Circulating miR-26b-5p and miR-451a as diagnostic biomarkers in medullary thyroid carcinoma patients.

PURPOSE/METHODS: The determination of tumour biomarkers is paramount to advancing personalized medicine, more so in rare tumours like medullary thyroid carcinoma (MTC), whose diagnosis is still challenging. The aim of this study was to identify non-invasive circulating biomarkers in MTC. To achieve this goal, paired MTC tissue and plasma extracellular vesicle samples were collected from multiple centres and microRNA (miRNA) expression levels were evaluated. RESULTS: The samples from a discovery cohort of 23 MTC patients were analysed using miRNA arrays. Lasso logistic regression analysis resulted in the identification of a set of circulating miRNAs as diagnostic biomarkers. Among them, miR-26b-5p and miR-451a, were highly expressed and their expression decreased during follow-up in disease-free patients in the discovery cohort. Circulating miR-26b-5p and miR-451a were validated using droplet digital PCR in a second independent cohort of 12 MTC patients. CONCLUSION: This study allowed the identification and validation of a signature of two circulating miRNAs, miR-26b-5p and miR-451a, in two independent cohorts reporting a significant diagnostic performance for MTC. The results of this study offer advancements in molecular diagnosis of MTC proposing a novel non-invasive tool to use in precision medicine.

Silent thyroiditis following vaccination against COVID-19: report of two cases.

PURPOSE: It is well established that thyroiditis and other thyroid disorders can be induced by COVID-19 infection, but there is limited information about the autoimmune/inflammatory syndrome induced by adjuvants (ASIA) after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. We report two cases of thyrotoxicosis following SARS-CoV-2 vaccine. METHODS AND RESULTS: Two young health care peoples (wife and husband) received a first dose of SARS-CoV-2 vaccine, and few weeks later developed clinical manifestations of thyroid hyperactivity, with increased thyroid hormone levels on thyroid function tests, suppressed thyroid-stimulating hormone and negative antithyroid antibodies, despite being healthy before vaccination. They were diagnosed at the 4th week after first dose of SARS-Cov-2 vaccine as silent thyroiditis and followed without treatment, since their symptoms were not severe. At the 6th week, the patients became wholly asymptomatic and their thyroid function returned to normal. CONCLUSIONS: Thyrotoxicosis can occur after SARS-CoV-2 vaccination probably related to silent thyroiditis.

Pregnancy-associated plasma protein A mRNA expression as a marker for differentiated thyroid cancer: results from a "surgical" and a "cytological" series.

PURPOSE: Pregnancy-associated plasma protein A (PAPPA) is a metalloproteinase initially described for its role during pregnancy. PAPPA regulates IGF ligands 1 (IGF1) bioavailability through the degradation of IGF-binding protein 4 (IGFBP4). After the cleavage of IGFBP4, free IGF1 is able to bind IGF1 receptors (IGF1R) triggering the downstream signaling. Recently, PAPPA expression has been linked with development of several cancers. No data have been published on thyroid cancer, yet. METHODS: We evaluated PAPPA, insulin-like growth factor (IGF1), IGF1 receptors (IGF1R) and IGF-binding protein 4 (IGFBP4) mRNA expression levels in a "Surgical series" of 94 thyroid nodules (64 cancers, 16 follicular adenomas and 14 hyperplastic nodules) and in a "Cytological series" of 80 nodules from 74 patients underwent to fine-needle aspiration cytology (FNAC). In tissues, PAPPA was also evaluated by western blot. RESULTS: We found that PAPPA expression was increased in thyroid cancer specimen at mRNA and protein levels and that, adenomas and hyperplastic nodules had an expression similar to normal tissues. When applied on thyroid cytologies, PAPPA expression was able to discriminate benign from malignant nodules contributing to pre-surgical classification of the nodules. We calculated a cut-off with a good specificity (91%) which reached 100% when combined with molecular biology. CONCLUSION: These results show that PAPPA could represent a promising diagnostic marker for differentiated thyroid cancer.

Baseline serum TSH levels predict the absence of thyroid dysfunction in cancer patients treated with immunotherapy.

