (12%) homozygous mutant (Ala/Ala). A significant negative relationship was observed between aging and the rise in serum TSH levels during LT4 withdrawal. However, this negative correlation found in WT was reduced in heterozygous and lost in mutant homozygous patients (Thr/Thr r = -0.45, p = 0.0002, 95% confidence interval [CI] -0.63 to -0.23; Ala/Thr r = -0.39, p = 0.0012, CI -0.60 to -0.67; and Ala/Ala r = -0.30, p = 0.2347; CI -0.70 to 0.20). Accordingly, when we compared the TSH measured in each patient to its theoretical value predicted from age, the TSH did not reach its putative target in 47% of WT patients, in 70% of Ala/Thr, and 76% of Ala/Ala carrying patients (p = 0.0036). This difference was lost in individuals older than 60 years, suggesting a decline of D2 associated with aging. The hypothesis that the pituitary D2 decreases with age was confirmed by the evidence that D2 mRNA and protein levels were lower in pituitary from old versus young mice. Conclusion: An age-related decline in TSH production in response to hypothyroidism was correlated with decreased D2 levels in pituitary. The presence of DIO2 homozygous Ala/Ala polymorphism was associated with a reduced level of TSH secretion in response to hypothyroidism, indicating a decreased pituitary sensitivity to serum thyroxine variation (Institutional Research Ethics board approval number no. 433/21).

Breast cancer is the most common cause of cancer-related deaths in the female population worldwide. To the best of our knowledge, breast cancer (BRCA)1/2 gene mutations have not been described yet on breast cancer cytological specimens. Here we describe the case of a 38-year old woman with a family and personal history for breast cancer, who underwent a fine needle aspiration (FNA) procedure for a novel 30 mm lesion located in the external quadrants of the contralateral (left) breast. Cytological findings and ancillary immunostaining confirmed the diagnosis of a triple negative NST carcinoma. BRCA1/2 molecular assessment was carried out on DNA extracted from cytological (November 2020), biopsy (December 2014) and surgical resection (July 2015) specimens, as well as on the resection of a benign fibroadenoma, by using a next generation sequencing approach. Molecular analysis showed a pathogenic BRCA1 insertion (c.5266dupC; p.Q1756PfsTer74) in the cytological specimen (allelic fraction 92.0%), biopsy (allelic fraction 84.2%), surgical resection (allelic fraction 87.8%) and fibroadenoma (58.9%), demonstrating a germinal BRCA mutated status.

Molecular testing has acquired a relevant role for diagnostic and prognostic stratification of indeterminate thyroid nodules. Besides the available commercial solutions marketed in the United States, various local testing strategies have been developed in the last decade. In this setting, the modern interventional cytopathologist, the physician who performs the both aspirate and the morphologic interpretation plays a key role in the correct handling of fine-needle aspiration (FNA) samples not only for microscopy but also for molecular techniques. This review summarizes experiences with local approaches to the molecular testing of thyroid FNA, highlighting the role of the modern interventional cytopathologist.

Fine needle cytology (FNC) is widely used as a first-line procedure in the diagnostic algorithm of lymphadenopathies. In a metastatic setting, a first-line diagnostic approach identifies non-haematopoietic malignancy; however, cytopathologists could also provide a second diagnostic level, identifying the origin of the primary tumour. This paper outlines a comprehensive and practical approach to the cytological diagnosis of lymph node metastases.Cytological diagnoses of lymph node metastases performed over a 10-year period were selected and divided into two groups. The first group, labelled "oncological," comprised patients with a previous history of malignancy; the second group, labelled "naïve," included patients with no relevant history. Pathology records were retrieved to record microscopic findings, namely, background appearance, group architecture, and specific cell features; data from cell block (CB) preparations were also collected.Overall, 982 cases were selected: 497 cases (50.61%) in the naïve group, and 485 (49.39%) in the oncological group. Overall, a second diagnostic level was achieved in 834/982 cases (84.92%); cases diagnosed as carcinoma not otherwise specified were more frequent in the naïve group than in the oncological group (17.51% vs. 8.04%, P lt; 0.01). Notably, although CB material was available in only 44.87% of the naïve cases, we were able to achieve a second diagnostic level thanks to the integration of clinical and cytomorphological findings, plus lymph node topography, in 82.49% of the cases.Our results confirmed that in a metastatic setting, FNC can reliably lead to the identification of the origin of the primary tumour.

