W. Todd Penberthy, PhD

Investigators at the Mount Sinai School of Medicine, Toronto, Canada, have been working on determining whether or not PD-L1 expression levels and tissue (sub)localization may correlate with aggressiveness and/or disease-free survival in thyroid cancer. Paul G. Walfish, MD, FRCP presented this research, October 21, 2015, at the 15th Annual International Thyroid Congress, Laek Buena Vista, Florida.

Walfish et al examined PD-L1 expression using formalin-fixed paraffin-embedded tissue sections with antiPD-L1 rabbit polyclonal antibodies. A total of 200 patients with papillary thyroid cancer ranging from benign to aggressive subtypes were analyzed. Two different cytologists independently performed double-blind immunohistochemical scoring on these samples. A score of 1 to 4 was given based on the percentage of cells stained with a value of 4 denoted: >70% of the cells stained. The intensity of staining was tallied with a value ranging from 0 to 3, with 3 represented intense staining. The sum of intensity plus percentage measures was made for all samples. Both cytologists independently observed clear increases in PD-L1 signal in tissues that directly correlated with disease progression.

Coexpression of PD-L1 with thyroid transcription factor-1 proved detection predominately restricted to thyroid tissues, but PD-L1 was also notably detectable in lymphocytes. Accordingly, Walfish cautioned that one must be very careful when interpreting PD-L1 histology if there are signs of Hashimoto&rsquo;s disease or lymphocytic thyroiditis because, even in the benign nodules, there is a high percentage of PD-L1containing cells.

More detailed analysis of PD-L1 subcellular localization also revealed a significantly greater amount of cytosolic PD-L1 in more aggressive thyroid cancers, with little change in nuclear PD-L1 levels. Kaplan Meier analysis of PD-L1 membrane-positive tumor cells revealed significantly shorter median disease free survival (DFS = 9.75 months) compared with PD-L1negative tumor cells (DFS = 166.5 months;

Walfish described high PD-L1 expression associated with PTC aggressiveness and emphasized that this may be useful for identifying patients that will need more aggressive surveillance and therapy. When assessing PD-L1 expression, the coexisting lymphocytic infiltration due to autoimmune thyroid disease can lead to false-positive results.

Walfish stressed that PD-L1 histology suggests a possibility as a prognostic marker of disease aggressiveness, particularly for metastatic disease stages III and IV. Moreover these results justify the administration of antiPD-L1 immunotherapy in patients that are unresponsive to radioiodine or other chemotherapy measures.

In the question and answer that followed, one audience member asked about fine needle aspiration (FNA) biopsy as a means of obtaining tissue for analysis. Walfish elaborated that it would be nice to be able to perform this analysis with FNA biopsy, but it is not so easy because you have to rule out infiltrating lymphocytes. These studies were all done using formalin-fixed paraffin-embedded tissue sections.

Walfish P. G.  Programmed death ligand 1 expression correlates with aggressive metastatic papillary thyroid carcinoma.&nbsp; Presented at the 15th International Thyroid Congress: Lake Buena Vista, Florida; October 21, 2015. Abstract #463.&nbsp;

Articles

Investigators at the Mount Sinai School of Medicine, Toronto, Canada, have been working on a potential correlation for aggressiveness and/or disease-free survival in thyroid cancer patients.

PD-L1 Strongly Correlates With Papillary Thyroid Cancer Aggressiveness

Researchers at the University of Pittsburgh have been working with the hypothesis that well-differentiated thyroid cancers harbor distinct molecular pathological profiles that may be useful as prognostic indicators of future aggressiveness.

Umamaheswar Duvvuri, MD, PhD, assistant professor of otolaryngology specializing in head and neck surgery at the University of Pittsburgh School of Medicine, presented work describing next-generation sequencing (NGS) of locally advanced, well differentiated thyroid cancers (WDTCs) during a session at the 15th International Thyroid Congress. Duvvuri highlighted that the incidence of WDTCs has increased at an alarming rate, and he presented data showing a near doubling of incidence over the past 6 years.

The University of Pittsburgh has put together a series of mutational panels focusing on the major signal-transducing players controlling papillary thyroid carcinoma oncogenes.  This started with a 7-gene panel in 2007, which led to a 15-gene panel by 2013, and then a 56-gene panel in 2014 that employs NGS. Through this process, they have gone from identifying mutations in 70% of the tumors to identifying mutations in over 95% of the tumors, according to Duvvuri. The researchers used various markers including pan-thyroid markers to ensure that they are looking at the correct tissues for their molecular analysis.

mutations are associated with more aggressive cancers, Duvvuri et al have observed that the most aggressive thyroid cancers tend to have multiple mutations including mutations in

. To study this finding, they identified a group of T4 WDTCs that did not have any anaplastic or poorly differentiated areas. All patients were treated with curative intent.

