Novel Approaches in the Treatment of Non-Small-Cell Lung Cancer

ABSTRACT: A wealth of data indicates that certain genetic abnormalitiescan target specific cytotoxic drugs and intervene at an early step as amechanism of resistance in the treatment of non-small-cell lung cancer.Therefore prescribing certain combinations of cytotoxic anticancer agents to avast majority of these patients is futile. Genetic abnormalities have been foundto be useful surrogate markers for response, particularly in colorectal cancer:thymidylate synthase mRNA and ERCC1 mRNA levels. In addition, b-tubulinmutations may also confer paclitaxel resistance in patients. An important targetto be explored for gemcitabine resistance is the assessment of a particularregion in chromosome 11p15.5 wherein lies the ribonucleotide reductase gene thatcould affect gemcitabine metabolism. Shedding light on this genetic framework,several proposed customized chemotherapy studies could help validate therelevance of these markers. [ONCOLOGY 15(Suppl 6):52-60, 2001]

Introduction

To date, the treatment of non-small-cell lung cancer (NSCLC) has been entirely empirically based. That is, if each drug has a 20% response rate, then the combination of drugs A and B easily predicts a 40% response rate and will be rapidly accepted if both drugs have different, nonoverlapping toxicity profiles. A plethora of new cytotoxic anticancer agents have increased response rates-either as a single drug or in combination. However, the results of a recent, four-arm randomized Eastern Cooperative Oncology Group study[1] indicate that no particular combination of either cisplatin (Platinol) with paclitaxel (Taxol), docetaxel (Taxotere), or gemcitabine (Gemzar) surface as a clear salient chemotherapy combination. Certainly new approaches need to be implemented in the treatment of non-small-cell lung cancer.

Rational Background: Genetic Markers In Colorectal Cancer

The Human Genome Project has almost been completed, and a vast majority of approximately 10⁵ human mRNA have been at least partly cloned and sequenced as cDNAs. When completed, these cDNAs will provide valuable tools to study the protein products of their respective mRNAs. These diverse sets of proteins could be selected for anticancer drug discovery programs.[2] New knowledge of normal and aberrant function of oncogenes and tumor suppressor genes provides unique opportunities to understand, and ultimately to control, the processes leading to malignancy. Thus, identification of the molecular and genetic events involved in each step of tumor progression may be central to understand NSCLC and to develop resistant markers and novel targets for pharmacologic intervention in this tumor.

Using laser capture microdissection and cDNA arrays, it has been found that most squamous cell carcinomas of the head and neck overexpress members of the wnt and notch growth and differentiation regulatory system. This suggests that the wnt and notch pathways may contribute to squamous cell carcinogenesis.[3] Also, high levels of expression of notch 3 (a member of the notch proto-oncogene family) have been observed in 25% of NSCLC patients tested. Overexpression was correlated with translocations, involving 19p in these lines.[4]

Gemcitabine Trial

A randomized trial showed that gemcitabine as a single agent yields the same response rate and survival as the cisplatin/etoposide combination but with much better tolerability. The response rate was 17.9% for the gemcitabine arm and 15.3% for the cisplatin/etoposide arm. Median survival times were 6.6 months for gemcitabine and 7.6 months for cisplatin/etoposide. The 1-year survival probability estimate was 26% for gemcitabine and 24% for cisplatin/etoposide. Also, patients in the gemcitabine arm had a greater probability of achieving a tumor response after 2 months (8% vs 0%) and lasting at least 6 months (73% vs 45%).[5]

Docetaxel, Paclitaxel

Treatment of non-small-cell lung cancer, however, remains an almost insurmountable pitfall for oncologists. In randomized studies,[6,7] docetaxel and paclitaxel produced major objective tumor regression in only 13% to 16%. But these single agents still induce significant survival improvement over best supportive care. In the paclitaxel randomized study, median time to progression was 4 months with paclitaxel and only 1.2 months for best supportive care alone (P = .001).[7] Recently, on the grounds of meta-analysis of individual data from a large colorectal cancer clinical trials dataset, Buyse et al[8] proposed that response rate could be a surrogate end point for survival advantage.