The application of next generation sequencing (NGS) technology to cytological samples has significantly modified molecular cytopathology practice. Cytological samples represent a valid source of high‐quality DNA for NGS analysis, especially for predicting patients' response to targeted treatments and for refining the risk of malignancy in indeterminate cytological diagnoses. However, several pre‐analytical factors may influence the reliability of NGS clinical analysis. Here, we briefly review the challenges of NGS in cytology practice, focusing on those pre‐analytical factors that may negatively affect NGS success rates and routine diagnostic applications. Finally, we address the future directions of the field., Molecular cytopathology is a rapidly evolving field. Modern molecular cytopathologists play a key role in bridging the gap between conventional microscopy and novel molecular technologies. Cytological samples represent a valid source of high‐quality DNA for next generation sequencing (NGS) analysis, especially for predicting patients’ response to targeted treatments and for refining the risk of malignancy in indeterminate cytological diagnosis.

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak was declared a pandemic, the magnitude of coronavirus disease 2019 (COVID-19) has continued to grow, putting an unprecedented strain on all medical fields. Its effects on cytopathology workloads have been dramatic. Indeed, despite the implementation of several laboratory biosafety recommendations, cytological screening activities and cytological sampling of patients at low risk of malignancy have been postponed to limit the risk of contagion and to lessen the strain on overwhelmed hospital facilities. In this scenario, a drastic reduction in the total number of cytological specimens has been observed worldwide. This review summarizes the current evidence of the impact of the COVID-19 pandemic on cytopathology practice by focusing on its impact on cytological sample workload.

AimsIn 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.MethodsThe 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.ResultsNexthyro 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.ConclusionOur 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.

BACKGROUND: The molecular characterization of thyroid nodules in cytological samples has so far been focused on discriminating between benign and malignant forms in a purely diagnostic setting. The evidence on the impact of molecular biomarkers to determine the risk of aggressiveness in cytologically “neoplastic” lesions is limited to genomic alterations (such as BRAF and TERT mutations). The aim of our study was to assess the preoperative role of microRNAs (miRNAs) in predicting the nodal status of patients with papillary thyroid cancer. METHODS: A pilot series of histological samples of papillary thyroid carcinoma with (6 cases) or without (6 cases) lymph node metastases, matched for other major clinical and pathological features, was analyzed for global miRNA expression in a screening phase. A set of miRNAs was then validated in a series of 63 consecutive cytological samples of papillary carcinomas: 48 pN-negative and 15 pN-positive at histology. RESULTS: Unsupervised cluster analysis segregated surgical pN-negative and pN-positive samples, except for 1 case. The 45 differentially expressed miRNAs in pN-positive versus pN-negative cases were predicted to regulate a wide range of cellular pathways, enriched for Wnt, gonadotropin-releasing hormone receptor, and cerulein/cholecystokinin receptor signaling. In agreement with their profiles in surgical samples, 4 miRNAs of the 10 selected for validation (miR-154-3p, miR-299-5p, miR-376a-3p, and miR-302E) had a significant differential expression in cytological samples of papillary carcinoma with lymph node metastases and predicted the positive nodal status with a relatively good performance. CONCLUSIONS: MiRNA profiling is a potential promising strategy to define papillary carcinoma aggressiveness in the preoperative setting.

Background: Rearranged during transfection (RET) gene fusions are detected in 10–20% of thyroid cancer patients. Recently, RET fusion-positive metastatic thyroid cancers have attracted much attention owing to the FDA approval of two highly selective anti-RET tyrosine kinase inhibitors, namely, selpercatinib, and pralsetinib. Areas covered: This review summarizes the available evidence on the biological and predictive role of RET gene fusions in thyroid carcinoma patients and the latest screening assays currently used to detect these genomic alterations in histological and cytological specimens. Expert opinion: Management of advanced thyroid carcinoma has significantly evolved over the last decade thanks to the approval of three multikinase inhibitors, i.e. sorafenib, lenvatinib, cabozantinib, and of two selective RET-tyrosine inhibitors, i.e. selpercatinib and pralsetinib. In this setting, the detection of RET-fusions in advanced thyroid cancer specimens through the use of next-generation sequencing has become a commonly used strategy in clinical practice to select the best treatment options.