Starting with 80 patients with DTC, the researchers excluded those with tumors that were not well differentiated and those for which insufficient tissue could be harvested. They procured, processed, and analyzed 25 samples from well-differentiated T4 thyroid carcinomas. The mean age was 58 years (29-86), and patients were followed for 5 years.  A total of 85% had

mutation. Only one patient out of 25 had no identifiable mutation, suggesting that most of the pertinent mutations were currently identifiable because 96% (24/25) of patients were identified.

Most interestingly, 50% of the patients had mutations in both

; however, only 4% of T1-3 tumors (5/103) demonstrated

comutations. Therefore, the researchers evaluated a control cohort that looked at 102 patients with early-stage cancer. A total of 45 patients had

. Duvvuri pointed out that this means 5% of early mutations have the

comutation, while advanced cancers have 50%

comutation, which may indicate that this comutation is more distinct in advanced thyroid cancer.

Next Duvvuri et al looked at the impact of these mutations on the likelihood for recurrence or death. They observed a roughly three-fold increase in the recurrence of T4 WDTCs in patients that possessed the

Duvvuri et al have found that advanced well-differentiated thyroid cancers do tend to have a characteristic molecular profile, which includes a high rate of BRAF with

comutations, and these subtypes have a higher recurrence rate. Most advanced WDTCs have a high rate of mutation with a particularly high rate of

comutation.  The University of Pittsburgh group is actively investigating how well this comutation correlates with the more aggressive phenotype.

Duvvuri U. Multi-gene next generation sequencing (thyroseq) assay on locally invasive T4 well differentiated thyroid cancer.  Presented at the 15th International Thyroid Congress: Lake Buena Vista, Florida; October 21, 2015. Abstract #461.

Researchers have posited that well-differentiated thyroid cancers harbor distinct molecular pathological profiles that may be useful as prognostic indicators of future aggressiveness.

Next-Generation Sequencing Suggests BRAF With TERT Comutation Occurs in Advanced Well-differentiated Thyroid Cancer

While medullary thyroid cancer (MTC) is not nearly as common as papillary or follicular thyroid cancer, progressive metastatic MTC prognosis does have a poorer prognosis than these more common forms of metastatic thyroid cancer.

One out of four MTCs are caused by germline mutations in the

gene. These are considered familial MTCs. While sporadic MTCs are not familial, they do most often possess acquired somatic

mutations that are detectable in some cells of sporadic MTC. Two tyrosine kinase inhibitors (TKIs) are currently approved and used for treatment of metastatic MTC. Still, prognosis remains poor for TKI-treated progressive metastatic MTC.

With the continuing development of diagnostic and prognostic molecular technologies, researchers based at the Institute of Pathology, University Hospital Essen, Essen, Germany are characterizing the molecular profile of the progressive metastatic MTC disease state and the desired vandetanib-responsive therapy state. The hope is to identify biomarkers that can be used to parse out patients early in the treatment phase. An early determination of likely drug responsiveness may assist in decisions to avoid unnecessary serious adverse events (AEs) associated with TKIs and inform medical oncologists that another drug should be considered. Alternatively, with a favorable early-response molecular profile, the therapeutic course would be maintained.

Presenting at the 15th annual International Thyroid Congress, University Hospital Essen physician Vera Tiedje, MD, described molecular profiling from 32 patients with MTC at different tumor stages (n = 10 pN0cM0; n = 9 pN1cM0; n=14 pN1p/cM1) with a median age of 56 years and a follow-up time of 42 months. Nine of the patients died over this observation period. Ten of the patients were started on vandetanib treatment due to progressive disease, based on RECIST criteria.

Molecular profiling comparisons were made between the patients&rsquo; own primary vs more progressive tumor tissues. Tiedje et al examined the mRNA expression level of 33 different tyrosine kinase genes using nCounter NanoString. Next-generation sequencing was used to determine the

mutations in 65% (21/32 samples) of patients. The

mutation is the highest risk mutation, and it was also the most common mutation detected, at 57% (8/14) of patients in the pN1p/cM1 MTC arm.

= .042) mRNA expression was significantly lower in MTC of the pN1cM0/pN1pcM1 arm compared with the pN0cM0 arm. By contrast,

= .026) mRNA expression was significantly higher in MTC of the pN1cM0/pN1pcM1 arm compared with the pN0cM0 arm.