Irinotecan, Oxaliplatin

Clinical trials in metastatic colorectal cancer have shown that any improvement in survival is modest albeit statistically significant, even with regimens containing new agents such as irinotecan (CPT-11, Camptosar,) or oxaliplatin (Eloxatine). Striking improvements in survival are unlikely until the complete response rate is high.[9] Interestingly, pharmacogenetically oriented treatment has started for colorectal tumors where response and survival have been associated with the thymidylate synthase (TS) mRNA concentrations, thymidine phosphorylase, and the dihydropyrimidine dehydrogenase. The three genes involved can be used to predict fluorouracil (5-FU) response and to customize adjuvant chemotherapy by using other available drugs, such as irinotecan, for patients expected to be fluorouracil resistant.[10,11]

Preliminary data suggest that gene expression levels of topoisomerase 1, p21, bcl2 may predict response to irinotecan therapy. Also, increased toxicity in patients treated with irinotecan may be explained by polymorphism in the UGT 1A1 gene. Recently, a new method for isolating mRNA and measuring TS gene expression from paraffin-embedded tissue section using TaqMan technology has been developed at the University of Southern California.[12] TS gene expression was also shown to influence the response to 5-FU-based chemotherapy and survival in patients with gastric cancer.[13]

Fluorouracil vs. Irinotecan

Trials have begun to be designed in which patients with intratumoral relative TS gene expression higher than 4´ 10^-3 are treated with irinotecan while patients with lower than 4´ 10^-3 are treated with 5-FU.[12] In fact, there are many common similarities between colorectal cancer and non-small-cell lung cancer. They have some seemingly equal genetic profiles characterized by multiple losses of heterozygosity (LOH) on chromosomes 5q, 17p (in the p53 region, among others). For example, the adenomatous polyposis coli (APC) gene is methylated at a very high frequency (> 95%) in esophageal adenocarcinoma. Those patients with high levels of methylated APC in their pretreatment serum had a significantly shorter survival time than patients with undetectable or low levels.

More importantly, analysis of several serum specimens obtained from several patients under treatment show that serum status of the marker correlated with the degree of tumor response. The Danenbergs have reported that lung tumors also harbor nearly a 95% frequency of methylated APC genes. The frequency of detection of methylated APC in the serum of lung cancer patients was 50%. Analysis of serum samples for stage I lung cancer patients show that methylated APC levels predicted treatment outcome.[14]

11p15.5 LOH as Potential Genetic Surrogate Marker

Several markers of lung cancer susceptibility have been identified.[15] We found a higher percentage of rare HRAS1 VNTR alleles in lung cancer patients than in unaffected controls.[16] In addition, lung cancer cells contain deletions of multiple chromosomal regions. Deletion mapping studies have defined more than 30 regions dispersed in 21 different chromosome arms as candidate tumor suppressor loci.[17]

Polymorphic DNA probes for chromosome 3p were used to compare tumor and constitutional genotypes of nine small-cell lung cancer patients, with loss of heterozygosity (LOH) a common feature in tumor DNA of all patients.[18] Since then, allele loss involving chromosome arm 3p is one of the most frequent and earlier known genetic events in lung cancer. Intriguingly, in a recent chromosome 3p allelotyping it was noted that most small-cell lung carcinomas (91%) and squamous cell carcinomas (95%) exhibited larger 3p segments of allele loss, whereas most (71%) of the adenocarcinomas had smaller chromosome, nearer 3p allele loss.[19]

Evaluating LOH

In our recent experience (unpublished) evaluating loss of heterozygosity, we used primers flanking dinucleotide microsatellite repeat polymorphisms spanning the chromosome 3p arm, which is located at chromosome 3p14 (D3S1285) and marker D3S1234 and at chromosome 9p21 (D9S1747) and p53 in specimens of resected NSCLC patients and in their serum DNA. We analyzed the data with customized software designed to facilitate color formatting and visualization of data. Microsatellite analysis for LOH in 54 resected NSCLC patients is depicted in Figure 1. 

For another excellent review about tumor suppressor genetic abnormalities in lung cancer, see Pitterle et al.[20] Beyond the potential prognostic value of LOH, especially in stage I patients, these numerous chromosomal deletions could be involved in drug resistance.

Recently, Bepler et al[21] reported that 11p15.5 LOH, including the gene for the enzyme ribonucleotide reductase, predicts poor survival. Survival of patients with stage I and LOH (23 patients) is equal to that of patients with stage II disease, whereas survival significantly improved (P = .003) in patients with stage I and no LOH (101 patients). The LOH 11A region is lost in 75% of lung cancers. This region is located on chromosome segment 11p15.5 between the b-hemoglobin (b-hhh) and insulin genes.[22] A number of imprinted genes with maternal expression have been identified at 11p15: BWR1A (or IMPT1/TSSC5), BWRIC (or IPL/TSSC3), and CDKNIC, which encodes the cyclin-dependent kinase inhibitor p57^KIP2 that is found mutated in some cases of Beckwith-Wiedemann syndrome (an excessive-growth genetic disorder that predisposes to Wilm’s tumor).

