Joseph I. Helman

Endothelial Cell-Initiated Signaling Promotes the Survival and Self-Renewal of Cancer Stem Cells

Recent studies have demonstrated that cancer stem cells play an important role in the pathobiology of head and neck squamous cell carcinomas (HNSCC). However, little is known about functional interactions between head and neck cancer stem-like cells (CSC) and surrounding stromal cells. Here, we used aldehyde dehydrogenase activity and CD44 expression to sort putative stem cells from primary human HNSCC. Implantation of 1,000 CSC (ALDH+CD44+Lin-) led to tumors in 13 (out of 15) mice, whereas 10,000 noncancer stem cells (ALDH-CD44-Lin-) resulted in 2 tumors in 15 mice. These data demonstrated that ALDH and CD44 select a subpopulation of cells that are highly tumorigenic. The ability to self-renew was confirmed by the observation that ALDH+CD44+Lin- cells sorted from human HNSCC formed more spheroids (orospheres) in 3-D agarose matrices or ultra-low attachment plates than controls and were serially passaged in vivo. We observed that approximately 80% of the CSC were located in close proximity (within 100-μm radius) of blood vessels in human tumors, suggesting the existence of perivascular niches in HNSCC. In vitro studies demonstrated that endothelial cell-secreted factors promoted self-renewal of CSC, as demonstrated by the upregulation of Bmi-1 expression and the increase in the number of orospheres as compared with controls. Notably, selective ablation of tumor-associated endothelial cells stably transduced with a caspase-based artificial death switch (iCaspase-9) caused a marked reduction in the fraction of CSC in xenograft tumors. Collectively, these findings indicate that endothelial cell-initiated signaling can enhance the survival and self-renewal of head and neck CSC.

Pilot Randomized Phase II Study of Celecoxib in Oral Premalignant Lesions

Purpose: Cyclooxygenase-2 (COX-2)–specific inhibition suppresses carcinogenesis in preclinical models and is a promising strategy for preventing oral cancer. In this pilot randomized phase II study, we evaluated the efficacy and safety of the COX-2 inhibitor celecoxib in patients with oral premalignant lesions (OPL). Experimental Design: Patients were randomly assigned to placebo (n = 18), celecoxib 100 mg twice daily (n = 17), or celecoxib 200 mg twice daily (n = 15) for 12 weeks. Six additional patients received celecoxib (400 mg twice daily) in an unblinded extension of the study. Biopsies were obtained at baseline and week 12. All patients entering the study were required to have at least one histologically confirmed early (atypical hyperplasia, atypical hyperkeratosis, or mild dysplasia) or advanced (moderate to severe dysplasia) OPL. Results: Forty-nine patients (46 of 50 randomized and 3 of 6 open label) were evaluable for efficacy analyses. There were no statistically significant differences between the response rates of the randomly assigned arms: placebo, 33.3% (6 of 18); celecoxib 100 mg twice daily, 41.2% (7 of 17); and celecoxib 200 mg twice daily, 20.0% (3 of 15). Two patients responded on celecoxib 400 mg twice daily. Celecoxib was generally well tolerated. Patients with higher baseline COX-2 mRNA levels had an increased risk of disease progression within 3 months. Conclusions: Celecoxib at 100 or 200 mg twice daily was ineffective in controlling OPLs in this randomized controlled trial. This result and cardiovascular toxicity results of other (large scale) randomized controlled trials of selective COX-2 inhibitors have discouraged the continued investigation of these agents in oral cancer chemoprevention. Better methods for identifying high-risk patients and more active interventions are needed for future oral cancer chemoprevention trials.

Cisplatin Induces Bmi-1 and Enhances the Stem Cell Fraction in Head and Neck Cancer

