Peter M. Sadow

Targeting BRAFV600E with PLX4720 Displays Potent Antimigratory and Anti-invasive Activity in Preclinical Models of Human Thyroid Cancer

The role of the B-RafV600E mutation in aggressive thyroid cancers is examined.

Renal Cell Carcinoma in Tuberous Sclerosis Complex

Renal cell carcinoma (RCC) occurs in 2% to 4% of patients with tuberous sclerosis complex (TSC). Previous reports have noted a variety of histologic appearances in these cancers, but the full spectrum of morphologic and molecular features has not been fully elucidated. We encountered 46 renal epithelial neoplasms from 19 TSC patients and analyzed their clinical, pathologic, and molecular features, enabling separation of these 46 tumors into 3 groups. The largest subset of tumors (n=24) had a distinct morphologic, immunologic, and molecular profile, including prominent papillary architecture and uniformly deficient succinate dehydrogenase subunit B (SDHB) expression prompting the novel term “TSC-associated papillary RCC (PRCC).” The second group (n=15) were morphologically similar to a hybrid oncocytic/chromophobe tumor (HOCT), whereas the last 7 renal epithelial neoplasms of group 3 remained unclassifiable. The TSC-associated PRCCs had prominent papillary architecture lined by clear cells with delicate eosinophilic cytoplasmic thread-like strands that occasionally appeared more prominent and aggregated to form eosinophilic globules. All 24 (100%) of these tumors were International Society of Urological Pathology (ISUP) nucleolar grade 2 or 3 with mostly basally located nuclei. Tumor cells from 17 of 24 TSC-associated PRCCs showed strong, diffuse labeling for carbonic anhydrase IX (100%), CK7 (94%), vimentin (88%), and CD10 (83%) and were uniformly negative for SDHB, TFE3, and AMACR. Gains of chromosomes 7 and 17 were found in 2 tumors, whereas chromosome 3p deletion and TFE3 translocations were not detected. In this study, we reported a sizable cohort of renal tumors seen in TSC and were able to identify them as different morphotypes, which may help to expand the morphologic spectrum of TSC-associated RCC.

A genome-wide screen for microdeletions reveals disruption of polarity complex genes in diverse human cancers.

In a genome-wide screen of 684 cancer cell lines, we identified homozygous intragenic microdeletions involving genes encoding components of the apical-basal cell polarity complexes. Among these, PARD3 is disrupted in cell lines and primary tumors from squamous carcinomas and glioblastomas. Reconstituting PARD3 expression in both cell types restores tight junctions and retards contact-dependent proliferation. Searching specifically for small intragenic microdeletions using high-resolution genomic arrays may be complementary to other genomic deletion screens and resequencing efforts in identifying new tumor suppressor genes.

Detection of Novel Actionable Genetic Changes in Salivary Duct Carcinoma Helps Direct Patient Treatment

Purpose: Salivary duct carcinomas (SDC) are a rare and aggressive subtype of salivary gland cancers for which cytotoxic chemotherapy has limited efficacy. We investigated whether genotyping analysis could detect novel tumor-specific mutations that would help direct SDC patient treatment using targeted agents. Experimental Design: We genotyped 27 SDC archival specimens from patients followed at Massachusetts General Hospital and Massachusetts Eye and Ear Infirmary (Boston, MA) between 2000 and 2011. These included the tumors of 8 patients who were tested prospectively. Targeted mutational analysis of 13 clinically relevant cancer genes was conducted using SNaPshot multiplexed genotyping. FISH was conducted to detect HER2 gene amplification. Patient medical records and tumor histopathologic features were retrospectively reviewed. Results: Mutually exclusive genetic aberrations were detected in 15 of 27 (56%) tumors, including 2 (7%) mutations in BRAF, 5 (19%) mutations in PIK3CA, and 8 (30%) cases of HER2 gene amplification. To our knowledge, this is the first time that BRAF and PIK3CA mutations have been reported in this tumor type. Prospective clinical testing of 8 patients with SDC identified actionable genetic alterations in 6 tumors and influenced therapeutic decisions for all 6 patients. Conclusion: SNaPshot molecular profiling identified novel genetic changes in SDCs, expanded the therapeutic options for patients with this rare tumor, and is changing SDC management at our institution. These findings highlight the importance of using broad-based genetic profiling to expedite the identification of effective-targeted therapies for patients with rare malignancies. Clin Cancer Res; 19(2); 480–90. ©2012 AACR.