PURPOSE: Immunotherapy against immune checkpoints has significantly improved survival both in metastatic and adjuvant setting in several types of cancers. Thyroid dysfunction is the most common endocrine adverse event reported. Patients who are at risk of developing thyroid dysfunction remain to be defined. We aimed to identify predictive factors for the development of thyroid dysfunction during immunotherapy. METHODS: This is a retrospective study including a total of 68 patients who were treated with immune checkpoint inhibitors (ICIs) for metastatic or unresectable advanced cancers. The majority of patients were treated with anti-PD1 drugs in monotherapy or in combination with anti-CTLA4 inhibitors. Thyroid function and anti-thyroid antibodies, before starting immunotherapy and during treatment, were evaluated. Thyroid ultrasound was also performed in a subgroup of patients at the time of enrolment in the study. RESULTS: Eleven out of 68 patients (16.1%) developed immune-related overt thyroid dysfunction. By ROC curve analysis, we found that a serum TSH cut-off of 1.72 mUI/l, at baseline, had a good diagnostic accuracy in identifying patients without overt thyroid dysfunction (NPV = 100%, p = 0.0029). At multivariate analysis, both TSH and positive anti-thyroid antibodies (ATAbs) levels, before ICIs treatment, were independently associated with the development of overt thyroid dysfunction during immunotherapy (p = 0.0001 and p = 0.009, respectively). CONCLUSIONS: Pre-treatment serum TSH and ATAbs levels may help to identify patients at high risk for primary thyroid dysfunction. Our study suggests guidance for an appropriate timely screening and for a tailored management of thyroid dysfunctions in patients treated with ICIs.

BROX haploinsufficiency in familial nonmedullary thyroid cancer.

BACKGROUND: The familial nonmedullary thyroid cancer (FNMTC) is suspected to be a Mendelian condition in up to 3-8% of thyroid cancers. The susceptibility chromosomal loci and genes of 95% of FNMTC cases remain to be characterized. The inheritance of FNMTC appears to be autosomal dominant with incomplete penetrance and variable expressivity. The finding of the causative gene of FNMTC and the identification of patients at risk that need genetic testing were our aim. METHODS: We analyzed by whole-exome sequencing patients and non-affected relatives of five families with at least two family members affected by papillary thyroid cancer, selecting for new or extremely rare variants with predicted pathogenic value. RESULTS: A family showed, in all three affected members, a new loss-of-function variant (frameshift deletion) in BROX gene at 1q41 that was absent from all internal and external databases. In a second family with three affected relatives, we found an additional new BROX variant. The smaller families presented no variants in BROX or in the other causative genes studied. CONCLUSIONS: BROX could be a new causative gene for FNMTC. Variants in BROX may result in the haploinsufficiency of a key gene involved in the morphogenesis of MVBs, in the endosomal sorting of cargo proteins, and in EGFR. Functional studies are needed to support this result. The thorough genomic analysis by NGS in all families with three or more affected members should become a routine approach to obtain a comprehensive genetic view and find confirmative second cases.

Indication for radioiodine remnant ablation in differentiated thyroid cancer patients: does 2018 Italian consensus change anything?

PURPOSE: We speculated that radioiodine remnant ablation (RRA) could be performed less frequently in differentiated thyroid cancer (DTC) patients, if the recommendations of the 2018 Italian Consensus (ITA) were applied in clinical practice. Therefore, we compared the ITA indications for RRA with the recommendations by the 2015 American Thyroid Association guidelines (ATA). METHODS: We retrospectively evaluated 380 consecutive DTC patients treated with surgery and RRA, followed at the Section of Endocrinology, University of Siena, Italy from January 2006 to December 2019. RESULTS: Using ITA a significant increase of DTC patients classified as low or high risk and a significant decrease of patients defined at intermediate risk were observed (p < 0.0001). Consequently, the percentage of patients without routinary indication for RRA (47.4%, versus 38.2%, p < 0.0001) and those with a definite indication for RRA (8.2 versus 1.8%, p < 0.0001) was significantly higher compared to ATA. Moreover, using ITA the percentage of patients with a selective use of RRA was lower in comparison to ATA (44.7% versus 60%, p < 0.0001). Nevertheless, the prevalence of distant metastases, at post-ablative whole body scan, in patients without indication for RRA, was not different using either ATA or ITA (2.1% and 1.1% respectively, p = 0.37). CONCLUSION: The use of ITA Consensus, in clinical practice, increases significantly the number of patients for whom RRA is not routinely indicated in comparison to ATA guidelines but without differences in delaying the diagnosis of distant metastatic disease.