Gene Clustering

A striking feature of imprinted genes in mammals is their tendency to cluster in the genome. A major imprinted gene cluster occurs in human chromosome 11p15.5. The centromeric and telomeric boundaries of the LOH 11A region is defined for the markers D11S988 and D11S860, which are spaced at an interval of 580-655 kb; the LOH 11A region contains the genes SSA/RO52, RRM1, and GOK.[21] Bepler et al[22] constructed a precise physical map containing the b-hemoglobin gene cluster region through the gene for ribonucleotide reductase subunit M1(RRM1) to the marker D11S860. They mapped the location of 6 polymorphic markers, 22 potential genes, and 18 transposable elements.

Previous studies of the Bepler group[23,24] show that LOH 11A (allele loss) is associated with metastasis formation in patients with lung cancer.[23] This unusual finding is explained by the presence of the RRM1 gene in the LOH 11A region that encodes the large subunit (M1) of the enzyme ribonucleotide reductase,[24] which is involved in the metabolism of gemcitabine.[25] Hence, we can speculate that reduced ribonucleotide reductase activity influences the dNTP pools that in turn can influence the incorporation of gemcitabine into DNA. The RRM1 gene occupies only 55 kb of the LOH region and additional genes may be involved in lung cancer.

RR Activity

Ribonucleotide reductase (RR) is an S-phase-specific, rate-limiting enzyme of the DNA synthesis pathway and is mainly responsible for the conversion of ribonucleoside diphosphate to deoxyribonucleoside triphosphate, which is essential for DNA synthesis. RR consists of two subunits-M1 and M2. The M1 subunit possesses a binding site for enzyme regulation, and the M2 subunit is involved with RR activity.[25] Because gemcitabine targets RR as a mechanism of cytotoxicity, alteration or deletion of RR may play a role in gemcitabine resistance.

The reliance of RR as a resistant marker should be tested in different clinical settings. Beyond conventional clinicopathologic prognostic factors in surgically resected non-small-cell lung cancer,[26] we have not started to analyze genetic markers that can be reliance markers of cytotoxic effect. This kind of research matches for neoadjuvant gemcitabine-based chemotherapy trials. For example, gemcitabine/cisplatin has been used as neoadjuvant treatment with a high response rate of 70% in stage IIIA N2 non-small-cell lung cancer patients. Downstaging N2 was obtained in 53% of instances. Nevertheless, resections were complete only in 71% of patients who underwent thoracotomy.[27]

K-ras Gene Mutation

Conversely, ras mutations have been associated with enhanced gemcitabine cytotoxic effect in human NSCLC cell lines.[28] Decrease in DNA levels during treatment seems to be a better prognostic marker.[29] Also treatment led to the disappearance of K-ras gene mutations in plasma DNA in six of nine pancreatic adenocarcinomas. Conversely, three patients with a persistently positive K-ras gene mutation in post-treatment plasma samples were likely to show early recurrence or to have progressive disease.[30]

We can hypothesize that customized chemotherapy (LOH 11A region and K-ras mutations) can increase response and survival. In addition, molecular assessment of response at regular intervals can provide (by means of serum DNA analysis)[31] the basis for new methods of response evaluation. If this is demonstrated in a pharmocogenetic-orientated clinical trial, we will need to reconsider the six to eight chemotherapy cycles suggested by the 1997 American Society of Clinical Oncology guidelines.[32]

Gemcitabine/Cisplatin Trial

Our Spanish Lung Cancer Group conducted a randomized trial comparing cisplatin with a gemcitabine dose at 1,250 mg/m² on days 1 and 8 to etoposide/cisplatin. The response rate for patients with gemcitabine/cisplatin was higher than for patients with etoposide/cisplatin (40.6% vs 21.9%; P = .02). This superior response rate was associated with a significant delay in time to disease progression (6.9 months vs 4.3 months; P = .01).[33]

A subsequent, large randomized trial has included 600 patients, confirming the efficacy of gemcitabine/cisplatin (response rate 40.4%) over a triplet of gemcitabine/cisplatin/vinorelbine (Navelbine) or in comparison with sequential noncisplatin doublets.[34] Hence, in our experience gemcitabine/cisplatin at a 21-day interval is an effective and well-tolerated combination.

Currently, we are trying to validate ras mutations and LOH 11A as important surrogate markers of tumor response in NSCLC patients treated with gemcitabine/cisplatin. We can take advantage of the SLCG experience to undertake a study of gemcitabine/cisplatin in metastatic NSCLC in which baseline serum DNA levels should be analyzed for K-ras mutations and loss of heterozygosity at 11p15.5 to determine the influence of these abnormalities in predicting gemcitabine resistance or sensitivity. We calculate that 240-300 patients could be treated with gemcitabine/cisplatin to analyze the different subgroups with zero, one, or two genetic abnormalities (Table 1).