Generally, predictive biomarker tests are clinically validated on histological formalin-fixed, paraffin-embedded (FFPE) samples. In addition to FFPE samples, cytological samples have also emerged as a useful approach to detect predictive biomarkers. However, as of today, despite the promising results reported in the recent literature, their full implementation in routine clinical practice is still lagging owing to a lack of standardized preparatory protocols, challenging assessments of cyto-histological correlation, and variable inter-observer agreement. The aim of this report was to explore the possibility of implementing a large-scale validation of predictive biomarker testing on cytological material. To this aim, we evaluated the technical feasibility of PD-L1 assessment on a cell block (CB)-derived tissue microarray (cbTMA). Consecutive and unselected CBs prepared from metastatic lymph node fine-needle cytology (FNC) samples were retrospectively collected and used for TMA construction. PD-L1 immunohistochemistry (IHC) was carried out on cbTMA sections with the companion diagnostic kit SP263 assay. TMA contained 33 CB-derived cores. A total of 20 sections were hematoxylin and eosin (H&E) stained. Overall, 29 (88%) samples were visible at least in one H&E-stained slide. Four cases out of five sections stained with the SP263 assay (4/29, 13.8%) showed PD-L1 positivity in neoplastic and/or immune cells; remarkably, no unspecific background was observed. Although our study was based on a limited and non-selected series, our findings do provide proof of concept for the use of cbTMA in predictive biomarker testing on cytological material in large-scale post-clinical trial validation studies, multicenter studies, and quality control programs.

Background: Immune-checkpoint inhibitors (ICIs) have increased and improved the treatment options for patients with non-oncogene-addicted advanced stage non-small cell lung cancer (NSCLC). However, the role of ICIs in oncogene-addicted advanced stage NSCLC patients is still debated. In this study, in an attempt to fill in the informational gap on the effect of ICIs on other driver mutations, we set out to provide a molecular landscape of clinically relevant oncogenic drivers in programmed death-ligand 1 (PD-L1) positive NSCLC patients. Methods: We retrospectively reviewed data on 167 advanced stage NSCLC PD-L1 positive patients (≥1%) who were referred to our clinic for molecular evaluation of five driver oncogenes, namely, EGFR, KRAS, BRAF, ALK and ROS1. Results: Interestingly, n = 93 (55.7%) patients showed at least one genomic alteration within the tested genes. Furthermore, analyzing a subset of patients with PD-L1 tumor proportion score (TPS) ≥ 50% and concomitant gene alterations (n = 8), we found that n = 3 (37.5%) of these patients feature clinical benefit with ICIs administration, despite the presence of a concomitant KRAS gene alteration. Conclusions: In this study, we provide a molecular landscape of clinically relevant biomarkers in NSCLC PD-L1 positive patients, along with data evidencing the clinical benefit of ICIs in patient NSCLC PD-L1 positive alterations.

Background: In a previous worldwide survey, the authors showed a drastic reduction in the number of cytological specimens processed during the coronavirus disease 2019 “lockdown” period along with an increase in malignancy rates. To assess the continued impact of the pandemic on cytological practices around the world, they undertook a second follow-up worldwide survey collecting data from the post-lockdown period (2020). Methods: Participants were asked to provide data regarding their cytopathology activity during the first 12 weeks of their respective national post-lockdown period (2020), which ranged from April 4 to October 31. Differences between the post-lockdown period and the corresponding 2019 period were evaluated, and the authors specifically focused on rates of malignant diagnoses. Results: A total of 29 respondents from 17 countries worldwide joined the survey. Overall, a lower number of cytological specimens (n = 236,352) were processed in comparison with the same period in 2019 (n = 321,466) for a relative reduction of 26.5%. The overall malignancy rate showed a statistically significant increase (12,442 [5.26%] vs 12,882 [4.01%]; P

