Reclassification Of Variant Research Articles

Cytological Diagnosis of Follicular Patterned Lesion of Thyroid Nodule and its Follow-up Histopathology

The follicular patterned lesion of thyroid nodules is an exciting topic because it is not clear whether the lesion is benign or malignant. It comprises hyperplastic/adenomatoid nodules, follicular adenoma, follicular carcinoma and follicular variant of papillary carcinoma. The cytological diagnosis of atypia of undetermined significance/Follicular lesion of undetermined significance, follicular neoplasm/suspicious for follicular neoplasm and suspicious for malignancy remains controversial in terms of management. The study aimed to examine the extent of invasion of malignant lesions in follow-up histopathology diagnosed as undetermined lesions cytologically. A total of 256 cases were included in this study; among these, 199 cases were benign lesions, 15 cases were non-diagnostic and 42 cases were diagnosed either of uncertainty for malignancy with cellular atypia or malignancy. Subsequent follow-up resection and histopathology were done with 48 cases (18.75%), which comprises 1 non-diagnostic, 10 benign lesions, 21 atypia of undetermined significance/Follicular lesion of undetermined significance, 7 Follicular neoplasm/Suspicious for a follicular neoplasm, 5 suspicious for malignancy and 4 malignancies. Also, 6 Follicular variant of papillary thyroid carcinoma, 6 classic papillary carcinoma, 5 follicular carcinoma and 1 anaplastic carcinoma were diagnosed in follow-up resection histopathology of 48 cases. Thirty eight cases were of undetermined significance of malignancy with cytological features of the follicular lesion. Four Follicular variant of papillary thyroid carcinoma, 2 follicular carcinoma and 1 papillary carcinoma were diagnosed in 21 resected cases cytologically reported as Atypia of undetermined significance/Follicular lesion of undetermined significance with malignancy risk of 33.33%. Three malignancies in follicular neoplasm/suspicious for a follicular neoplasm with a risk of 42.85% and 4 malignancies in suspicious malignancy group with a risk of 80% were found. The average risk of malignancy of these three undetermined categories was reduced to 24.24% from 42.42% upon reclassification of the Follicular variant of papillary thyroid carcinoma. All the Follicular variants of papillary thyroid carcinoma diagnosed by histopathology were non-invasive regarding lymph node involvement and microvascular or capsular invasion. Mediscope 2023;10(2): 68-77

Combining clinical and molecular characterization of CDH1: a multidisciplinary approach to reclassification of a splicing variant.

Pathogenic germline variants (PGVs) in the CDH1 gene are associated with diffuse gastric and lobular breast cancer syndrome (DGLBC) and can increase the lifetime risk for both diffuse gastric cancer and lobular breast cancer. Given the risk for diffuse gastric cancer among individuals with CDH1 PGVs is up to 30-40%, prophylactic total gastrectomy is often recommended to affected individuals. Therefore, accurate interpretation of CDH1 variants is of the utmost importance for proper clinical decision-making. Herein we present a 45-year-old female, with lobular breast cancer and a father with gastric cancer of unknown pathology at age 48, who was identified to have an intronic variant of uncertain significance in the CDH1 gene, specifically c.833-9C > G. Although the proband did not meet the International Gastric Cancer Linkage Consortium (IGCLC) criteria for gastric surveillance, she elected to pursue an upper endoscopy where non-targeted gastric biopsies identified a focus of signet ring cell carcinoma (SRCC). The proband then underwent a total gastrectomy, revealing numerous SRCC foci, but no invasive diffuse gastric cancer. Simultaneously, a genetic testing laboratory performed RNA sequencing to further analyze the CDH1 intronic variant, identifying an abnormal transcript from a novel acceptor splice site. The RNA analysis in conjunction with the patient's gastric foci of SRCC and family history was sufficient evidence for reclassification of the variant from uncertain significance to likely pathogenic. In conclusion, we report the first case of the CDH1 c.833-9C > G intronic variant being associated with DGLBC and illustrate how collaboration among clinicians, laboratory personnel, and patients is crucial for variant resolution.