Recent evidence has unveiled a subpopulation of highly tumorigenic, multipotent cells capable of self-renewal in head and neck squamous cell carcinomas (HNSCCs). These unique cells, named here cancer stem cells (CSCs), proliferate slowly and might be involved in resistance to conventional chemotherapy. We have shown that CSCs are found in perivascular niches and rely on endothelial cell-secreted factors [particularly interleukin-6 (IL-6)] for their survival and self-renewal in HNSCC. Here, we hypothesized that cisplatin enhances the stem cell fraction in HNSCC. To address this hypothesis, we generated xenograft HNSCC tumors with University of Michigan-squamous cell carcinoma 22B (UM-SCC-22B) cells and observed that cisplatin treatment increased (P = .0013) the fraction of CSCs [i.e., aldehyde dehydrogenase activity high and cluster of differentiation 44 high (ALDH(high)CD44(high))]. Cisplatin promoted self-renewal and survival of CSCs in vitro, as seen by an increase in the number of orospheres in ultralow attachment plates and induction in B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) and octamer-binding transcription factor 4 expression. Cisplatin-resistant cells expressed more Bmi-1 than cisplatin-sensitive cells. IL-6 potentiated cisplatin-induced orosphere formation generated when primary human HNSCC cells were sorted for ALDH(high)CD44(high) immediately after surgery and plated onto ultralow attachment plates. IL-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation (indicative of stemness) was unaffected by treatment with cisplatin in UM-SCC-22B cells, whereas IL-6-induced extracellular signal-regulated kinase (ERK) phosphorylation (indicative of differentiation processes) was partially inhibited by cisplatin. Notably, cisplatin-induced Bmi-1 was inhibited by interleukin-6 receptor blockade in parental and cisplatin-resistant cells. Taken together, these results demonstrate that cisplatin enhances the fraction of CSCs and suggest a mechanism for resistance to cisplatin therapy in head and neck cancer.

Bisphosphonate-related Osteonecrosis of the Jaw: A Pictorial Review

Bisphosphonate-related osteonecrosis of the jaw (ONJ) is characterized by nonhealing exposed bone in the maxillofacial region in patients who have undergone bisphosphonate treatment. The underlying etiology is unclear and may be multifactorial. The diagnosis is primarily clinical. Diagnostic tissue sampling may exacerbate the process and is typically avoided, necessitating other diagnostic approaches. The appearance of ONJ at diagnostic imaging is variable and includes sclerotic, lytic, or mixed lesions with possible periosteal reaction, pathologic fractures, and extension to soft tissues. There is a spectrum of signal intensity changes on T1- and T2-weighted magnetic resonance (MR) images with variable enhancement, findings that may correspond to the clinical and histopathologic stage of the process. Bone scintigraphy is sensitive with increased uptake in the area of the lesion. Although the imaging findings are nonspecific, there appears to be a role for imaging in the management of ONJ. Radiography is relatively insensitive but typically employed as the first line of radiologic investigation. Computed tomography and MR imaging are more precise in demonstrating the extent of the lesion. A number of imaging modalities have revealed lesions that may be associated with bisphosphonate exposure in asymptomatic individuals or in the context of nonspecific symptoms. The risk of these lesions advancing to overt clinical disease is unknown at this time. The radiologist should be aware of ONJ and include it in the differential diagnosis when evaluating patients with a history of bisphosphonate therapy without jaw irradiation, so as to avoid potentially harmful biopsies.

Endothelial interleukin-6 defines the tumorigenic potential of primary human cancer stem cells.

Head and neck squamous cell carcinomas (HNSCC) contain a small subpopulation of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self‐renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)−6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of Interleukin‐6 (IL‐6), we observed a direct correlation between IL‐6 levels in tumor‐associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell‐IL‐6 enhanced orosphere formation, p‐STAT3 activation, survival, and self‐renewal of human CSC. Notably, a humanized anti‐IL‐6R antibody (tocilizumab) inhibited primary human CSC‐mediated tumor initiation. Collectively, these data demonstrate that endothelial cell‐secreted IL‐6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL‐6/IL‐6R signaling. Stem Cells 2014;32:2845–2857

Characterization of tumorigenic cell lines from the recurrence and lymph node metastasis of a human salivary mucoepidermoid carcinoma

