Distinct roles of PIK3CA in the enrichment and maintenance of cancer stem cells in head and neck squamous cell carcinoma.

Recurrence and metastasis are the major causes of mortality in head and neck squamous cell carcinoma (HNSCC). It is suggested that cancer stem cells (CSCs) play pivotal roles in recurrence and metastasis. Thus, a greater understanding of the mechanisms of CSC regulation may provide opportunities to develop novel therapies for improving survival by controlling recurrence or metastasis. Here, we report that overexpression of the gene encoding the catalytic subunit of PI3K (PIK3CA), the most frequently amplified oncogene in HNSCC, promotes epithelial-to-mesenchymal transition and enriches the CSC population. However, PIK3CA is not required to maintain these traits and inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway paradoxically promotes CSC population. Molecular analysis revealed that overexpression of PIK3CA activates multiple receptor tyrosine kinases (RTKs), in which ephrin receptors (Ephs), tropomyosin receptor kinases (TRK) and mast/stem cell growth factor receptor (c-Kit) contribute to maintain CSC population. Accordingly, simultaneous inhibition of these RTKs using a multi-kinase inhibitor ponatinib has a superior effect at eliminating the CSC population and reduces metastasis of PIK3CA-overexpressing HNSCC cells. Our result suggests that co-targeting of Ephs, TRKs and the c-Kit pathway may be effective at eliminating the PI3K-independent CSC population, thereby providing potential targets for future development of a novel anti-CSC therapeutic approach for HNSCC patients, particularly for patients with PIK3CA amplification.

Bilateral Ear Canal Cholesteatoma with Underlying Type I First Branchial Cleft Anomalies.

OBJECTIVES:: To describe a case of bilateral ear canal cholesteatomas in the setting of underlying first branchial cleft cyst anomalies and to review the pathophysiology underlying the development of external auditory canal cholesteatomas from branchial cleft cyst abnormalities. METHODS AND RESULTS:: We present a case study of a 61-year-old man who presented with chronic right-sided hearing loss and left-sided postauricular drainage. Clinical evaluation, radiographic work-up, and pathologic analysis confirmed a diagnosis of bilateral ear canal cholesteatoma in the setting of underlying first branchial cleft cyst anomalies. The patient's clinical course, surgical treatment, and management considerations are discussed here. CONCLUSION:: Ear canal cholesteatoma represents a rare clinical disease entity deserving a thorough initial assessment. Careful consideration of underlying diseases that result in chronic inflammation, such as branchial cleft lesions, should be included in the differential diagnosis of idiopathic canal cholesteatoma in the absence of prior otologic surgery or trauma.

Cooperation Between Pten and Smad4 in Murine Salivary Gland Tumor Formation and Progression.

Salivary gland tumor (SGT) is a rare tumor type, which exhibits broad-spectrum phenotypic, biological, and clinical heterogeneity. Currently, the molecular mechanisms that cause SGT pathogenesis remain poorly understood. A lack of animal models that faithfully recapitulate the naturally occurring process of human SGTs has hampered research progress on this field. In this report, we developed an inducible keratin 5-driven conditional knockout mouse model to delete gene(s) of interest in murine salivary gland upon local RU486 delivery. We have deleted two major tumor suppressors, Pten, a negative regulator of the PI3K pathway, and Smad4, the central signaling mediator of TGFß pathway, in the murine salivary gland. Our results have shown that deletion of either Pten or Smad4 in murine salivary gland resulted in pleomorphic adenomas, the most common tumor in human SGT patients. Deletion of both Pten and Smad4 in murine salivary gland developed several malignancies, with salivary adenoid cystic carcinoma (SACC) being the most frequently seen. Molecular characterization showed that SACC exhibited mTOR activation and TGFß1 overexpression. Examination of human SGT clinical samples revealed that loss of Pten and Smad4 is common in human SACC samples, particularly in the most aggressive solid form, and is correlated with survival of SACC patients, highlighting the human relevance of the murine models. In summary, our results offer significant insight into synergistic role of Pten and Smad4 in SGT, providing a rationale for targeting mTOR and/or TGFß signaling to control SGT formation and progression.

