Jonathan S. T. Sham

Preliminary Results of a Randomized Study on Therapeutic Gain by Concurrent Chemotherapy for Regionally-Advanced Nasopharyngeal Carcinoma: NPC-9901 Trial by the Hong Kong Nasopharyngeal Cancer Study Group

PURPOSE This randomized study compared the results achieved by concurrent chemoradiotherapy (CRT) versus radiotherapy (RT) alone for nasopharyngeal carcinoma (NPC) with advanced nodal disease. PATIENTS AND METHODS Patients with nonkeratinizing/undifferentiated NPC staged T1-4N2-3M0 were randomized to CRT or RT. Both arms were treated with the same RT technique and dose fractionation. The CRT patients were given cisplatin 100 mg/m2 on days 1, 22, and 43, followed by cisplatin 80 mg/m2 and fluorouracil 1,000 mg/m2/d for 96 hours starting on days 71, 99, and 127. RESULTS From 1999 to January 2004, 348 eligible patients were randomly assigned; the median follow-up was 2.3 years. The two arms were well-balanced in all prognostic factors and RT parameters. The CRT arm achieved significantly higher failure-free survival (72% v 62% at 3-year, P = .027), mostly as a result of an improvement in locoregional control (92% v 82%, P = .005). However, distant control did not improve significantly (76% v 73%, P = .47), and the overall survival rates were almost identical (78% v 78%, P = .97). In addition, the CRT arm had significantly more acute toxicities (84% v 53%, P < .001) and late toxicities (28% v 13% at 3-year, P = .024). CONCLUSION Preliminary results confirmed that CRT could significantly improve tumor control, particularly at locoregional sites. However, there was significant increase in the risk of toxicities and no early gain in overall survival. Longer follow-up is needed to confirm the ultimate therapeutic ratio.

Improvement in Treatment Results and Long-Term Survival of Patients With Esophageal Cancer: Impact of Chemoradiation and Change in Treatment Strategy

Objective To identify prognostic factors and reasons for improved survival over time in patients with esophageal cancer. Summary Background Data Management strategies for esophageal cancer have evolved with time. The impact of chemoradiation in the overall treatment results has not been adequately studied. Methods From 1990 to 2000, 399 (62.4%) of 639 patients with intrathoracic squamous cancers underwent resection. Two study periods were analyzed: period I (01/1990–06/1995), and period II (07/1995–12/2000); during period II, chemoradiation was introduced. Prognostic factors were identified by multivariate analysis and the 2 periods compared. Results Hospital mortality rate after resection decreased from 7.8% to 1.2%, P = 0.002. Five favorable prognostic factors were identified: female gender (female vs. male, HR = 0.66), infracarinal tumor location (infra vs. supra-carinal, HR = 0.63), low pTNM stage (III/IV vs. 0/I/II/T0N1, HR = 1.76), pM0 stage (M1a/b vs. M0, HR = 1.56), and R0 category (R1/2 vs. R0, HR = 2.49). Median survival was 15.8 and 25.6 months in periods I and II, respectively, P = 0.02. More R0 resections were evident in period II, being possible in 63% (period I) and 79% (period II) of patients, P = 0.001. This was attributed to tumor downstaging by chemoradiation and more stringent patient selection for resection in period II. Performing less R1/2 resections in period II coincided with using primary chemoradiation in treating advanced tumors. In patients treated without resection, survival also improved from 3 (period I) to 5.8 months (period II), P < 0.01. Conclusions Survival has improved; chemoradiation enabled better patient selection for curative resections and also resulted in more R0 resections by tumor downstaging. This treatment strategy led to overall better outcome for the whole patient cohort, even in those treated by nonsurgical means.

Factors affecting risk of symptomatic temporal lobe necrosis: significance of fractional dose and treatment time.

PURPOSE To study the factors affecting the risk of symptomatic temporal lobe necrosis after different fractionation schedules. METHODS AND MATERIALS One thousand thirty-two patients with T1-2 nasopharyngeal carcinoma treated with radical radiotherapy in Hong Kong during 1990-1995 were studied. They were treated at four different centers with similar techniques but different fractionation schedules: 984 patients were given 1 fraction daily throughout (q.d.), and 48 patients were irradiated twice daily (b.i.d.) for part of the course. The median total dose was 62.5 Gy (range 50.4-71.2), dose per fraction was 2.5 Gy (range 1.6-4.2), and overall treatment time (OTT) was 44 days (range 29-70). In addition, 500 patients received supplementary doses for parapharyngeal extension, 113 received booster doses by brachytherapy, and 114 received sequential chemotherapy using cisplatin-based regimes. RESULTS Altogether, 24 patients developed symptomatic temporal lobe necrosis: 18 from the q.d. group and 6 from the b.i.d. group. The 5-year actuarial incidence ranged from 0% (after 66 Gy in 33 fractions within 44 days) to 14% (after 71.2 Gy in 40 fractions within 35 days). Multivariate analyses showed that the risk was significantly affected by the fractional effect of the product of total dose and dose per fraction (hazard ratio [HR] = 1.04, 95% confidence interval [CI] 1.02-1.05), OTT (HR 0.88, 95% CI 0.80-0.97), and b.i.d. scheduling (HR 13, 95% CI 3-54). Repeating the analyses for patients treated with the q.d. schedules confirmed the independent significance of OTT in addition to the product of total dose and dose per fraction. CONCLUSION The tentative results suggest that in addition to fractional dose, the OTT also had significant impact on the risk of temporal lobe necrosis, and b.i.d. scheduling increased the hazard further.

