David Y. Zhang

Human papillomavirus may be common within nasopharyngeal carcinoma of Caucasian Americans: investigation of Epstein-Barr virus and human papillomavirus in eastern and western nasopharyngeal carcinoma using ligation-dependent polymerase chain reaction.

Nasopharyngeal carcinoma (NPC), particularly those tumors endemic to the Far East, commonly harbor Epstein‐Barr virus (EBV), thought to serve as an important oncogenic promoter. Human papillomavirus (HPV) is associated with a proportion of upper aerodigestive tract carcinomas. We hypothesized that HPV might also contribute to the pathogenesis of NPC, and we queried whether geographic and racial distinctions may be identified between NPC of the Far East versus those diagnosed in Caucasian American patients with regard to the interrelationship of histologic subtype and viral infection.

Recurrent TERT promoter mutations in urothelial carcinoma and potential clinical applications

Increased telomerase activity is associated with almost all types of advanced human cancers with unknown molecular mechanism(s). Two recurrent point mutations in the promoter region of telomerase reverse transcriptase (TERT)--the key subunit of telomerase--have recently been identified in melanoma as well as a small sample of bladder cancer cell lines. However, the incidence and clinical-pathological significance of these mutations in urothelial carcinoma have not been well established yet. We collected 86 specimens of urothelial carcinoma including upper and lower urinary tract: high grade and low grade, invasive and noninvasive, and primary and metastatic. We also included some matched benign urothelium and common benign bladder lesions: cystitis, nephrogenic adenoma, and inverted papilloma. In addition, we collected urine samples for urothelial carcinoma workup; blood samples from patients underwent cystectomy with extensive lymphovascular invasion. All specimens were subject to polymerase chain reaction amplification and bidirectional Sanger sequencing for the TERT promoter mutations: C228T and C250T. We found that 64 (74%) of 86 carcinoma samples harbored 1 of the 2 TERT promoter mutations (C228T, n = 54; C250T, n = 10); the incidences were roughly equal regardless of site of origin, histologic grade, and invasive status. All matched benign and benign lesion samples showed wild-type sequence. These TERT promoter mutations are the most common genetic alterations in urothelial carcinoma and are not associated with tumor locations, grade, or invasiveness. Importantly, the feasibility of detecting these mutations in urine samples may provide a novel method to detect urothelial carcinoma in urine.

Constitutive and LPS-Induced Expression of MCP-1 and IL-8 by Human Uveal Melanocytes In Vitro and Relevant Signal Pathways

PURPOSE Melanocytes are one of the major cellular components in the uvea. Interleukin-8/CXCL8 and monocyte chemoattractant protein-1 (MCP-1/CCL2) are the two most important proinflammatory chemokines. We studied the constitutive and lipopolysaccharide (LPS)-induced expression of IL-8 and MCP-1 in cultured human uveal melanocytes (UM) and explored the relevant signal pathways. METHODS Conditioned media and cells were collected from UM cultured in medium with and without stimulation of LPS. Interleukin-8 and MCP-1 proteins and mRNAs were measured using an ELISA kit and RT-PCR, respectively. Nuclear factor (NF)-κB in nuclear extracts and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases1/2 (ERK1/2), and c-Jun N-terminal kinase1/2 (JNK1/2) in cells cultured with and without LPS were measured by ELISA kits. Inhibitors of p38 (SB203580), ERK1/2 (UO1026), JNK1/2 (SP600125), and NF-κB (BAY11-7082) were added to the cultures to evaluate their effects. RESULTS Low levels of IL-8 and MCP-1 proteins were detected in the conditioned media in UM cultured without serum. Lipopolysaccharide (0.01-1 μg/mL) increased IL-8 and MCP-1 mRNAs and proteins levels in a dose- and time-dependent manner, accompanied by a significant increase of phosphorylated JNK1/2 in cell lysates and NF-κB in nuclear extracts. Nuclear factor-κB and JNK1/2 inhibitors significantly blocked LPS-induced expression of IL-8 and MCP-1. CONCLUSIONS This is the first report on the expression and secretion of chemokines by UM. The data suggest that UM may play a role in the pathogenesis of ocular inflammatory diseases.