Granular cell tumor presenting in the scrotum of a pediatric patient: a case report and review of the literature.

BACKGROUND: Granular cell tumors are neoplasms of Schwann cell origin. They typically arise in the head and neck of adults, with the tongue being the most common location; granular cell tumors of male genitalia are exceedingly rare. We identified only eight prior cases of scrotal granular cell tumor in the literature, and only one was in a child. Herein, we report a second case of childhood scrotal granular cell tumor and provide a review of the most relevant literature. CASE PRESENTATION: A fifteen-year-old hispanic boy was referred to our hospital's pediatric surgery service for a painless and firm scrotal mass. Clinical impression was that of an epidermal inclusion cyst. There was no evidence of associated medical problems from the clinical history and physical examination. Surgical enucleation of the lesion demonstrated a solid nodule with morphological and immunohistochemical features consistent with a benign granular cell tumor. CONCLUSIONS: This is the second case reported of a scrotal granular cell tumor in a child. Although genital granular cell tumors are rare, and most are benign, careful clinical examination, complete surgical excision, expert histologic evaluation, and a close follow-up are recommended for accurate diagnosis and to rule out eventual malignancy.

Radiation-induced dedifferentiated chondrosarcoma with orbital invasion.

PURPOSE: To report 2 interventional cases of dedifferentiated chondrosarcoma with orbital involvement after radiotherapy performed in childhood and to review the literature on chondrosarcoma in the orbit following radiation treatment. METHODS: Retrospective analysis of medical records of 2 patients with chondrosarcoma of the orbits with review of the literature. RESULTS: The first patient developed chondrosarcoma of the orbital and maxillary sinus 36 years after external beam radiation therapy to the OS to treat retinoblastoma. The second patient developed a large orbital chondrosarcoma 35 years after external beam radiation therapy in the treatment of craniofacial fibrous dysplasia. CONCLUSIONS: These cases highlight the risk of secondary chondrosarcoma in patients following radiotherapy and the importance of lifetime monitoring.

Male primary retroperitoneal mucinous cystadenoma.

Primary retroperitoneal mucinous cystadenoma of borderline malignancy is a rare disease, especially in male patients. Often these tumors are not incidentally found due to abdominal symptoms. We present the radiologic abdominal computed tomography scan, surgical, and pathologic images of this unique, rare condition in a male patient. Surgical treatment is recommended to establish diagnosis and treatment.

Colliding tumor of the paranasal sinus.

Sinonasal malignant neoplasms comprise only 3% of all head and neck malignancies. Synchronous and metachronous tumors of the head and neck have been described, but rarely have there been reports of a single tumor with two distinct histologies. Here, we describe a case of a sinonasal malignant neoplasm with two distinct histologies. A case report and literature review was performed. We present a case of paranasal sinus neoplasm involving two malignant cell types. An 83-year-old woman presented with a 2-year history of symptoms suggestive of chronic sinusitis, which included nasal congestion and intermittent midface pressure. More recently, her symptoms progressed with the development of left-side epistaxis and she was found to have a mass in the left maxillary and ethmoid regions. A biopsy of the maxillary sinus mass revealed a moderately differentiated squamous cell carcinoma (SCC). She underwent complete resection of the lesion through an extended endoscopic approach. Final pathological analysis showed a malignant neoplasm with two distinct malignant morphologies; a moderately differentiated SCC and small cell neuroendocrine carcinoma. Appropriate diagnosis and treatment of head and neck malignancy depends on accurate tumor classification and staging. We present a case of a sinonasal tumor with two distinct malignant entities and review the available literature on the subject. Additionally, we discuss the etiologic theories and challenges in planning the optimal approach to management in this scenario.

A patient tumor transplant model of squamous cell cancer identifies PI3K inhibitors as candidate therapeutics in defined molecular bins.