THY1 is a candidate tumour suppressor gene with decreased expression in metastatic nasopharyngeal carcinoma

Using oligonucleotide microarray analysis, THY1, mapping close to a previously defined 11q22–23 nasopharyngeal carcinoma (NPC) critical region was identified as showing consistent downregulated expression in the tumour segregants, as compared to their parental tumour-suppressing microcell hybrids (MCHs). Gene expression and protein analyses show that THY1 was not expressed in the NPC HONE1 recipient cells, tumour segregants, and other NPC cell lines; THY1 was exclusively expressed in the non-tumourigenic MCHs. The mechanism of THY1 gene inactivation in these cell lines was attributed to hypermethylation. Clinical study showed that in 65% of NPC specimens there was either downregulation or loss of THY1 gene expression. Using a tissue microarray and immunohistochemical staining, 44% of the NPC cases showed downregulated expression of THY1 and 9% lost THY1 expression. The frequency of THY1 downregulated expression in lymph node metastatic NPC was 63%, which was significantly higher than in the primary tumour (33%). After transfection of THY1 gene into HONE1 cells, a dramatic reduction of colony formation ability was observed. These findings suggest that THY1 is a good candidate tumour suppressor gene in NPC, which is significantly associated with lymph node metastases.

THE EFFECT OF LOCO-REGIONAL CONTROL ON DISTANT METASTATIC DISSEMINATION IN CARCINOMA OF THE NASOPHARYNX: AN ANALYSIS OF 1301 PATIENTS

PURPOSE This study evaluated the effect of loco-regional control on incidence of distant metastases in patients with nasopharyngeal carcinoma. METHODS AND MATERIALS Retrospective analysis was performed on 1301 patients with nasopharyngeal carcinoma treated from 1976-1989 in our hospital. The effect of different prognostic indicators on distant metastases free survival (DMFS) was analyzed, including T stage, N stage, sex, and loco-regional control. The significance of loco-regional control in distant metastatic dissemination was further studied with period and subgroup analysis. Cox regression was performed to identify the independent prognostic variables. RESULTS Patients with loco-regional relapse had significantly higher distant metastases rate than patients with loco-regional control (time-adjusted distant metastases rate at 5 years was 40.7% vs. 29.4%, p = 0.0012). By period analysis, the effect of loco-regional control on distant metastases was found to be significant in the first 2 years from diagnosis. Subgroup analysis showed that loco-regional relapse was associated with significantly higher distant metastases rate in patients with T1, N0, and N1 disease (p = 0.001, 0.001, 0.0226, respectively). The Cox regression model also confirmed loco-regional control as an independent prognostic indicator of distant metastases (p = 0.0001) besides T stage (p = 0.0006) and N stage (p = 0.0001). CONCLUSION Loco-regional relapse is a significant risk factor for development of distant metastases. Further effort should be made to improve loco-regional control and to eradicate distant metastases.

Quantitative Plasma Hypermethylated DNA Markers of Undifferentiated Nasopharyngeal Carcinoma

Purpose: Gene-specific methylation is common in primary undifferentiated nasopharyngeal carcinoma (NPC). DNA released from apoptotic or necrotic cell death including those aberrantly methylated promoter DNA of cancer cells is absorbed into the circulation as cell-free plasma DNA of the patient. This study aims at evaluation of the potential use of methylated gene promoter DNA as a serological tumor marker of primary and potentially salvageable local or nodal recurrent NPC. Experimental Design: The quantity of plasma hypermethylated gene promoters of CDH1, DAPK1, p15, p16, RASSF1A, and MLH1 of 41 NPC patients before treatment and 43 normal individuals were studied using real-time quantitative PCR. The post-treatment plasma hypermethylated CDH1, DAPK1,and p16were also measured in 13 NPC patients with locoregional recurrence and 17 patients in remission. Results: Concentrations of cell-free circulating DNA were significantly higher in NPC patients than normal controls (28.79 ng/ml versus 16.57 ng/ml, respectively). There was no significant difference in plasma DNA concentration of EBV-positive and -negative normal individuals. Methylated DNA was detectable in plasma of NPC patients before treatment including 46% for CDH1,42% for p16,20% for DAPK1,20% for p15,and 5% for RASSF1A.Hypermethylated MLH1was not detected in plasma of all of the NPC patients and normal individuals. Aberrantly hypermethylated promoter DNA of at least one of the five genes was detectable in 29 of 41 (71%) plasma of NPC patients before treatment. Hypermethylated promoter DNA of at least one of the three genes (CDH1, DAPK1, and p16) was detectable in post-treatment plasma of 5 of 13 (38%) recurrent NPC patients and none of the patients in remission. Conclusions: Our results suggested that cell-free circulating methylated gene promoter DNA is a possibly useful serological marker in assisting in screening of primary and potentially salvageable local or regional recurrent NPC.