The indeterminate thyroid nodules diagnosed by fine-needle aspiration cytology (FNAC)represents a problem for both cytopathologists and clinicians. The former sometimes use this diagnostic category as a sort of basket, putting in cases that they do not know exactly how to classify. The latter are faced with a highly variable risk of malignancy and consequently the management remains a challenge. On the histopathological side, the new WHO classification of tumors of the thyroid introduced the concept of tumors with uncertain and low malignant potential, and the concept of non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), whose prognosis and management are still to be completely elucidated. While the risk of malignancy of the indeterminate diagnostic category has decreased due to the re-classification of certain types of papillary thyroid carcinomas of the follicular variant into a low malignant potential form (the NIFTP), cases diagnosed cytologically as indeterminate will probably increase in the future to avoid false positive diagnosis. Thus, the indeterminate thyroid diagnostic category still remains a challenge, both at the diagnostic level and for its management. The new version of the Bethesda system for reporting thyroid cytopathology suggests managing these patients with a repeat FNA, diagnostic lobectomy and/or molecular testing.

Thyroid cancer is the most common type of endocrine cancer, and its prevalence continue to rise. Non-metastatic thyroid cancer patients are successfully treated. However, looking for new therapeutic strategies is of great importance for metastatic thyroid cancers that still lead to death. With respect to this, the tumor microenvironment (TME), which plays a key role in tumor progression, should be considered as a new promising therapeutic target to hamper thyroid cancer progression. Indeed, thyroid tumors consist of cancer cells and a heterogeneous and ever-changing niche, represented by the TME, which contributes to establishing most of the features of cancer cells. The TME consists of extracellular matrix (ECM) molecules, soluble factors, metabolites, blood and lymphatic tumor vessels and several stromal cell types that, by interacting with each other and with tumor cells, affect TME remodeling, cancer growth and progression. Among the thyroid TME components, cancer-associated fibroblasts (CAFs) have gained more attention in the last years. Indeed, recent important evidence showed that thyroid CAFs strongly sustain thyroid cancer growth and progression by producing soluble factors and ECM proteins, which, in turn, deeply affect thyroid cancer cell behavior and aggressiveness. Hence, in this article, we describe the thyroid TME, focusing on the desmoplastic stromal reaction, which is a powerful indicator of thyroid cancer progression and an invasive growth pattern. In addition, we discuss the origins and features of the thyroid CAFs, their influence on thyroid cancer growth and progression, their role in remodeling the ECM and their immune-modulating functions. We finally debate therapeutic perspectives targeting CAFs.

Background During the current coronavirus disease 2019 (COVID‐19) pandemic, the cytopathology workload has decreased remarkably worldwide as all screening and elective procedures have been postponed to prioritize the clinical management of patients at high oncological risk. In the current study, the authors provide data on the lasting impact of COVID‐19 on cytopathology practice during the initial phases of the Italian postlockdown period. Methods The percentages of the cytological sample types processed at the University of Naples Federico II during the first 12 weeks of the Italian postlockdown period were compared with those of the same period in 2019. The study period was divided into four 3‐week periods. Differences in the rates of malignant diagnoses were also assessed. Results During the 12‐week study period, the overall cytological sample workload decreased by 41.6% in comparison with 2019. In particular, the workload significantly declined for each sample type: Pap smears, –33.3%; urine, –42.8%; serous fluids, –14.4%; thyroid, –54.5%; breast, –43%; lymph node, –27.3%; and salivary gland, –61%. By contrast, the overall malignancy rate was significantly increased (P = .0011). Conclusions The reduction in the cytological sample workload during the postlockdown period still represents an ongoing effect of the COVID‐19 pandemic. On the other hand, the rise in the overall malignancy rate reflects the importance of prioritizing diagnostic procedures for patients at high oncological risk., The reduction in the cytological sample workload during the postlockdown period represents an ongoing effect of the COVID‐19 pandemic. On the other hand, the rise in the overall malignancy rate reflects the importance of prioritizing diagnostic procedures for patients at high oncological risk.