Targeted therapy development in head and neck squamous cell carcinoma (HNSCC) is challenging given the rarity of activating mutations. Additionally, HNSCC incidence is increasing related to human papillomavirus (HPV). We sought to develop an in vivo model derived from patients reflecting the evolving HNSCC epidemiologic landscape, and use it to identify new therapies. Primary and relapsed tumors from HNSCC patients, both HPV+ and HPV-, were implanted on mice, giving rise to 25 strains. Resulting xenografts were characterized by detecting key mutations, measuring protein expression by IHC and gene expression/pathway analysis by mRNA-sequencing. Drug efficacy studies were run with representative xenografts using the approved drug cetuximab as well as the new PI3K inhibitor PX-866. Tumors maintained their original morphology, genetic profiles and drug susceptibilities through serial passaging. The genetic makeup of these tumors was consistent with known frequencies of TP53, PI3KCA, NOTCH1 and NOTCH2 mutations. Because the EGFR inhibitor cetuximab is a standard HNSCC therapy, we tested its efficacy and observed a wide spectrum of efficacy. Cetuximab-resistant strains had higher PI3K/Akt pathway gene expression and protein activation than cetuximab-sensitive strains. The PI3K inhibitor PX-866 had anti-tumor efficacy in HNSCC models with PIK3CA alterations. Finally, PI3K inhibition was effective in two cases with NOTCH1 inactivating mutations. In summary, we have developed an HNSCC model covering its clinical spectrum whose major genetic alterations and susceptibility to anticancer agents represent contemporary HNSCC. This model enables to prospectively test therapeutic-oriented hypotheses leading to personalized medicine.

Programmed death-1+ T cells and regulatory T cells are enriched in tumor-involved lymph nodes and associated with aggressive features in papillary thyroid cancer.

CONTEXT: Recurrent metastatic lymph node (LN) disease is common in patients with papillary thyroid cancer (PTC). Novel prognostic markers may be helpful in guiding a therapeutic approach. Our previous studies revealed that immune suppression is evident in PTC and associated with more severe disease. OBJECTIVE: To characterize the immune response to metastatic PTC, we assessed CD4(+) T cell polarization in LN. In addition, we investigated the role of programmed death-1 (PD-1) and T cell exhaustion. DESIGN: Uninvolved (UILN) and tumor-involved lymph nodes (TILN) were sampled ex vivo by fine-needle biopsy. T cell subsets were identified by flow cytometry. In parallel, archived TILN specimens were characterized by immunofluorescence. SETTING: The study was conducted at the University of Colorado Hospital. PATIENTS: Data were collected on 94 LN from 19 patients with PTC undergoing neck dissection. MAIN OUTCOME: T cell subset frequencies were compared in UILN and TILN and assessed for correlation with recurrent disease and extranodal invasion. RESULTS: Regulatory CD4(+) T cells (Treg) were enriched in TILN compared with UILN and further elevated in TILN from patients with recurrent disease. PD-1(+) T cells were present at high frequency in TILN and markedly enriched in TILN that showed evidence of extranodal invasion. In TILN, Treg frequency correlated with PD-1(+) T cell frequencies. Although PD-1(+) T cells produced interferon-γ, they failed to fully down-regulate CD27 and were not actively proliferating. CONCLUSIONS: Increased Treg and PD-1(+) T cell frequencies in LN may be indicative of aggressive recurrent PTC. Future prospective studies are necessary to determine the prognostic and therapeutic value of these findings in PTC.

Prospective, randomized study of conventional versus HybridKnife endoscopic submucosal dissection methods for the esophagus: an animal study.

