Recent approval of immune checkpoint inhibitors for the treatment of melanoma and lung cancer has generated a new excitement in the field of cancer therapeutics. PD-1/ PD-L1 pathway is an important regulator of immune tolerance in the tumor microenvironment. Pembrolizumab is highly selective, humanized monoclonal IgG4-kappa antibody against PD-1 receptor that promotes anti-tumor immune response by preventing interaction of PD-1 with its ligands PD-L1 and PD-L2. The FDA granted accelerated approval for Keytruda (pembrolizumab) in October 2015 to treat patients with advanced nonsmall-cell lung cancer (NSCLC) whose disease has progressed after other treatments and with tumors that express PD-L1 as determined by a companion diagnostic, the PD-L1 IHC 22C3 pharmDx test. This approval was based on the demonstration of durable objective response rate and acceptable safety in the NSCLC subgroup of a large phase I clinical trial (Keynote-001). Subsequently, the Keynote-010 study also demonstrated overall survival benefit in a randomized study comparing pembrolizumab to docetaxel in PD-L1 positive previously treated advanced NSCLC. In this article, we review the completed trials and ongoing clinical development of pembrolizumab in NSCLC.

Lung cancer is the leading cause of cancer death world-wide.

An estimated 221,200 new cases of lung cancer were expected to be diagnosed in the United States in 2015 with 158,040 deaths estimated.

There have been advances in treatment of the subset of patients with driver mutations such as epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) rearrangement. Despite these advances, the 5-year survival for patients with stage IV (metastatic) disease remains dismal. Platinum doublet therapy continues to be the standard first-line treatment for advanced nonsmall-cell lung cancer (NSCLC) patients without driver mutations.

In 2015, immune checkpoint inhibitors were added to the armamentarium for treatment of NSCLC.

Cancer cells multiply by evading immune response. One such mechanism of interest is upregulation of pro- grammed cell death protein ligands (PDL-1 and PDL-2) on tumor cells and tumor-invading inflammatory cells that bind to PD-1 receptors on cytotoxic T lymphocytes causing down regulation of the immune response.

Pembrolizumab (Keytruda) is a highly selective, humanized monoclonal IgG4-kappa isotype antibody against PD-1. It was developed by grafting a high affinity mouse antihuman PD-1 antibody into a human IgG4 with a stabilizing S228 P Fc alteration. It disrupts the binding of PD-1 with its ligands (PDL-1 and PDL-2) causing recognition of tumor cells by cytotoxic T cells.

Keynote-001: Phase I Trial in Advanced Treatment Refractory NSCLC

Keynote-001 trial is a landmark phase I trial that was granted breakthrough therapy designation in October 2014 and accelerated FDA approval in October 2015 for pembrolizumab as a second line treatment for patients with NSCLC.

It is approved after progression of disease on or after platinum-based chemotherapy in NSCLC (EGFR receptor mutation negative and no ALK rearrangement) whose tumors exhibit high-level PD-L1 expression (greater than 50% based on 22C3 IHC assay). The dose approved by FDA is 2mg/kg every 3 weeks until disease progression or unacceptable toxicity.

In this Phase I trial, 495 patients were assigned to receive pembrolizumab at a dose of 2mg or 10mg every 3 weeks or 10mg every 2 weeks. The adverse effects didn&rsquo;t vary according to dose or schedule and included fatigue, decreased appetite, and pruritis. The overall incidence of pneumonitis was less than 4%. The overall response rate (ORR) evaluated by ECIST criteria was 19.4% (95% confidence interval [CI], 16, and 23.2) and median duration of response was 12.5 months with a median OS of 12 months. The ORR in previously treated patients was 18% (95% CI 14.4 to 22.2) and 24.8% (95% CI 16.7 to 34.3) in previously untreated patients. The response rate was similar across different doses, schedules, or histologic subtypes. Current or former smokers had ORR of 22.5% and never smokers had an ORR of 10.3%.

Keynote 010: Randomized Phase II/III Trial in Previously Treated NSCLC

Keynote 010 was a recent randomized, multicentre, open label phase II/III trial.

In this trial, previously treated NSCLC patients with PDL-1 expression on at least 1% of tumor cells were randomized to receive pembrolizumab 2mg/kg, pembrolizumab 10mg/kg, or docetaxel 75mg/m2 every 3 weeks. The primary end- points were overall survival (OS) and progression-free survival (PFS) for total population and in patients with PD-L1 expression on at least 50% of tumor cells. The median OS was 10.4 and 12.7 months for pembrolizumab 2mg/kg and 10mg/kg respectively and 8.5 months for the docetaxel arm. OS was significantly longer for pembrolizumab 2mg/kg versus docetaxel [Hazard ratio (HR) 0.71, 95% CI 0.58-0.88;

= 0.0008] and pembrolizumab 10mg/kg versus docetaxel (HR 0&middot;61, 95% CI 0&middot;490&middot;75;

<0&middot;0001). Median PFS for unselected patients was not significantly different in the three arms. Patients with at least 50% of tumor cells showing expression of PDL-1 had significantly higher OS with pembrolizumab 2 mg/kg than with docetaxel (median 14&middot;9 months vs 8&middot;2 months; HR 0&middot;54, 95% CI 0&middot;380&middot;77;

= 0&middot;0002) and with pembrolizumab 10 mg/kg than with docetaxel (17&middot;3 months vs 8&middot;2 months; 0&middot;50, 0&middot;36 0&middot;70;