Familial non-medullary thyroid cancer: a critical review.

BACKGROUND: Familial non-medullary thyroid carcinoma (FNMTC), mainly of papillary histotype (FPTC), is defined by the presence of the disease in two or more first-degree relatives in the absence of other known familial syndromes. With the increasing incidence of PTC in the recent years, the familial form of the disease has also become more common than previously reported and constitutes nearly 10% of all thyroid cancers. Many aspects of FNMTC are debated, concerning both clinical and genetic aspects. Several studies reported that, in comparison with sporadic PTCs, FPTCs are more aggressive at disease presentation, while other authors reported no differences in the clinical behavior of sporadic and familial PTCs. For this reason, recent guidelines do not recommend screening of family members of patients with diagnosis of differentiated thyroid cancer (DTC). FNMTC is described as a polygenic disorder associated with multiple low- to moderate-penetrance susceptibility genes and incomplete penetrance. At the moment, the genetic factors contributing to the development of FNMTC remain poorly understood, though many putative genes have been proposed in the recent years. PURPOSE: Based on current literature and our experience with FNMTC, in this review, we critically discussed the most relevant controversies, including its definition, the genetic background and some clinical aspects as screening and treatment.

Thr92Ala polymorphism in the type 2 deiodinase gene: an evolutionary perspective.

PURPOSE: In the past, a role of thyroid hormones in human evolution has been hypothesized. T3, the metabolically active form, derives from extrathyroidal conversion of T4 by deionidase 2 (D2) enzyme encoded by DIO2 gene. In thyroid-deficient patients, decreased levels of free T3 have been associated with the polymorphism rs225014 A/G in DIO2, which causes the substitution of Threonine with Alanine (p.Thr92Ala) at protein level. METHODS: We compared DNA and protein sequences of D2 from archaic human subspecies with those of contemporary humans. RESULTS: Neanderthals and Denisovans displayed only the G allele at the rs225014 polymorphism, which encodes for an Alanine on the amino acid level. These data suggest that these hominines were homozygous for the Ala amino acid. These arcaic humans often lived in condition of iodine deficiency and thus, defective mechanisms of T3 biosynthesis could be life threatining. A reduced D2 activity is likely to cause decreased T3 levels, which could be critical for those individuals. Neanderthals and Denisovans were hunters/gatherers, and their diet was mainly based on the consumption of meat, with a low intake of carbohydrates. The need for circulating T3 is reduced at such alimentary conditions. On the basis of our genome comparisons the A allele, corresponding to Threonine and associated with higher levels of circulating T3 in thyroid-deficient patients, appeared for the first time during evolution in Anatomically Modern Humans during the Upper Pleistocene and has been conserved during the Neolithic age. With the advent of agriculture and herding, individuals carrying A allele might have a higher probability for surviving and reproducing. Thus, the variant was positively selected during the evolution. CONCLUSION: Here we present an evolutionary perspective for p.Thr92Ala variant of D2 from Neanderthals to Anatomically Modern Humans.

Features and outcome of differentiated thyroid carcinoma associated with Graves' disease: results of a large, retrospective, multicenter study.

BACKGROUND: Whether differentiated thyroid cancer (DTC) occurring concomitantly with Graves' disease (GD) is more aggressive and bound to a less favorable outcome is controversial. OBJECTIVE: Aim of this multicenter retrospective study was to compare baseline features and outcome of DTC patients with GD (DTC/GD+) or without GD (DTC/GD-). PATIENTS: Enrolled in this study were 579 patients referred to five endocrine units (Cagliari, Pavia, Pisa, Siena, and Varese) between 2005 and 2014: 193 patients had DTC/GD+ , 386 DTC/GD-. Patients were matched for age, gender and tumor size. They underwent surgery because of malignancy, large goiter size, or relapse of hyperthyroidism in GD. RESULTS: Baseline DTC features (histology, lymph node metastases, extrathyroidal extension) did not differ in the two groups, except for multifocality which was significantly more frequent in DTC/GD+ (27.5% vs. 7.5%, p < 0.0001). At the end of follow-up (median 7.5 years), 86% of DTC/GD+ and 89.6% DTC/GD- patients were free of disease. Patients with persistent or recurrent disease (PRD) had "biochemical disease" in the majority of cases. Microcarcinomas were more frequent in the DTC/GD+ group (60% vs. 37%, p < 0.0001) and had an excellent outcome, with no difference in PRD between groups. However, in carcinomas ≥ 1 cm, PRD was significantly more common in DTC/GD+ (24.4% vs. 11.5%; p = 0.005). In the whole group, univariate and multivariate analyses showed that GD+ , lymph node involvement, extrathyroidal invasion, multifocality and tall cell histotype were associated with a worse outcome. Female gender and microcarcinomas were favorable features. No association was found between baseline TSH-receptor antibody levels and outcome. Graves' orbitopathy (GO) seemed to be associated with a better outcome of DTC, possibly because patients with GO may early undergo surgery for hyperthyroidism. CONCLUSIONS: GD may be associated with a worse outcome of coexisting DTC only if cancer is ≥ 1 cm, whereas clinical outcome of microcarcinomas is not related to the presence/absence of GD.