BACKGROUND: Endoscopic submucosal dissection (ESD) increases en bloc and histologically complete resection rate of neoplastic mucosal tumors but is technically more demanding than EMR. Limited data are available comparing the efficacy and safety of a new ESD designed to overcome these limitations and conventional ESD (C-ESD) techniques. OBJECTIVE: To compare the safety, efficacy, and operation time of the new HybridKnife ESD (HK-ESD) with C-ESD in the esophagus. DESIGN: Prospective, randomized, controlled study. SETTING: Animal research laboratory. SUBJECTS: Seventeen anesthetized Yorkshire pigs. INTERVENTIONS: Removal of a 4-cm length of half-circumference esophageal mucosa by C-ESD with Hook knife or Flexknife versus HK-ESD. MAIN OUTCOME MEASUREMENTS: Procedure time, en bloc and complete resection rate, and complications (bleeding and perforation). RESULTS: All resections were completed en bloc. Procedure time was shorter in C-ESD. However, it was similar after 12 procedures. Significantly more bleeding occurred during C-ESD (28 vs 12, P = .0007). Histological muscularis propria injuries occurred with equal frequency (16 vs 17) and were mostly seen during the first 11 procedures. There were 3 perforations (2 endoscopic, 1 histological), all with C-ESD. LIMITATIONS: Nonsurvival study, use of 2 conventional knives, no training period for a new procedure. CONCLUSIONS: The HK-ESD technique was equally effective as the C-ESD technique for successful en bloc resection and was safer with less bleeding and perforation. Although procedure time was longer in HK-ESD, the difference became nonsignificant after 12 procedures.

Vesiculobullous eruptions of the oral cavity.

The spectrum of vesiculobullous eruptions of the oral cavity is wide and rich, with different disease entities that encompass different etiologies, pathogenesis, clinical manifestations, treatment plans, and prognostic ends. Trying to present all these entities in a comprehensive fashion is challenging, but in this article, most of the important entities pertaining to this topic have been encompassed in a concise manner.

Tumor-associated lymphocytes and increased FoxP3+ regulatory T cell frequency correlate with more aggressive papillary thyroid cancer.

CONTEXT: Ten to 30% of patients with papillary thyroid cancer (PTC) develop recurrent disease and may benefit from innovative adjuvant therapies. Immune-based therapies are under investigation to treat many types of cancer. The role of the immune system in PTC is poorly understood. OBJECTIVE: We investigated whether tumor-associated lymphocytes (TAL), in the absence of background thyroiditis (LT), contribute to disease severity. We hypothesized that the type of lymphocytes associated with PTC would correlate with parameters of disease. DESIGN: This retrospective study analyzed archived PTC samples for the presence of TAL and/or LT. A group of patients with TAL was evaluated for lymphocyte subsets by immunohistofluorescence. PATIENTS AND SETTING: One hundred PTC patients were analyzed for LT and TAL, and 10 PTC patients with TAL were assessed for lymphocyte subsets at University of Colorado Hospital. MAIN OUTCOME: We assessed correlations between disease and the presence of TAL, LT, and lymphocyte subset frequency. RESULTS: Patients with TAL exhibited higher disease stage and increased incidence of invasion and lymph node metastasis compared with patients without lymphocytes or with LT. CD4(+) T cell frequency correlated with tumor size (r = 0.742; P = 0.017). FoxP3(+) regulatory T cell (Treg) frequency correlated with lymph node metastases (r = 0.858; P = 0.002), and CD8 to Treg ratio correlated inversely with tumor size (r = -0.804; P = 0.007). CONCLUSIONS: TAL and high Treg frequency in primary thyroid tumors correlates with more aggressive disease. Future prospective studies may identify Treg frequency as a predictive factor in PTC, and the suppressive effects of Treg should be considered in the design of immune-based therapies.

Retinoid and thiazolidinedione therapies in melanoma: an analysis of differential response based on nuclear hormone receptor expression.