SCF β-TRCP suppresses angiogenesis and thyroid cancer cell migration by promoting ubiquitination and destruction of VEGF receptor 2

The E3 ubiquitin ligase β-TRCP, acting in concert with casein kinase I, drives ubiquitination and degradation of VEGFR2, and renders human papillary thyroid cancer cells resistant to the VEGFR2 inhibitor sorafenib.

Regulation of expression of thyroid hormone receptor isoforms and coactivators in liver and heart by thyroid hormone

Autoregulation of thyroid hormone (TH) receptors (TRs) is a mechanism whereby a cell can regulate its responsiveness to TH. Nuclear coactivators (NCoAs) modulate TH action and may also be important for regulation of TR expression. We have determined the effect of TH withdrawal and treatment on the expression of different isoforms of TR as well as expression of the NCoAs SRC-1, TIF-2 and SRC-3 using quantitative real time polymerase chain reaction. In order to identify the effect that each TR isoform exerts over the expression of the other, NCoA and TR transcripts were measured in liver and heart tissue from wild type mice or mice with deletion of either TR isoform or SRC-1 genes. In liver, regulation of TR beta1 and TR alpha2 subtype expression is inversely related to TH levels and the regulation of TR beta expression is, in part, controlled by TR alpha. In the heart, the opposite is the case, regulation of TR alpha2 and TR beta1 isoform expression is directly related to TH levels and this regulation is primarily controlled by TR alpha. Although NCoAs are, in general, increased in response to hypothyroidism or in states of TH resistance, SRC-1 specifically does not regulate TR isoform expression. We have demonstrated that TR isoforms and NCoAs are autoregulated transcription factors with tissue specificity.

Malignant struma ovarii.

Background A 25-year-old woman presented to her gynecologist with pelvic pain. Pelvic ultrasonography showed a 9 cm left ovarian mass. The patient underwent left oophorectomy, omental biopsy, and lymph node sampling. The ovarian mass proved to be a struma ovarii with numerous microscopic foci of papillary thyroid carcinoma. The patient had no symptoms of hyperthyroidism, and her thyroid function and serum thyroglobulin levels were normal.Investigations Investigations included a pelvic ultrasound scan, histological examination of the ovarian mass and omental nodules, and lymph node sampling.Diagnosis Malignant struma ovarii.Management The patient was referred to an endocrinology clinic for further investigations. Serum levels of TSH, thyroglobulin and thyroglobulin antibodies were measured. In addition, the patient underwent thyroid ultrasonography, which showed a 1 cm nodule that proved benign on biopsy. She was treated with thyroxine to reduce TSH secretion. Follow-up pelvic ultrasonography 1 year later showed no evidence of recurrent disease, and her serum thyroglobulin levels remained normal.

p53 constrains progression to anaplastic thyroid carcinoma in a Braf-mutant mouse model of papillary thyroid cancer