Exploring the impact of the reclassification of a hereditary cancer syndrome gene variant: emerging themes from a qualitative study

The complexity of genetic variant interpretation means that a proportion of individuals who undergo genetic testing for a hereditary cancer syndrome will have their test result reclassified over time. Such a reclassification may involve a clinically significant upgrade or downgrade in pathogenicity, which may have significant implications for medical management. To date, few studies have examined the psychosocial impact of a reclassification in a hereditary cancer syndrome context. To address this gap, semi-structured telephone interviews were performed with eighteen individuals who had a BRCA1, BRCA2 or Lynch syndrome-related (MLH1, MSH2, MSH6 or PMS2) gene variant reclassified. The interviews were analysed utilising an inductive, qualitative approach and emergent themes were identified by thematic analysis. Variable levels of recall amongst participants were found. Common motivations for initial testing included a significant personal and/or family history of cancer and a desire to “find an answer”. No individual whose uncertain result was upgraded reported negative psychosocial outcomes; most reported adapting to their reclassified result and appraised their genetic testing experience positively. However, individuals whose likely pathogenic/pathogenic results were downgraded reported feelings of anger, shock and sadness post reclassification, highlighting that additional psychosocial support may be required for some. Genetic counselling issues and recommendations for clinical practice are outlined.

UNRAVELING THE CAUSE OF A KINDRED WITH CHRONIC CANDIDIASIS

<h3>Introduction</h3> Chronic mucocutaneus candidiasis (CMC) is a clinical syndrome characterized by invasive chronic fungal infections involving skin, nails, and mucosal tissues. Advancements in genetic testing have aided in identification of underlying molecular causes; however challenges arise when variants of uncertain significance are identified in genes of interest. Our case highlights the importance of family history in genotype-phenotype correlation leading to reclassification of a novel variant in a rare immune deficiency. <h3>Case Description</h3> A 67-year-old female is referred for evaluation of chronic candidiasis including vaginitis, onychomycosis, stomatitis, gingivitis requiring teeth removal, and recurrent esophageal structuring requiring balloon dilation. She developed fluconazole resistance and treatment includes a combination of nystatin, clotrimazole, itraconazole, and in severe cases, amphotericin. She had no clinically significant history of bacterial or viral infections. Medical history included melanoma, cervical and breast cancers. Immune evaluation including HIV testing, tetanus and pneumococcal titers, total complement, lymphocyte flow cytometry and mitogen/antigen proliferation were unremarkable. Her immunoglobulins were notable for a low IgM of 43mg/dL (reference 57-237mg/dL), and diphtheria titer was low at 0.036IU/mL (reference >/= 0.100IU/mL). Gene sequencing revealed a variant of uncertain significance in STAT1 c.200A>C (p.Gln67Pro). Family history was notable for CMC in multiple relatives across four generations. Family sequencing was positive for the same STAT1 mutation in all affected members and negative for one unaffected member. <h3>Discussion</h3> Dominant STAT1 gain-of-function mutations, often in the coiled-coil domain, are an established cause of CMC. The outlined case represents a novel N-terminal domain mutation, initially classified as a variant, but reclassified as pathogenic following kindred sequencing.

Upgrading an intronic TMEM67 variant of unknown significance to likely pathogenic through RNA studies and community data sharing.

A couple of Ashkenazi Jewish descent was referred for an early anatomy scan at 14+2weeks of gestation following a previous pregnancy termination due to posterior encephalocele and enlarged kidneys. The index pregnancy was also positive for several fetal abnormalities, including enlarged kidneys with cystic dysplasia and abnormal cerebellar morphology highly suggestive of Joubert syndrome. Trio exome sequencing revealed compound heterozygosity for variants in the TMEM67 gene: a known pathogenic maternally inherited variant found in trans with a paternal intronic variant of unknown significance. RNA analysis revealed that the intronic variant creates a cryptic acceptor splice site in intron 12, leading to the insertion of 22 bp and causing a frameshift with a premature stop codon. This analysis enabled the reclassification of the intronic variant to likely pathogenic. This information empowered the couple to make informed reproductive choices and opt for preimplantation genetic testing (PGT) for future pregnancies.

CAPN3 c.1746-20C>G variant is hypomorphic for LGMD R1 calpain 3-related.

