Richard B. Lanman

A Large Multicenter Correlation Study of Thyroid Nodule Cytopathology and Histopathology

BACKGROUND Fine-needle aspiration (FNA) biopsies are the cornerstone of preoperative evaluation of thyroid nodules, but FNA diagnostic performance has varied across different studies. In the course of collecting thyroid FNA specimens for the development of a molecular diagnostic test, local cytology and both local and expert panel surgical pathology results were reviewed. METHODS Prospective FNAs were collected at 21 clinical sites. Banked FNAs were collected from two academic centers. Cytology and corresponding local and expert panel surgical pathology results were compared to each other and to a meta-review of 11 recently published U.S.-based thyroid FNA studies. RESULTS FNA diagnostic performance was comparable between the study specimens and the meta-review. Histopathology malignancy rates for prospective clinic FNAs were 34% for cytology indeterminate cases and 98% for cytology malignant cases, comparable to the figures found in the meta-review (34% and 97%, respectively). However, histopathology malignancy rates were higher for cytology benign cases in the prospective clinic FNA subcohort (11%) than in the meta-review (6%, with meta-review rates of 10% at community sites and 2% at academic centers, p < 0.0001). Resection rates for prospective clinic FNAs were also comparable to the meta-review for both cytology indeterminate cases (62% vs. 59%, respectively) and cytology malignant cases (82% vs. 81%, respectively). Surgical pathology categorical disagreement (benign vs. malignant diagnosis) was higher between local pathology and a consensus of the two expert panelists (11%) than between the two expert panelists both pre- (8%) and postconferral (3%). CONCLUSIONS Although recent guidelines for FNA biopsy and interpretation have been published, the rates of false-positive and false-negative results remain a challenge. Two-thirds of cytology indeterminate cases were benign postoperatively and may decrease with the development of an accurate molecular diagnostic test. High disagreement rates between local and expert panel histopathology diagnosis suggests that central review for surgical diagnoses should be used when developing diagnostic tests based on resected thyroid specimens.

Molecular Classification of Thyroid Nodules Using High-Dimensionality Genomic Data

OBJECTIVE We set out to develop a molecular test that distinguishes benign and malignant thyroid nodules using fine-needle aspirates (FNA). DESIGN We used mRNA expression analysis to measure more than 247,186 transcripts in 315 thyroid nodules, comprising multiple subtypes. The data set consisted of 178 retrospective surgical tissues and 137 prospectively collected FNA samples. Two classifiers were trained separately on surgical tissues and FNAs. The performance was evaluated using an independent set of 48 prospective FNA samples, which included 50% with indeterminate cytopathology. RESULTS Performance of the tissue-trained classifier was markedly lower in FNAs than in tissue. Exploratory analysis pointed to differences in cellular heterogeneity between tissues and FNAs as the likely cause. The classifier trained on FNA samples resulted in increased performance, estimated using both 30-fold cross-validation and an independent test set. On the test set, negative predictive value and specificity were estimated to be 96 and 84%, respectively, suggesting clinical utility in the management of patients considering surgery. Using in silico and in vitro mixing experiments, we demonstrated that even in the presence of 80% dilution with benign background, the classifier can correctly recognize malignancy in the majority of FNA samples. CONCLUSIONS The FNA-trained classifier was able to classify an independent set of FNAs in which substantial RNA degradation had occurred and in the presence of blood. High tolerance to dilution makes the classifier useful in routine clinical settings where sampling error may be a concern. An ongoing multicenter clinical trial will allow us to validate molecular test performance on a larger independent test set of prospectively collected thyroid FNAs.

Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA.

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

A prospective assessment defining the limitations of thyroid nodule pathologic evaluation

BACKGROUND Clinical management of thyroid neoplasms is based on light microscopic diagnosis, but its accuracy and precision are poorly defined. OBJECTIVE To assess inter- and intraobserver variability of preoperative cytopathologic and postoperative histopathologic thyroid diagnoses. DESIGN Samples were collected in a prospective, multicenter trial validating a gene expression classifier between June 2009 and December 2010. SETTING 14 academic and 35 community clinical sites. PATIENTS 653 patients with 776 surgically resected thyroid nodules of 1 cm or greater. MEASUREMENTS Intraobserver concordance among 2 or more central histopathologists who independently read histopathology slides was calculated. Interobserver concordance between the diagnoses made by the central histopathologists and those made by local pathologists were calculated. Intra- and interobserver concordance for cytopathology was similarly calculated by comparing diagnoses made by local pathologists with those made by a central panel of 3 cytopathologists. RESULTS Concordance on the histopathologic distinction between benign and malignant diagnoses was 91% comparing local with central histopathologists and 90% comparing 2 central histopathologists. Using the 6-category Bethesda System, 64.0% of diagnoses made by local and central cytopathologists and 74.7% of intraobserver diagnoses were concordant. Central cytopathologists made fewer indeterminate diagnoses than local pathologists (41.2% vs. 55.0%). LIMITATIONS Many local pathologists did not use the Bethesda System, so their reports were translated to allow comparison. The study required histopathology, and the study population and specimens did not encompass all newly evaluated patients with a thyroid nodule. CONCLUSION Substantial inter- and intraobserver variability exists in the cytopathologic and histopathologic evaluation of thyroid nodules, confirming an inherent limitation of visual microscopic diagnosis. PRIMARY FUNDING SOURCE Veracyte.