AimsLung cancer predictive biomarker testing is essential to select advanced-stage patients for targeted treatments and should be carried out without delays even during health emergencies, such as the coronavirus (COVID-19) outbreak.MethodsFifteen molecular laboratories from seven different European countries compared 4 weeks of national lockdown to a corresponding period in 2019, in terms of tissue and/or plasma-based molecular test workload, analytical platforms adopted, number of cases undergoing programmed death-ligand1 (PD-L1) expression assessment and DNA-based molecular tests turnaround time.ResultsIn most laboratories (80.0%), tissue-based molecular test workload was reduced. In 40.0% of laboratories (6/15), the decrease was >25%, and in one, reduction was as high as 80.0%. In this instance, a concomitant increase in liquid biopsy was reported (60.0%). Remarkably, in 33.3% of the laboratories, real-time PCR (RT-PCR)-based methodologies increased, whereas highly multiplexing assays approaches decreased. Most laboratories (88.9%) did not report significant variations in PD-L1 volume testing.ConclusionsThe workload of molecular testing for patients with advanced-stage lung cancer during the lockdown showed little variations. Local strategies to overcome health emergency-related issues included the preference for RT-PCR tissue-based testing methodologies and, occasionally, for liquid biopsy.

Immune-checkpoint inhibitors (ICIs) play a key role in the treatment of advanced stage colorectal cancer (CRC) patients featuring a deficient DNA mismatch repair (dMMR) system or a high microsatellite instability (MSI-H) profile. However, beyond the established role in CRC patients, ICIs have highly proven efficacy in other solid tumors featuring MSI-H/dMMR status represented by endometrial, gastric, ovarian, prostatic, and pancreatic carcinomas (EC, GC, OC, PrC, and PaC). Our aim was to compare the concordance rates among the Idylla™ MSI test, TapeStation 4200, and immunohistochemical (IHC) analysis in assessing MSI-H/dMMR status in EC, GC, OC, PrC, and PaC patients. The Sanger sequencing-based Titano MSI test was used in discordant cases. One hundred and eighty-five cases (n = 40 PrC, n = 39 GC, n = 38 OC, n = 35 PaC, and n = 33 EC) were retrospectively selected. MMR protein expression was evaluated by IHC. After DNA quality and quantity evaluations, the IdyllaTM and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Remarkably, compared to IHC, the Idylla™ platform achieved a global concordance rate of 94.5% (154/163) for the microsatellite stable (MSS)/proficient MMR (pMMR) cases and 77.3% (17/22) for the MSI-H/dMMR cases. Similarly, a global concordance rate of 91.4% (149/163) and 68.2% (15/22) for MSS/pMMR and MSI-H/dMMR cases was also identified between IHC and the TapeStation 4200 microfluidic system. In addition, a global concordance of 93.1% (148/159) and 69.2% (18/26) for MSS/pMMR and MSI-H/dMMR cases was observed between the Idylla™ and TapeStation 4200 platforms. Discordant cases were analyzed using the Titano MSI kit. Overall, our data pinpointed a central role for molecular techniques in the diagnostic evaluation of dMMR/MSI-H status not only in CRC patients but also in other types of solid tumors.

Although gene fusions occur rarely in non-small-cell lung cancer (NSCLC) patients, they represent a relevant target in treatment decision algorithms. To date, immunohistochemistry and fluorescence in situ hybridization are the two principal methods used in clinical trials. However, using these methods in routine clinical practice is often impractical and time consuming because they can only analyze single genes and the quantity of tissue material is often insufficient. Thus, novel technologies, able to test multiple genes in a single run with minimal sample input, are being under investigation. Here, we discuss the utility of next-generation sequencing and nCounter technologies in detecting simultaneous gene fusions in NSCLC patients.

Gene fusions represent novel predictive biomarkers for advanced non-small cell lung cancer (NSCLC). In this study, we validated a narrow NGS gene panel able to cover therapeutically-relevant gene fusions and splicing events in advanced-stage NSCLC patients. To this aim, we first assessed minimal complementary DNA (cDNA) input and the limit of detection (LoD) in different cell lines. Then, to evaluate the feasibility of applying our panel to routine clinical samples, we retrospectively selected archived lung adenocarcinoma histological and cytological (cell blocks) samples. Overall, our SiRe RNA fusion panel was able to detect all fusions and a splicing event harbored in a RNA pool diluted up to 2 ng/&micro, L. It also successfully analyzed 46 (95.8%) out of 48 samples. Among these, 43 (93.5%) out of 46 samples reproduced the same results as those obtained with conventional techniques. Intriguingly, the three discordant results were confirmed by a CE-IVD automated real-time polymerase chain reaction (RT-PCR) analysis (Easy PGX platform, Diatech Pharmacogenetics, Jesi, Italy). Based on these findings, we conclude that our new SiRe RNA fusion panel is a valid and robust tool for the detection of clinically relevant gene fusions and splicing events in advanced NSCLC.

