Chung-Hsin Chen

Aristolochic acid-associated urothelial cancer in Taiwan

Aristolochic acid, a potent human carcinogen produced by Aristolochia plants, is associated with urothelial carcinoma of the upper urinary tract (UUC). Following metabolic activation, aristolochic acid reacts with DNA to form aristolactam (AL)-DNA adducts. These lesions concentrate in the renal cortex, where they serve as a sensitive and specific biomarker of exposure, and are found also in the urothelium, where they give rise to a unique mutational signature in the TP53 tumor-suppressor gene. Using AL-DNA adducts and TP53 mutation spectra as biomarkers, we conducted a molecular epidemiologic study of UUC in Taiwan, where the incidence of UUC is the highest reported anywhere in the world and where Aristolochia herbal remedies have been used extensively for many years. Our study involves 151 UUC patients, with 25 patients with renal cell carcinomas serving as a control group. The TP53 mutational signature in patients with UUC, dominated by otherwise rare A:T to T:A transversions, is identical to that observed in UUC associated with Balkan endemic nephropathy, an environmental disease. Prominent TP53 mutational hotspots include the adenine bases of 5′AG (acceptor) splice sites located almost exclusively on the nontranscribed strand. A:T to T:A mutations also were detected at activating positions in the FGFR3 and HRAS oncogenes. AL-DNA adducts were present in the renal cortex of 83% of patients with A:T to T:A mutations in TP53, FGFR3, or HRAS. We conclude that exposure to aristolochic acid contributes significantly to the incidence of UUC in Taiwan, a finding with significant implications for global public health.

Mutational Signature of Aristolochic Acid Exposure as Revealed by Whole-Exome Sequencing

The mutational signature of aristolochic acid exemplifies how genome-wide sequencing can be used to identify environmental exposures leading to cancer. Carcinogen AAlert Aristolochic acid (AA) is a natural compound derived from plants in the Aristolochia genus. For centuries, Aristolochia has been used throughout Asia to treat a variety of ailments as a component of traditional Chinese medicine. In recent years, however, a more sinister side of this herb has come to light when it was linked to kidney damage and cancers of the urinary tract. Now, two studies by Poon et al. and Hoang et al. present a “molecular signature” of AA-induced DNA damage, which helps to explain the mutagenic effects of AA and may also be useful as a way to detect unsuspected AA exposure as a cause of cancer. The molecular signature seen in AA-associated tumors is characterized by a predominance of A:T-to-T:A transversions, a relatively unusual type of mutation that is infrequently seen in other types of cancer, including those caused by other carcinogens. These mutations concentrate at splice sites, causing the inappropriate inclusion or exclusion of entire exons in the resulting mRNA. The overall mutation rate is another notable feature of AA-associated cancers, because it is several times higher than the rate of mutations caused by other carcinogens such as tobacco and ultraviolet light. In both studies, the authors also used the molecular signature to discover that AA was a likely cause of tumors previously attributed to other carcinogens. In one case, a urinary tract cancer that had been attributed to smoking and, in the other case, a liver cancer previously attributed to a chronic hepatitis infection were both identified as having the telltale signature of AA mutagenesis. The identification of a specific molecular signature for AA has both clinical and public health implications. For individual patients, the molecular signature could help physicians identify which tumors were caused by AA. Although this information cannot yet be used to optimize the treatment of individual patients, those who are diagnosed with AA-associated cancers could be monitored more closely for the appearance of additional tumors. Meanwhile, a better understanding of the mutagenic effects of AA should also help to strengthen public health efforts to decrease exposure to this carcinogenic herb. In humans, exposure to aristolochic acid (AA) is associated with urothelial carcinoma of the upper urinary tract (UTUC). Exome sequencing of UTUCs from 19 individuals with documented exposure to AA revealed a remarkably large number of somatic mutations and an unusual mutational signature attributable to AA. Most of the mutations (72%) in these tumors were A:T-to-T:A transversions, located predominantly on the nontranscribed strand, with a strong preference for deoxyadenosine in a consensus sequence (T/CAG). This trinucleotide motif overlaps the canonical splice acceptor site, possibly accounting for the excess of splice site mutations observed in these tumors. The AA mutational fingerprint was found frequently in oncogenes and tumor suppressor genes in AA-associated UTUC. The AA mutational signature was observed in one patient’s tumor from a UTUC cohort without previous indication of AA exposure. Together, these results directly link an established environmental mutagen to cancer through genome-wide sequencing and highlight its power to reveal individual exposure to carcinogens.