Real-world efficacy and safety of lenvatinib: data from a compassionate use in the treatment of radioactive iodine-refractory differentiated thyroid cancer patients in Italy.

BACKGROUND: Lenvatinib is a multi-kinase inhibitor approved for patients with radioactive iodine (RAI)-resistant differentiated thyroid cancer (DTC). Before the drug approval from the Italian National Regulatory Agency, a compassionate use programme has been run in Italy. This retrospective study aimed to analyse data from the first series of patients treated with lenvatinib in Italy. METHODS: The primary aim was to assess the response rate (RR) and progression-free survival (PFS). Secondary end-points include overall survival (OS) and toxicity data. RESULTS: From November 2014 to September 2016, 94 patients were treated in 16 Italian sites. Seventeen percent of patients had one or more comorbidities, hypertension being the most common (60%). Ninety-eight percent of patients were treated by surgery, followed by RAI in 98% of cases. Sixty-four percent of patients received a previous systemic treatment. Lenvatinib was started at 24 mg in 64 subjects. Partial response and stable disease were observed in 36% and in 41% of subjects, respectively; progression was recorded in 14% of patients. Drug-related side-effects were common; the most common were fatigue (13.6%) and hypertension (11.6%). Overall, median PFS and OS were 10.8 months (95% confidence interval [CI], 7.7-12.6) and 23.8 months (95% CI, 19.7-25.0) respectively. CONCLUSION: Lenvatinib is active and safe in unselected, RAI-refractory, progressive DTC patients in real-life setting. RR and PFS seem to be less favourable than those observed in the SELECT trial, likely due to a negative selection that included heavily pretreated patients or with poor performance status.

Variants in MCT10 protein do not affect FT3 levels in athyreotic patients.

PURPOSE: Several single-nucleotide polymorphisms in genes encoding for transporters have been associated with serum thyroid hormone concentrations with inconsistent results. The aim of this study was to assess the clinical significance of the rs17606253 in SLC16A10 gene alone and in combination with the DIO2 Thr92Ala variation in athyreotic patients. METHODS: One-hundred patients submitted to total thyroidectomy and treated with levothyroxine were included. Pre- and post surgical serum TSH levels did not differ by more than ± 0.5 mIU/l. RESULTS: Both patients carrying the wild-type allele or heterozygous for rs17606253 in SLC16A10 gene had a significant reduction in FT3 post surgical levels (p = 0.01 and p < 0.0001, respectively) while Thr92Ala in DIO2 gene was associated with reduced FT3 levels for heterozygous and rare homozygous patients (p < 0.0001 and p = 0.01, respectively). We identified two groups ("FT3 unchanged" and "FT3 reduced") using a cutoff of at least 0.5 pg/ml as a significant variation between pre- and post surgical FT3 values. In this case, the rs17606253 was not statistically associated with reduced FT3 levels at genotype and allele levels. On the contrary, the Thr92Ala in DIO2 gene was confirmed statistically associated with reduced FT3 levels after surgery with a p = 0.035 at genotype level and p = 0.014 at allele level. CONCLUSIONS: We confirmed the role of DIO2 Thr92Ala polymorphism on T3 levels. On the contrary, SLC16A1 rs17606253 polymorphism did not impair hormone levels in athyreotic patients treated with levothyroxine therapy.