UNLABELLED The long-term outcome of patients with mucoepidermoid carcinoma is poor. Limited availability of cell lines and lack of xenograft models is considered a major barrier to improved mechanistic understanding of this disease and development of effective therapies. OBJECTIVE To generate and characterize human mucoepidermoid carcinoma cell lines and xenograft models suitable for mechanistic and translational studies. METHODS Five human mucoepidermoid carcinoma specimens were available for generation of cell lines. Cell line tumorigenic potential was assessed by transplantation and serial in vivo passaging in immunodeficient mice, and cell line authenticity verified by short tandem repeat (STR) profiling. RESULTS A unique pair of mucoepidermoid carcinoma cell lines was established from a local recurrence (UM-HMC-3A) and from the metastatic lymph node (UM-HMC-3B) of the same patient, 4 years after surgical removal of the primary tumor. These cell lines retained epithelial-like morphology through 100 passages in vitro, contain the Crtc1-Maml2 fusion oncogene (characteristic of mucoepidermoid carcinomas), and express the prototypic target of this fusion (NR4A2). Both cell lines generated xenograft tumors when transplanted into immunodeficient mice. Notably, the xenografts exhibited histological features and Periodic Acid Schiff (PAS) staining patterns that closely resembled those found in human tumors. STR profiling confirmed the origin and authenticity of these cell lines. CONCLUSION These data demonstrate the generation and characterization of a pair of tumorigenic salivary mucoepidermoid carcinoma cell lines representative of recurrence and lymph node metastasis. Such models are useful for mechanistic and translational studies that might contribute to the discovery of new therapies for mucoepidermoid carcinoma.

Clinical application of the temporoparietal-galeal flap in closure of a chronic oronasal fistula: review of the anatomy, surgical technique, and report of a case.

The reconstructing surgeon has a variety of options when closing defects of the hard and soft palate. Techniques can be as simple as a palatal island flap or as complex as the radial forearm flap. We present here a method of closing a medium to large defect of the palate using the temporoparietal-galeal (TPG) flap, a regional and very reliable flap for reconstructing defects of the oral cavity and face. The TPG is a thin and pliable flap with an axial blood supply and a good arc of rotation to the oral cavity. The flap can be used as a fascial flap, a fasciocutaneous flap, or an osteofascial flap. It provides an excellent source of vascularized tissue. Anatomy

Effect of erlotinib on epidermal growth factor receptor and downstream signaling in oral cavity squamous cell carcinoma

The purpose of this study was to determine if there are differences in biomarker modulation and epidermal growth factor receptor (EGFR) degradation between the tumor and the normal mucosa after treatment with an EGFR inhibitor, erlotinib, in head and neck cancer.

ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas

A small sub-population of cells characterized by increased tumorigenic potential, ability to self-renew and to differentiate into cells that make up the tumor bulk, has been characterized in some (but not all) tumor types. These unique cells, namedcancer stem cells, are considered drivers of tumor progression in these tumors. The purpose of this work is to understand if cancer stem cells play a functional role in the tumorigenesis of salivary gland mucoepidermoid carcinomas. Here, we investigated the expression of putative cancer stem cell markers (ALDH, CD10, CD24, CD44) in primary human mucoepidermoid carcinomas by immunofluorescence, in vitro salisphere assays, and in vivo tumorigenicity assays in immunodeficient mice. Human mucoepidermoid carcinoma cells (UM-HMC-1, UM-HMC-3A, UM-HMC-3B) sorted for high levels of ALDH activity and CD44 expression (ALDHhighCD44high) consistently formed primary and secondary salispheres in vitro, and showed enhanced tumorigenic potential in vivo (defined as time to tumor palpability, tumor growth after palpability), when compared to ALDHlowCD44low cells. Cells sorted for CD10/CD24, and CD10/CD44 showed varying trends of salisphere formation, but consistently low in vivo tumorigenic potential. And finally, cells sorted for CD44/CD24 showed inconsistent results in salisphere formation and tumorigenic potential assays when different cell lines were evaluated. Collectively, these data demonstrate that salivary gland mucoepidermoid carcinomas contain a small population of cancer stem cells with enhanced tumorigenic potential and that are characterized by high ALDH activity and CD44 expression. These results suggest that patients with mucoepidermoid carcinoma might benefit from therapies that ablate these highly tumorigenic cells.

Current Management Strategies for Verrucous Hyperkeratosis and Verrucous Carcinoma

Proliferative verrucous leukoplakia, a lesion of unknown origin with no strictly defined diagnostic criteria, is worthy of great clinical concern and scrutiny. Treatment options vary, with no consensus regarding the most efficient and effective strategy. Further complicating the unusual biology and behavior of this lesion is its usual multifocal presentation and progression, with lifelong vigilance required after treatment. Verrucous carcinoma is a progressive lesion with high recurrence and 5-year survival rates. Surgery remains the preferred treatment. Further investigation into the combination of surgery and antiviral agents may bring additional improvement in patient care.