Stopping smoking might reduce tumour recurrence in nonmuscle-invasive bladder cancer

To evaluate effects of stopping smoking on the outcome of nonmuscle‐invasive bladder cancer, as cigarette smoking is a risk factor for bladder cancer and little is known about whether stopping smoking reduces the risk of recurrence or progression.

Aristolochic acid-induced upper tract urothelial carcinoma in Taiwan: clinical characteristics and outcomes.

Aristolochic acid (AA), a component of all Aristolochia‐based herbal medicines, is a potent nephrotoxin and human carcinogen associated with upper urinary tract urothelial carcinoma (UUC). To investigate the clinical and pathological characteristics of AA‐induced UUC, this study included 152 UUC patients, 93 of whom had been exposed to AA based on the presence of aristolactam‐DNA adducts in the renal cortex. Gene sequencing was used to identify tumors with A:T‐to‐T:A transversions in TP53, a mutational signature associated with AA. Cases with both aristolactam‐DNA adducts and A:T‐to‐T:A transversions in TP53 were defined as AA‐UUC, whereas patients lacking both of these biomarkers were classified as non‐AA‐UUC. Cases with either biomarker were classified as possible‐AA‐UUC. Forty (26%), 60 (40%), and 52 (34%) patients were classified as AA‐UUC, possible‐AA‐UUC and non‐AA‐UUC, respectively. AA‐UUC patients were younger (median ages: 64, 68, 68 years, respectively; p=0.189), predominately female (65%, 42%, 35%, respectively; p=0.011), had more end‐stage renal disease (28%, 10%, 12%, respectively; p=0.055), and were infrequent smokers (5%, 22%, 33%, respectively; p=0.07) compared to possible‐AA‐UUC and non‐AA‐UUC patients. All 14 patients who developed contralateral UUC had aristolactam‐DNA adducts; ten of these also had signature mutations. The contralateral UUC‐free survival period was shorter in AA‐UUC compared to possible‐ or non‐AA‐UUC (p=0.019 and 0.002, respectively), whereas no differences among groups were observed for bladder cancer recurrence. In conclusion, AA‐UUC patients tend to be younger and female, and have more advanced renal disease. Notably, AA exposure was associated with an increased risk for developing synchronous bilateral and metachronous contralateral UUC.

Biomonitoring of Aristolactam-DNA Adducts in Human Tissues Using Ultra-Performance Liquid Chromatography/Ion-Trap Mass Spectrometry

Aristolochic acids (AAs) are a structurally related family of nephrotoxic and carcinogenic nitrophenanthrene compounds found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. AAs have been implicated in the etiology of so-called Chinese herbs nephropathy and of Balkan endemic nephropathy. Both of these disease syndromes are associated with carcinomas of the upper urinary tract (UUC). 8-Methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I) is a principal component of Aristolochia herbs. Following metabolic activation, AA-I reacts with DNA to form aristolactam (AL-I)-DNA adducts. We have developed a sensitive analytical method, using ultraperformance liquid chromatography-electrospray ionization/multistage mass spectrometry (UPLC-ESI/MS(n)) with a linear quadrupole ion-trap mass spectrometer, to measure 7-(deoxyadenosin-N(6)-yl) aristolactam I (dA-AL-I) and 7-(deoxyguanosin-N(2)-yl) aristolactam I (dG-AL-I) adducts. Using 10 μg of DNA for measurements, the lower limits of quantitation of dA-AL-I and dG-AL-I are, respectively, 0.3 and 1.0 adducts per 10(8) DNA bases. We have used UPLC-ESI/MS(n) to quantify AL-DNA adducts in tissues of rodents exposed to AA and in the renal cortex of patients with UUC who reside in Taiwan, where the incidence of this uncommon cancer is the highest reported for any country in the world. In human tissues, dA-AL-I was detected at levels ranging from 9 to 338 adducts per 10(8) DNA bases, whereas dG-AL-I was not found. We conclude that UPLC-ESI/MS(n) is a highly sensitive, specific and robust analytical method, positioned to supplant (32)P-postlabeling techniques currently used for biomonitoring of DNA adducts in human tissues. Importantly, UPLC-ESI/MS(n) could be used to document exposure to AA, the toxicant responsible for AA nephropathy and its associated UUC.