ERCC1 mRNA as Potential Surrogate of Cisplatin Response

Genetic understanding can help explain why three of seven patients previously treated with gemcitabine/cisplatin also had partial responses to second-line gemcitabine.[35] In the Crinò et al study,[34] all three patients attained a partial response to the first-line gemcitabine/cisplatin regimen. Similarly, noncisplatin combinations[36] could be relevant in cases of cisplatin resistance. Cisplatin has been crucial in non-small-cell lung cancer combinations and also surfaces as an independent prognostic factor.[37] However, alterations in drug uptake or efflux and intracellular quenching of cisplatin molecules contribute to cisplatin resistance.

The relative mRNA level of the excision repair cross-complementing ERCC1 gene was associated with response and survival in cisplatin-treated gastric cancer patients.[38] The human DNA repair gene ERCC1 is involved in the excision repair of platinum-DNA adducts. Originally it was observed that ovarian cancer patients whose tumors were clinically resistant to therapy showed greater levels of ERCC1 mRNA compared with tumor tissues from patients clinically sensitive to cisplatin-based chemotherapy.[39] On the same lines, ERCC1 gene expression levels have been associated with survival after neoadjuvant chemotherapy in esophageal cancer.[40] Now, ERCC1 mRNA is being investigated in NSCLC patients treated with gemcitabine/cisplatin.

Chemo-Resistance

The ERCC1 gene exhibits homology to the yeast RAD10 gene. Other molecular processes that would contribute to differences in chemo- and radioresistance include variations in DNA repair mechanisms. Overexpression of wild-type RAD51 has been found in 66% of human pancreatic cancer cells.[41] Overexpression of other genes has been involved in chemoresistance, particularly tumor cell lines with acquired resistance to cisplatin-overexpressed metallothionein.[42,43] HOXA9 expression in predicting chemotherapy response as measured by quantitative PCR should be tested in non-small-cell lung cancer. In fact, the homeobox gene HOXA9 was the single most highly correlated gene of the 6,812 genes overexpressed in acute leukemia patients with treatment failure.[44] Also, BRCA1 overexpression is linked to cisplatin resistance.[45] Table 2 summarizes several key genes whose expression levels can be determined by quantitative PCR.

Shedding of tumor cells into the circulation has been described in the order of 0% to 27% using PCR methodology and 0% to 5% using immunocytochemistry.[46] However, measuring blood transcripts could be an important surrogate for circulating cancer cells[47] as well as crucial to determine mRNA levels of chemoresistance-related genes. Using real-time quantitative PCR, we can measure mRNA by means of a real-time quantitative PCR assay (Figure 2).

The demonstration that tumor-derived DNA is detectable in the serum of cancer patients[48-50] raises the possibility that methylation assays can be performed in serum DNA samples. Transcription of many genes are commonly silenced by a mechanism of DNA methylation.[45,50] Methylation of CpG in the promoter region invariably correlates with the absence of gene expression.[50,51] It has been observed that hMLH1 promoter methylation could play a significant role in determining the sensitivity of ovarian tumors to cisplatin as well as to other chemotherapeutic agents.[52,53]

Direct sequencing of sodium bisulfite-modified genomic DNA enables us to identify the methylation state of all the CpG sites in the amplified sequences (57 for the hMLH1). The level of methylation of the CpG sites (57 sites) is judged from the pattern of the electropherogram (Figure 3).[54] Because the single methylation of a single site is unlikely to suppress transcription, only cases with more than two methylated CpG sites are judged as methylation positive. The extent of methylation of a CpG site measured from electropherogram data can indicate the relative degree of chemoresistance.

Methylation protocol is rather sensitive and was originally described by Clark et al.[55,56] Examples of target genes for methylation that can contribute to confer resistance are shown in Table 2. Recently, a human ras effector homologue (RASSF1) has been identified andlocated at 3p21.3 that is frequently methylated in non-small-cell lung cancer.[57]

We have examined the presence of cell-free DNA in the bloodstream of treated NSCLC patients at different points of follow-up and sequenced the tubulin gene for the presence of mutations. Our working hypothesis was that tubulin mutations could be a marker for survival due to its important role in cell division. b-tubulin mutations were detected in serum DNA from 55/131 (42%) patients and in none of the control subjects.[58] b-tubulin mutations were described in approximately 30% of cell lines and resulted in paclitaxel resistance.[59,60] In the clinical setting, b-tubulin mutations were found in the same frequency and were also related to paclitaxel resistance.[61]