The Impact of Benign Gene Expression Classifier Test Results on the Endocrinologist–Patient Decision to Operate on Patients with Thyroid Nodules with Indeterminate Fine-Needle Aspiration Cytopathology

BACKGROUND Seventy-five percent of thyroid nodules with indeterminate fine-needle aspiration (FNA) cytology are found to be benign postoperatively. A novel genomic test, the Afirma gene expression classifier (AGEC), has been available for clinical use in the United States, since late 2010. In 2010, two modest-sized validation studies showed that the AGEC could identify a benign gene expression signature in indeterminate cytology thyroid FNA samples with a negative predictive value >95%. The objective of this study was to evaluate how the AGEC impacted the joint decision of the endocrinologist and patient to operate when FNA cytology was indeterminate, but the AGEC reading of the nodule was benign. METHODS In this cross-sectional cohort study, data were contributed retrospectively by 51 endocrinologists at 21 practice sites that had previously obtained ≥3 benign AGEC readings in ≥1 cm nodules with indeterminate FNA cytology readings. Information regarding demographic data, nodule size and location, decision to operate, surgery type (hemithyroidectomy [HT] or total thyroidectomy [TT]), and reason for recommending surgery was retrospectively collected. RESULTS Compared to a 74% previous historical rate of surgery for cytologically indeterminate nodules, the operative rate fell to 7.6% during the period that AGEC were obtained in the clinical practices, a highly significant reduction in the decision to operate (p<0.001). The rate of surgery on cytologically indeterminate nodules that were benign by the AGEC reading did not differ from the historically reported rate of operation on cytologically benign nodules (p=0.41). The four primary reasons reported by the physicians for operating on nodules with a benign AGEC reading, in descending order: large nodule size (46.4%), symptomatic nodules (25.0%), rapidly growing nodules (10.7%), or a second suspicious or malignant nodule in the same patient (10.7%). These reasons are concordant with those typically given for operation on cytologically benign nodules. CONCLUSIONS In a substantial group of medical practices, obtaining an AGEC test in patients with cytologically indeterminate nodules was associated with a striking reduction in the rate of diagnostic thyroidectomy. Approximately, one surgery was avoided for every two AGEC tests run on thyroid FNAs with indeterminate cytology.

Increases in thyroid nodule fine-needle aspirations, operations, and diagnoses of thyroid cancer in the United States.

BACKGROUND To provide population-based estimates of trends in thyroid nodule fine-needle aspirations (FNA) and operative volumes, we used multiple claims databases to quantify rates of these procedures and their association with the increasing incidence of thyroid cancer in the United States. METHOD Private and public insurance claims databases were used to estimate procedure volumes from 2006 to 2011. Rates of FNA and thyroid operations related to thyroid nodules were defined by CPT4 codes associated with International Classification of Diseases, Ninth Revision Clinical Modification codes for nontoxic uni- or multinodular goiter and thyroid neoplasms. RESULTS Use of thyroid FNA more than doubled during the 5-year study period (16% annual growth). The number of thyroid operations performed for thyroid nodules increased by 31%. Total thyroidectomies increased by 12% per year, whereas lobectomies increased only 1% per year. In 2011, total thyroidectomies accounted for more than half (56%) of the operations for thyroid neoplasms in the United States. Thyroid operations became increasingly (62%) outpatient procedures. CONCLUSION Thyroid FNA and operative procedures have increased rapidly in the United States, with an associated increase in the incidence of thyroid cancer. The more substantial increase in number of total versus partial thyroid resections suggests that patients undergoing thyroid operation are perceived to have a greater risk of cancer as determined by preoperative assessments, but this trend could also increase detection of incidental microcarcinomas.

Nivolumab for previously treated unresectable metastatic anal cancer (NCI9673): a multicentre, single-arm, phase 2 study