AimsFollowing the development for liquid biopsies of the SiRe next-generation sequencing (NGS) panel that covers 568 clinical relevant mutations in EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRa genes, in this current study, we apply this small NGS panel on tissue samples of lung cancer.MethodsA total of 322 specimens were prospectively tested. Technical parameters were analysed on both cytological and histological samples. In a subset of 75 samples, the EGFR SiRe results were compared with those generated by the European Community (CE)–IVD EGFR assay on Idylla platform. Clinical outcomes of 11 patients treated, on the basis of SiRe results, were also evaluated.ResultsOnly 28 (8.7%) specimens failed to produce a library; out of the 294 remaining samples, a total of 168 somatic mutations were found. In nearly all instances (74/75–99%), the EGFR SiRe results were confirmed by Idylla. In general, SiRe analytical parameters were excellent. However, histological and cytological specimens differed in relation to average reads for sample, mean number of mapped reads, median read length and average reads for amplicon. Treatment outcome evaluation in 11 patients showed a partial response in 82 % (9/11) patients with a median progression-free survival of 340 days.ConclusionsThe small gene panel SiRe is a clinically relevant tool useful to widespread the adoption of NGS in predictive molecular pathology laboratories.

In the advanced stages of non-small cell lung cancer (NSCLC), molecular testing is often performed on archival cytological smears. The nCounter system (NanoString Technologies) is a new promising multiplex digital colour-coded barcode technology. However, its feasibility to evaluate the RNA expression of clinical relevant biomarkers on routine cytological smears is still uncertain. To this end, RNA was extracted from 12 NSCLC routine stained cytological smears, and nCounter analysis performed by using a 48-gene panel. Overall, 11/12 (92%) of the smears were adequate for the secondary analysis, fulfilling the quality check parameter analysis of nSolver software. This pilot study shows that RNA nCounter analysis is feasible on routine cytological smears preparing the field for the implementation of this technology in the routine setting.

Background: When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA. Methods: Our panel (‘SiRe') covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice. Results: SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression. Conclusions: SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.

Background: We have recently reported the downregulation of the Metallophosphoesterase-domain-containing protein 2 (MPPED2) gene and its cognate long non-coding RNA, MPPED2-AS1, in papillary thyroid carcinomas. Functional studies supported a tumor suppressor role of both these genes in thyroid carcinogenesis. We then decided to investigate their role in breast carcinogenesis. Methods: In order to verify MPPED2 expression, 45 human breast carcinoma samples have been investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Then, MPPED2 has been transfected in several human breast carcinoma cell lines, analyzing its role in cell proliferation, migration and invasion. To study the regulation of MPPED2 expression the methylation of its promoter was investigated by targeted bisulfite sequencing. Results: MPPED2 expression was decreased in breast cancer samples, and this was confirmed by the analysis of data available in The Cancer Genome Atlas (TCGA). Interestingly, the hypermethylation of MPPED2 promoter likely accounted for its downregulation in breast cancer. Additionally, MPPED2-AS1 was also found downregulated in breast cancer tissues and, intriguingly, its expression decreased the hypermethylation of the MPPED2 promoter by inhibiting DNA methyltransferase 1 (DNMT1). Furthermore, the restoration of MPPED2 expression reduced cell proliferation, migration and invasion capability of breast carcinoma cell lines. Conclusion: Taken together, these results propose MPPED2 downregulation as a critical event in breast carcinogenesis.

