Diffuse C-Cells Hyperplasia Is the Source of False Positive Calcitonin Measurement in FNA Washout Fluids of Thyroid Nodules: A Rational Clinical Approach to Avoiding Unnecessary Surgery

Purpose: The FNA-CT is useful for the diagnosis of MTC. The aim of this study was to evaluate the performance of FNA-CT in TNs coexisting with CCH. Methods: This study retrospectively reviewed the records of 11 patients with TNs submitted to thyroidectomy on the basis of elevated basal and/or stimulated serum CT values, which at histology were not confirmed to be MTC. The results obtained in this group were compared with those of a previously reported group of histologically proven MTC patients submitted to an identical presurgical evaluation. All patients, negative for known mutations in the RET proto-oncogene, were preoperatively submitted to neck ultrasound, FNA-cytology, and FNA-CT. Results: Approximately 6 of 11 patients showed increased (>36 ng/mL, as established in previous studies not involving patients with CCH) FNA-CT. All these patients showed diffuse CCH at histology in the thyroid lobe submitted to FNA; 5 of them were benign at histology, while only one was malignant (papillary thyroid carcinoma, PTC). The remaining 5 of 11 patients had low FNA-CT (<36 ng/mL), and all of them showed only focal CCH in the lobe submitted to FNA; three of them were malignant (2 PTC, 1 follicular carcinoma), while two were benign. Conclusions: Employing the currently proposed cut-off values, false-positive FNA-CT results may be observed in benign/malignant TNs with coexisting diffuse CCH. FNA-CT must therefore be cautiously used in the diagnostic approach for patients with TNs and a slightly increased basal or stimulated serum CT concentration in order to avoid unnecessary surgery

Functional characterization of a CDKN1B mutation in a Sardinian kindred with multiple endocrine neoplasia type 4 (MEN4)

Inactivating germline mutations of the CDKN1B gene, encoding for the nuclear cyclin-dependent kinase inhibitor p27kip1 protein, have been reported in patients with multiple endocrine neoplasia type 4 (MEN4), a MEN1-like phenotype without MEN1 mutations. The aim of this study was to in vitro characterize the germline CDKN1B mutation c.374_375delCT (S125X) we detected in a patient with MEN4. The proband was affected by multiglandular primary hyperparathyroidism and gastro-entero-pancreatic tumors. We carried out subcellular localization experiments transfecting into eukaryotic HeLa and GH3 cell lines plasmid vectors expressing the CDKN1B wild type (wt) or mutant cDNA. Western blot studies showed that fusion proteins were expressed at equal levels. The mutated protein was shorter compared to the wt protein and lacked the highly conserved C-terminal domain, which includes the bipartite nuclear localization signal at amino acids 152/153 and 166/168. In HeLa and GH3 cells wt p27 localized in the nucleus whereas the p27_S125X protein was retained in the cytoplasm predicting the loss of tumor suppressive function. The proband's tumoral parathyroid tissue did not show allelic loss, since wt and mutant alleles were both present by sequencing the somatic DNA. Immunohistochemistry showed a complete loss of nuclear p27 expression in the parathyroid adenoma removed by the patient at the second surgery. In conclusion, our study confirms the pathogenic role of the c.374_375delCT CDKN1B germline mutation in a patient with MEN4

In vitro phenotypic characterisation of two genotype I African swine fever viruses with genomic deletion isolated from Sardinian wild boars

African swine fever virus (ASFV) causes a devastating disease affecting domestic and wild pigs. ASF was first introduced in Sardinia in 1978 and until 2019 only genotype I isolates were identified. A remarkable genetic stability of Sardinian ASFV isolates was described, nevertheless in 2019 two wild boar isolates with a sustained genomic deletion (4342 base pairs) were identified (7303WB/19, 7212WB/19). In this study, we therefore performed in vitro experiments with monocyte-derived macrophages (moMФ) to unravel the phenotypic characteristics of these deleted viruses. Both 7303WB/19 and 7212WB/19 presented a lower growth kinetic in moMФ compared to virulent Sardinian 26544/OG10, using either a high (1) or a low (0.01) multiplicity of infection (MOI). In addition, flow cytometric analysis showed that both 7303WB/19 and 7212WB/19 presented lower intracellular levels of both early and late ASFV proteins. We subsequently investigated whether deleted virus variants were previously circulating in wild boars in Sardinia. In the four years preceding the last genotype I isolation (February 2015–January 2019), other eight wild boar isolates were collected, all belonging to p72 genotype I, B602L subgroup X, but none of them presented a sustained genomic deletion. Overall, we observed the deleted virus isolates in Sardinia only in 2019, at the end of a strong eradication campaign, and our data suggest that it might possess an attenuated phenotype in vivo. A better understanding of ASFV evolution in endemic territories might contribute to development of effective control measures against ASF

Autonomous use of a visual biofeedback in early rehabilitation after total knee replacement. Pilot study design

Patients undergoing Total Knee Replacement (TKR) improve functional capabilities, but a lot of them show strength deficits and asymmetric patterns of movement. Early rehabilitation within the first five days improve short-term outcomes, in particular recovery of functional capabilities and levels of physical activity. Biofeedback in early rehabilitation after TKR is effective to improve gait symmetry, reduce pain and increase levels of physical activity. This pilot study aims to evaluate the effects of the autonomous use of a visual biofeedback, based on the use of inertial sensor (KARI, CoRehab, Trento, Italy), on patient outcomes in very short post-TKR period

RET/PTC rearragements in Hashimoto’s thyroiditis nodules.