The investigated intronic CAPN3 variant NM_000070.3:c.1746-20C>G occurs in the Central and Eastern Europe with a frequency of >1% and there are conflicting interpretations on its pathogenicity. We collected data on 14 patients carrying the CAPN3 c.1746-20C>G variant in trans position with another CAPN3 pathogenic/likely pathogenic variant. The patients compound heterozygous for the CAPN3 c.1746-20C>G variant presented a phenotype consistent with calpainopathy of mild/medium severity. This variant is most frequent in the North/West regions of Russia and may originate from that area. Molecular studies revealed that different splicing isoforms are produced in the muscle. We hypothesize that c.1746-20C>G is a hypomorphic variant with a reduction of RNA and protein expression and only individuals having a higher ratio of abnormal isoforms are affected. Reclassification of the CAPN3 variant c.1746-20C>G from variant with a conflicting interpretation of pathogenicity to hypomorphic variant explains many unidentified cases of limb girdle muscular dystrophy R1 calpain 3-related in Eastern and Central Europe.

Evaluation for Retinal Therapy for RPE65 Variation Assessed in hiPSC Retinal Pigment Epithelial Cells.

Human induced pluripotent stem cells (hiPSCs) generated from patients and the derivative retinal cells enable the investigation of pathological and novel variants in relevant cell populations. Biallelic pathogenic variants in RPE65 cause early-onset severe retinal dystrophy (EOSRD) or Leber congenital amaurosis (LCA). Increasingly, regulatory-approved in vivo RPE65 retinal gene replacement therapy is available for patients with these clinical features, but only if they have biallelic pathological variants and sufficient viable retinal cells. In our cohort of patients, we identified siblings with early-onset severe retinal degeneration where genomic studies revealed compound heterozygous variants in RPE65, one a known pathogenic missense variant and the other a novel synonymous variant of uncertain significance. The synonymous variant was suspected to affect RNA splicing. Since RPE65 is very poorly expressed in all tissues except the retinal pigment epithelium (RPE), we generated hiPSC-derived RPE cells from the parental carrier of the synonymous variant. Sequencing of RNA obtained from hiPSC-RPE cells demonstrated heterozygous skipping of RPE65 exon 2 and the introduction of a premature stop codon in the mRNA. Minigene studies confirmed the splicing aberration. Results from this study led to reclassification of the synonymous variant to a pathogenic variant, providing the affected patients with access to RPE65 gene replacement therapy.

Abstract P2-09-04: Reclassification of variant of unknown significance in BRCA1 and BRCA2 genes based on loss of heterozigosity assay

Abstract Variants of unknown significance (VUS) occur in approximately 10% of BRCA1 and BRCA2 genetic testing. Definition of VUS as pathogenic or of benign potential is a crucial step in the reporting and counseling process. Thus limiting the clinical utility of a genetic test and impairs the correct patient management. BRCA1 and 2 are tumor suppressor genes and loss of heterozygosity (LoH) as a second hit for inactivating wildtype allele is a relatively common event in tumor. Previously, in a multi-institutional effort, AC Camargo Cancer Center (Brazil), Barretos Cancer Hospital (Brazil) and UTMD Anderson Cancer Center (USA) compiled a total of 332 BRCA1/2 VUS supposedly from unrelated carriers (46 of BRCA1 and 286 of BRCA2). From them, 256 were distinct VUS (37 in BRCA1 and 219 in BRCA2). Breast cancer sub-classification were collected from medical records and in silico analyzes were performed using four software (SIFT, Polyphen2, CADD and REVEL). Based on this analysis, VUS were categorized into five classes: very high, high, medium, low and very low risk of pathogenicity, according to agreement level among the four in silico software. However, all these analysis are not sufficient for supporting the reclassification of VUS. Therefore we proposed to analyze LoH in matched tumor tissue to assess the pathogenic potential of BRCA1 and 2 VUS. Normal (leucocytes or normal breast tumor tissue) samples, when available, were used for correcting PCR amplification bias between the two alleles. DNA was extracted and subjected to PCR reaction. Amplicons libraries were performed according to manufacturing protocol (Ion Plus Fragment Library Kit) and sequenced on the Ion Proton™ System (ThermoFisher Scientific). Specific variants were called in the TVC program (ThermoFisher Scientific) with standard criteria. A total of 12 VUS were investigated (5 in BRCA1 and 7 in BRCA2). LoH was detected in 7 (60%) tumor samples (3 in BRCA1 and 4 in BRCA2). Of these, 4 VUS were classified as very high or high risk of pathogenicity [2 in BRCA1 (BR1p.Y1703C; BR1p.S1655P) and 2 in BRCA2 (BR2p.S2670L; BR2p.E3002K)]. Variant of allele frequency (VAF) of 94, 65, 70 and 75%, respectively, were detected in tumor tissues. Additionally, recent functional assays described these 4 VUS as non-functional or deleterious variants showing complete agreement with the current LoH results. According to ACMG guidelines considering all available data, LoH, deleterious function and concordant pathogenicity probability. These 4 VUS might be reclassified as pathogenic variants. On the other hand, the same combined analysis did not allow reclassification of 3 other VUS. Although VAF were 74, 73 and 70% respectively, they were previously classified as very or low risk of pathogenicity and there are no functional assays describing them until now. Our results showed that LoH analyses can substantially contribute to VUS reclassification, improving the management and counseling of BRCA1/2 VUS-carriers. Citation Format: Dirce M Carraro, Edenir I Palmero, Carolina M Berra, Giovana T Torrezan, Rafael C Brianese, Gabriela C Fernandes, Angelica M Gutierrez Barrera, Henrique C Galvo, Maria NC Formiga, Banu K Arun. Reclassification of variant of unknown significance in BRCA1 and BRCA2 genes based on loss of heterozigosity assay [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-09-04.