Melanoma is one of the most aggressive solid tumors and includes a stromal microenvironment that regulates cancer growth and progression. The components of stromal microenvironment such as fibroblasts, fibroblast aggregates and cancer-associated fibroblasts (CAFs) can differently influence the melanoma growth during its distinct stages. In this work, we have developed and studied a stromal microenvironment model, represented by fibroblasts, proto-myofibroblasts, myofibroblasts and aggregates of inactivated myofibroblasts, such as spheroids. In particular, we have generated proto-myofibroblasts from primary cutaneous myofibroblasts. The phenotype of proto-myofibroblasts is characterized by a dramatic reduction of &alpha, smooth muscle actin (&alpha, SMA) and cyclooxygenase-2 (COX-2) protein levels, as well as an enhancement of cell viability and migratory capability compared with myofibroblasts. Furthermore, proto-myofibroblasts display the mesenchymal marker vimentin and less developed stress fibers, with respect to myofibroblasts. The analysis of crosstalk between the stromal microenvironment and A375 or A2058 melanoma cells has shown that the conditioned medium of proto-myofibroblasts is cytotoxic, mainly for A2058 cells, and dramatically reduces the migratory capability of both cell lines compared with the melanoma-control conditioned medium. An array analysis of proto-myofibroblast and melanoma cell-conditioned media suggests that lower levels of some cytokines and growth factors in the conditioned medium of proto-myofibroblasts could be associated with their anti-tumor activity. Conversely, the conditioned media of melanoma cells do not influence the cell viability, outgrowth, and migration of proto-myofibroblasts from spheroids. Interestingly, the conditioned medium of proto-myofibroblasts does not alter the cell viability of both BJ-5ta fibroblast cells and myofibroblasts. Hence, proto-myofibroblasts could be useful in the study of new therapeutic strategies targeting melanoma.

In the era of personalized medicine, cytopathology reports must be standardized to provide clinicians with clear information regarding relevant diagnostic, prognostic, and predictive tumor features. Since 2010, the international Molecular Cytopathology Meeting has been held annually in Naples, Italy, to disseminate recent breakthroughs and ongoing investigations in the field of modern cytopathology. In particular, the seventh annual meeting, which took place on November 26 through 27, 2018, focused on novel cytological and molecular classifications and novel DNA sequencing techniques, as well as on the possibility of using cytological rather than tissue specimens for lung cancer biomarker testing. The present review aims to update cytopathologists on the more recent achievements in modern cytopathology and to identify key research questions that still remain unanswered.

AimsIn everyday practice, epidermal growth factor receptor (EGFR) testing is centralised in referral laboratories that receive paucicellular cytological specimens. Ideally, EGFR testing should be carried out in the centre where the patient is diagnosed such that the most cellular slide can be selected from in-house collected cytological material. However, available techniques are little standardised and difficult to be implemented in settings with little expertise in molecular testing. The Idylla EGFR prototype assay is a rapid and fully automated test which may easily be adopted by a wider number of pathological centres. This study assessed whether an Idylla EGFR prototype assay can be reliably applied to cytological lung cancer specimens.MethodsThe limit of detection (LOD) of the Idylla EGFR prototype assay was assessed by cell line dilution studies. A total of 10 ng was directly placed inside an Idylla EGFR prototype assay cartridge. Idylla results were compared with fragment length (exon 19 del) and Taqman assays.ResultsThe Idylla EGFR prototype assay showed an LOD of 1% mutant allele and yielded valid results in 74/76 (97.3%) samples, detecting all the mutant cases (n=32) identified by standard techniques; in addition, Idylla detected two low abundance EGFR exon 19 deletions and two G719X exon 18 point mutations, not covered by our standard reference method.ConclusionsIdylla EGFR prototype assay is sensitive on extracted DNA and can reliably be applied on cytological samples, enabling implementation of EGFR testing even in less experienced diagnostic units.

The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) International joint committee has recently updated the previous 2013 molecular guidelines version. Here we summarize these changes which demonstrate that molecular cytopathology is here to stay.

Background Neutrophil functions have long been regarded as limited to acute inflammation and the defense against microbes. The role(s) of neutrophils in cancer remain poorly understood. Neutrophils infiltrate tumors and are key effector cells in the orchestration of inflammatory responses. Thyroid cancer (TC) is the most recurrent endocrine malignant tumor and is responsible for 70% of deaths due to endocrine cancers. No studies are so far available on the role of neutrophils in TC. Objective Our purpose was to study the involvement of tumor-associated neutrophils in TC. Methods Highly purified human neutrophils (>99%) from healthy donors were stimulated in vitro with conditioned media derived from TC cell lines TPC1 and 8505c (TC-CMs). Neutrophil functions (e.g., chemotaxis, activation, plasticity, survival, gene expression, and protein release) were evaluated. Results TC-derived soluble factors promoted neutrophil chemotaxis and survival. Neutrophil chemotaxis toward a TC-CM was mediated, at least in part, by CXCL8/IL-8, and survival was mediated by granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, each TC-CM induced morphological changes and activation of neutrophils (e.g., CD11b and CD66b upregulation and CD62L shedding) and modified neutrophils' kinetic properties. Furthermore, each TC-CM induced production of reactive oxygen species, expression of proinflammatory and angiogenic mediators (CXCL8/IL-8, VEGF-A, and TNF-α), and a release of matrix metalloproteinase 9 (MMP-9). Moreover, in TC patients, tumor-associated neutrophils correlated with larger tumor size. Conclusions TC cell lines produce soluble factors able to "educate" neutrophils toward an activated functional state. These data will advance the understanding of the molecular and cellular mechanisms of innate immunity in TC.