<0&middot;0001). In this subgroup of patients with high PDL-1 expression, PFS was also significantly longer with pembrolizumab 2 mg/kg than with docetaxel (median 5&middot;0 months vs 4&middot;1 months; HR 0&middot;59, 95% CI 0&middot;44 0&middot;78;

= 0&middot;0001) and with pembrolizumab 10 mg/kg than with docetaxel (5&middot;2 months vs 4&middot;1 months; 0&middot;59, 0&middot;450&middot;78;

<0&middot;0001). Pembrolizumab had lower incidence of grade 35 adverse effects (AEs) compared to docetaxel (13% and 16% respectively in pembrolizumab 2 mg/kg arm and 10 mg/kg arm, and 35% in docetaxel arm). Both doses of pembrolizumab showed better OS with a more favorable adverse effect profile compared to docetaxel. The benefit seemed to be magnified for patients with more than 50% of tumor cells showing PDL-1 expression and translated into a significant difference in PFS in this subset.

Two large international phase III trials, Keynote-02415 and Keynote-042,

are comparing pembrolizumab to standard of care platinum-based chemotherapy in the first-line setting. Both these trials are randomizing patients with previously untreated NSCLC without EGFR mutations or ALK translocation with tumors showing PDL-1 expression (&ge;1%) to receive pembrolizumab (200mg fixed dose every 3 weeks) or platinum-based doublet therapy (carboplatin/cisplatin plus paclitaxel/gemcitabine or carboplatin plus premetrexed for non-squamous histology). The Keynote-024 trial is accruing patients with high level PDL-1 expression (&ge;50%) only, whereas the Keynote-042 will enroll patients with &ge;1% PD-L1 expression and stratify them based on weak (1- 49%) or strong (&ge;50%) PDL-1 expression. Chemotherapy will be given for maximum of 6 cycles and may be followed by optional pemetrexed maintenance in patients with non-squamous histology. The patients randomized to Pembrolizumab will be treated for up to 35 cycles or disease progression. Crossover from standard of care platinum doublet to pembrolizumab is allowed for disease progression.

The Keynote-024 trial will continue until approximately 300 patients with metastatic NSCLC are randomized. The primary endpoint is PFS; secondary endpoints are ORR, OS, and safety. The primary endpoint for Keynote-042 trial is OS in the PD-L expression &ge;50% stratum and secondary endpoints include PFS for patients with PD-L expression &ge;50% and PFS and OS for all the patients. The trial will continue until the target of 1240 patients is reached. The primary endpoint is OS, and the secondary endpoint is PFS.

Combination Trials with Chemo, Immuno, or Targeted Therapy

Keynote 021 is multicohort phase I/II study of pembrolizumab in combination with chemotherapy or immunotherapy in patients with stage IIIB or metastatic NSCLC.

Cohorts A and C are evaluations of pembrolizumab plus platinum-based doublet chemotherapy, cohort E and F are evaluations of pembrolizumab and erlotinib or geftinib, and cohort D is evaluation of pembrolizumab and ipilimumab. The results from cohort A and C and those of Cohort D were reported in two separate abstracts at ASCO 2015. Treatment-naive NSCLC patients were randomized 1:1 to receive pembrolizumab (2mg/kg or 10mg/kg every 3 weeks) plus carboplatin (AUC of 6) and paclitaxel (200mg/m2) (cohort A = 20 patients) or carboplatin (AUC of 5) and premetrexed (500mg/m2) (Cohort C = 24 patients with nonsquamous histology). Four cycles of combination chemo/immunotherapy was followed by pembrolizumab monotherapy or maintenance therapy with pemetrexed and pembrolizumab. The ORR was 28% in cohort A and 58% in cohort C. The disease control rate with pembrolizumab of 10mg/kg was 80% in cohort A and 100% in cohort C, and with Pembrolizumab 2mg/ kg was 60% in cohort A and 92% in cohort C. One dose-limiting toxicity: Grade 3 rash requiring hospitalization and drug discontinuation was reported in cohort C with pembrolizumab dose of 10mg/kg. Grade 3-4 AEs were reported in 32% patients in cohort A and 38% patients in cohort C. Immune-related AEs were reported in 2 (8%) patients in cohort A (one case each of grade 3 rash and grade 2 colitis) and 4 (17%) in cohort C (one case each of grade 3 colitis, grade 2 hypothyroidism, and grade 1 colitis and hyperthyroidism). Based on its acceptable safety profile and encouraging clinical activity, the combination of pembrolizumab, carboplatin, and pemetrexed is being evaluated in larger cohort. Efficacy was not significantly different across different pembrolizumab dose levels, suggesting a flat dose/exposure relationship. Phase II cohorts will examine a flat dose of pembrolizumab 200mg in combination with chemotherapy.

Cohort D was a phase I study that examined the combination of pembrolizumab (10mg/kg or 2mg/kg) with anti-CTLA-4 antibody ipilimumab (0.3, 1 or 3mg/kg) every 3 weeks as second-line treatment in advanced NSCLC patients who have progressed on 2 or fewer lines of treatment. Combination immunotherapy for 4 cycles was followed by pembrolizumab monotherapy until progression or up to 2 years. Based on initial safety data from other immunotherapy combination trials, the protocol was amended to enroll patients on a pembrolizumab 2mg/kg and ipilimumab 1mg/kg every 3 weeks regimen. Three grade 3 events (17%) were reported: adrenal insufficiency, drug eruption, and maculopapular rash. Overall incidence of drug-related AEs was 33% and two treatment related-discontinuations were reported. There was no dose-limiting toxicity at the pembrolizumab 2mg/kg and ipilimumab 1mg/kg dose. In 18 response-evaluable patients, ORR was 39% and included 1 patient with complete response. Based on these results, the combination is being evaluated in an expansion cohort of 32 patients.