An association between Hashimoto thyroiditis (HT) and thyroid carcinoma, particularly papillary thyroid carcinoma (PTC), has been postulated for decades, although definitive molecular links remain elusive. The majority of PTC is characterized by alterations or rearrangements along the MAPK signaling cascade. Activation of RET/PTC oncogene has been reported in both non-neoplastic and neoplastic HT thyroid follicular cells (Tallini et al., 2006), but the issue is still controversial. We have investigated cells from HT nodules for RET rearrangement, using interphase fluorescence in situ hybridization (I-FISH). Dual-color I-FISH experiments, using home-brew breakapart DNA probe, able to detect RET disruptions were performed on 207 fine needle aspiration biopsies on unselected nodules from 168 patients and 38 corresponding surgical samples. Normal tissue nuclei cutoff value (mean ±3DS) was ≥ 3%. In addition, RET/PTC was investigated by RT-PCR. Coexistent overt autoimmune thyroid disease (AITD) was assessed by serological (antithyroid autoantibodies, ATA), clinical and echographic data. RET breakage (FISH+) was observed in 13/207 (6.3%) samples. RET/PTC was confirmed by RT-PCR (RT-PCR+) in 2/12 available FISH+ nodules (16.6%). RT-PCR+ corresponded to FISH+ value ≥ 8%. No correlation between RET rearrangement and ATA (5/92 patients ATA +; 8/115 ATA-), or between AITD (6/78 AITD+; 7/129 AITD-) was observed. Nodules with RET disruption were observed along the whole cytological spectrum, with prevalence significantly higher in suspect or malignant cytology (13.6% in 66 TIR 3-5 vs 3.1% in 129 TIR 2 nodules: p=0.005 by X2test). 7 of 13 FISH+ nodules were surgically removed and available for I-FISH. RET breakage was confirmed in 3 samples, all of which were classified as classic PTC type. According to our results, RET rearrangement does not seem to correlate with the HT phenotype. The number of FISH+ nodules within the cytological classes, increases according to the risk of malignancy. Sensitivity of breakapart I-FISH strategy, able to detect gene disruption in single nuclei, could explain the difference between I-FISH and RT-PCR results. Since in our experimental condition, RET-PTC m-RNA transcript can be detected in ≥ 8% FISH+ samples , the oncogenic potential of nodules below this value remains to be clarified, and FISH+ nodules with benign cytology should be considered for a careful follow-up

Functioning glucagonoma associated with primary hyperparathyroidism: multiple endocrine neoplasia type 1 or incidental association?

<p>Abstract</p> <p>Background</p> <p>Diagnosis of multiple endocrine neoplasia type 1 (MEN1) is commonly based on clinical criteria, and confirmed by genetic testing. In patients without known MEN1-related germline mutations, the possibility of a casual association between two or more endocrine tumors cannot be excluded and subsequent management may be difficult to plan. We describe a very uncommon case of functioning glucagonoma associated with primary hyperparathyroidism (pHPT) in which genetic testing failed to detect germline mutations of <it>MEN-1</it> and other known genes responsible for MEN1.</p> <p>Case presentation</p> <p>The patient, a 65-year old woman, had been suffering for more than 1 year from weakness, progressive weight loss, angular cheilitis, glossitis and, more recently, skin rashes on the perineum, perioral skin and groin folds. After multidisciplinary investigations, functioning glucagonoma and asymptomatic pHPT were diagnosed and, since family history was negative, sporadic MEN1 was suspected. However, genetic testing revealed neither <it>MEN-1</it> nor other gene mutations responsible for rarer cases of MEN1 (<it>CDKN1B</it>/p27 and other cyclin-dependent kinase inhibitor genes <it>CDKN1A</it>/p15, <it>CDKN2C</it>/p18, <it>CDKN2B</it>/p21). The patient underwent distal splenopancreatectomy and at the 4-month follow-up she showed complete remission of symptoms. Six months later, a thyroid nodule, suspected to be a malignant neoplasia, and two hyperfunctioning parathyroid glands were detected respectively by ultrasound with fine needle aspiration cytology and ^99mTc-sestamibi scan with SPECT acquisition. Total thyroidectomy was performed, whereas selective parathyroidectomy was preferred to a more extensive procedure because the diagnosis of MEN1 was not supported by genetic analysis and intraoperative intact parathyroid hormone had revealed “adenoma-like” kinetics after the second parathyroid resection. Thirty-nine and 25 months after respectively the first and the second operation, the patient is well and shows no signs or symptoms of recurrence.</p> <p>Conclusions</p> <p>Despite well-defined diagnostic criteria and guidelines, diagnosis of MEN1 can still be challenging. When diagnosis is doubtful, appropriate management may be difficult to establish.</p

RET/PTC rearrangement in Hashimoto’s thyroiditis nodules: contribution to an ongoing debate.

