Assessment of DNA Repair Mechanisms to Determine the Susceptibility of Non-small Cell Lung Cancer to Alkylating Agents

Lung cancer continues to be the leading cause of cancer-related deaths in the US, with a 5-year survival rate of only 15.7% [1]. NSCLC encompasses the largest group within this category and represents an important population for which to target efforts to increase the size of the chemotherapeutic arsenal. Currently, platinum-based chemotherapy results in a statistically significant survival benefit as compared to best supportive care in all stages of NSCLC [2]. However, the various platinum combinations have generated a plateau in overall response and survival benefit.


Introduction
Lung cancer continues to be the leading cause of cancer-related deaths in the US, with a 5-year survival rate of only 15.7% [1]. NSCLC encompasses the largest group within this category and represents an important population for which to target efforts to increase the size of the chemotherapeutic arsenal. Currently, platinum-based chemotherapy results in a statistically significant survival benefit as compared to best supportive care in all stages of NSCLC [2]. However, the various platinum combinations have generated a plateau in overall response and survival benefit.
Temozolomide is an oral alkylating agent that is routinely used in high grade gliomas. The FDA approval of temozolomide followed a clinical trial that showed that the addition of temozolomide improved median survival in patients with glioblastoma multiforme from 12.1 to 14.6 months and improved the 2-year survival rate from 10.4% to 26.5%, as compared to radiation therapy alone [3]. An accompanying study showed that the patients that benefited from temozolomide were those that had the MGMT gene silenced by promoter methylation [4]. MGMT is a repair enzyme, which reverses the temozolomide-induced DNA damage and thus, its activity is a mechanism of resistance to this agent [5]. Promoter methylation results in gene silencing and abrogates this resistance mechanism [6][7].
Temozolomide-induced damage leads to cell death via the activity of the MMR system. This system is composed of a family of proteins, orthologs of the prokaryotic MutS and MutL proteins. They assemble at the site of a DNA mismatch, which leads directly to cell cycle arrest and cell death. If the MMR system is disabled, alkylation damage accumulates without triggering cell killing [8]. Acquisition of mutations in the MMR system is another mechanism of resistance to alkylating agents [9].
The functional status of the MMR system and of the MGMT enzyme can be assessed by commercially available molecular tests. MMR deficiency is diagnosed by a high degree of instability in regions of repetitive DNA, a phenotype called microsatellite instability (MSI). MGMT silencing is detected by the presence of methyl groups in the MGMT promoter sequences. We conducted MGMT methylation and MSI tests on paraffin-embedded NSCLC samples, in order to determine the proportion of patients who may derive benefit from temozolomide or other alkylating agents.

MGMT methylation test
Detection of promoter methylation of the MGMT gene was conducted as previously published [10]. Briefly, genomic DNA was extracted from sections using the classical phenol/chloroform extraction method. DNA was then subjected to bisulfate treatment using the EZ DNA Modification Kit (ZYmo, Orange, CA). Unmethylated cytosines, but not methylated cytosines, are modified into uracil by this treatment. Methylation-specific polymerase chain reactions (PCR) was performed for the MGMT promoter sequence using specific primers and the Amplifluor assay format on a ABI Prism 7900HT instrument (Applied Biosystems, Foster City, CA). β-actin was used as a reference gene in the assay using primers outside any CpG islands. The MGMT target sequence is located on chromosome 10 between positions 131155505 and 131155619, while the β-actin target sequence is located on chromosome 7 between positions 5538428 and 5538325 (RefSeq, NM_002412). The amplicon sizes for the meth-MGMT and theβ-actin analytes are 136 bp and 125 bp, respectively.
The amplicon copy number for MGMT and β-actin were generated using the SDS 2.2 software (Applied Biosystems, Foster City, CA) as previously described [10]. Briefly, this program determines the cycle number at which the amplification curves cross the threshold value set automatically by the software and then uses it to calculate the copy number based on a linear regression of the values plotted on a standard curve. To compensate for variations in copy number due to sample differences, the derived meth-MGMT copy numbers were divided by the copy number of the control analyte β-actin for the same sample. The resulting value was multiplied by 1000 to produce a ratio value. Ratio values above 10 are considered methylated samples.

