From Bedside to Bench

I have no basic science research experience, but when the opportunity to join a laboratory project was offered to me as a clinical fellow, I joined simply because I was interested in the experience and what I could potentially learn from it. Currently, I am involved in a research project assessing whether or not microRNAs (miRNAs) can predict brain metastases in patients with triple-negative and HER2-positive breast cancer. The question originated from observations in the clinic that brain metastases are more common in patients with this subtype of breast cancer.

The concept of miRNA is relatively new. MiRNAs are short, non-encoding ribonucleic acids (RNAs) that regulate the transcription of messenger RNA. As I began working in the lab, I quickly gained insight into the other side of medicine: the laboratory! There, I extracted total RNA with pipettes for accurate measurement of different reagents and used a stopwatch to determine the multiple incubation periods in varying temperatures. As with any scientific endeavor, challenges have arisen. Difficulties obtaining older tissue samples is one. Obtaining suboptimal final products on polymerase chain reaction after several hours of working is another. However, approaching these challenges with perseverance and resourcefulness has made this project move forward, albeit slowly.

The EGFR Story in Lung Cancer

I do not have results to share yet, but later goals include identifying the individual targets and mechanisms of miRNAs that give rise to brain metastases. Future implications include identifying higher-risk patients by using miRNA as a biomarker in serum or tissue, and perhaps using these as targets for treatment. It’s bedside to bench and back again! Reverse translational medicine is not novel. Perhaps the best examples in oncology are those involving the epidermal growth factor receptor (EGFR).In May of 2003, the FDA approved gefitinib, an orally administered EGFR tyrosine kinase inhibitor, as therapy for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) who were refractory to established cancer treatments (both a platinum drug and docetaxel). Tumor response rate was used as a surrogate marker. The overall response rate to gefitinib was around 10%.¹

In other words, most patients treated with gefitinib did not respond. However, in about 10% of patients, a dramatic and rapid response was observed. Subsequently, a phase 3 trial was designed to determine the survival advantage of gefitinib in the NSCLC population as a whole. The ISEL (Iressa Survival Evaluation in Lung Cancer) trial enrolled 1692 patients with previously treated, locally advanced, or metastatic NSCLC, and randomized the patients to gefitinib or placebo.

The results were disappointing. Gefitinib provided no significant survival advantage over placebo (5.6 months vs 5.1 months), although further analysis did reveal improved median survival in 2 subgroups in favor of the gefitinib arm: 1) never-smokers (8.9 months vs 6.1 months; P = .012), and 2) Asian patients (9.5 months vs 5.5 months; P = .01).²

Following a second clinical trial that failed to demonstrate a survival benefit, the FDA revised the labeling for gefitinib in 2005. This limited the use of the drug to patients currently receiving and benefitting from, or who previously received and benefitted from, gefitinib.¹

Researchers were curious why gefitinib produced a survival advantage in particular subgroups (never-smokers and patients of Asian origin).² For this reason, Lynch and colleagues examined the molecular mechanisms behind these results and noted somatic mutations in the tyrosine kinase domain of the EGFR gene in 8 of 9 patients with gefitinib-responsive lung cancer; such mutations were found in 0 of the 7 non-responders. The results of their work suggest that screening for such mutations in patients with lung cancer may help identify those who will respond to gefitinib.³ Other groups of investigators have published similar results independently.⁴

The EGFR Story in Colorectal Cancer

The Iressa Pan-Asia Study (IPASS) was designed to compare gefitinib against carboplatin-paclitaxel as first-line treatment in clinically selected East Asian patients with advanced NSCLC. The patients were never-smokers or former light smokers, and therefore more likely to have EGFR mutations. EGFR mutation status was assessed in patients who consented to have their tumors checked for this and other biomarkers. EGFR mutation status could only be evaluated in 36% of patients. In the subgroup of patients who were positive for the EGFR mutation, progression-free survival (PFS) was significantly higher in those who received treatment with gefinitib rather than with carboplatin-paclitaxel (hazard ratio [HR], 0.48; P <.001). However, in the subgroup of patients without EGFR mutation, chemotherapy conferred a PFS benefit over gefitinib (HR, 2.85; P <.001).⁵Cetuximab and panitumumab are monoclonal antibodies that inhibit EGFR activation. Both cetixumab and panitumumab have demonstrated single-agent efficacy in metastatic colorectal cancer when compared against supportive care. However, response rates are low.^6-9 Is it the EGFR status or the level of EGFR expression that predicts a response to one of these agents?

