Medication regimens
Out of the 75 patients with HBV-related HCC, only 12 had a history of treatment with nucleoside/tide drugs. Of these, seven had received LAM, three received ADV and two received ENT. Three of the patients on LAM were later shifted to ENT, and 1 on ADV initially was shifted to ENT.
HBV DNA was extracted by the standardized manual phenol-chloroform method described, with slight modifications. The polymerase gene was amplified in a 25 mL PCR reaction mixture, containing 2 mL of the DNA sample, 10 mM of each dNTP, 20 ng of each primer and 1U of Taq DNA polymerase. Amplification was carried out in a thermal cycler (Biometra, Gottingen, Germany) for 30 cycles that were comprised of denaturation at 95°C for 60 sec, annealing at 67.5°C for 45 sec and extension at 72°C for 45 sec, with a final extension step at 72°C for 10 min. The above steps were accompanied by an initial denaturation at 95°C for 10 min. The primer sequences used were 5′-CTGTAACACGAGAAGGGGTCCTAG-3′ and 5′-GTGGGGTCACCATATTCTTGGG-3′. A 2-mL aliquot of the PCR products was resolved in a 2% agarose gel containing ethidium bromide and observed under UV light. The PCR products of interest were purified for sequencing using the Perfectprep Gel Clean-up Kit (Eppendorf, Westbury, NY, USA), as per the manufacturer’s instructions. The PCR amplicons with a sharp band were sent for commercial sequencing, as per the guidelines of the company whose services were hired.
The PCR products for the HBV polymerase gene isolated from the HBV-related HCC cases were sequenced using the commercially available sequencing services from Macrogen, South Korea. The sequences obtained from Macrogen were submitted to Genbank under the accession number BankIt1600355: (61). The polymerase gene sequences were analyzed with the software http://www.hepseq.org/Public/Tool/annotator_tool.php and the drug-resistant mutations detected were studied in detail.
A total of 4 viz. (L80I (LAM), I169T (ENT), N236T (ADV) and A181V LAM/ADV)) mutations were observed in three patients. Two of the patients were harboring dual-drug resistant mutation. The drug-resistant mutants are shown in the Figures S1–S3, along with the results of statistical analysis.
Mutations such as the L80I observed in this study have been documented as associated with LAM resistance, and characterized as a compensatory mutation for M204V/I.6 They stated that the main M204V/I drug-resistant mutation and additional compensatory mutations such as L80I/V, V173L and L180M are associated with high-level resistance to LAM (3TC), telbivudine (L-dT) and emtricitabine (FTC). In addition, the importance of this particular mutation observed in our study needs to be further assessed since this novel mutation is not located in the supposed drug binding domain. The ability of rtL80I to mediate LAM resistance is of poignant general interest and deserves the utmost attention primarily for two reasons. First, the fact that mutations in the periphery of the nucleos(t)ide binding site may lead to resistance has to be taken into consideration when developing, applying and assessing resistance testing approaches. Second, it may also have an association with other drugs available for HBV treatment.7
Similarly, dual-drug-resistant mutations were observed in two of the HBV-related HCC cases in our study of patients from North India. The first case harbored the L80I and N236T mutations. L80I is a LAM mutation, while N236T is associated with ADV resistance.2 The main M204V/I drug-resistant mutation and the additional compensatory mutations–L80I/V, V173L and L180M–are associated with high-level resistance to LAM, L-dT and FTC.6 The other patient with dual-drug mutation was found to be harboring the I169T and A181V mutations. I169T has been associated with the ENT resistance mutations in LAM-resistant patients, and is believed to play a secondary role in ENT resistance; meanwhile, A181V/T is associated with resistance to ADV and/or LAM.2,7,8 These mutants were expected, as few of the HBV-related HCC cases in our study had been under nucleotide/nucleoside therapy in the past. The documentation of dual-drug mutants observed in the current study can be explained, however, as many patients (while on treatment) developed LAM mutations and were either switched to ADV or ENT.
This current report confirms the presence of previously reported drug-resistant mutations in the hepatitis B viral genome infecting a North Indian population. Its association with the advanced stage of the disease and to genotype D, in particular, needs to be studied in depth. Although, drug resistance was not observed in the genome of HBV genotype C, the polymerase gene of this genotype needs careful investigation as it has been reported to be more aggressive than the other genotype.9 The current study did not encounter cases with co-infection of the different genotypes of HBV, as reported by Datta S, et al, because most of the cases selected (50, data not shown) were from North and South India, where the predominant circulating HBV genotypes are D and A.9 However, the authors of this manuscript do not rule out the possibility of integration of HBV genomes in cases where genotype C has been reported (from Northeast India). Future prospective study on drug-resistant mutants, with respect to different genotypes of HBV, will provide more information and may well provide scientific data in support of the school of belief that HBV genotype D is more prone to drug resistance. The present findings will also be of immense use for framing health programmes to fight against HBV infection in India, as the dominant genotype in this part of the globe is HBV genotype D.