The combination of pembrolizumab (2mg/kg) with erlotinib 150mg daily or gefitinib 250mg daily is being evaluated in NSCLC patients with EGFR mutations. Efficacy results are not currently available for these cohorts.

Various other combinations of pembrolizumab with standard platinum chemotherapy, radiation therapy, post-surgery in adjuvant setting, with EGFR and ALK inhibitor and other targeted drugs, are being studied in several ongoing or soon-to-start phase I/II/III studies. Some of these trials are studying the role of pembrolizumab outside of the advanced/ metastatic NSCLC setting.

Pembrolizumab Activity in Subgroups of NSCLC

Hellmann et al reported activity of pembrolizumab in key subgroups of NSCLC at the 16th World Conference of Lung Cancer (WCLC) in Denver, CO.

Advanced NSCLC patients (n = 550) treated with different doses of pembrolizumab 2 mg/kg every 3 weeks, 10 mg/kg every 3 weeks, or 10 mg/kg every 2 weeks in the Key- note-001 trial were included. Patients with squamous histology (n=95) had ORR of 26.3% (50% for PD-L1 ex- pression tumor proportion score [TPS] &ge;50%, 17.2% for TPS 1-49%, and 0% for TPS <1%) compared to non-squamous histology (n = 446) with ORR of 19.1% (35.9% for TPS &ge;50%, 11.1% for TPS 1-49%, and 12.3% for TPS <1%). Median PFS for patients with squamous histology was 6.1 months (10.3 months for TPS &ge;50% and 6 months for TPS 1-49%) compared to 3 months (4.3 months for TPS &ge;50% and 2.3 months for TPS 1-49%) for patients with non-squamous histology. Patients with prior smoking history had OS of 14.3 months (26.8 months for TPS &ge;50% and 10.6 months for TPS 1-49%) compared to 8.8 months (8.3 months for TPS &ge;50% and 6.9 months for TPS 1-49%) for never smokers. Patients with EGFR mutation (n = 77) had ORR of 7.8% (20% for TPS &ge;50%, 8.7% for TPS 1-49%) compared to ORR of 21.6% (39.8% for TPS &ge;50%, 12.2% for TPS 1-49%) for EGFR wild type (n = 450). Similar results have been reported in other trials suggesting better activity for smokers and squamous histology compared to never smokers and patients with positive EGFR mutation.

Brain metastases are common among patients with advanced NSCLC and pose a treatment challenge due to the concern for the ability of drugs to penetrate the bloodbrain barrier. Patients with untreated brain metastases are generally excluded from most trials. Goldberg et al reported preliminary data at WCLC 2015 from an ongoing phase II trial evaluating response to pembrolizumab in 18 patients with PDL-1 expression and small, asymptomatic, brain metastases that are previously untreated or progressing after local treatment.

Brain metastases response rate and systemic response rate were 33%. All patients with systemic response also had response in brain mets except for 1 patient who progressed in brain after a transient response and required radiation therapy. There were no neurological aderse events greater than grade 1.

In an unselected population, approximately 1 in 5 patients show durable response to checkpoint inhibitors.

Developing reliable, reproducible biomarkers to identify this subset of patients who are likely to show a response remains a lucrative target. In the Keynote-001 trial, there was an observed relationship between high level PD-L1 and response to pembrolizumab, so the initial protocol was amended to include a coprimary endpoint to assess the efficacy of pembrolizumab in patients with a high PDL-1 expression. The ORR was 45.2% (95% CI, 33.5-57.3) in patients with a proportional score of at least 50%, compared to 16.5% (95% CI, 9.9-25.1) in patients with performance status (PS) of 1-49% and 10.7% (95% CI, 2.3-28.2) for those with a PS score of less than 1%. The higher response rate for patients with a PS score of greater than 50 was sustained when divided by previously treated patients (

= 0.01). Of all the patients who had samples that could be evaluated, 23.2 % of patients had a proportion score of at least 50%, 37.6% had a score of 1-49%, and 39.2% had a score of less than 1%. This finding of higher PD-L1 expression and response to pembrolizumab was also reported by Herbst et al as mentioned above. In a meta-analysis, Wang et al reported that high PD-L1 expression was significantly associated with poor differentiation of tumor and worse OS.

There are several limitations in assessing the role of PD-L1 expression as a biomarker. Many different assays and definitions of positivity have been used to express PD-L1 positivity for different checkpoint inhibitors, which makes it difficult to compare results across trials. Moreover, PDL-1 expression can be variable within a tumor or in an individual at a different site of metastatic disease. As the tumor cells develop resistance and multiply post-treatment, it is likely that the expression of PD-L1 will change over time. In addition to the tumor cells, the tumor-invading dendritic and macrophage cells express PD-L1 and do not show uniform tumor invasion. Consequently, depending on the biopsy, the specimen might not be a true representative of actual PDL-1 expression.

Occasionally, interpretation of clinical benefit could be more complex with pembrolizumab or other checkpoint inhibitors as pseudoprogression can result in an increase in size of the target lesion before actual response evaluation criteria in solid tumors (RECIST) response occurs. Systematic criteria, defined as immune-related response criteria (irRC) have been created to capture these additional response patterns seen with immune therapy beyond those described by RECIST criteria. irRC takes into account total tumor burden and allows treatment even in the presence of new lesion and initial increase in the target lesion. Pseudoprogression has been encountered less frequently with NSCLC compared to melanoma and early rescanning in 4 to 6 weeks helps to differentiate pseudoprogression from true progression.