Revised criteria for diagnosis of NIFTP reveals a better correlation with tumor biological behavior.

The recent reclassification of a follicular variant of papillary thyroid carcinoma (FVPTC), subset as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), aims to avoid overtreatment of patients with an indolent lesion. The diagnosis of NIFTP has recently been revisited using more rigid criteria. This study presents histological and molecular findings and a long clinical follow-up of 94 FVPTC, 40 cases of follicular adenoma (FTA) and 22 cases of follicular carcinoma (FTC) that were classified before the advent of the NIFTP reclassification. All slides were reviewed using these rigid criteria and analysis of numerous sections of paraffin blocks and reclassified as 7 NIFTPs, 2 EFVPTCs, 29 infiltrative FVPTC (IFVPTCs), 57 invasive EFVPTC (I-EFVPTCs), 39 FTAs and 22 FTCs. Remarkably, EFVPTC and NIFTP patients were all free of disease at the end of follow-up and showed no BRAF mutation. Only one NIFTP sample harbored mutations, an NRAS Q61R. PAX8/PPARG fusion was found in I-EFVPTCs and FTC. Although additional studies are needed to identify a specific molecular profile to aid in the diagnosis of lesions with borderline morphological characteristics, we confirmed that the BRAF V600E mutation is an important tool to exclude the diagnosis of NIFTP. We also show that rigorous histopathological criteria should be strongly followed to avoid missing lesions in which more aggressive behavior is present, mainly via the analysis of capsule or vascular invasion and the presence of papillary structures.

Distinguishing non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) from classic and invasive follicular-variant papillary thyroid carcinomas based on cytologic features

An international panel recently recommended reclassification of non-invasive follicular variant of papillary thyroid carcinoma (PTC) to non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). NIFTPs have little or no risk of recurrence and can be treated with lobectomy alone. Preoperative distinction of NIFTP from PTC will help avoid overtreatment. All thyroid tumors with a histologic diagnosis of PTC and preceding diagnostic cytology (n = 299) over a 5-year period were identified. Cases meeting criteria for NIFTP were reclassified as such. All NIFTP cases with available cytology (n = 6) and a similar number of randomly selected invasive follicular variant of papillary thyroid carcinoma (IFVPTC; n = 9) and classic PTC (cPTC, n = 11) were evaluated for 18 cytologic features. A total of 35 (12%) lesions were reclassified as NIFTP, 194 (65%) were cPTC, and 70 (23%) were IFVPTC. The NIFTPs had a preceding cytologic interpretation of benign (31%), atypia of undetermined significance (34%), follicular neoplasm (9%), suspicious for malignancy (12%), or malignant(14%). Cytologically, NIFTP was distinguished from cPTC by absence of any architectural features in all 6 cases, and by absence of pseudoinclusions (P < 0.001) and multinucleated giant cells (P = 0.027) in nearly all. Nuclear pseudoinclusions (P = 0.001), marginal micronucleoli (P = 0.018), irregular branching sheets (P = 0.025), and linear arrangement (P = 0.025) favored IFVPTC over NIFTP. NIFTPs were originally assigned to a variety of cytologic categories. There are several cytologic differences between NIFTP and cPTC or IFVPTC. Our findings support restricting the definitive diagnosis of PTC to cases with architectural features of PTC and/or intranuclear pseudoinclusions.