An association between Hashimoto thyroiditis (HT) and thyroid carcinoma, has been postulated for decades, although definitive molecular links remain elusive. Activation of RET/PTC oncogene (typical of papillary thyroid carcinoma) has been reported in both non-neoplastic and neoplastic HT thyroid follicular cells (Tallini et al., 2006), but the issue is still controversial. To contribute new insight to this issue, we have investigated RET rearrangement on 207 fine needle aspiration biopsies on unselected nodules from 168 patients and 38 corresponding surgical samples. I-FISH with a homebrew-breakpart DNA probe was used. Normal tissue nuclei cutoff value (mean ± 3DS) was 3%. In addition, RET/PTC was investigated by RT-PCR. Coexistent overt autoimmune thyroid disease (AITD) was assessed by serological (antithyroid autoantibodies, ATA), clinical and echographic data. RET breakage (FISH+) was observed in 6.3% samples. RET/PTC was confirmed by RT-PCR (RT-PCR+) in 23% FISH+ nodules. RT-PCR+ corresponded to FISH+ value ≥ 6,8%. No correlation between RET rearrangement and ATA (5/92 nodules ATA +; 8/115 ATA-), or between AITD (6/78 AITD+; 7/129 AITD-) was observed. Nodules with FISH+ were observed along the whole cytological spectrum, with prevalence significantly higher in suspect or malignant cytology (13.6% TIR 3-5 vs 3.1% TIR 2 nodules). 7/13 FISH+ nodules were surgically removed and available for I-FISH. FISH+ was confirmed in 3 samples, classified as PTC classic type. Our results suggest no association between RET rearrangement and HT. The percentage of FISH+ nodules increases according to the risk of malignancy. Difference between I-FISH and RT-PCR results was observed, presumibly due to the sensitivity of I-FISH strategy. Since we detected RET-PTC m-RNA transcript in ≥ 6,8% FISH+ samples, the oncogenic potential of nodules below this value remains to be clarified, and FISH+ nodules with benign cytology should be considered for a careful follow-up

A Naturally Occurring Microhomology-Mediated Deletion of Three Genes in African Swine Fever Virus Isolated from Two Sardinian Wild Boars

African swine fever virus (ASFV) is the etiological agent of a lethal disease of domestic pigs and wild boars. ASF threatens the pig industry worldwide due to the lack of a licensed vaccine or treatment. The disease has been endemic for more than 40 years in Sardinia (Italy), but an intense campaign pushed it close to eradication; virus circulation was last detected in wild boars in 2019. In this study, we present a genomic analysis of two ASFV strains isolated in Sardinia from two wild boars during the 2019 hunting season. Both isolates presented a deletion of 4342 base pairs near the 5′ end of the genome, encompassing the genes MGF 360-6L, X69R, and MGF 300-1L. The phylogenetic evidence suggests that the deletion recently originated within the Sardinia ecosystem and that it is most likely the result of a non-allelic homologous recombination driven by a microhomology present in most Sardinian ASFV genomes. These results represent a striking example of a genomic feature promoting the rapid evolution of structural variations and plasticity in the ASFV genome. They also raise interesting questions about the functions of the deleted genes and the potential link between the evolutionary timing of the deletion appearance and the eradication campaign

A Deeper Insight into Evolutionary Patterns and Phylogenetic History of ASFV Epidemics in Sardinia (Italy) through Extensive Genomic Sequencing

African swine fever virus (ASFV) is the etiological agent of the devastating disease African swine fever (ASF), for which there is currently no licensed vaccine or treatment available. ASF is defined as one of the most serious animal diseases identified to date, due to its global spread in regions of Africa, Europe and Asia, causing massive economic losses. On the Italian island of Sardinia, the disease has been endemic since 1978, although the last control measures put in place achieved a significant reduction in ASF, and the virus has been absent from circulation since April 2019. Like many large DNA viruses, ASFV mutates at a relatively slow rate. However, the limited availability of whole-genome sequences from spatial-localized outbreaks makes it difficult to explore the small-scale genetic structure of these ASFV outbreaks. It is also unclear if the genetic variability within outbreaks can be captured in a handful of sequences, or if larger sequencing efforts can improve phylogenetic reconstruction and evolutionary or epidemiological inference. The aim of this study was to investigate the phylogenetic patterns of ASFV outbreaks between 1978 and 2018 in Sardinia, in order to characterize the epidemiological dynamics of the viral strains circulating in this Mediterranean island. To reach this goal, 58 new whole genomes of ASFV isolates were obtained, which represents the largest ASFV whole-genome sequencing effort to date. We provided a complete description of the genomic diversity of ASFV in terms of nucleotide mutations and small and large indels among the isolates collected during the outbreaks. The new sequences capture more than twice the genomic and phylogenetic diversity of all the previously published Sardinian sequences. The extra genomic diversity increases the resolution of the phylogenetic reconstruction, enabling us to dissect, for the first time, the genetic substructure of the outbreak. We found multiple ASFV subclusters within the phylogeny of the Sardinian epidemic, some of which coexisted in space and time