Out of 10 patients treated with vandetanib, just 5 showed a partial response. All of these responders had the

=.042) mRNA expression was distinctly higher in primary tumor tissues of patients responding to vandetanib therapy.

Together these are encouraging results, which suggest that molecular profiling may be useful to determine the likelihood of progressive metastatic MTC development. Most significantly, the early determination of whether or not the cancer is responding to vandetanib therapy. Data indicates that the early monitoring of

may offer early hints about whether or not the patient should stay on vandetanib therapy.

Tiedje et al identified distinct characteristic changes in the levels of tyrosine kinases associated with progression from primary tumor to metastatic progression encoding mRNAs for

Of even more immediate practical significance, changes in the level of

were seen in the patients that favorably responded to vandetanib therapy.

Tiedje V. Prognostic markers and response to vandetanib therapy in sporadic medullary thyroid cancer patients. Presented at the 15th International Thyroid Congress: October 21, 2015. Abstract #492.

While medullary thyroid cancer is not common as other thyroid cancers, it does have a poorer prognosis than these more common forms of metastatic thyroid cancer.

Molecular Profiling of Medullary Thyroid Cancer Identifies Prognostic Markers for Vandetanib

is essential to the survival of many tumors. Accordingly,

has been the focus of targeted therapeutics like the BRAF V600E-selective inhibitor, vemurafenib, which has been used for papillary thyroid carcinomas (PTCs).

While BRAF V600E and angiogenesis inhibitors are frequently used, a lack of response has been observed in many human cancer cells. At the 15th International Thyroid Congress, Carmelo Nucera, MD, PhD, assistant professor at Harvard Medical School presented data from his studies that explored how the combination of antiangiogenesis/tyrosine kinase inhibitors (TKIs) may impact known BRAF inhibitors.

Nucera et al used cell cultures of human metastatic/recurrent

-PTCs and tested the effects of vemurafenib (selective inhibitor of

) versus sorafenib (antiangiogenesis/TKI) used as single agents or in combination. To date, they have used predominately three types of assays. This includes biochemical assays of enzyme activity along with two dynamic cellular assays, such as measures of cell-cycle proliferation and in vitro microenvironmental analysis: migration and motility.

-PTCs responded to the BRAF inhibitor vemurafenib more than to the TKI sorafenib. By contrast, if you use wild-type BRAF cells, then the BRAF inhibitor does not work and sorafenib works very well. Ultimately the combination overlaps in activity, according to Nucera.

Nucera states that it is all about tumor heterogeneity, therefore, the tumor microenvironment needs to be considered. Macrophages, for example, are an important component in the anaplastic pericyte microenvironment. We know that macrophages and endothelial cells exist in this microenvironment.

Nucera&rsquo;s group has observed that the pericytes act like stem cells, interacting with endothelial cells to regulate angiogenesis. Pericytes spread after exposure to various signaling molecules like PDGFR-&beta;, according to Nucera. Pericytes have a strong capability to produce an extracellular matrix, which may ultimately confer advantage to PTC cells to promote their migration and invasion.

The researchers observed higher PDGFR-&beta; in

cells than in the wild-type equivalent, but addition of a BRAF inhibitor caused down-regulation of PDGFR-&beta; activation. No response was seen after sorafenib treatment, while the combination belies the treatment of the single sorafenib agent.

Nucera&rsquo;s study preliminarily suggests that the combination of an antiangiogenic compound and a BRAF inhibitor may be more effective than either agent alone, not only in blocking the proliferation of PTC cells, but also in preventing cell types like pericytes in the microenvironment from promoting otherwise reproductive advantages to PTC cells.

Nucera highlighted that these results indicate for the first time that PTC cells express VEGFR2, and this dual combination treatment with anti-BRAF V600E and antiangiogenesis therapy is likely to improve therapeutic response in metastatic BRAF V600E-positive PTC cells by directly reducing cell survival and migratory capacities via pERK1/2 and  pPDGFR-&beta; down-regulation.

Richard Kloos, MD, cochair for the session and professor of medicine at Ohio State University, stated that the value of adding vemurafenib must be that the BRAF activity is not strong enough. Kloos then asked whether or not the sorafenib effect is really a dose effect working through BRAF. Nucera elaborated that the BRAF inhibitor is working on the BRAF system. Therefore, if you use pan tyrosine kinase inhibitor like sorafenib, you can improve the activity to kill any tumor cell, according to Nucera. The rationale for using an antiangiogenic agent is that you have a human system with many different cells with many different TK targets. In Nucera&rsquo;s observations the BRAF inhibition with a pan TKI will fundamentally work on any tumor cell.