MSI Test
Detection of microsatellite instability in our samples was conducted as previously described [11]. Briefly, tissue was microdissected from tumor and normal tissue according to the H&E slide. DNA was isolated using xylene/ethanol deparaffinization followed by Proteinase K digestion and heat inactivation. Amplification of six microsatellite markers, TGFBRII, BAT26, BAT25, D5S346, D17S250 and D2S123, was accomplished using specific primer sets (LabCorp, RTP, NC) and phosphoramidite fluorescent labels (IDT, IA). Labeled PCR products were detected using the ABI 310 Genetic Analyzer and GeneScan collection software (Applied Biosystems, Foster City, CA). The identification of normal or tumor allele amplicon sizes was accomplished by examining the appropriate electropherogram (normal and tumor) and determining the predominant amplican size(s) for each allele at each locus, i.e. the greatest peak height(s). If the tumor specimen has novel amplicon lengths in relation to the patient's normal amplicon length(s) at a specific locus, that locus was considered positive for MSI. Samples were scored as following: MSI-high included samples were 2 loci or more exhibited microsatellite instability, MSI-low included samples were 1 loci exhibited microsatellite instability and MSI-stable included all samples where no loci exhibited microsatellite instability.
Loss of heterozygosity (LOH) was also determined for the microsatelliate markers. LOH was defined as complete or partial loss of one of two alleles at a heterozygous locus in tumor-derived DNA compared with normal DNA. Scoring of the alleles was based on comparison of the peak height ratio of a heterozygous matching normal and tumor sample. The peak height ratio was obtained by dividing the height of allele 1 by the height of allele 2. This was done for both normal and the tumor samples. The T/N ratio of the ratios was then calculated as: [(A1T/A2T)/ (A1N/A2N)]. LOH was scored if the T/N ratio deviates more that 30% from the reference value of 1.

Results
We determined the MGMT promoter methylation status and the microsatellite instability of one hundred and eight (108) tumor tissue samples from 96 patients that were treated at the Maine Center for Cancer Medicine for a diagnosis with Non-small cell lung cancer. The characteristics of this patient population are noted on Table 1. Eighty (80) of those patients had methylation levels below 10 ("unmethylated"), 9 patients had above-threshold methylation levels ("methylated"), 5 patients had methylation levels above 10 but below threshhold ("unmethylated"), and 3 patients had an "invalid" result. There were 11 patients with 2 or more tissue blocks. Results of those duplicate samples were all concordant with one exception; two samples from the same patient were found, in one case to have unmethylated promoter and in the other case to have an "invalid" result. The overall rate of methylated MGMT promoter among our NSCLC patients was 9.6%, indicating that this cancer type is not especially susceptible to temozolomide (Table 2).
Ninety one (91) of the patients had tumors that displayed microsatellite stability (MSIS), 2 patients had a low level of microsatellite instability (MSIL), 2 patients had a high level of microsatellite instability (MSIH), and 1 patient had no normal tissue for comparison purposes and therefore had an invalid result. There were 11 patients with 2 or more tissue blocks. Results of those duplicate samples were concordant except in two cases. In both those cases, one sample showed some degree of instability and the other had a stable result. The overall rate of microsatellite instability among our NSCLC patients was 3.7% indicating that this cancer type is unlikely to be resistant to alkylating agents. There were no patients with both MGMT promoter methylation and microsatellite instability (

Bronchioalveolar 6
Large cell 3 Mixed 2 NSC not further specified 12 There were no detectable associations between the status of these repair mechanisms and smoking history, histology or tumor grade. Also, we did not discern any remarkable differences in the response or survival between the patients that had aberrations in either MGMT or MSI and those that did not. The average progression free survival among patients with MGMT promoter methylation was 21 months, and the median was 13 months. For those with MSI, the average progression free survival was 17 months while the median was 16 month. For the reminder of the NSCLC population, the average progression free survival was 16 month and the median was 11 months. The small number of NSCLC patients with MGMT promoter methylation or a positive MSI test precluded obtaining statistically significant differences.