Objective: In our Pathology Department, fine needle aspiration (FNA) of palpable thyroid nodules isperformed by cytopathologists who ensure correct sample management and rapid on-site evaluation (ROSE).Conversely, ultrasound (US)-guided FNAs have traditionally been carried out by endocrinologists andradiologists in outside clinics, where the presence of a cytopathologist is not always feasible. To overcome thislimitation, cytopathologists have started to perform US-guided FNAs themselves. This study retrospectivelyevaluates 1 year of this novel practice.Methods: A total of 2225 US-guided FNAs were performed in our clinic by cytopathologists, whereas 1490aspirates were taken by a group of non-cytopathologists. Among these, 756 FNAs were taken by a singleexperienced endocrinologist. The distribution of the Bethesda classification categories was evaluated in each ofthese groups.Results: FNAs performed by cytopathologists were more often diagnostic and better prepared than thosetaken by non-cytopathologists, including those taken by the experienced endocrinologist (P < 0.01). The latteroperator yielded a higher rate of suspicious and malignant FNAs, reflecting a more appropriate clinical triageof worrisome nodules.Conclusion: Although the endocrinologist’s evaluation is crucial to select clinically relevant thyroid nodules,cytopathologists can reliably perform US guidance in addition to their traditional expertise in sampling,specimen preparation and ROSE.Keywords: ultrasound-guided fine needle aspiration, thyroid, rapid on-site evaluation, interventionalpathologistIntroductionThyroid fine needle aspiration (FNA) is a reliableand cost-effective diagnostic tool, as long as high-quality samples, representative of the lesion, areobtained for accurate microscopic interpretation. Theideal physician to perform thyroid FNA should beone who is skilled in obtaining representative mate-rial and in the preparation of slides. As these skillsare strengthened by the microscopic observation ofsamples, it is not surprising that the best results onpalpable nodules are obtained when the FNA is per-formed or triaged by the same cytopathologist whowill eventually make the diagnosis.

Myofibroblasts are activated fibroblasts involved in tissue repair and cancer. They are characterized by de novo expression of α-smooth muscle actin (α-SMA), immunoregulatory phenotype and paracrine interaction with normal and tumorigenic cells leading to cell proliferation. At the end of wound-healing myofibroblasts undergo apoptotic cell death, whereas in vitro-activated fibroblasts are also subjected to a programmed necrosis-like cell death, termed nemosis, associated with cyclooxygenase-2 (COX-2) expression induction and inflammatory response. Furthermore, myofibroblasts form clusters during wound healing, fibrotic states and tumorigenesis. In this study, we generated and analysed clusters such as spheroids from human primary cutaneous myofibroblasts, which represent a part of stromal microenvironment better than established cell lines. Therefore, we evaluated apoptotic or necrotic cell death, inflammation and activation markers during myofibroblasts clustering. The spheroids formation did not trigger apoptosis, necrotic cell death and COX-2 protein induction. The significant decrease of α-SMA in protein extracts of spheroids, the cytostatic effect exerted by spheroids conditioned medium on both normal and cancer cell lines and the absence of proliferation marker Ki-67 after 72 h of three-dimensional culture indicated that myofibroblasts have undergone a deactivation process within spheroids. The cells of spheroids reverted to adhesion growth preserved their proliferation capability and can re-acquire a myofibroblastic phenotype. Moreover, the spontaneous formation of clusters on plastic and glass substrates suggests that aggregates formation could be a physiological feature of cutaneous myofibroblasts. This study represents an experimental model to analyse myofibroblasts deactivation and suggests that fibroblast clusters could be a cell reservoir regulating tissues turnover.