Low-pass genome sequencing: a validated method in clinical cytogenetics.

Clinically significant copy-number variants (CNVs) known to cause human diseases are routinely detected by chromosomal microarray analysis (CMA). Recently, genome sequencing (GS) has been introduced for CNV analysis; however, sequencing depth (determined by sequencing read-length and read-amount) is a variable parameter across different laboratories. Variating sequencing depths affect the CNV detection resolution and also make it difficult for cross-laboratory referencing or comparison. In this study, by using data from 50 samples with high read-depth GS (30×) and the reported clinically significant CNVs, we first demonstrated the optimal read-amount and the most cost-effective read-length for CNV analysis to be 15 million reads and single-end 50 bp (equivalent to a read-depth of 0.25-fold), respectively. In addition, we showed that CNVs at mosaic levels as low as 30% are readily detected, furthermore, CNVs larger than 2.5 Mb are also detectable at mosaic levels as low as 20%. Herein, by conducting a retrospective back-to-back comparison study of low-pass GS versus routine CMA for 532 prenatal, miscarriage, and postnatal cases, the overall diagnostic yield was 22.4% (119/532) for CMA and 23.1% (123/532) for low-pass GS. Thus, the overall relative improvement of the diagnostic yield by low-pass GS versus CMA was ~ 3.4% (4/119). Identification of cryptic and clinically significant CNVs among prenatal, miscarriage, and postnatal cases demonstrated that CNV detection at higher resolutions is warranted for clinical diagnosis regardless of referral indications. Overall, our study supports low-pass GS as the first-tier genetic test for molecular cytogenetic testing.

Novel biomarkers of nasopharyngeal carcinoma metastasis risk identified by reverse phase protein array based tumor profiling with consideration of plasma Epstein-Barr virus DNA load.

PURPOSE: In patients with Epstein-Barr virus (EBV) associated nasopharyngeal carcinoma (NPC), intertumor heterogeneity causes interpatient heterogeneity in the risk of distant metastasis. We aimed to identify novel biomarkers of metastasis risk using reverse phase protein array (RPPA) profiling of NPC patients at risk for metastasis and considering plasma EBV DNA load. EXPERIMENTAL DESIGN: A total of 98 patients with NPC with and without metastasis after treatment, matched with respect to clinical parameters, are enrolled. Total protein expression is measured by RPPA, and protein functions are analyzed by pathway bioinformatics. RESULTS: The RPPA analysis revealed a profile of 70 proteins that are differentially expressed in metastatic and nonmetastatic tumors. Plasma EBV DNA load after treatment correlated with protein expression level better than plasma EBV DNA load before treatment did. The biomarkers of NPC metastasis identified by proteomics regulate signaling pathways involved in cell cycle progression, apoptosis, and epithelial-mesenchymal transition. The authors identified 26 biomarkers associated with 5-year distant failure-free survival in univariate analysis; five biomarkers remained significant in multivariate analysis. CONCLUSIONS AND CLINICAL RELEVANCE: A comprehensive RPPA profiling study is warranted to identify novel metastasis-related biomarkers and further examine the activation state of signaling proteins to improve estimation of metastasis risk for patients with EBV-associated NPC.

Pretreatment Diffusion-Weighted MRI Can Predict the Response to Neoadjuvant Chemotherapy in Patients with Nasopharyngeal Carcinoma.

PURPOSE: To explore the potential of diffusion-weighted (DW) magnetic resonance imaging (MRI) using apparent diffusion coefficient (ADC) for predicting the response to neoadjuvant chemotherapy in nasopharyngeal carcinoma (NPC). METHODS AND MATERIALS: Ninety-two consecutive patients with NPC who underwent three cycles of neoadjuvant chemotherapy were retrospectively analyzed. DW and anatomical MRI were performed before and after neoadjuvant chemotherapy prior to radiotherapy. Pretreatment ADCs and percentage increases in ADC after chemotherapy were calculated for the primary lesions and metastatic adenopathies. Receiver operating characteristic curve analysis was used to select optimal pretreatment ADCs. RESULTS: Pretreatment mean ADCs were significantly lower for responders than for nonresponders (primary lesions, P = 0.012; metastatic adenopathies, P = 0.013). Mean percentage increases in ADC were higher for responders than for nonresponders (primary lesions, P = 0.008; metastatic adenopathies, P < 0.001). The optimal pretreatment primary lesion and metastatic adenopathy ADCs for differentiating responders from nonresponders were 0.897 × 10(-3) mm(2)/sec and 1.031 × 10(-3) mm(2)/sec, respectively. CONCLUSIONS: NPC patients with low pretreatment ADCs tend to respond better to neoadjuvant chemotherapy. Pretreatment ADCs could be used as a new pretreatment imaging biomarker of response to neoadjuvant chemotherapy.