Clinicopathological Characteristics and Survival Outcome of Arsenic Related Bladder Cancer in Taiwan

PURPOSE We compared clinicopathological characteristics and outcomes in patients with bladder cancer who were exposed to graded arsenic levels in drinking water. MATERIALS AND METHODS From 1993 through 2006, 977 patients with bladder cancer in Taiwan were studied retrospectively. Patients were from 3 areas, including the core zone (arsenic related blackfoot disease endemic area with a well water arsenic level of 350 to 1,100 ng/ml), zone 1 (a well water arsenic level of 350 ng/ml or greater but not a blackfoot disease endemic area) and zone 2 (a well water arsenic level of less than 350 ng/ml). Clinicopathological characteristics and survival outcome were compared among the groups. RESULTS Of these patients 81 (8.3%), 246 (25.2%) and 650 (66.5%) lived in the core zone, and zones 1 and 2, respectively. More high grade and high stage tumors were observed in core zone patients than in those in zones 1 and 2, including high grade in 48.7% vs 41.4% and 39.2% of patients, advanced disease in 39.5% vs 31.0% and 18.5% and nodal metastasis in 8.6% vs 3.3% and 3.4%, respectively. Median overall and cancer specific survival in core zone patients was significantly shorter than in patients in zones 1 and 2, including 69 vs 119 and 113-month overall survival and for the 75th percentile of cancer specific survival 34.5 vs 119 and 113 months, respectively. On multivariate analysis with adjustment for tumor grade and stage the zonal difference was not a significant factor for overall or cancer specific survival. CONCLUSIONS Patients with arsenic related bladder cancer may have decreased overall and cancer specific survival because they have more unfavorable tumor phenotypes than patients in other areas in Taiwan.

A cocktail regimen of intravesical mitomycin-C, doxorubicin, and cisplatin (MDP) for non-muscle-invasive bladder cancer

OBJECTIVE To compare the efficacy and toxicity profiles of 3 intravesical regimens, including doxorubicin alone, bacillus Calmette-Guerin (BCG), and a cocktail regimen, in the prevention of bladder cancer recurrence. MATERIALS AND METHODS Two hundred ninety patients with newly diagnosed non-muscle-invasive bladder cancer treated with transurethral resection (TUR) between March 1996 and December 2004 were analyzed retrospectively. Each cycle of the cocktail regimen contained 30 mg each of sequential weekly intravesical mitomycin-C (MMC), doxorubicin, and cisplatin (MDP). Two cycles of MDP were given within the first 6 weeks of TUR, followed by 1 cycle each at 3, 6, and 12 months, and every 6 months until 36 months after a negative cystoscopy. Doxorubicin and BCG alone was given at similar time points as the MDP and BCG protocols. RESULTS There were no demographic differences among the 3 groups. The median follow-up duration was 50 months. Dropout rates due to intolerance and/or poor compliance with the BCG, doxorubicin, and MDP protocols were 22.5%, 16.8%, and 11.0%, respectively. The MDP and BCG groups had similar bladder recurrence rates (37.9% vs. 33.9% at 5 years, respectively; P = 0.69). The doxorubicin group had significantly more recurrences than the BCG or MDP groups (HR = 1.9 (vs. BCG; P = 0.02) and 1.8 (vs. MDP; P = 0.01)). MDP was associated with less major adverse events than BCG (5.8% vs. 15.0%, respectively; P = 0.02). CONCLUSIONS Compared with maintenance BCG, the MDP group had a similar recurrence rate but less side effects. Large randomized study is warranted to further determine the benefit of MDP adjuvant intravesical therapy.