Should familial disease be considered as a negative prognostic factor in micropapillary thyroid carcinoma?

PURPOSE: An increased aggressiveness of familial papillary thyroid carcinoma (FPTC) compared with sporadic form has been reported. On the contrary, the biological behavior of familial microPTC (FmPTC) is still debated. To assess if familial diseases should be considered as a negative prognostic factor in mPTC, the clinical presentation and outcome of FmPTC and sporadic mPTC (SmPTC) were compared. METHODS: We retrospectively analyzed 291 mPTC (SmPTC n = 248, FmPTC n = 43) patients followed for a median follow-up of 8.3 years. FmPTC was defined as the presence of PTC in two or more first-degree relatives, after excluding hereditary syndromes associated with PTC. RESULTS: FmPTC patients had more frequently bilateral tumor (32.6% versus 16.5%, p = 0.01) and lymph node metastases at diagnosis (30.2% versus 14.9%, p = 0.02). At the first follow-up, FmPTC patients had a higher rate of structural disease and a lower rate of remission compared to SmPTC (p = 0.01). Also in a multivariate model, using a "CHAID tree-building algorithm", familial disease correlated with a worse clinical presentation and outcome of mPTC patients. Familial disease was associated with a higher rate of intermediate risk patients in non incidental mPTC and with a higher rate of structural incomplete response in mPTC without lymph node metastases (p = 0.01). CONCLUSIONS: Like in macroPTC, the familial form of the diseases has been shown to be a negative prognostic factor also in mPTC, therefore, it should be highly regarded in the management of mPTC patients.

MicroRNA expression profile of thyroid nodules in fine-needle aspiration cytology: a confirmatory series.

INTRODUCTION: MiRNAs are small endogenous non-coding RNAs implicated with gene expression regulation. Changes in miRNA levels have been reported in thyroid cancer. Fine-needle aspiration cytology (FNAC) is the most reliable tool for differential diagnosis of thyroid nodules. METHODS: We have analyzed 174 FNAC from 168 patients with thyroid nodules for expression levels of 11 miRNAs (miRNA197; -187; -181b-3p; -181b-5p; -224; -181a; 146b; -221; -222; -155 and miRNA183) known to be up-regulated in cancer tissues compared to benign lesions. Expression of miRNAs was analyzed in FNA samples calculating the fold change of miRNA expression relative to normal thyroid tissue after normalization to an endogenous control. RESULTS: In FNAC, miRNA expression was confirmed to be higher in malignant or suspicious for malignancy nodules compared to benign, only for miRNA146b, -222 and -221 (fold change expression ≥ 5). CONCLUSION: In this study, we confirmed that a limited set of miRNAs can be used for the differential diagnosis of thyroid nodules.

Autoimmune thyroid diseases are more common in patients with prolactinomas: a retrospective case-control study in an Italian cohort.

BACKGROUND: Prolactin may exert immunological effects. Over the years, a higher prevalence of autoimmune thyroid diseases (ATD) has been reported in patients with prolactinomas (PRLs) in areas with sufficient iodine intake. PURPOSE: The aim of our study was to evaluate the prevalence of ATD [Graves' disease (GD) and chronic autoimmune thyroiditis (AIT)] in a retrospective cohort of Italian patients with PRLs compared to a sex-matched control group, represented by subjects with non-functioning pituitary adenoma (NFPA) or empty sella (ES). MATERIALS AND METHODS: We enrolled 149 patients (108 F/41 M) with PRLs (110 micro/39 macro) and 143 subjects (100 F/43 M) with NFPA (n = 96, 56 micro/40 macro) or ES (n = 47), with normal serum prolactin. Neck ultrasound and thyroid function tests (anti-thyroid antibodies, TSH, FT3 and FT4) were performed in all patients. RESULTS: In PRLs, median serum prolactin was significantly higher (98.3 vs. 8.9 ng/ml, p ≤ 0.0001), while age was lower (34 vs. 46 years, p ≤ 0.001) compared to controls. The prevalence of ATD was 13.4% (20/149) in PRLs (1 GD and 19 AIT) compared to 6.3% (9/143) in the controls (p = 0.042). At the multivariate analysis, serum prolactin was the only independent factor predicting ATD. Thyroid volume (12.5 ± 5.9 ml vs. 12.8 ± 10 ml, p = 0.47) and the presence of uni- or multinodular goiter (29.5% vs. 35%, p = 0.35) did not differ between PRLs and control groups. CONCLUSIONS: Our data in an area with mild iodine deficiency confirm a higher prevalence of ATD in patients with prolactinomas.