Prognostic value of classifying parapharyngeal extension in nasopharyngeal carcinoma based on magnetic resonance imaging.

PURPOSE: To subclassify parapharyngeal extension in nasopharyngeal carcinoma (NPC) and investigate its prognostic value and staging categories based on magnetic resonance imaging (MRI). METHODS AND MATERIALS: Data from 1504 consecutive NPC patients treated with definitive-intent radiotherapy were analyzed retrospectively. Sites of parapharyngeal extension were defined by MRI. Overall survival (OS), local relapse-free survival (LRFS), and distant metastasis-free survival (DMFS) were calculated by the Kaplan-Meier method and compared with the log-rank test. Hazard consistency and hazard discrimination were determined by multivariate analysis with Cox proportional hazards models. RESULTS: 1104 patients (73.4%) had parapharyngeal extension; 1.7-63.8% had involvement of various anatomic sites. The hazard ratio for death was significantly higher with extensive parapharyngeal extension (lateral pterygoid muscle of masticator space and beyond or parotid space) than with mild extension (medial pterygoid muscle of masticator space, or carotid, prestyloid, and prevertebral or retropharyngeal space). OS, LRFS, and DMFS with extensive parapharyngeal extension were similar to those in T4 disease; OS, LRFS, and DMFS with mild parapharyngeal extension were significantly higher than in those T3 disease (all P ≤ 0.015). CONCLUSIONS: Parapharyngeal extension in NPC should be subclassified as mild or extensive, which should be regarded as stages T2 and T4 diseases, respectively.

Clinical implications of hepatitis B viral infection in Epstein-Barr virus-associated nasopharyngeal carcinoma.

BACKGROUND: Little is known about the clinical implication of hepatitis B virus (HBV) infection in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC). OBJECTIVE: This study aimed to investigate the clinical characteristics and prognostic factors in patients with newly-diagnosed NPC with HBV infection. STUDY DESIGN: A total of 722 patients with pathologically-diagnosed NPC who received comprehensive treatment at First People's Hospital of Foshan between June 2006 and December 2011 were enrolled in this retrospective study; 79 and 643 patients were HBsAg(+) and HBsAg(-), respectively. The correlations between HBV (HBsAg status and HBV DNA load) and EBV DNA were analyzed, further long-term survival and prognostic factors also were explored. RESULTS: We reported NPC patients with HBsAg(+) represented worse outcome, and distant-failure especially liver metastasis was more common in these patients. HBV infection was more frequent in younger patients and male patients. No correlation was observed between the pre-treatment plasma EBV DNA load (cutoff, 1500 copies/ml) and HBsAg status (positive or negative; r=-0.036, P=0.392), or the pre-treatment plasma EBV DNA load and HBV DNA load (r = 0.042, P = 0823). CONCLUSIONS: Both HBV and EBV infection is an independent negative prognostic factor for long-term survival, distant metastasis, especially liver metastasis, was more common in NPC patients with HBsAg(+), and it seemed no link between EBV DNA load and HBsAg status in NPC.

Molecular markers to assess short-term disease local recurrence in nasopharyngeal carcinoma.