BACKGROUND: Metastatic melanoma has a high mortality rate and suboptimal therapeutic options. Molecular targeting may be beneficial using the rexinoid LGD1069, a retinoid x receptor selective agonist, and thiazolidinediones (TZD), PPARgamma selective ligands, as novel treatments. RESULTS: Mouse xenograft models with human melanoma cell lines [A375(DRO) or M14(5-16)] were treated for 4 weeks with daily vehicle, RXR agonist (rexinoid, LGD1069, 30 mg/kg/d), PPARgamma agonist (TZD, rosiglitazone, 10 mg/kg/d) or combination. A375(DRO) tumor growth was significantly inhibited by either ligand alone and the combination had an additive effect. M14(5-16) tumors only responded to LGD1069 100 mg/kg/day. A375(DRO) sublines resistant to rexinoid, TZD and combination were generated and all three sublines had reduced PPARgamma expression but preserved RXR expression. shRNA knockdown of PPARgamma or RXRgamma attenuated the rexinoid, TZD and combination ligand-mediated decreased proliferation in A375(DRO) cells. Rexinoid (LGD1069) and retinoid (TTNPB) treatment of M14(5-16) cells resulted in decreased proliferation that was additive with combination of both rexinoid and retinoid. shRNA knockdown of RXRgamma resulted in a decreased response to either ligand. CONCLUSION: A375 (DRO) melanoma cell growth is inhibited by rexinoid and TZD treatment, and this response is dependent on RXR and PPARgamma receptor expression. M14 (5-16) melanoma cell growth is inhibited by rexinoid and retinoid treatment, and this response is dependent on RXR expression. These findings may help guide molecular-based treatment strategies in melanoma and provide insight for mechanisms of resistance to nuclear receptor targeted therapies in certain cancers.

Suppression of radiation-induced salivary gland dysfunction by IGF-1.

BACKGROUND: Radiation is a primary or secondary therapeutic modality for treatment of head and neck cancer. A common side effect of irradiation to the neck and neck region is xerostomia caused by salivary gland dysfunction. Approximately 40,000 new cases of xerostomia result from radiation treatment in the United States each year. The ensuing salivary gland hypofunction results in significant morbidity and diminishes the effectiveness of anti-cancer therapies as well as the quality of life for these patients. Previous studies in a rat model have shown no correlation between induction of apoptosis in the salivary gland and either the immediate or chronic decrease in salivary function following gamma-radiation treatment. METHODOLOGY/PRINCIPAL FINDING: A significant level of apoptosis can be detected in the salivary glands of FVB mice following gamma-radiation treatment of the head and neck and this apoptosis is suppressed in transgenic mice expressing an activated mutant of Akt (myr-Akt1). Importantly, this suppression of apoptosis in myr-Akt1 mice preserves salivary function, as measured by saliva output, three and thirty days after gamma-radiation treatment. In order to translate these studies into a preclinal model we found that intravenous injection of IGF1 stimulated activation of endogenous Akt in the salivary glands in vivo. A single injection of IGF1 prior to exposure to gamma-radiation diminishes salivary acinar cell apoptosis and completely preserves salivary gland function three and thirty days following irradiation. CONCLUSIONS/SIGNIFICANCE: These studies suggest that apoptosis of salivary acinar cells underlies salivary gland hypofunction occurring secondary to radiation of the head and neck region. Targeted delivery of IGF1 to the salivary gland of patients receiving head and neck irradiation may be useful in reducing or eliminating xerostomia and restoring quality of life to these patients.

Bilateral multinodular oncocytomas of the parotid arising in a background of bilateral oncocytic nodular hyperplasia.

Oncocytic tumors of the major salivary glands are rare, accounting for less than 1% of all salivary gland tumors. When they do occur, these neoplasms typically present as solitary nodules that affect only one major salivary gland, usually the parotid. Multiple bilateral multinodular tumors are rare. We report the case of a 53-year-old woman with a case of synchronous multiple bilateral multinodular oncocytomas that arose in a background of bilateral oncocytic nodular hyperplasia in the parotid glands. The patient underwent superficial parotid resections, and at the 4-year follow-up, she exhibited no evidence of recurrence.

Estrogen and progesterone receptor and p53 gene expression in adenoid cystic cancer.