The treatment of NSCLC is changing rapidly and immune check point inhibitors such as pembrolizumab show promising results in the second-line setting with tolerable toxicities. Several ongoing phase II/III trials will help identify the role of pembrolizumab in the first-line setting alone or in combination with chemotherapy or CTLA-4 inhibitor. In multiple trials with pembrolizumab or other checkpoint inhibitors, there seems to be greater effect on OS compared to PFS, indicating continued benefit after treatment discontinuation with subsequent therapy. It is possible that PFS may not be an appropriate endpoint for clinical trials involving checkpoint inhibitors. As more data from ongoing clinical trials become available, it is likely that subsets within the NSCLC will be identified with more or less likely to benefit with pembrolizumab. For example, the subset analysis of Keynote 001 trial revealed that patients with sensitizing EGFR mutation-positive NSCLC are not the most likely group of patients to benefit from pembrolizumab. The toxicity profile of pembrolizumab is quite favorable compared to traditional chemotherapy and similar to other checkpoint inhibitors. Immune related adverse effects can occur any time during the treatment and have also been reported after treatment discontinuation. Most serious immune reactions can be adequately managed with discontinuation of treatment and prompt administration of corticosteroids. Future trials will likely explore other biomarkers in associaion with PD-L1 to enrich the patient population likely to benefit from this treatment.

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Articles

Clinical Articles

In this article, Priyanka Bhateja, MD, and Neelesh Sharma, MD, PhD review the completed trials and ongoing clinical development of pembrolizumab in NSCLC.

Pembrolizumab in Non-Small Cell Lung Cancer

Next-generation sequencing of papillary thyroid cancers has identified genomic alterations for which targeted therapy has proven effective. While thyroid stimulating hormone receptor (TSHR) mutations are relatively common in benign thyroid nodules, rare reports of TSHR mutations in malignant thyroid tumors are noted. Thyroid stimulating hormone (TSH) suppression is a standard of care for treatment of patients diagnosed with thyroid cancer, but for cancers harboring TSHR mutations, the effectiveness of this intervention is uncertain.

We present a case of a 49-year-old woman with a pT4aN1B papillary thyroid cancer who underwent radical thyroidectomy with bilateral neck dissection and RAI I-131 ablation, but poorly tolerated TSH suppression therapy due to tachycardia. Rising thyroglobulin levels and lymph node mapping showed recurrent/progressive disease in the thyroid bed. Genomic characterization of the tumor using a CLIA-certified next-generation sequencing assay identified an activating TSHR mutation, A623V. Clinical course and genomic profiling results were discussed in the setting of a multidisciplinary, molecular tumor board to generate therapeutic recommendations. As the TSHR A623V mutation may be associated with increased receptor sensitivity to TSH, further suppression of TSH with exogenous thyroid hormone was recommended. To achieve tolerability, beta-blocker was concurrently titrated with an increasing levothyroxine dose to control tachycardia while maximally suppressing TSH. Subsequent lymph node mapping showed no evidence of suspicious neck lymph nodes. Based on the results in this patient, aggressive therapeutic TSH suppression may represent an important therapeutic approach for patients with papillary thyroid cancer with TSH-sensitive TSHR mutations.

Since 1980, the number of new cases of thyroid cancer more than doubled, resulting in its rank as the fifth most common malignancy in women in 2015. Thyroid cancer represents 6% of new cancer cases in women, and 60% of affected individuals are under age 55.1 Although most thyroid cancers are the well-differentiated, papillary, or follicular subtypes that carry a good prognosis, papillary thyroid cancers with poor prognostic features show the highest risk for recurrence.

Metastatic papillary thyroid cancer (PTC) can be managed effectively for prolonged periods with intermittent use of radioactive iodine (RAI) therapy, although the number of doses can be limiting. Once PTC becomes RAI-resistant or shows limited RAI accumulation, few effective treatments are available.

Thyroid stimulating hormone receptor (TSHR) and its endogenous ligand thyroid stimulating hormone (TSH) are pivotal factors in physiologic function and pathophysiology of benign and malignant thyroid diseases. Measurement of hormones involved in the thyroid axis function are used for screening and diagnosis of hyperthyroidism, hypothyroidism, and autonomously functioning nodules as well as for treatment and monitoring for thyroid cancer. TSHR is a G-protein coupled membrane receptor with an extracellular, leucine rich, ligand binding domain for TSH, 3 extracellular loops, and 3 intracellular loops linking the 7 helical structures comprising the transmembrane domain.

Constitutive activating mutations of TSHR, neuroblastoma RAS viral oncogene homolog (NRAS), and Guanine Nucleotide Binding Protein Alpha (GNAS) occur frequently in thyrotoxic disorders.

Different frequencies of TSHR mutations ranging from 20% to 86% have been reported for benign thyroid diseases including toxic follicular adenomas, nodules, and multinodular goiter. TSHR mutations occur across all age ranges from infants to adults at similar frequencies, although the spectrum of mutations differs.

Activating TSHR mutations have also been detected exclusively in hyper-functioning areas of the thyroid not presenting as discrete nodules, but not in nonfunctioning areas of the same gland.

The spectrum of mutations spanning the TSHR gene has been described, but the majority occurs in the transmembrane domain. In one family with familial non-autoimmune hyperthyroidism, a transmembrane region mutation, specifically TSHR L665F in helix 7, resulted in constitutive activation of TSHR and downstream activation of Gs proteins.