Role of Human Papillomavirus and p16 Staining in a Patient With Head and Neck Cancer Presenting With a Synchronous Lung Nodule: A Case Report and Review of the Literature

Case Report A 67-year-old man with a 50 pack-year smoking history, who quit smoking 30 years before, presented complaining of dysphagia, odynophagia, hemoptysis, and a 20-pound weight loss over 3 months. A positron emission tomography/computed tomography scan showed a 2-cm, [F]fluorodeoxyglucose (FDG) –avid, left upper lobe nodule, a 3-cm FDG-avid left tonsillar mass, and a 2-cm FDG-avid left level IIa lymph node. The patient underwent a transoral biopsy that revealed squamous cell carcinoma (SCC), nonkeratinizing type, with diffuse immunohistochemical staining for p16 as well as strongly positive in situ hybridization for human papillomavirus 16 (HPV16). Fine-needle aspiration of the lung nodule was suggestive of SCC. The patient underwent a minimally invasive thoracic surgery and left upper lobe wedge resection with a level 5 mediastinal lymph node dissection. Histologic examination of the lung nodule showed a poorly differentiated SCC that was morphologically dissimilar to the tonsillar tumor (Fig 1). The tumor was 1.4 cm in largest diameter, and the margins and mediastinal lymph nodes were negative. Immunostaining for p16 was diffusely positive (Fig 2). Polymerase chain reaction (PCR) demonstrated that the tumor was negative for HPV16, HPV18, and other HPV genotypes such as 6, 11, 31, 33, 35, 45, 51, 52, 56, 58, 59, and 68. Discussion Second primary tumors of the upper aerodigestive tract are one of the main causes of death after treatment of early-stage head and neck cancer. It is often challenging to distinguish between a second primary and a lung metastasis in patients currently presenting with or with a previous history of head and neck squamous cell carcinoma (HNSCC) who are found to have a synchronous or metachronous lung nodule. Heavy tobacco exposure is a strong negative predictor for overall survival in both HPV-positive oropharyngeal cancer (HPVOPC) as well as HPV-negative OPC, reinforcing the known increased risk of second cancers and noncancer-related death. Furthermore, there is a difference in the temporal pattern of distant relapse on the basis of HPV status. For instance, patients with HPVpositive SCC are at risk of distant relapses for 5 years after treatment and longer. In contrast, the rate of distant relapses in patients with HPV-negative disease remains relatively low and stable after 2 years of follow-up. These observations suggest not only a difference in the tumor biology, but have implications regarding therapeutic decisions in patients with HPVOPC. During the staging process for newly diagnosed HNSCC, synchronous distant metastases are found in 2% to 17% of patients. Synchronous second primary tumors of the lung have been reported in 2% of patients. A retrospective study that assessed the effectiveness of using thoracic computed tomography scans for the staging of patients presenting with newly diagnosed HNSCC found that the overall rate of synchronous pulmonary tumors was 4.3%. HPV is the direct cause of several types of cancer, including HPVOPC, which is now the most common HPV-related malignancy in the United States and increasing rapidly in incidence. OPC caused by HPV16 has a distinct behavior and significantly better prognosis compared with HPV-negative tumors. HPVOPCs overexpress the Fig 1. Fig 2. JOURNAL OF CLINICAL ONCOLOGY D I A G N O S I S I N O N C O L O G Y VOLUME 33 NUMBER 3 JANUARY 2

Recent Advances in Probe Amplification Technologies

Oligonucleotide probes provide a useful tool for the detection of target nucleic acids by the formation of a double helical structure between complementary sequences. The stringent requirements of Watson–Crick base pairing make hybridization extremely specific. However, the detection of target sequence by hybridization is often insensitive due to the limited number of signal molecules that can be labeled on the probe. In general, the analytical sensitivity of probe hybridization is of the order 106 molecules. Therefore, it cannot meet the needs of most clinical diagnostic applications. Many technologies have been developed to improve the detection sensitivity by amplifying the probe sequence bound to the target. All probe amplification technologies are developed based on the recent advancement in molecular biology and the understanding of in vivo nucleic acid synthesis (i.e., ligation, polymerization, transcription, digestion/cleavage, etc.). A fundamental advantage of probe amplification technologies ascribes to their isothermal nature, (i.e., accomplishing amplification at a constant temperature with the exception of LCR, which requires temperature cycling). Isothermal amplification allows the test to be done using a simple instrument and makes quality control of the instrument easier. In order for the probe to be amplified, the probes have to be specially designed or synthesized. For example, in rolling circle amplification (RCA), a circularized probe is used, whereas the Invader assay employs an overlapping structure within the probes. Finally, maximum amplification is achieved by generating new DNA products (RCA, RAM, SMART, Q-beta replicase, etc.), although some of the technologies (i.e., LCR and CPT) use existing DNA primers without a net increase of DNA products. In addition to amplification of probe sequence to achieve a desired sensitivity, each technology has its unique features, thus unique clinical applications. For example, Invader technology is very useful for single nucleotide polymorphism (SNP) scoring due to specific recognition by the enzyme cleavase to the overlapping structure of two probes. On the other hand, RCA is probably the only technology that can be used for on-chip amplification due to the attachment of product to the primer sequence linked on the chip surface. Therefore, in order to select a