Nucera C. Combinatorial targeting with anti-BRAFV600E and anti-angiogenesis therapy enhances cell death and suppresses cell migration in metastatic papillary thyroid carcinoma. Presented at the 15th International Thyroid Congress: Lake Buena Vista, Florida; October 20, 2015. Abstract #106.

BRAF V600E is essential to the survival of many tumors and has been the focus of targeted therapeutics like a new BRAF V600E-selective inhibitor, which has been used for papillary thyroid carcinomas.

Anti-BRAF V600E Plus Antiangiogenesis May Possess Greater Activity Against Metastatic Papillary Thyroid Carcinoma

At the 2015 International Thyroid Congress, Keith Bible, MD, PhD, Mayo Clinic professor of oncology described the current status of using VEGFR tyrosine kinase inhibitors to treat thyroid cancer:

Most tyrosine kinase inhibitors are promiscuous, having a footprint across the kinome that is very messy and seems to have activity for thyroid cancer, but I think we are still struggling to know how that footprint varies. It is striking to me that we can often go from one kinase inhibitor to the next and get sequential responses, when in theory they are all at least primarily targeting VEGFR. So I think we have a lot to learn about off-target effects and what is really most important in these agents and what is most beneficial in these patients.

Investigators are attempting to identify markers that can help with early prediction of outcomes within short time frames of treatment initiation, &ldquo;so as not to have to continue treatment for a longer period of time,&rdquo; according to Bible. On behalf of the Mayo Phase II Consortium, Bible presented data that focused on the primary endpoint of changes in serum thyroglobulin (Tg) post-initiation of pazopanib treatment to determine whether or not Tg changes may be used as an early (&le;4 weeks) predictor of treatment-mediated success for patients with progressive, metastatic radioactive iodine-refractory differentiated thyroid cancer (RAIR-DTC). Analysis of Response Evaluation Criteria In Solid Tumors (RECIST) response was analyzed as a secondary endpoint.

Sixty patients with RAIR-DTC under the Mayo Clinic Phase II Consortium from across the USA, Asia, and Australia were treated with pazopanib between May 2009 and December 31, 2011. Three of these patients went off of the study without tumor burden re-evaluation. Bible pointed out that hypertension grade 3 and above was the most common adverse event (AE) of pazopanib treatment for patients with RAIR-DTC, with 53% of these patients requiring additional therapy. Other side effects listed in order from most common to least frequent included: fatigue (n=8), decreased neutrophil count (n=8), diarrhea (n=7), hand-foot syndrome (n=7), and increased AST/ALT liver enzymes (n=6).  Overall, the AEs were consistent with expectation. Bible emphasized, most favorably no deaths were attributed to pazopanib therapy.

In the final analysis, changes in Tg did not serve as a predictor of outcome, but rather there was only a coincidental decrease that correlated with general treatment, but not favorable outcome. Twenty two (37%; 95% CI: 24.6-50.1%) patients had partial RECIST responses. This outcome result was considered by Bible to be &ldquo;a high level.&rdquo; Of the sixty patients that were treated and evaluated, over 90% had previously received systemic pazopanib therapy in addition to radioiodine. Prior pazopanib treatment did not correlate with treatment success either. Mutations in

were identified in 11 of 16 patients examined.  However, none of these mutations would correlate with treatment response.

Bible stated that we have not been able to confirm that induced changes in thyroglobulin can be a robust predictor of response to pazopanib therapy. Moreover, neither mutational analysis nor prior treatment served as a predictor of future success either. Nonetheless, pazopanib was confirmed to have significant clinical activity in progressive, RAI-refractory DTC. Accordingly, investigators continue to work toward identifying, on an empirical level, which markers can serve as a useful indicator of whether or not a given VEGFR tyrosine kinase inhibitor working in a particular individual is likely to provide effective therapeutic benefit.

Bible KC. Pazopanib in patients with progressive, radioactive iodine-refractory, metastatic differentiated thyroid cancer lacking thyroglobulin antibodies: Mayo Phase 2 Consortium Study Results. Presented at the 2015 International Thyroid Congress: October 21, 2015. Abstract #103.

Keith Bible, MD, PhD, Mayo Clinic professor of oncology goes into detail about the current status of using VEGFR tyrosine kinase inhibitors to treat thyroid cancer.

Pazopanib Treatment Outcome Not Predicted by Thyroglobulin Status

At the 2015 International Thyroid Conference, Nicole O. Vietor, MD of the Walter Reed National Military Medical Center, Bethesda, presented a report assessing practice discordances related to previously published guidelines published from the American Thyroid Association (ATA; 2009, 2015) and the American Association of Clinical Endocrinologists (AACE; 2010).