Genetic Lessons From ECOG Study

The accumulated genetic information could have important clinical implications for future customized treatment. Which lessons can be learned from Eastern Cooperative Oncology Group (ECOG) study E1594?[1] Almost 1,200 patients included in this ECOG study were randomized to receive one of four regimens: cisplatin/paclitaxel, cisplatin/gemcitabine, cisplatin/docetaxel, or carboplatin/docetaxel. Response rates were as follows: 21%, 21%, 17%, and 15%, respectively. Time to progression was 3.5, 4.5, 3.6, and 3.3 months, respectively, and 1-year survival was 31%, 36%, 31%, and 35%, respectively. These are rather meager results that fall short of a winning regimen.

How can the ECOG study’s therapeutic results be improved? There were three arms in study E1594 (Table 3): A, C, and D used either cisplatin or carboplatin (arm D) plus either paclitaxel or docetaxel (arm C). Taking advantage of screening serum DNA, those patients with tubulin mutations (30% at GTP binding sites) should receive nontaxane combinations such as gemcitabine/cisplatin. Conversely, for arm B (cisplatin/gemcitabine) if there is LOH 11A, then a combination of paclitaxel or docetaxel would be preferred. Finally, all four arms used cisplatin or carboplatin (arm D). If ERCC1 overexpression is noted at baseline, then a noncisplatin combination such as gemcitabine/paclitaxel or docetaxel could be chosen.

Irinotecan

Irinotecan offers another choice. A new combination of irinotecan/5-FU/leucovorin was compared with the standard treatment of 5-FU/leucovorin in two trials in metastatic colorectal cancer. There was significant improvement in the response rate for the irinotecan-containing regimen (35% and 39% for irinotecan/5-FU/leucovorin vs 21% and 22% for 5-FU/leucovorin). Both studies showed a modest but highly significant improvement in median survival of 2 and 3 months.[62,63]

Customized Chemotherapy

More recently we collaborated with investigators from UC Davis Cancer Center and Norris Comprehensive Cancer Center to create the Genotypic International Lung Trial (GILT). Our goal was twofold. One, to test tubulin mutations and ERCC1 mRNA expression at baseline. And second, to randomize stage IV non-small-cell lung cancer patients to a control arm of docetaxel/cisplatin or one of four experimental groups in the genotypic arm according to the presence of tubulin mutations, ERCC1 mRNA overexpression, or both abnormalities. A fourth group, in which none of these alterations is observed, will be treated with the same regimen as the control arm (Table 4). The control arm receiving docetaxel/cisplatin will validate the role of tubulin mutations and ERCC1 expression.

Based on the presence of tubulin mutations, patients will receive in the genotypic arm gemcitabine/cisplatin; if ERCC1 overexpression is present, they will receive gemcitabine/docetaxel; if both alterations are present, patients will receive gemcitabine/irinotecan. The role of docetaxel has been associated with significant prolongation of time to progression and survival at a dose of 75 mg/m² in previously treated non-small-cell lung cancer patients.[64,65]

Pemetrexed Disodium

Pemetrexed disodium (Alimta, LY231514, MTA) targets several folate requiring enzymes including thymidylate synthase (TS), dihydrofolate reductase, aminoimidazole carboxamide ribonucleotide fomyltransferase (AICARFT), and others. However, pemetrexed is 30 to 200 times more potent as an inhibitor of thymidylate synthase than of either AICARFT or GARFT (glycinamide ribonucleotide formyltransferase), suggesting that its clinical effects are mediated predominantly through TS inhibition.[66] Pemetrexed plus cisplatin has been evaluated in a phase II study with 39% partial response rate and a median survival of 10.9 months.[67]

The Role of Thymidylate Synthase

As previously noted, gene expression levels of TS may predict clinical outcome. Also, genomic polymorphism of the TS gene may be associated with response and toxicity to 5-FU chemotherapy. A triple repeat on the 5-UTR region of the TS gene is significantly associated with higher intratumoral gene expression of TS.[12]

Table 5 shows four genetic markers that urgently need to be validated. Demonstrating that they could be reliable surrogate markers of response could contribute directly to improving NSCLC outcome as well as to change imaging intensity on a positron-emission tomography scan.[68,69] The latter could be a more reliable predictor of response than the standard computerized tomography assessment.

Conclusion

In the pharmacological arsenal, gemcitabine plays a pivotal role in new chemotherapy combinations in lung cancer management. Insights into molecular mechanisms are shedding light on potential genetic predictive markers of response that can lead to individualized chemotherapy for non-small-cell lung cancer patients. This article has discussed several guidelines for choosing cytotoxic drug combinations.

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