Such activation was thought to arise from structural changes in the receptor. Mutations in other genes such as NRAS and GNAS have also been reported for hyperfunctioning thyroid adenomas, though at variable frequencies.

evaluated a series of benign thyroid tumors, but only one GNAS R201C mutation in a Hurthle cell adenoma and one Gi2 alpha mutation R179C in a follicular adenoma were observed.

TSHR mutations are rare in malignant thyroid tumors, although individual cases have been reported including a hyper-functioning Hurthle cell carcinoma, an autonomously functioning follicular carcinoma, and papillary thyroid cancer in the setting of hyperthyroidism.

Genes commonly mutated in papillary thyroid cancers include BRAF, NRAS, KRAS, and HRAS,

suggesting that most of these cancers are driven by activation of the RAS-RAF-MEK signaling pathway. Moreover, the presence of mutations in the RAS-RAF-MEK pathway is associated with those papillary thyroid cancers having poor prognostic features.

Understanding the relationship between TSHR activating mutations, dependence on TSH, and development of thyroid cancer would aide in the therapeutic approach to these tumors. Herein, we describe a case of a papillary thyroid cancer found to contain the A623V activating mutation in TSHR and lacking mutations in either BRAF or NRAS. This mutation has been associated with increased sensitivity of receptor to TSH. Aggressive TSH suppression with aggressive management of symptoms resulted in clinical benefit and tumor regression.

The patient was a 48-year-old woman with no family history of cancer and no known exposure to hazardous materials. She was in her usual state of health when a palpable thyroid nodule was noted on a routine physical exam. Laboratory studies showed a euthyroid state with TSH = 4.04 mIU/L (normal range 0.35-5.50m IU/L) and free T4 = 1.04 ng/dL (normal range 0.8- 1.8 ng/dL). Fine needle aspirations of a right upper thyroid nodule and a left jugular chain lymph node, identified on neck ultrasound, each showed malignant cells consistent with papillary carcinoma. She underwent thyroidectomy and right modified neck dissection. Pathology showed multifocal papillary thyroid carcinoma in the right lobe, isthmus and left lobe measuring up to 1.5 cm in greatest dimension with extension into bilateral skeletal muscle, multiple foci in adipose and fibro-connective tissue regional to the cricoid cartilage, and involvement of the bilateral re- current laryngeal nerves. No lymphovascular invasion was identified. Ten of a total of 41 lymph nodes demonstrated metastatic disease at multiple levels in the neck. Her disease was thus staged as pT4a N1b. Her postoperative course was complicated by vocal cord paralysis, extended ventilation, and MSSA tracheobronchitis resulting in a two-month recouperation with recovery of vocal cord function. A subsequent, additional left modified neck dissection showed metastatic papillary thyroid carcinoma in three of 35 lymph nodes. Unfortunately, this post-operative course was further complicated by a thoracic duct leak, chylous effusion, and bronchiolitis obliterans organizing pneumonia resulting in baseline hypoxemia and tachycardia. Replacement levothyroxine was started shortly after surgery, and 6 months post-operatively, TSH was 0.46 mIU/L.

Eight months after surgery, her TSH rose to 11.45 mIU/L and an I-123 scan with thyrogen stimulation showed a focal area of uptake at the anterior mid to lower neck with 24-hour radioiodine neck uptake value 0.8%. She completed thyroid ablation with 145 mCi I-131 one month later. A post-ablation scan 1 week later showed focal accumulation in the midline and neck, but no definite suspicious uptake outside of the neck. She was placed on levothyroxine 125mcg daily and TSH reached 0.63 mIU/L at 10 months after surgery, during which she experienced daily episodes of palpitations and tachycardia to greater than 110 beats per minute (bpm), dry skin, dry mouth, alopecia, and increased sensitivity to cold. Reducing the dose reduced the side effects but resulted in weight gain and increasing thyroglobulin from 1 ng/ mL to 8.2 ng/mL (normal range 1.5-38.5 ng/mL).

One year after initial diagnosis, she experienced an episode of palpitations, dizziness, headache, and shortness of breath. Cardiac evaluation was unremarkable with the exception of tachycardia. A chest X-ray showed a shadow within the right medial chest wall. TSH was 0.33 mIU/L and T4 was 9.2 (normal range 5.0-12.0 mcg/dL). A follow-up chest CT showed a resolving left lower lobe infiltrate and a new anterior superior mediastinal soft-tissue mass at the thoracic inlet presumed to be recurrence of the thyroid cancer. A PET/CT scan one month later revealed an asymmetric hypermetabolic focus with a maximum SUV of 6.5 situated posterolateral to the vocal cords on the right side. Relatively low-level FDG uptake was associated with the anterior soft tissue density mass reported on a prior CT scan, measuring 5.5 x 2.5 cm.

Comprehensive genomic profiling was performed on routine formalin-fixed, paraffin-embedded thyroid tissue from her thyroidectomy using the CLIA-certified FoundationOne platform.

Targeted sequencing of the entire coding sequence was performed for 315 genes and introns of 28 genes involved in fusions at a depth of 500-1000X. Tumor genomic profiling from tissue from her thyroidectomy demonstrated two mutations, TSHR A623V and MDM4 S367L, but no evidence of BRAF or NRAS alterations. Her case was discussed at our institutional, multidisciplinary molecular tumor board. As the TSHR A623V mutation may result in activation in part through increased receptor sensitivity to TSH, the recommendation was to increase her levothyroxine to achieve maximal TSH suppression. Her multidisciplinary team concomitantly used beta-blockers, metoprolol 25 mg orally twice daily, and dropped her heart rate to 79 bpm, while increasing thyroid replacement. An additional RAI ablation was planned but the I123 preliminary scan showed no RAI uptake in the previously positive area in the neck and only physiologic uptake in the salivary glands. Therefore, ablation was aborted, and shortly thereafter, her levothyroxine dose was increased from 125 &mu;g to 137 &mu;g. Following the titration of beta-blockers, her heart rate remained below 100 bpm and she experienced no further limitations or episodes of tachycardia and palpitations.