Characterization of HBV integrants in 14 hepatocellular carcinomas: association of truncated X gene and hepatocellular carcinogenesis.

Although the integration of hepatitis B virus (HBV) into human DNA has been found to be associated with the development of hepatocellular carcinoma (HCC), the molecular mechanism remains unclear. In order to obtain additional insight into the correlation of HBV integration and HCC development, integrated HBV in 14 primary HCC cases was isolated and characterized by sequencing analysis. Our findings in this study showed that: (1) none of the known cellular oncogene or tumor suppressor gene was affected by the HBV integration; (2) although the integration of HBV is random, the integration site was often within or close to human repetitive sequences; (3) integrated HBV may possess the capacity to transpose to another chromosome region through reintegration; (4) rearrangements of HBV sequence were observed in all the 14 integrants, involving (most frequently) X (12/14 integrants), P (8/14), S (7/14), and C (7/14) genes; and (5) 3′-deleted X gene and consequent C-terminal truncated X protein caused by HBV integration was observed in 10 cases. These deletions cause the losses of p53-dependent transcriptional repression binding site, transcription factor Sp1 binding site, and growth-suppressive effect domain, leading to cell proliferation and transformation. This finding suggests that 3′-deleted X gene caused by the HBV integration may play an important role in the HCC development.

Array-based comparative genomic hybridization analysis identified cyclin D1 as a target oncogene at 11q13.3 in nasopharyngeal carcinoma

Nasopharyngeal carcinoma is highly prevalent in Southern China and Southeast Asia. To unveil the molecular basis of this endemic disease, high-resolution comparative genomic hybridization arrays were used for systematic investigation of genomic abnormalities in 26 nasopharyngeal carcinoma samples. A comprehensive picture of genetic lesions associated with tumorigenesis of nasopharyngeal carcinoma was generated. Consistent chromosomal gains were frequently found on 1q, 3q, 8q, 11q, 12p, and 12q. High incidences of nonrandom losses were identified on chromosomes 3p, 9p, 11q, 14q, and 16q. In addition to previously characterized regions, we have identified several novel minimal regions of gains, including 3q27.3-28, 8q21-24, 11q13.1-13.3, and 12q13, which may harbor candidate nasopharyngeal carcinoma-associated oncogenes. In this study, gain of 11q13.1-13.3 was the most frequently detected chromosomal aberration and a 5.3-Mb amplicon was delineated at this region. Within this 11q13 amplicon, concordant amplification and overexpression of cyclin D1 (CCND1) oncogene was found in nasopharyngeal carcinoma cell lines, xenografts, and primary tumors. Knockdown of cyclin D1 by small interfering RNA in nasopharyngeal carcinoma cell lines led to significant decrease of cell proliferation. The findings suggest that cyclin D1 is a target oncogene at 11q13 in nasopharyngeal carcinoma and its activation plays a significant role in nasopharyngeal carcinoma tumorigenesis.

Long-term follow-up of potentiation of radiotherapy by cis-platinum in advanced cervical cancer

Between January 1982 and January 1983 all patients with advanced cervical cancer were randomized into three groups: radiotherapy only (group I), radiotherapy plus weekly cis-platinum (group II), and radiation plus twice-weekly cis-platinum (group III). Better initial central control was observed in group III patients. Long-term survival was similar in the three groups. Potentiation of radiotherapy with cis-platinum failed to show any significant improvement in long-term survival.

TSLC1 Is a Tumor Suppressor Gene Associated with Metastasis in Nasopharyngeal Carcinoma

In up to 87% of nasopharyngeal carcinoma (NPC) clinical tumor specimens, there was either down-regulation or loss of TSLC1 gene expression. Using a tissue microarray and immunohistochemical staining, the frequency of down-regulated or loss of expression of TSLC1 in metastatic lymph node NPC was 83% and the frequency of loss of expression of TSLC1 was 35%, which was significantly higher than that in primary NPC (12%). To examine the possible growth-suppressive activity of TSLC1 in NPC, three NPC cell lines, HONE1, HNE1, and CNE2, were transfected with the wild-type TSLC1 gene cloned into the pCR3.1 expression vector; a reduction of colony formation ability was observed for all three cell lines. A tetracycline-inducible expression vector, pETE-Bsd, was also used to obtain stable transfectants of TSLC1. There was a dramatic difference between colony formation ability in the presence or absence of doxycycline when the gene is shut off or expressed, respectively, with the tetracycline-inducible system. Tumorigenicity assay results show that the activation of TSLC1 suppresses tumor formation in nude mice and functional inactivation of this gene is observed in all the tumors derived from tumorigenic transfectants. Further studies indicate that expression of TSLC1 inhibits HONE1 cell growth in vitro by arresting cells in G(0)-G(1) phase in normal culture conditions, whereas in the absence of serum, TSLC1 induced apoptosis. These findings suggest that TSLC1 is a tumor suppressor gene in NPC, which is significantly associated with lymph node metastases.