An important challenge in nasopharyngeal carcinoma (NPC) research is to develop effective predictors of tumor recurrence following treatment to determine whether immediate adjuvant therapy is necessary. We retrospectively analyzed archived specimens collected from 45 patients with paired samples of primary NPC (pNPC) and recurrent NPC (rNPC). Clinical samples were collected from the Cancer Center Databases of the First People's Hospital of Foshan and Shantou Central Hospital (affiliates of Sun Yat-Sen University) between 2001 and 2012. Expression levels of phosphor-Stat3 (p-Stat3), signalosome complex subunit 5 (Jab1/Csn5), Akt1, C/EBP homologous protein (CHOP), Ki-67, and apoptosis were determined by immunohistochemistry in pNPC and rNPC samples from the same patients. Differences in these markers between the short-term interval to recurrence (ITR) group (ITR <18 months) and long-term ITR group (ITR ≥18 months) were further analyzed. In Cox's regression analysis, the ITR was significantly associated as an independent­negative prognostic factor for overall survival (hazard ratio, 0.211; 95% confidence interval, 0.053-0.841; P=0.027). p-Stat3 was increased in the short-term ITR group (ITR <18 months) and tended to be lower in the long-term ITR group (ITR ≥18 months). In the short-term ITR group, nuclear Akt expression was significantly increased in paired rNPC (P=0.028). In the long-term ITR group, the expression of nuclear Jab1/Csn5 (P=0.047) and assessment of apoptosis measured with TdT-mediated dUTP nick end­labeling (TUNEL) (P=0.003) was significantly increased in paired rNPC. The results suggest that differences between short- and long-term ITR may predict outcome in rNPC. Furthermore, the overexpression of Jab1/Csn5 and Akt may contribute to the carcinogenesis of rNPC, and Akt seems to promote the progression of short-term ITR. Intra-individual changes of Jab1/Csn5, Akt, and TUNEL may help to identify short-term ITR.

Prognostic significance of circulating CD19+ B lymphocytes in EBV-associated nasopharyngeal carcinoma.

In this retrospective study, the correlation between pre- and post-treatment plasma Epstein-Barr virus (EBV) DNA and circulating immune subsets as well as the prognostic implications was investigated in nasopharyngeal carcinoma (NPC) patients. Patients (n=356) were diagnosed and received comprehensive treatment at the First People's Hospital of Foshan from 2006 to 2010. Pre- and post-treatment plasma EBV DNA load and circulating immune subsets (percentage of CD3+ T cell, CD3+ CD4+ T cells, CD3+ CD8+ T cells, CD19+ B cells and CD56+ NK cells) were analyzed by real-time PCR and flow cytometry. Patient age correlated negatively with CD3+ T cells (r=-0.264, P=0.001) and positively with CD56+ NK cells (r=0.272, P=0.001). Pre-treatment plasma EBV DNA correlated negatively with CD19+ B cells (r=-0.223, P=0.009) and CD4/CD8 ratio (r=-0.177, P=0.047). Patients with low CD19+ B cell had poorer 5-year progression-free survival (PFS) (66.6 vs. 81.8%, P=0.036) and 5-year overall survival (OS) (70.5 vs. 81.5%, P=0.097) than patients with high CD19+ B cells. Low CD19+ B cells was identified as a negative prognostic factor for 5-year PFS (hazard ratio [HR] 0.487; P=0.040), but not for 5-year OS (HR 0.550; P=0.102) in multivariate analysis. Post-treatment plasma EBV DNA was the most important prognostic factor for 5-year PFS (HR 2.983; P=0.006) and 5-year OS (HR 3.927; P<0.001). This study demonstrates the clinical value of circulating CD19+ B cell measurements in NPC patients.

Prognostic value of grading masticator space involvement in nasopharyngeal carcinoma according to MR imaging findings.

PURPOSE: To derive a suitable method for grading masticator space invasion in nasopharyngeal carcinoma on the basis of magnetic resonance (MR) images and to determine its prognostic value in patients undergoing intensity-modulated radiation therapy. MATERIALS AND METHODS: After institutional review board approval and informed consent were acquired, 808 patients with nasopharyngeal carcinoma who were treated with definitive intensity-modulated radiation therapy were analyzed retrospectively. The anatomic sites of masticator space involvement were identified with MR imaging. Overall survival, local relapse-free survival, and distant metastasis-free survival were calculated by using the Kaplan-Meier method and were compared by using the log-rank test. Potential prognostic factors were identified by means of multivariate analysis. RESULTS: Masticator space involvement was diagnosed in 163 of 808 patients (20.2%). Patients with lateral invasion (involvement of the lateral pterygoid muscle of the masticator space and beyond) had significantly poorer overall survival and distant metastasis-free survival than those with medial invasion (involvement of the medial pterygoid muscle of the masticator space) (P = .035 and P = .026, respectively). Furthermore, their overall survival, local relapse-free survival, and distant metastasis-free survival were significantly poorer compared with patients with stage T2 or T3 disease (all P ≤ .023) but similar to patients with stage T4 disease. The grade of masticator space involvement was an independent prognostic factor for overall survival, local relapse-free survival, and distant metastasis-free survival (all P ≤ .023). CONCLUSION: Masticator space involvement in nasopharyngeal carcinoma should be graded as medial (stage T2 disease) or lateral (stage T4 disease). This can facilitate staging of nasopharyngeal carcinoma and may be a suitable prognostic indicator of survival.