Evaluation 18 months post-diagnosis revealed resolution of tachycardia and a heart rate of 89 bpm. She denied any new symptoms and felt significantly better on the increased dose of levothyroxine (137 mcg daily). Her dose of metoprolol ER was escalated to 50 mg twice daily. TSH was 0.34 mIU/L. She underwent a neck ultrasound for lymph node mapping, which showed no abnormally enlarged or abnormal-appearing right-sided lymph nodes. A left-sided 15 x 6 x 9 mm lymph node in the superior jugular chain was more prominent than other lymph nodes but it appeared to have a hilum and showed normal fat echo. At 23 months post-diagnosis, her levothyroxine dose was adjusted again to 150 mcg daily and metoprolol to 75 mg twice daily with a TSH of <0.1 mIU/L and no tachycardia.

We present a patient with a TSHR A623V-mutated, pT4aN1B papillary thyroid cancer who underwent a radical thyroidectomy with bilateral neck dissection and suffered severe post-operative pulmonary complications. Following clinical recovery, she received I-131 ablation. Symptomatic tachycardia limited TSH suppression and there was evidence of disease recurrence on suboptimal TSH suppression as shown by imaging. TSH suppression was recommended by the institutional molecular tumor board, given the potential for enhanced sensitivity of this TSHR-mutated thyroid cancer to endogenous TSH. Achieving side effect control with beta-blocker while increasing the levothyroxine dose for TSH suppression resulted in subjective and objective clinical benefit. Most TSHR mutated thyroid cancer in the literature has been associated with a hyper-functioning carcinoma or hyperthyroidism.

This patient was in a euthyroid state and had no histological evidence to suggest a hyper-functioning gland. However, the relationship between autonomously functioning thyroid cancer and presence of a TSHR mutation has not been systematically evaluated.

TSHR is characterized by a high level of constitutive activity in its wild-type state, but the physiologic relevance of this is not well understood.

hypothesized that TSHR activating mutations result in higher levels of basal cAMP, which, in turn, lead to downstream pathway activation that promote the development of toxic follicular thyroid adenomas. Malignant thyroid tumors could also be hypothesized to arise with the same mechanism, although a second genomic alteration may be necessary for transformation. Given the relatively high frequency of TSHR mutations in non-malignant thyroid diseases but low frequency in thyroid cancer, this suggests that most papillary thyroid cancers do not arise from TSHR-mutated benign nodules.

Despite the recurrent observation of TSHR mutations in thyroid disease, debate still exists as to the relevance of TSHR mutations in the pathophysiology and clinical behavior of benign and malignant thyroid disorders. Although exon 10 is the most commonly interrogated exon for genomic evaluation of TSHR, somatic mutations affecting the third intracellular loop, sixth transmembrane segment, and second extracellular loop in addition to other receptor regions have also been described.

The TSHR mutation observed in this papillary thyroid cancer occurs in the third intracellular loop of the transmembrane domain but has not been implicated as a contact residue responsible for oligomerization and contacts between transmembrane domains.

Most TSHR activating mutations have been reported to be constitutively active.

However, review of the literature suggests that only a limited number have unequivocal data supporting a link of constitutive activation.

Moreover, the data do not clearly address whether mutated TSH receptors are truly ligand-independent as opposed to ligand &ldquo;hypersensitive.&rdquo; In fact, some TSHR mutations may confer constitutive activity but will either not respond to TSH or will respond similar to wild-type TSHR. In contrast, others may introduce structural changes leading to receptor stimulation at lower levels of TSH with or without change in basal TSHR activity.

The TSHR A623V, found in the papillary thyroid cancer of our patient, has been reported as occurring in well-differentiated thyroid cancer.

This mutation has been shown to be oncogenic, as overexpression of TSHR A623V led to transformation of NIH3T3 cells.

Intriguingly, although this mutated receptor was transforming by itself, suggesting constitutive activity, this increased activity may itself be dependent on some basal level of TSH found in normal serum conditions or due to intrinsically higher basal receptor activity. TSHR A623V transformation resulting in activation of the MAPK pathway validated findings by others that TSHR A623V showed higher basal activity as compared to wild-type TSHR, and also demonstrated greater dose-dependent, TSH-induced cAMP production as compared to wild-type TSHR.42 The latter supports the hypothesis that TSHR A623V displays ligand-dependent activity and may require some level of exogenous TSH for full activation.