Clinical Outcome of Taiwanese Men With Metastatic Prostate Cancer Compared With Other Ethnic Groups

OBJECTIVES Prostate cancer incidence varies significantly among different ethnic groups. However, little is known about the survival outcome among groups. We sought to compare the survival outcome in patients with metastatic prostate cancer among different ethnic groups and to identify independent prognostic factors affecting overall survival in Taiwanese patients. METHODS From January 1996 to February 2005, 482 men with newly diagnosed metastatic prostate adenocarcinoma were enrolled from five major medical centers in Taiwan. The cohort accounted for about 11.5% of all patients with metastatic disease during the period in Taiwan. The demographics, tumor characteristics, and survival outcome were compared with several published Western and Japanese series. Five series were selected from MEDLINE: the Southwest Oncology Group; Detroit Metropolitan Surveillance, Epidemiology, and End Results Program Registry; American College of Surgeons; National Cancer Registry in Sweden; and Gurma and Nagasaki University Group in Japan. RESULTS The Taiwanese patients were the oldest among the selected series. The median overall survival of our patients was 38.4 months (95% confidence interval 33 to 45 months), which was longer than that in the Western series (median 25 to 32 months) and similar to those in the Japanese series (median 36 months). In a multivariate analysis with age adjustment, bone pain, Gleason score 8 or greater, and visceral metastases independently predicted a reduced overall survival in our series compared with each favorable strata (hazard ratio 2.22, 1.96, and 1.51, respectively; all P <0.05). CONCLUSIONS Taiwanese men with metastatic prostate cancer might have a better survival compared with Western men.

Characteristics of female non-muscle-invasive bladder cancer in Taiwan: association with upper tract urothelial carcinoma and end-stage renal disease.

OBJECTIVES Urothelial carcinogenic mechanisms may differ between men and women. We investigated gender differences in the pathological characteristics and clinical outcomes of non-muscle-invasive bladder urothelial carcinoma (UC). METHODS We retrospectively reviewed a total of 413 consecutive patients, 285 men (69.0%) and 128 women (31.0%), with primary non-muscle-invasive bladder UC, treated with transurethral resection between January 1997 and July 2005. A single pathologist reviewed all pathology data. All statistical comparisons were two-sided. RESULTS Only a small proportion (2.3%) of female patients were current smokers compared with a larger proportion of males (48.1%). Female patients had more end-stage renal disease (ESRD) (20.3%) at diagnosis and/or previous or synchronous upper tract UC (34.4%) than males (5.6% and 14.7% for two disease conditions, respectively). There were no gender differences in pathological phenotypes such as tumor multiplicity, tumor grade, or stage. In a Cox proportional hazard model, female patients had more metachronous upper tract UC than males but had similar chances of recurrence and progression as males. Combining both genders, tumor multiplicity, stage, and continued smoking were independent prognostic factors predicting bladder recurrence. CONCLUSIONS There were no gender differences in clinicopathological characteristics such as tumor multiplicity, grade, stage, or risk of recurrence or progression. Smoking is not a major risk factor for women in Taiwan. Female patients with bladder cancer tended to have more upper tract UC and ESRD than males.

Aristolochic acid in the etiology of renal cell carcinoma

Background: Aristolochia species used in the practice of traditional herbal medicine contains aristolochic acid (AA), an established human carcinogen contributing to urothelial carcinomas of the upper urinary tract. AA binds covalently to genomic DNA, forming aristolactam (AL)–DNA adducts. Here we investigated whether AA is also an etiologic factor in clear cell renal cell carcinoma (ccRCC). Methods: We conducted a population-based case–control study to investigate the linkage between Aristolochia prescription history, cumulative AA consumption, and ccRCC incidence in Taiwan (5,709 cases and 22,836 matched controls). The presence and level of mutagenic dA-AL-I adducts were determined in the kidney DNA of 51 Taiwanese ccRCC patients. The whole-exome sequences of ccRCC tumors from 10 Taiwanese ccRCC patients with prior exposure to AA were determined. Results: Cumulative ingestion of more than 250 mg of AA increased risk of ccRCC (OR, 1.25), and we detected dA-AL-I adducts in 76% of Taiwanese ccRCC patients. Furthermore, the distinctive AA mutational signature was evident in six of 10 sequenced ccRCC exomes from Taiwanese patients. Conclusions: This study strongly suggests that AA contributes to the etiology of certain RCCs. Impact: The current study offers compelling evidence implicating AA in a significant fraction of the RCC arising in Taiwan and illustrates the power of integrating epidemiologic, molecular, and genetic data in the investigation of cancer etiology. Cancer Epidemiol Biomarkers Prev; 25(12); 1600–8. ©2016 AACR.