Molecular Features Of Hvptc Compared With Ptc And Atc/pdtc
Figure 3: The association of DNA quality and total mutational load among all samples. Violin plot in left panel show the number of selected mutations and selected low AF mutations between high-quality and low-quality samples. Boxplot in right panel show the insert size and library complexity between high-quality and low-quality samples. Correlation between sample collection date and insert size, library complexity, number of mutations, number of low AF mutations illustrated by scatterplot.
All mutations identified were summarized in Figure 4A where samples were grouped by subtype, patient, and quality group. Mutations were color-coded by their mutation allele frequency. Mutation status was further combined onto gene level and patient level in Figure 4B. Thirty-seven mutation variant types were identified among 11 genes across all samples. All details about mutations identified can be found in Supplementary Table 3. BRAF V600E mutation was the most common mutation identified, which is present in almost all HVPTC samples , most CPTC samples , and none of the ATC and PDTC samples. HVPTC harbored an even higher BRAF V600E mutation rate than CPTC, although the difference was not statistically significant.
Figure 4: Mutational profiling of HVPTC compared with PTC and ATC/PDTC. Heatmap of mutations for all samples grouped by subtype and data quality. Representation of the mutation status by Gene-level for all samples grouped by subtype.
Clinical And Pathologic Characteristics
Demographic information and clinical data were obtained from the electronic medical record. Surgical pathology specimens were reviewed for tumor size and lymph node involvement, as well as the presence of multifocality, lymphovascular invasion, necrosis, and extrathyroidal extension. If multiple foci of tumor were present, the largest tumor dimension was noted. Tumor staging was based on the AJCC 7th edition TNM staging .
Papillary Thyroid Carcinoma With Focal Hobnail Features
Medical Radiological Research Center, Obninsk, Kaluga Region, Russia.
Background: Papillary thyroid carcinoma , the most common endocrine malignancy, is a tumor of indolent biological and clinical behaviour that usually has a good prognosis. Only a small percentage of patients are affected by aggressive variants of PTC: tall, columnar cell and diffuse sclerosing variants. These aggressive tumor subtypes are characterized by higher rate of local recurrence, regional and distant metastases than classic papillary PTC. Recently another clinically aggressive variant of PTC with hobnail features has been described. However prognostic significance of such focal hobnail component in classic papillary PTC has not been defined.
Conclusion: PTC with focal hobnail component is characterized by the high frequency of regional metastases at the moment of primary tumor manifestation. Obviously that prevalence of BRAFT1799A point mutation in PTC with focal hobnail features is similar as in classic papillary PTC without hobnail component.
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Braf And Snapshot Mutational Analysis
All available tumors were assessed for the BRAFV600E mutation, which leads to valine being substituted for by glutamate at codon 600. In 9 out of 10 patients in which BRAFV600E testing was performed, genotyping analysis was carried out using the SNaPshot platform as previously described . In two patients, BRAFV600E testing was ordered clinically at the discretion of the surgeon. One of those cases was tested at our institution using the SNaPshot platform, and the other tumor was tested at an outside facility for the BRAFV600E mutation. In brief, total nucleic acid was isolated from formalin-fixed paraffin-embedded tissue and subject to multiplex PCR followed by a single nucleotide primer extension step that generates allele-specific fluorescently labeled probes . The SNaPshot tumor genotyping assay was developed at the MGH Translational Research Laboratory for clinical tumor genotyping. It can detect low-level somatic mutations in TNA extracted from archival material and was designed to query 68 hotspot loci in 14 genes commonly mutated in cancer .
Dna Isolation And Library Preparation
DNA of FFPE samples was extracted and the DNA concentration was measured by QubitdsDNA assay. The gDNA quality was assessed to make sure that A260/A280 is within the range of 1.8 to 2.0. Shearing fragmentation by sonication was conducted, followed by end repair, phosphorylation and adaptor ligation. Fragments of size 200-400bp were selected by bead , followed by hybridization with the capture probes baits, hybrid selection with magnetic beads, and PCR amplification. A bioanalyzer high sensitivity DNA assay was then performed to assess the quality and size of the fragments. All indexed samples were then sequenced on NextSeq500 with pair-end reads.
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Treatment And Patients Outcome
All patients underwent total thyroidectomy, and lymph node dissection was performed on 47 of them . According to the latest ATA risk stratification system , 17 patients were low risk , 68 were intermediate risk , and 11 were high risk . In three cases, there were no sufficient data to determine the ATA risk category. As expected, PTC with hobnail features were associated with ATA low risk since patients with HVPTC cannot be classified as low risk . For a subset of patients , follow-up data were available including 16 HVPTC and 23 PTC with hobnail features. The median follow-up was 1 year after surgery. Thirty-three patients received radioiodine ablation, and three of them also underwent external beam radiation therapy. No differences between HVPTC and PTC with hobnail features were observed in terms of rate of lymphadenectomy and radioiodine ablation . Among patients with follow-up data, 19 had excellent response to therapy , eight had structural incomplete response, and 12 had indeterminate response . Structural persistence was associated with HVPTC patients , while no differences were observed when considering structural or biochemical persistence together .