Significance We generated a thyroid-specific CreER transgenic mouse and used this strain to model progression of v-raf murine sarcoma viral oncogene homolog B (BRAF)-mutant papillary thyroid cancer to anaplastic thyroid cancer (ATC). These murine tumors recapitulated the temporal progression and molecular hallmarks of human ATC. We demonstrated that combined mapk/Erk kinase (MEK) and BRAF inhibition resulted in enhanced antitumor activity vs. single-agent BRAF inhibitors in this preclinical model. This model represents a previously lacking mouse model of BRAF-mutant ATC and adds to the experimental armamentarium of a highly lethal disease in need of scientific advances. These data also suggest that potent inhibition of the MAPK pathway may improve outcomes in advanced thyroid cancers. Anaplastic thyroid carcinoma (ATC) has among the worst prognoses of any solid malignancy. The low incidence of the disease has in part precluded systematic clinical trials and tissue collection, and there has been little progress in developing effective therapies. v-raf murine sarcoma viral oncogene homolog B (BRAF) and tumor protein p53 (TP53) mutations cooccur in a high proportion of ATCs, particularly those associated with a precursor papillary thyroid carcinoma (PTC). To develop an adult-onset model of BRAF-mutant ATC, we generated a thyroid-specific CreER transgenic mouse. We used a Cre-regulated BrafV600E mouse and a conditional Trp53 allelic series to demonstrate that p53 constrains progression from PTC to ATC. Gene expression and immunohistochemical analyses of murine tumors identified the cardinal features of human ATC including loss of differentiation, local invasion, distant metastasis, and rapid lethality. We used small-animal ultrasound imaging to monitor autochthonous tumors and showed that treatment with the selective BRAF inhibitor PLX4720 improved survival but did not lead to tumor regression or suppress signaling through the MAPK pathway. The combination of PLX4720 and the mapk/Erk kinase (MEK) inhibitor PD0325901 more completely suppressed MAPK pathway activation in mouse and human ATC cell lines and improved the structural response and survival of ATC-bearing animals. This model expands the limited repertoire of autochthonous models of clinically aggressive thyroid cancer, and these data suggest that small-molecule MAPK pathway inhibitors hold clinical promise in the treatment of advanced thyroid carcinoma.

Late Intervention with anti-BRAFV600E Therapy Induces Tumor Regression in an Orthotopic Mouse Model of Human Anaplastic Thyroid Cancer

Human anaplastic thyroid cancer (ATC) is a lethal disease with an advanced clinical presentation and median survival of 3 months. The BRAF(V600E) oncoprotein is a potent transforming factor that causes human thyroid cancer cell progression in vitro and in vivo; therefore, we sought to target this oncoprotein in a late intervention model of ATC in vivo. We used the human ATC cell line 8505c, which harbors the BRAF(V600E) and TP53(R248G) mutations. Immunocompromised mice were randomized to receive the selective anti-BRAF(V600E) inhibitor, PLX4720, or vehicle by oral gavage 28 d after tumor implantation, 1 wk before all animals typically die due to widespread metastatic lung disease and neck compressive symptoms in this model. Mice were euthanized weekly to evaluate tumor volume and metastases. Control mice showed progressive tumor growth and lung metastases by 35 d after tumor implantation. At that time, all control mice had large tumors, were cachectic, and were euthanized due to their tumor-related weight loss. PLX4720-treated mice, however, showed a significant decrease in tumor volume and lung metastases in addition to a reversal of tumor-related weight loss. Mouse survival was extended to 49 d in PLX4720-treated animals. PLX4720 treatment inhibited cell cycle progression from 28 d to 49 d in vivo. PLX4720 induces striking tumor regression and reversal of cachexia in an in vivo model of advanced thyroid cancer that harbors the BRAF(V600E) mutation.

Steroid Receptor Coactivator-1 Deficiency Causes Variable Alterations in the Modulation of T3-Regulated Transcription of Genes in Vivo

Thyroid hormone exerts its biological effect by binding to a TR. Both liganded and unliganded TRs regulate the transcription of T3-responsive genes. Cofactors with activating or repressing function modulate the transcriptional regulation by TRs. We showed that steroid receptor coactivator 1 (SRC-1)-deficient mice (SRC-1−/−) exhibit partial resistance to thyroid hormone at the level of the pituitary thyrotrophs. To determine whether SRC-1 deficiency affects globally T3-dependent transcriptional regulation, we studied the effects of thyroid hormone deprivation and replacement on the expression of several genes in different tissues of SRC-1−/− and wild-type mice (SRC-1+/+). Thyroid hormone deficiency was induced by a low iodine diet (LoI) supplemented with propylthiouracil (PTU) for 2 wk. l-T3 was injected ip for the last 4 d in one group (PTU+T3 group), and another group (PTU group) received only vehicle. Levels of mRNAs for T3-responsive genes were determined by Northern blotting: GH and TSHβ in pituitary;...