Pretreatment Epstein-Barr virus DNA load and cumulative cisplatin dose intensity affect long-term outcome of nasopharyngeal carcinoma treated with concurrent chemotherapy: experience of an institute in an endemic area.

BACKGROUND: We retrospectively compared the long-term efficacy of concurrent chemoradiotherapy (CCRT) regimens (docetaxel vs. cisplatin), total dose intensity of cisplatin (> 200 vs. ≤ 200 mg/m2) and pretreatment plasma levels of Epstein-Barr virus (EBV) DNA for nasopharyngeal carcinoma (NPC), and investigated the prognostic factors. METHODS: We enrolled 214 patients diagnosed with NPC and treated with CCRT. 41 patients received weekly docetaxel and 173 weekly cisplatin. 62 received cumulative cisplatin of ≤ 200 mg/m2 and 111, > 200 mg/m2. Pretreatment levels of EBV DNA were available for 155 patients. RESULTS: Patients receiving concurrent weekly docetaxel and cisplatin had similar 5-year rates for overall survival (OS) (p = 0.306), progression-free survival (PFS) (p = 0.133), distant failure-free survival (DFS) (p = 0.110), and locoregional failure-free survival (LFS) (p = 0.452). Cumulative cisplatin of > 200 mg/m2 improved the 5-year rates of PFS (p = 0.018) and DFS (p = 0.042) significantly in comparison with cumulative cisplatin of ≤ 200 mg/m2. EBV DNA levels of ≥ 1,500 copies/ml was closely associated with poor DFS (p = 0.011), PFS (p = 0.006), and OS (p = 0.004). CONCLUSIONS: Weekly cisplatin was well tolerated in CCRT, during which cumulative cisplatin of > 200 mg/m2 improved PFS and DFS. The long-term efficacy of concurrent docetaxel was similar to that of concurrent cisplatin. The EBV DNA level was the most significant prognostic factor.

Concurrent weekly docetaxel chemotherapy in combination with radiotherapy for stage III and IVA-B nasopharyngeal carcinoma.

BACKGROUND AND PURPOSE: Cisplatin is the most common chemotherapeutic agent for loco-regionally advanced nasopharyngeal carcinoma (NPC); however, toxicity is a limiting factor for some patients. We retrospectively compared the efficacy and toxicity of weekly docetaxel-based and cisplatin-based concurrent chemoradiotherapy in loco-regionally advanced NPC. METHODS AND MATERIALS: Eighty-four patients with Stage III and IVA-B NPCs, treated between 2007 and 2008, were retrospectively analyzed. Thirty received weekly docetaxel-based concurrent chemotherapy, and 43 were given weekly cisplatin-based concurrent chemotherapy. Radiotherapy was administered using a conventional technique (seven weeks, 2.0 Gy per fraction, total dose 70-74 Gy) with 6-8 Gy boosts for some patients with locally advanced disease. RESULTS: Median follow-up time was 42.3 months (range, 8.6-50.8 months). There were no significant differences in the 3-year loco-regional failure-free survival (85.6% vs. 92.3%; p=0.264), distant failure-free survival (87.0% vs. 92.5%; p=0.171), progression-free survival (85.7% vs. 88.4%; p=0.411) or overall survival (86.5% vs. 92.5%, p=0.298) of patients treated concurrently with docetaxel or cisplatin. Severe toxicity was not common in either group. CONCLUSIONS: Weekly docetaxel-based concurrent chemoradiotherapy is potentially effective and has a tolerable toxicity; however, further investigations are required to determine if docetaxel is superior to cisplatin for advanced stage NPC.