OBJECTIVES: The current study examined the role of estrogen receptors (ER), progesterone receptors (PR) and p53 expression in adenoid cystic carcinoma (ACC) to determine if simple expression or possible overexpression of these products might influence the development and natural course of this cancer. STUDY DESIGN: ER and PR status and p53 overexpression were retrospectively evaluated utilizing immunohistochemical evaluation of 47 ACC specimens. METHODS: Formalin-fixed paraffin-embedded tissues from 47 ACC specimens and 47 samples of normal salivary gland tissue were evaluated histochemically for the presence of ER, PR and p53. Immunoreactivity was scored using a 0 to +3 scale in which staining was either (0) negative, (+1) spotty, (+2) weakly positive, or (+3) strongly positive. RESULTS: ER was expressed in 8 of 47 tumors while PR was expressed in 4 of 47 tumors. p53 aberrations were demonstrated in 26 of 47 tumors. Tumors showed varying degrees of immunopositivity ranging from 0 to +3. CONCLUSIONS: These studies suggest that p53 aberrations may be involved in ACC tumor progression and that ER and PR may play a role in ACC development.

Phase I trial of gefitinib in combination with radiation or chemoradiation for patients with locally advanced squamous cell head and neck cancer.

PURPOSE: To establish the safety and toxicity profile of daily gefitinib with radiation alone or with concurrent chemoradiotherapy in previously untreated patients with locally advanced squamous cell head and neck cancer (LAHNC). PATIENTS AND METHODS: Patients with intermediate-stage LAHNC were treated with concomitant boost radiation (RT) alone with escalating doses of daily gefitinib (250 or 500 mg; cohort I). Once a safety profile was determined with RT alone, patients with high-risk disease were then treated with daily gefitinib (250 or 500 mg), weekly cisplatin (CDDP; 30 mg/m2), and once-daily RT (cohort II). Patients also received post-RT gefitinib at 250 mg daily for a period of up to 2 years. RESULTS: Twenty-three patients were enrolled and assessable for toxicity. No dose-limiting toxicities (DLTs) were observed in patients treated in cohort I at either 250 or 500 mg of gefitinib daily with concomitant boost RT to 72 Gy. In patients receiving chemoradiotherapy and gefitinib (cohort II), DLTs included one grade 4 diarrhea and one grade 4 neutropenic fever. Fifteen patients started maintenance gefitinib, and eight (53%) experienced grade 1 to 2 acne-like skin rash and diarrhea, but no grade 3 or 4 toxicity occurred. CONCLUSION: Gefitinib (250 or 500 mg daily) was well tolerated with concomitant boost RT or concurrent chemoradiotherapy with weekly CDDP. Protracted administration of gefitinib for up to 2 years at 250 mg daily was also tolerated well.

Overexpression of cyclin D1 and cortactin is primarily independent of gene amplification in salivary gland adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) of the salivary glands exhibits persistent growth, invasion and metastasis. Chromosome 11q13 amplification is a frequent event associated with tumor progression in a number of carcinomas and is associated with poor prognosis. Two genes within the 11q13 amplicon that are overexpressed as a result of 11q13 amplification are the cell cycle regulatory protein cyclin D1 (CCND1) and cortactin (CTTN), a protein involved cell motility and invasion. To determine the expression and gene status of cyclin D1 and cortactin in ACC, we evaluated 39 ACC cases by immunohistochemistry (IHC) for cyclin D1 and cortactin expression. Amplification of CCND1 and CTTN was determined by fluorescent in situ hybridization (FISH). Cyclin D1 overexpression was present in 90% (35/39) and cortactin expression in 62% (24/39) of evaluated cases, although CCND1 and CTTN levels were elevated in only two cases (5%) as determined by FISH. Our results indicate that chromosome 11q13 amplification is uncommon in ACC, but that cyclin D1 and cortactin are frequently overexpressed and may therefore contribute to the growth and invasive potential of ACC.

Malignant hidradenoma of the medial canthus with orbital and intracranial extension.

A 70-year-old woman presented with signs and symptoms consistent with nasolacrimal duct obstruction. MRI revealed a medial canthal mass with orbital and intracranial extension. The patient was treated with wide surgical excision, chemotherapy, and radiation. A diagnosis of malignant hidradenoma of the medial canthus was confirmed by histopathology. Despite aggressive therapy, the patient developed metastatic disease and died within 1 year.