BACKGROUND Squamous cell carcinoma of the anal canal (SCCA) is a rare malignancy associated with infection by human papillomavirus (HPV). No consensus treatment approach exists for the treatment of metastatic disease. Because intratumoral HPV oncoproteins upregulate immune checkpoint proteins such as PD-1 to evade immune-mediated cytotoxicity, we did a trial of the anti-PD-1 antibody nivolumab for patients with metastatic SCCA. METHODS We did this single-arm, multicentre, phase 2 trial at ten academic centres in the USA. We enrolled patients with treatment-refractory metastatic SCCA, who were given nivolumab every 2 weeks (3 mg/kg). The primary endpoint was response according to Response Evaluation Criteria in Solid Tumors, version 1.1, in the intention-to-treat population. At the time of data cutoff, the study was ongoing, with patients continuing to receive treatment. The study is registered with ClinicalTrials.gov, number NCT02314169. RESULTS We screened 39 patients, of whom 37 were enrolled and received at least one dose of nivolumab. Among the 37 patients, nine (24% [95% CI 15-33]) had responses. There were two complete responses and seven partial responses. Grade 3 adverse events were anaemia (n=2), fatigue (n=1), rash (n=1), and hypothyroidism (n=1). No serious adverse events were reported. INTERPRETATION To our knowledge, this is the first completed phase 2 trial of immunotherapy for SCCA. Nivolumab is well tolerated and effective as a monotherapy for patients with metastatic SCCA. Immune checkpoint blockade appears to be a promising approach for patients with this orphan disease. FUNDING National Cancer Institute/Cancer Therapy Evaluation Program, the HPV and Anal Cancer Foundation, the E B Anal Cancer Fund, The University of Texas MD Anderson Moon Shots Program, and an anonymous philanthropic donor.

Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients

Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.

Analytical Performance Verification of a Molecular Diagnostic for Cytology-Indeterminate Thyroid Nodules

OBJECTIVE Our objective was to verify the analytical performance of the Afirma gene expression classifier (GEC) in the classification of cytologically indeterminate thyroid nodule fine-needle aspirates (FNAs). DESIGN Analytical performance studies were designed to characterize the stability of RNA in FNAs during collection and shipment, analytical sensitivity as applied to input RNA concentration and malignant/benign FNA mixtures, analytical specificity (i.e. potentially interfering substances) as tested on blood and genomic DNA, and assay performance studies including intra-nodule, intraassay, inter-assay, and inter-laboratory reproducibility. RESULTS RNA content within FNAs preserved in FNAProtect is stable for up to 6 d at room temperature with no changes in RNA yield (P = 0.58) or quality (P = 0.56). FNA storage and shipping temperatures were found to have no significant effect on GEC scores (P = 0.55) or calls (100% concordance). Analytical sensitivity studies demonstrated tolerance to variation in RNA input (5-25 ng) and to the dilution of malignant FNA material down to 20%. Analytical specificity studies using malignant samples mixed with blood (up to 83%) and genomic DNA (up to 30%) demonstrated negligible assay interference with respect to false-negative calls, although benign FNA samples mixed with relatively high proportions of blood demonstrated a potential for false-positive calls. The test is reproducible from extraction through GEC result, including variation across operators, runs, reagent lots, and laboratories (sd of 0.158 for scores on a >6 unit scale). CONCLUSIONS Analytical sensitivity, analytical specificity, robustness, and quality control of the GEC were successfully verified, indicating its suitability for clinical use.

Does Addition of BRAF V600E Mutation Testing Modify Sensitivity or Specificity of the Afirma Gene Expression Classifier in Cytologically Indeterminate Thyroid Nodules

OBJECTIVE The purpose of this study was to determine the frequency of BRAF mutation in cytologically indeterminate thyroid nodules and to investigate whether adding the BRAF test improves diagnostic accuracy of the Afirma Gene Expression Classifier (GEC). DESIGN BRAF V600E mutational status was determined for DNA extracted from cytologically benign (n = 40), indeterminate (n = 208), and malignant (n = 48) fine-needle aspiration specimens previously categorized by GEC as molecularly Benign or Suspicious. Analytical performance of the BRAF assay was assessed to establish reproducibility and limits of detection. Molecular testing results were correlated with blinded expert histopathological diagnoses. RESULTS The BRAF assay detected mutations reproducibly to 2.5% mutant allele frequency. The prevalence of BRAF mutations in cytologically benign specimens was 2 of 40 (5.0%, 95% confidence interval [CI], 0-16) and in cytologically malignant specimens was 36 of 48 (75.0%, 95% CI, 60-86). In the cytologically indeterminate category, 10.1% of specimens were BRAF+: 2 of 95 were subcategorized as atypia of undetermined significance or follicular lesion of undetermined significance (2.1%, 95% CI, 0-7); 1 of 70 as follicular neoplasm or suspicious for follicular neoplasm (1.4%, 95% CI, 0-9); and 18 of 43 as suspicious for malignancy (41.9%, 95% CI, 27-58). All BRAF+ specimens were classified as Suspicious by the GEC. CONCLUSIONS BRAF mutations are uncommon in nodules with atypia of undetermined significance or follicular lesion of undetermined significance or follicular neoplasm or suspicious for follicular neoplasm cytology. Most cytologically indeterminate nodules that proved to be malignant were also BRAF-, and all nodules that were false-negative by GEC were also BRAF-. Similarly, all BRAF+ specimens were also GEC Suspicious. Neither GEC test sensitivity nor specificity was improved by addition of BRAF mutation testing.