Primary nasopharyngeal adenocarcinoma: a review.

Primary nasopharyngeal adenocarcinoma (NAC) accounts for approximately 0.5% of all nasopharyngeal cancer. The diagnosis, staging and treatment of NAC has not been well described. This article presents a literature review on NAC and identifies its characteristics and management. The NAC group of diseases contains various pathological types and has a series of specific clinical characteristics, including slow progression, a low incidence of neck masses and frequent cranial neuropathy. The Epstein-Barr virus may not play an important role in NAC carcinogenesis. The rarity of the disease makes the staging classification and treatment strategies of NAC parallel to those recommended for nasopharyngeal squamous carcinoma. Some patients might benefit from surgery, and radiotherapy using precise techniques might achieve good control for treating NAC, but the roles of chemotherapy and target therapy are not clear. The proper staging system and optimal treatment strategies need to be established in NAC.

Radiologic criteria of retropharyngeal lymph node metastasis in nasopharyngeal carcinoma treated with radiation therapy.

PURPOSE: To determine the appropriate radiologic criteria of metastatic retropharyngeal lymph nodes (RLNs) in patients with nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: After institutional review board approval and informed consent, 303 consecutive NPC patients treated with definitive radiation therapy were examined after completion of therapy. RLNs were classified as metastatic on the basis of the results of magnetic resonance (MR) imaging follow-up.The receiver operating characteristic curve and area under the curve were determined to assess the accuracy of different size criteria in the diagnosis of RLN metastasis. RESULTS: Initial MR images revealed 523 RLNs in 265 patients. Two hundred sixty-four (50.5%) RLNs positive for malignant involvement were confirmed in 177 patients. The remaining 259 (49.5%) nodes were classified as negative for benign process at follow-up. The minimal axial diameter was found to be more accurate than the maximal axial diameter for assessing metastatic RLNs. The most accurate size criterion of metastatic RLNs was a minimal axial diameter of 6 mm or larger, resulting in an accuracy of 87.5% (457 of 522). Central necrosis or groups of two or more of RLNs had a 100% specificity for diagnosis of RLN metastases. CONCLUSION: The radiologic criteria that should be used for assessment of RLN metastases in NPC patients are nodes with a minimal axial diameter 6 mm or larger, any node with central necrosis, groups of two or more RLNs, or any medial RLN; these criteria may be useful in tumor staging and treatment planning.

[Application of 99mTc-DTPA in evaluation of blood-brain barrier permeability in patients receiving whole brain irradiation].

OBJECTIVE: To study the pattern of blood-brain barrier (BBB) permeability changes during whole brain radiotherapy (WBRT) for metastatic brain tumor. METHODS: Twenty patients with metastatic brain tumors receiving WBRT by 6 MV X-ray underwent (99)mTc-DTPA brain SPECT before and during WBRT (20, 40 Gy) and at 2 weeks after the end of irradiation. A frame of transverse (99)mTc-DTPA brain SPECT image that best displayed the brain metastasis was chosen, and the regions of interest (ROI) were defined in the tumor foci (T), the contralateral normal brain tissue (N) and the background outside the soft tissues around the cranium (B). The radioactive counts of every ROI were measured and the ratios of the total counts (T/B and N/B) before and during WBRT (20 Gy, 40 Gy) and at 2 weeks after the irradiation were calculated. RESULTS: The average T/B and N/B in the 20 patients with 30 brain metastases was 142.2-/+51.1 and 82.6-/+42.3 before WBRT, 260.3-/+121.5 and 150.7-/+72.5 during 20 Gy WBRT, 251.6-/+118.3 and 161.8-/+68.4 during 40 Gy WBRT, and 250.3-/+117.2 and 158.6-/+73.5 at 2 weeks after the irradiation, respectively. The measurements during WBRT (20 and 40 Gy) and at 2 weeks after the irradiation group underwent no significant variations (P>0.05), but showed significant differences from those before WBRT (P<0.05). CONCLUSIONS: Irradiation causes direct damage of the BBB function, and the permeability of the BBB increases significantly during and within 2 weeks following 20 and 40 Gy WBRT, which provides the optimal time window for interventions with chemotherapy.