Discussion
The disabling of repair mechanisms occurs as part of the process of cancer progression. It leads to the so-called "mutator phenotype", which results in the rapid accumulation of mutations and, over time, the selection of tumor clones that are progressively more aggressive and chemoresistant. However, this phenomenon at times opens windows in which the tumor becomes susceptible to specific chemotoxic agents. In the case of alkylating agents such as temozolomide, silencing of the MGMT gene by epigenetic changes in promoter methylation, renders a tumor susceptible to this agent, but only as long as the MMR system remains functional. If MMR becomes disabled, the opportunity to kill tumor cells with this class of agents closes.
Early results indicated that as many as 20% of lung cancers display MGMT promoter methylation [12]. Later studies have determined that percentage to be lower [13]. Our results indicate that approximately 10% of NSCL tumors have the MGMT gene silenced by promoter methylation. This is in contrast with the case in glioblastoma multiforme (GBM), where as many as 45% of tumors display this epigenetic change [4]. For this reason, benefit to temozolomide could be observed in unselected GBM populations.
The proportion of NSCLC cases that display inactivation of MMR was unknown and said to range between 0 to 70% [14]. Our results indicate that this percentage is very low (4%). The determination of this second number is arguably more important than the frequency of MGMT silencing. This is because if MMR is disabled, there are no known manipulations to re-activate it. However, there are inhibitors currently in clinical trials that can lower MGMT expression [15]. In other words, the proportion of NSCLC with a functional MMR system would set the upper limit for the number of patients that may benefit from temozolomide. Indeed the percentage of GBM patients with MMR deficiency is very low [16], in contrast with that in found in the colorectal cancer population where MMR deficiency, either inherited or sporadic, is much more prevalent [17].
Given the dramatic benefit derived from temozolomide in glioma patients with MGMT promoter methylation [3], the results of this translational study argue for the pursuit of early phase clinical trials of temozolomide in MGMT-deficient NSCLC patients. Moreover, if ongoing clinical trials to test the efficacy of MGMT inhibitors prove positive in GBM patients, it would open the door for similar studies in NSCLC. Interestingly, temozolomide has been studied in patients with brain metastasis from NSCLC and other solid tumors [18,19]. Although these studies showed a trend towards improved survival, the results did not reach statistical significance. We could argue that, in the case of NSCLC, this may be due to a low rate of MGMT promoter methylation and the resulting low sensitivity to temozolomide. Two approaches based on our results may lead to improved outcomes: 1) preselection of the 10% of NSCLC patients with MGMT methylation, 2) coadministration of temozolomide with MGMT inhibitors. Translational studies such as the present study provide background molecular data for more rational clinical trial design. samples that displayed defects in either of these two repair mechanisms. The first column indicates the patient sample ID and the second column indicates the tumor stage and histology of each sample. The copy number of the MGMT promoter amplicon is shown on the third column, while the copy number of the reference, b-Actin, amplicon in shown on the fourth column. The fifth column indicates whether the sample was categorized as having the MGMT promoter region methylated or unmethylated. Columns 6-11 show the instability of 6 microsatellite regions. Negative (Neg) indicates stable regions, MSI indicates regions that displayed instability, and LOH indicates regions with loss of heterozygosity. The last column indicates whether a sample was categorized as MSI-stable (MSIS) or samples with no regions of instability, MSI-high (MSIH) or samples were 2 loci or more exhibited microsatellite instability, or MSI-low(MSIL) or samples were 1 loci exhibited microsatellite instability.

MGMT Promoter Methylation Test
Microsatellite Instability Test