Chung et al reviewed medical records and identified 16 colorectal cancer patients who had irinotecan-based chemotherapy-refractory disease, were EGFR-negative, and received cetuximab in a non-research setting. An objective response was noted in 25% of these patients. The authors concluded that the current routine practice of using the results of EGFR immunohistochemistry testing for patient selection may exclude patients who could potentially benefit from anti-EGFR therapy with cetuximab.⁶

If not the EGFR status, what factors predict clinical benefit from anti-EGFR inhibition? For the answer, a group of researchers further assessed the question in the lab. Lièvre et al hypothesized that genetic alterations in EGFR-related pathways affect the response to cetuximab. The researchers tested tumor specimens from 30 patients with cetuximab- treated metastatic colorectal cancer for EGFR copy number as well as mutations in the protein-coding genes KRAS, BRAF, and PIK3CA. Among the 11 patients who responded to cetuximab, none tested positive for a KRAS mutation. On the other hand, a KRAS mutation was detected in 13 of the 19 non-responders. It was concluded that the KRAS mutation bestows resistance to anti-EGFR therapy.⁷

Based on these findings, Amado et al looked at the role of KRAS as a selection marker in chemotherapy-refractory metastatic colorectal cancer patients who enrolled in a phase 3 trial that compared panitumumab against supportive care. The investigation was done retrospectively. KRAS mutations in codons 12 and 13 were identified by real-time polymerase chain reaction testing conducted on formalinfixed, paraffin-embedded tumor sections collected for the trial. KRAS mutations were detected in 43% of patients. PFS, the primary end point of the trial, was significantly greater in the wild-type (WT), or nonmutated, KRAS population compared with that in the mutant KRAS population (12.3 weeks vs 7.4 weeks). Response rates were also much better in the WT-KRAS group (17% vs 0%). The authors concluded that WT-KRAS is required for panitumumab efficacy. In addition, the investigators noted, “These are the first results arising from a randomized, controlled trial showing that the state of a signaling molecule downstream of a target plays a crucial role in predicting clinical benefit to a targeted therapeutic.”⁸

Similar findings were published in the same year by Karapetis et al, who retrospectively determined the KRAS status of patients who participated in the phase 3 CO.17 study. The CO.17 study’s primary objective was to assess the effect of cetuximab on overall survival (OS) and PFS in patients with advanced colorectal cancer who had failed multiple lines of chemotherapy. The retrospective analysis by Karapetis et al showed that in WT-KRAS tumors, treatment with cetuximab improved OS (9.5 months vs 4.8 months) and PFS (3.7 months vs 1.9 months) compared with best supportive care. In the mutant KRAS population, cetuximab provided no benefit over supportive care.⁹

The results of these studies were practice changing. Based on these retrospective analyses, the FDA changed the product labels of cetuximab and panitumumab in 2009.¹⁰ My research project is currently not as ambitious, and I am definitely not going to be a physician-scientist after this experience. However, it is exciting to know that I may be able to contribute to the growing pool of molecular knowledge in oncology.

References

1. Information for healthcare professionals: gefitinib (marketed as Iressa). FDA website. http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/ DrugSafetyInformationforHeathcareProfessionals/ ucm085197.htm. Accessed July 21, 2014.
2. Thatcher N, Chang A, Parikh P, et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced nonsmall- cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet. 2005;366(9496):1527-1537.
3. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-smallcell lung cancer to gefitinib. N Engl J Med. 2004;350(21):2129-2139.
4. Ledford H. Translational research: the full cycle. Nature. 2008;453 (7197):843-845.
5. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009;361(10):947-957.
6. Chung KY, Shia J, Kemeny NE, et al. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol. 2005;23(9):1803-1810.
7. Lièvre A, Bachet JB, Le Corre D, et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res. 2006;66(8):3992-3995.
8. Amado RG, Wolf M, Peeters M, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol. 2008;26(10):1626-1634.
9. Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras mutations and benefit from cetuximab in advanced cancer. N Engl J Med. 2008; 359(17):1757-1765.
10. Cetuximab (Erbitux) and panitumumab (Vectibix). FDA website. http:// www.fda.gov/AboutFDA/CentersOffices/OfficeofMedicalProducts andTobacco/CDER/ucm172905.htm. Accessed July 27, 2014.