Well-differentiated thyroid cancer retains dependence on TSH signaling for growth. Thus, TSH suppression therapy aimed at achieving TSH levels <0.1 mIU/L, if clinically feasible, have become a generally accepted guideline in the postoperative setting for intermediate and high-risk thyroid cancer. Adequate TSH suppression therapy often becomes suboptimal for patients such as the one reported here due to side effects. As the patient showed evidence of disease progression in the setting or suboptimal TSH suppression, this further raised the possibility that this TSHR A623V-mutated thyroid cancer may be hypersensitive to TSH. However, in this high-risk patient with recurring disease, we achieved suppression through concurrent management of tachycardia. Recently, Wang et al

recognized that levothyroxine-induced TSH suppression to <0.05mIU/L and current recommended duration may cause potential harm even at normal TSH ranges. The comparison of patients with levothyroxine-induced TSH suppression and those not receiving levothyroxine in the setting of low-risk thyroid cancer showed no difference in tumor recurrence or disease-free survival over 6.5 years follow-up between these groups. Recognition of this finding resulted in a change in the 2015 ATA guidelines, whereby post-thyroidectomy TSH suppression is strongly recommended only for high-risk patients, but remains based only on &ldquo;moderate-quality evidence.&rdquo;

While this patient meets criteria for high-risk disease from the outset, escalating thyroglobulin and RAI uptake, coupled with the presence of the TSHR mutation, further drove support for more aggressive TSH management. The long-term risk for osteoporosis was acknowledged for this patient, who we placed on both calcium and vitamin D supplementation.

Notable in this case of papillary thyroid cancer is the absence of other oncogenic mutations, commonly identified by other groups through next-generation sequencing, including BRAF V600E, NRAS, HRAS, and select fusions.

For the 399 cases of papillary thyroid cancers reported in TCGA, only two mutations in TSHR, ie M453T and I640V, were reported.

The papillary thyroid cancer with TSHR I640V demonstrated concomitant NRAS Q61R mutation, but neither showed an MDM4 mutation. Mutations affecting TSHR and proteins of the MAPK pathway do not appear to be mutually exclusive in thyroid cancer where comutations in both TSHR and NRAS or TSHR and KRAS have been reported.

speculated that the more aggressive tumor in their patient was a result of such a co-event. Occurrence of both BRAF and TSHR mutations in a single case has not yet been described; however, this may be due to other mechanisms affecting TSHR. For example, evidence from methylation-specific PCR suggests that epigenetic silencing of the TSHR promoter through hypermethylation occurs in roughly 73% of thyroid cancers harboring the BRAF V600E mutation, and that this correlated with elevated TSH levels.

BRAF mutations have also been associated with poor prognostic features in papillary thyroid cancer and RAI resistance.

Evaluation for associations between TSHR mutations and thyroid cancer prognosis or RAI refractory phenotype requires further evaluation.

In BRAF or RAS wild-type thyroid cancers, testing for TSHR mutation may be of greater importance for determining therapeutic actionability. However, data from this patient cannot rule out the potential benefit of testing for TSHR mutation in BRAF- and RAS-mutated papillary thyroid cancers as well. The probability of identifying a TSHR-mutated papillary thyroid cancer would be low as only 1% of papillary thyroid cancer in TCGA were TSHR-mutated.

While most commercially-available tumor genomic profiling panels include BRAF and RAS, only a subset include TSHR in their panel and clinicians should consider reflex testing for TSHR, particularly in BRAF or RAS wild-type papillary thyroid cancer and in those patients with progression on suboptimal TSH suppression.

As a result of the absence of other actionable driver mutations in the RAS-RAF-MEK pathway in our case, recommendations were made for increased TSH suppression. Although the clinical significance of TSHR mutations in etiology of, and treatment of, hot thyroid nodules remains unresolved,

this case demonstrates the association between targeting signaling through the TSH receptor and clinical response of a TSHR-mutated papillary thyroid cancer over a 6-month time period. Ideally, extended follow-up would be of interest for this patient as the longer-term benefit of TSH suppression in the setting of TSHR-mutated papillary thyroid cancer is not yet known. TSH-mediated signaling through a mutant TSHR is implicated as driving tumor growth in this case based on the temporal relationships between TSH suppression, thyroglobulin, and clinical benefit in the absence of other thyroid-directed therapies. This case raises important questions regarding more tailored use of TSH suppression therapy for specific thyroid cancers based on oncogenic drivers.

Tumor genomic profiling of well-differentiated thyroid cancer identifies potentially actionable genomic alterations

Mutations in TSH receptor (TSHR) are found in a subset of malignant thyroid tumors and may be associated with non-hyperfunctioning gland

Maximal TSH suppression may be effective in some malignant thyroid tumors with TSHR mutations

Hugs for Brady, The Val Skinner Foundation, NIH P30CA072720. This research was supported by a generous gift to the Genetics Diagnostics to Cancer Treatment Program of the Rutgers Cancer Institute of New Jersey and RUCDR Infinite Biologics.

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We present a case of a 49-year-old woman with a pT4aN1B papillary thyroid cancer who underwent radical thyroidectomy with bilateral neck dissection and RAI I-131 ablation, but poorly tolerated TSH suppression therapy due to tachycardia. Rising thyroglobulin levels and lymph node mapping showed recurrent/progressive disease in the thyroid bed.

Oncogenic TSHR Mutation in Papillary Thyroid Cancer

Basal cell carcinoma (BCC) is the most commonly diagnosed human cancer, with more than 2 million cases per year in the United States. While most of these cases are amenable to local excision, many progress to locally advanced or metastatic disease that precludes surgical intervention.

Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, and other human tumors are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumor suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh)-secreted protein signal, and oncogenic mutations affecting these proteins cause excessive activity of the Hh response pathway.

Clinical trials have shown exceptional decrements in tumor burden as well as resolution of metastatic disease with Hh pathway inhibitors as the sole treatment, thus reducing the need for surgery and associated morbidity and mortality. Reported response rates to Hh inhibitors therapy vary from 43% to 86%. The most commonly reported side effects include dysgeusia, muscle spasms, alopecia, weight loss, and fatigue.