Evaluating the projected surgical impact of reclassifying noninvasive encapsulated follicular variant of papillary thyroid cancer as noninvasive follicular thyroid neoplasm with papillary-like nuclear features

The reclassification of noninvasive encapsulated follicular variant of papillary thyroid cancer to noninvasive follicular thyroid neoplasm with papillary-like nuclear features will reduce nonefficacious and potentially harmful care. Reclassification is estimated in 18.6% of patients with papillary thyroid carcinoma; we aimed to quantify the implications of this change. Pathology reports from April 2006 to April 2016 were reviewed to isolate cases that would have been designated as neoplasm with papillary-like nuclear features. Of the 1,335 cases of papillary thyroid carcinomas, 194 cases (14.5%) met criteria. Cases in which neoplasm with papillary-like nuclear features was found in combination with other thyroid malignancies (n = 25) and cases of prior thyroid lobectomy (n = 5) were excluded. Demographic, pathologic, treatment, and follow-up data were assessed for the remaining 164 potential neoplasm with papillary-like nuclear features cases. Logistic regression analysis was performed to evaluate association between fine-needle aspiration result and index procedure. Of the 164 patients with tumors who met neoplasm with papillary-like nuclear features criteria, fine-needle aspiration results were nondiagnostic (2%), benign (18%), atypia/follicular lesion of undetermined significance (26%), follicular neoplasm or suspicious for follicular neoplasm (20%), suspicious for malignancy (19%), malignant (6%), and not obtained (9%). Eighty-five (52%) patients underwent total thyroidectomy. A "suspicious for malignancy" fine-needle aspiration result was associated with undergoing total thyroidectomy versus thyroid lobectomy (P = .006). Thyroid lobectomy was the index procedure for 79 patients (48%); of these patients, 54% (n = 43, 3.2% of all patients with papillary thyroid carcinomas) underwent subsequent total thyroidectomy, and 24% received postoperative radioactive iodine treatment. There were no recurrences among the 125 patients with >3 months of follow-up. The reclassification of noninvasive encapsulated follicular variant of papillary thyroid cancer as neoplasm with papillary-like nuclear features will decrease nonefficacious treatment and reduce costs. However, the impact of this change with regard to extent of surgery was limited to 3.2% of patients with papillary thyroid carcinomas compared with the projected potential impact on 18.6%.

A modified reporting approach for thyroid FNA in the NIFTP era: A 1-year institutional experience.

The reclassification of noninvasive follicular variant of papillary thyroid carcinoma as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) has created diagnostic and management issues for thyroid fine-needle aspiration (FNA). In response to these challenges, the authors' laboratory adopted a NIFTP policy including 1) stringent criteria (requiring pseudo-inclusions, papillae, and/or psammoma bodies) for a malignant diagnosis of papillary carcinoma to limit false-positive results due to NIFTP and 2) the use of explanatory notes in cases with cytomorphologic features suggestive of possible NIFTP to encourage lobectomy over thyroidectomy. This study examined the effects of this policy on FNA classification and subsequent surgical management. All thyroid FNAs performed at Brigham and Women's Hospital (n = 1300) during a 1-year period were evaluated for changes in the use of diagnostic categories, explanatory NIFTP notes, and surgical follow-up in comparison with historical controls. The use of specific Bethesda categories did not significantly change. Only a single case of NIFTP was mistakenly classified as malignant. NIFTP was seldom suspected prospectively (17 of 1300; 1.3%); when NIFTP was suspected, cases were reported as suspicious for a follicular neoplasm/follicular neoplasm (n = 10) or suspicious for malignancy (SUS; n = 7). Five of the 7 SUS cases (71%) underwent partial thyroidectomy, compared to 19% of those classified as SUS without an explanatory NIFTP note (P < .02). Thyroid FNA reporting modifications due to NIFTP affect only a small subset of specimens. When NIFTP is suspected, an explanatory note promotes limited surgical excision. More stringent criteria for malignancy affect few cases while potentially limiting false-positive diagnosis due to NIFTP. Cancer Cytopathol 2017;125:854-64. © 2017 American Cancer Society.

Reclassification of Noninvasive Follicular Variant of Papillary Thyroid Carcinoma As a Benign Condition Will Reduce the Incidence of Malignancy in the Indeterminate FNA Categories

Reclassification of Noninvasive Follicular Variant of Papillary Thyroid Carcinoma As a Benign Condition Will Reduce the Incidence of Malignancy in the Indeterminate FNA Categories