Table 2 Multivariate logistic regression model to identify predictors of structural persistence.
Demographics And Clinical Characteristics
Twelve patients met our inclusion criteria, constituting 1% of PTC at our institution during the same time period . Nine out of 12 patients were female, with a mean age of 54.1±18.8 years . Seven out of 12 patients were Stage III or IV at presentation. Eleven patients were treated with total thyroidectomy and one with lobectomy. Ten out of 12 patients had radioactive iodine .
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Study Selection And Eligibility Criteria
Studies examining the prognostic significance of the HVPTC were included. Specific inclusion criteria included examination of HVPTC with defined diagnostic criteria patient demographic data including age and sex and pathological tumor data. We elected to examine all HVPTC cases with any proportion of tumor with hobnail features, as Asioli et al. demonstrated even those HVPTCs with < 30% hobnail features demonstrate a worse pattern of disease. Exclusion criteria include: non-human studies reviews non-English language incomplete or non-extractable data and data represented in another study. Two reviewers independently assessed titles and abstracts of all studies to screen for articles satisfying the inclusion criteria. Articles not satisfying inclusion criteria were then excluded. Full-text assessments were then performed to ensure satisfaction with inclusion criteria, and any discrepancies were then reviewed by a third author for determination.
Critical Appraisal And Quality Assessment
Articles were critically appraised to assess the level of evidence using the Oxford Center for Evidence-Based Medicine criteria . The risk of publication bias was assessed according to the Cochrane Handbook for Systematic Reviews of Interventions 5.1.0 . Two authors performed a pilot assessment on three studies to check for consistency of assessment. Both then performed independent risk assessment on the remaining studies. All disagreements were resolved by the way of discussion with a third author . Risk of bias items included the following: bias due to confounding, selection of participants into the study, classification of interventions, deviations from intended interventions, missing data, measurement of outcomes, and selection of the reported result. The risk of bias for each aspect is graded as low, unclear, or high. .
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Quality Assessment Of The Targeted Sequencing Data
Deep sequencing with the 18-gene panel was performed to achieve an average of 1947×coverage among all 73samples profiled. An average of 97.3% of all reads was successfully mapped to the human genome and 55.3% of all reads were mapped to our designed target regions , indicating a high capture efficiency of the designed probes. Figure 2B shows the coverage depth distribution in each sample, where all73 samples have a median coverage depth of more than 500×. Such high coverage enables us to assess mutations present in only a small portion of the tumor cells.
Figure 2: Quality assessment of the targeted sequencing data among 75 samples. Histogram show the percentage of all mapped reads and percentage of reads mapped to target regions for every sample. Bar plot of The median coverage depth among all the target regions for each sample.
Clinicalpathological And Molecular Features
From 2015 to 2020, a total of 9,162 PTC were diagnosed at our institution. Among them, 99 PTC had hobnail areas and were included in the study . Thirty-four PTC with hobnail areas met the criteria of HVPTC , while 65 cases were PTC with hobnail areas between 5% and 25%, including 45 classic variants , 15 tall cell variants , 4 solid-trabecular variants , 1 clear cell variant .
Figure 1 Histological features of hobnail variant papillary thyroid carcinoma hematoxylin and eosin staining. A case of HVPTC: low magnification intermixed papillary structures lined by neoplastic cells 40× magnification of papillary structures. Hobnail cells are evident details of nuclear atypia in hobnail-shaped cells a case of papillary thyroid carcinoma with hobnail features .
Overall, the mean age was 49.8 years, the median tumor size was 1.8 cm , and the female-to-male ratio was 1.4. Only 3 PTC were encapsulated non-invasive. On the contrary, aggressive features were often present, including 40.4% of extra-thyroidal extension , 49.5% of vascular invasion, and 32.3% of lymph node metastases . BRAF mutation was present in 69 out of 88 analyzed cases. Six tumors harbored a RET/PTC rearrangement, including 4 RET/PTC1 and 2 RET/PTC3. TERT promoter mutation was successfully tested in 53 cases, and 8 of them presented the C228T mutation.
Detailed clinicalpathological and mutational data are reported in Table 1.
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Mutational Analysis And Ret/ptc Rearrangements
Tumor was available in 10 out of 12 patients for mutational testing. One case had conventional BRAF testing, and nine tumors were subject to SNaPshot analysis. Eight out of 10 tumors tested had the BRAFV600E mutation, including all three patients without coexistent TCF that were tested . The mean percentage of BRAFV600E alleles was 25.3%, ranging from 19% to 31%. Other than identifying BRAFV600E in the eight patients undergoing SNaPshot analysis, there were no other mutations identified in the additional 13 genes studied. Eight out of 12 patients were tested for RET/PTC1 and RET/PTC3 rearrangements, and the two tumors that were BRAF wild type were both found to be positive for RET/PTC1 .
Targeted Dna Panel Design
The capture probe baits were designed to cover 140kb human genomic loci from 186 target regions, including selected exons and introns from 18 genes , so that we were able to detect all point mutation, indel, and CNV events as well as fusion events of important genes with any partner. The SureSelect reagents were prepared using the Agilent eArray platform and the probes were manufactured by Agilent.