Hedgehog pathway inhibitors are a promising group of drugs to be used in cancer chemotherapy and are an excellent example of disease modulation at the biochemical level. They are a new, target-specific treatment alternative associated with high tumor responses. Treatment patterns, clinical outcomes, health care resource use, and definitions of advanced and complex/difficult- to-treat disease are not uniformly adopted in the literature and clinical practice. Larger cohort studies are needed to confirm the long-term efficacy and safety of Hh pathway inhibitors and their role in neoadjuvant use for cytoreduction prior to planned surgical intervention. The purpose of this article is to discuss the efficacy, indications for use, and safety of oral Hh pathway inhibitors for locally advanced BCC (LABCC) and for metastatic BCC (MBCC) through a review of the literature.

Basal cell carcinoma (BCC) is the most commonly diagnosed human cancer, accounting for more than 2 million cases each year in the United States. While most of these cases can be treated by local excision, many progress to locally advanced BCC (LABCC) or metastatic BCC (mBCC) that rule out surgery.

Basal cell cancer comprises most non-melanoma skin cancers (NMSCs) and is more common than all other human malignancies combined. Evidence suggests that the worldwide incidence of BCCs is increasing. In the US, the diagnosis and treatment of NMSCs have increased dramatically, with a growth rate of 77% over the past 2 decades.

Overall, the reasons for this dramatic growth have been postulated to include the aging population, changes in sun exposure habits, environmental changes, migration patterns, and, to a lesser extent, increased prevalence of immunosuppressant use.

Besides environmental risk factors, host susceptibility factors have been described in several familial cancer syndromes, including nevoid basal cell carcinoma syndrome (Gorlin syndrome), which is an autosomal dominant familial cancer syndrome. This form of BCC predisposes patients to the early development of multiple BCCs with a higher likelihood of becoming locally advanced or even metastatic as surgery becomes less likely because of the multiplicity of lesions.

More than 2.8 million new cases of BCC are diag- nosed each year in the United States, alone and are estimated to result in more than 3,000 deaths.

Fortunately, BCCs are usually diagnosed and treated early.

Nevertheless, reported 5-year recurrence rates are estimated to be 2% to 3 %.

BCCs that are extensive and infiltrate structures below the skin or abut vital structures, such as the brain or eyes, may be difficult to surgically clear without significant morbidity. Many of these may become locally advanced or metastasize; those that metastasize to either local or distant lymph nodes or distant organs would best be addressed through systemic therapy.

While accurate estimates of the incidence of LABCCs are difficult to obtain, in part because of the lack of widespread use of a staging system by dermatologists and the lack of uniform reporting requirements for NMSCs, LABCCs are thought to represent 1% to 10 % of all BCCs, with mBCC accounting for 0.0028% to 0.5%.

From our clinical experience, patients presenting with LABCC, appear to fall into 2 categories: those who present after a delay in accessing medical attention, or those who have BCCs that are intrinsically aggressive, are refractory, or recur after treatment. Included in this latter group are those patients with chronic immunosuppression after organ transplant and some patients with Gorlin syndrome. Therapeutic options for such patients are limited.

Research into the reactivation of embryonic molecular pathways in adult tissues has led to the discovery of novel antineoplastic agents capable of redefining systemic treatment for LABCC and mBCC.

The Hh pathway was initially identified as a critical developmental regulator of embryonic segment polarity in Drosophila in 1980.

This, and related developmental work in fruit fly body patterning, was recognized by the Nobel Prize in Physiology and Medicine in 1995. Vertebrate homologs of the Drosophila Hedgehog ligand were first reported in 1993, and definition of central components of the mammalian signaling pathway followed in the late 1990s and early 2000s.

The first definitive linkage of mutation in this pathway to cancer, that is, to development of BCC, was made in 1996.

Hedgehog signaling regulates embryonic development, ensuring that tissues reach their correct location, size, and cellular content. The Hh pathway normally remains inactive in adult tissues, but reactivation of this pathway results in malignant transformation and tumorigenesis.

More than 90% of all BCCs have identified abnormal Hh pathway signaling. There are 2 classic mechanisms of Hh pathway reactivation: mutation-driven signaling (ligand independent) and abnormal signaling in the tumor microenvironment (ligand dependent).

In mutation-driven signaling, tumor cells harbor mutations in key components of the Hh pathway, allowing for independent activation of the biomolecular system and subsequent stimulation of unregulated proliferation.

In ligand-dependent activation, tumor cells overexpress Hh ligands that activate surrounding stromal cells, leading to uncontrolled growth.

Mutations in 2 major receptor proteins, Patched-1 (PTCH1), and Smoothened (SMO), may result in abnormal activation of the Hh pathway and are the focus of proposed interventions.

Hh pathway activation occurs via binding of an extracellular Hh ligand (Shh) to PTCH1, a transmembrane receptor. Once bound, PTCH1 is internalized and degraded, allowing for release of the transmembrane protein SMO from the inhibitory effect of PTCH1.

SMO can then move from the intracellular compartment to the cell membrane of the cilium and activate the Gli family of transcription factors. Activated Gli factors enter the nucleus and initiate the transcription of target genes.

The purpose of this article is to discuss the efficacy, indications for use, and safety of oral Hh pathway inhibitors for locally advanced BCC (LABCC) and for metastatic BCC (MBCC) through a review of the literature.

February 2016 | The Journal of Targeted Therapies in Cancer

Pembrolizumab in Non-Small Cell Lung Cancer

Oncogenic TSHR Mutation in Papillary Thyroid Cancer

Smoothened Inhibitors and the Hedgehog Pathway: Applications in Basal Cell Carcinoma and Beyond