Small papillary thyroid carcinoma with minimal extrathyroidal extension should be managed as ATA low-risk tumor.

PURPOSE: According to American Thyroid Association (ATA) guideline, papillary thyroid cancer (PTC) with minimal extrathyroidal extension (mETE) is classified at "intermediate risk" of persistent/recurrent disease. However, the impact of mETE per se on patients' outcome is not fully understood. The aim of our study was to evaluate the prognostic significance of mETE in patients with PTC not submitted to therapeutic or prophylactic lymph node dissection, according to tumor size and other prognostic factors. PATIENTS AND METHODS: We retrospectively evaluated a total of 514 PTC patients: 127 (24.7%) had mETE (pT3Nx) and 387 (75.3%) had negative margins (pT1-2Nx). At a median follow-up of 9.1 years, patients were divided in two groups: patients with "good outcome" (no evidence of disease) and patients with "poor outcome" (persistent structural disease or recurrent disease or tumor-related death). RESULTS: The rate of patients with "poor outcome" was significantly higher in patients with mETE compared with patients with negative margins (11.8 versus 5.1%; OR 2.4576, 95% CI 1.2178-4.9594, p = 0.01). However, mETE was significantly associated with poor outcome only in patients with tumors larger than 1.5 cm. CONCLUSIONS: mETE is an unfavorable prognostic factor in tumors larger than 1.5 cm, suggesting that, in the absence of other unfavorable characteristics, smaller tumors with mETE should be classified and managed as "low risk" tumors.

Chronic lymphocytic thyroiditis (CLT) has a positive prognostic value in papillary thyroid cancer (PTC) patients: the potential key role of Foxp3+ T lymphocytes.

BACKGROUND: An impact of chronic lymphocytic thyroiditis (CLT) on papillary thyroid cancer (PTC) outcome has long been advocated but it is still controversial. PURPOSE: The aim of this study was to evaluate the prognostic value of CLT in a retrospective cohort of PTC patients and to characterize the lymphocytic subpopulations and infiltrate (LI). MATERIALS AND METHODS: We assessed 375 PTC patients, aged 45.2 ± 16.4 years, and treated with thyroidectomy and radioiodine remnant ablation, with a mean follow-up of 6.28 ± 3.86 years. In a subgroup of patients (n = 81) tissue sections were reviewed for the presence of CLT or lymphocytes associated with tumor in absence of background thyroiditis (TAL); cytotoxic CD8+/regulatory Foxp3+ T lymphocyte (CD8+/Foxp3+) ratio was characterized by immunohistochemistry: a low ratio is suggestive of a less effective anti tumor immune response. RESULTS: Seventy-five/375 patients (20%) had a histological diagnosis of CLT and showed at the last follow-up a significantly better outcome compared to those with no CLT (cure rate: 91.8 versus 76.3%, p = 0.003). LI was characterized in 81 PTC patients (24 with CLT and 57 with TAL): the peri-tumoral CD8+/Foxp3+ ratio was lower in patients not cured at the final evaluation. CONCLUSIONS: Our data suggest that concurrent CLT has a protective effect on PTC outcome and that the imbalance between cytotoxic and regulatory T lymphocytes in the peri-tumoral TAL may affect the tumor-specific immune response favoring a more aggressive behavior of cancer.

HABP2 G534E variation in familial non-medullary thyroid cancer: an Italian series.

INTRODUCTION: Thyroid cancer may have a familial predisposition and may occur in the context of hereditary syndromes or as isolated tumor. Recently, the G534E variant in the HABP2 gene has been suggested as causative mutation for familial thyroid cancer, but other studies gave contradictory results. METHODS: We have analyzed the G534E variant in an Italian series of 63 familial thyroid cancer patients and 41 unaffected family members with end-point PCR, DHPLC and direct sequencing. RESULTS: All samples analyzed displayed a pattern typical of the homozygous wild type revealing the absence of the G534E variant. CONCLUSION: In this study, HABP2 G534E variant is not correlated with the familial form of PTC.