To perform the study, Vietor et al collected survey data from 897 respondents from the ATA, AACE, and The Endocrine Society (TES).  Most of the respondents were from North America (63%) followed by Europe (12%), Asia (6.5%), the Middle East/Africa (5.6%), and Oceania (1.9%).&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;

Vietor et al presented an index case describing a 52-year-old with no previous history of thyroid disease, smoking, or poor health.  A 1.5-cm thyroid nodule was detected. Which test would respondents perform? Consistent with current guidelines 99% of respondents indicated that they would do a TSH test. However, there were big demographic differences for whether or not to do a calcitonin test for any nodule. Roughly 32% of European respondents would do the calcitonin test, while just 2.3% of respondents from North America would do a calcitonin test. Asia, Latin America, and the Middle East ranged in between these values, with 14%, 9%, and 6% respectively. Concurrent with current guidelines, a little over 50% responded that they would perform a thyroid ultrasound.&nbsp;

Investigators examined the methods and procedures of performing fine needle aspirations (FNAs).  FNAs were performed mostly by endocrinologists (57%), but also radiologists performed FNAs frequently (32%) while pathologists (6.3%) performed the procedure in some cases. Ultrasound was used for 83% of FNAs. A 25-gauge needle was selected most commonly (44%) followed by 27 gauge (15%). Three passes was recorded as the most common (28%) number of passes performed, followed by four passes (23%).&nbsp; Approximately 33% of respondents did not use any pain reduction methods for the FNA procedure, while some physicians used topical gel (9%), ice pack (8%), or ethyl chloride spray (8%) to decrease pain. Interpretation of FNA results was categorically performed by cytopathologists (73%) more than any other specialist. Assessment of FNA was also frequently performed by a pathologist (18%), a commercial laboratory (8%), or in rare cases, an endocrinologist (1%).&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;

Survey participants were asked whether or not they would perform an FNA on the following different nodules: (1) 1.5-cm hypoechoic nodule, (2) 0.7-cm hypoechoic nodule with calcifications, (3) 1.4-cm complex-cystic nodule, (4) 1.2-cm isoechoic nodule, (5) 1.8-cm spongiform nodule, or 6) a 2.5-cm purely cystic nodule.  Participants responded in kind, with 94%, 67%, 55%, 42%, 37%, and 24% respectively, stating that they would perform FNA for the respective nodule criteria 1 through 6. The guidelines however, only recommend FNA for the first condition, 1.5-cm echoic nodule.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;

To address the geriatric factor, respondents were asked whether or not they would reconsider performing the FNA procedure on an 82-year-old person with otherwise similar criteria involving no previous history of thyroid disease, but a 1.5-cm thyroid nodule. Approximately 64% would no longer perform the FNA given the advanced age.  Respondents were then asked about pregnancy issues, for example, whether or not physicians would reconsider performing FNA in a 22-year-old woman at 8 weeks gestation with a 1.5-cm nodule.&nbsp; Vietor said, &ldquo;Results were split, with 34% stating that they would postpone the procedure and 37% stating they would perform the procedure now.&rdquo;

&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;

Molecular testing is increasingly incorporated into practices. For follicular lesions of undetermined significance, the majority of respondents (38%) would use molecular analysis, followed by repeated cytology (20%) and lobectomy (20%). For a follicular neoplasm, about 47% would perform a lobectomy with 29% performing molecular analysis and 15% performing a total thyroidectomy. For a suspicious malignancy the results were &ldquo;much more split,&rdquo; according to Vietor, with 43% performing total thyroidectomy and another 43% performing lobectomy. Roughly 9.55% would perform molecular analysis. Examination of international molecular testing reveals that the United States performs the most molecular tests in the world (74% of respondents).This was followed by Europe, where 43% perform molecular analysis.  &nbsp;

This was the first study documenting current management of thyroid nodules. A high level of variation in the use of diagnostic tests and treatment procedures was observed, particularly, FNA procedural application was discordant with recommended guidelines.  More FNA procedures are currently being performed than are currently recommended by clinical guidelines. Wide variation in the management of pregnant females and/or FNAs on follicular lesions of undetermined significance, follicular neoplasms, or suspicious malignancies was also observed. Of potential significance, international differences in the use of calcitonin and molecular testing were discovered.&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;

Dr. Nicole O. Vietor presented a report assessing practice discordances related to previously published guidelines published from the American Thyroid Association and the American Association of Clinical Endocrinologists.