November 1, 2015 (Vol. 35, No. 19)

Non-Small Cell Lung Carcinoma: ROSter for Genomic Analysis and Therapies for ALKoholics

Those of you who have a working knowledge of applied Latin can do a quick and easy translation and deduce that pons asinorum literally means the “bridge of donkeys.” Traditionally, it refers to the proof of the isosceles triangle theorem in Euclid’s Elements, and connotes a challenge that separates the nimble thinkers from the simple minds. The goal of this gazette will be to provide insights that will help us all function as nimble thinkers in the rapidly evolving field of diagnostic genomics.

Multiple significant updates/advances have occurred in the field of lung cancer during the past weeks. The American Society of Clinical Oncology (ASCO) updated guidelines for advanced stage non-small cell lung cancer (NSCLC) diagnosis that have an impact on the clinical genotyping of these tumors and basic science studies elucidated mechanisms in ALK signal transduction that can impact efficacy of targeted therapeutics and the emergence of resistance. The increased understanding of ALK signaling mechanisms provide insights for therapy for targeting multiple critical elements in EML4-ALK positive NSCLC tumor cells.

Guidelines for the selection of patients with NSCLC for treatment with tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) and the anaplastic lymphoma kinase (ALK) published in November 2014 (Leighl et al. J Clin Oncol 2014;32:3673-9) broadened the spectrum of testing to detect sensitizing mutations in EGFR and activating rearrangements of tyrosine kinases associated with responsiveness to these agents. In addition to testing of tumors from patients with advanced disease (stage IV), characterization of the status of EGFR and ALK genes in patients with stage I, II, and III disease was encouraged. In addition, the importance of testing for rearrangements in other genes encoding tyrosine kinases, specifically ROS and RET, each of which occur in approximately 1% of patients, was recognized.

Analysis for EGFR mutations should include all sensitizing mutations with a frequency ≥1% of total mutations in this gene and be sufficiently sensitive to detect the presence of the T790M mutation associated with resistance to EGFR TKIs in 5% of tumor cells. Testing of either primary tumors or metastasis is appropriate and a turnaround time of two weeks was recommended.

The challenge of analyzing small tissue specimens was considered in two recommendations. In the face of evidence of squamous differentiation or mixed histopathology, genomic analysis of biopsies in which the presence of adenocarcinoma could not be excluded was supported, particularly in nonsmokers and younger patients. Immunohistochemistry (IHC) with monoclonal antibodies to frequently occurring EGFR mutations (the most common [15 base pair] deletion in exon 19 and L858R, which constitute over 85% of sensitizing sequence variants) and for ALK expression was considered as an analytical back up method when tissues were not sufficient for genomic analysis, and the IHC assays were carefully validated. Finally, the pathologist’s role in preanalytical steps was emphasized.

ASCO guidelines for stage IV NSCLC were published in late August (Masters et al. J Clin Oncol 2015;62:1342).The major impact on genomic analysis is the recommendation for single-agent therapy with crizotinib for patients with rearrangements in the ROS1 gene, in the absence of known sensitizing EGFR mutations and activating ALK gene rearrangements. This recommendation was based on the results of an early trial of 50 patients that showed a significant response rate: 72% overall response with mean duration 17.6 months and 19.2 months median progression-free survival.

The FDA conferred Breakthrough Therapy Designation for crizotinib for this indication. Of course, testing for ROS1 gene rearrangements is implicit in this recommendation but no companion diagnostic has been approved by the FDA. Thus it appears that the FDA requirement for co-development of companion diagnostics will not delay the availability of this drug. In a similar vein, it is not clear that the assays for sensitizing mutations in EGFR that have been approved by the FDA meet the guidelines listed above that were recommended by ASCO in November 2014, because all common sensitizing sequence variants and the T790 resistance mutation are not covered.

Mechanistic insights into signaling cascades in lung cancer with the EML-4-ALK driver rearrangement demonstrate potential therapeutic approaches that target multiple elements in the RAS-MAPK pathway. Current targeted therapy with crizotinib or ceritinib is directed to the constitutively activated ALK kinase, resulting in significant effects that are temporally limited by the onset of resistance. The mechanisms for resistance currently recognized include secondary mutations in the ALK kinase domain, amplification of the rearranged EML-4-ALK oncogene, mutations in alternative lung cancer driver genes, such as EGFR and KRAS, and activation of normal receptor tyrosine kinases, including EGFR, HER2, and KIT.

A recent study by Hrustanovic and co-workers (Hrustanovic et al. Nature Medicine 2015; 21:1038-47) describes an additional novel mechanism for resistance: amplification of wild type KRAS, a downstream component of the relevant signaling pathway. Although ALK signaling involves multiple pathways in normal cells, including RAS-MAPK(kinase), PI3K(kinase)-AKT, and JAK-STAT, the proliferative effects of the EML4-ALK oncogene were found to be dependent upon RAS-MAPK signaling.

Inhibition of RAS-MAPK signaling, but not PI3K-AKT or JAK-STAT signaling, was as effective as inhibition of ALK kinase activity in suppressing the growth of EML4-ALK lung cancer cells. Comparison of growth inhibition of lung cancer cells with various driver oncogenes, demonstrated that EML4-ALK cells were more sensitive to inhibition of MAPK signaling with trametinib (a MEK inhibitor approved by the FDA for single agent therapy and subsequently in combination with dabrafenib, a BRAF antagonist, for treatment of patients with metastatic melanoma carrying the BRAF V600E mutation) than cells with an activated EGFR driver gene.

Inhibition of the RAS-MAPK pathway with trametinib in EML4-ALK lung cancer cells enhanced the therapeutic effect of crizotinib in culture and in mouse xenograft models. A gain in the number of KRAS gene copies was detected in EML4-ALK NSCLC tumors with acquired resistance to crizotinib in a subset of patients, but not in tumor tissues from the initial diagnosis (prior to therapy).

In summary, these studies provide evidence that a combined therapeutic approach with direct inhibition of ALK signaling with crizotinib or ceritinib and inhibition of the critical downstream pathway, KRAS-MAPK, with trametinib, a component of the dual target drug therapy approved by the FDA for treatment of patients with metastatic melanoma, may result in enhanced therapeutic effects and interference with mechanisms involved in the emergence of resistance to ALK TKIs.

The pons asinorum, or bridge of donkeys, originally referred to a proposition, the isosceles triangle theorem, from Euclid’s Elements. Being able to prove the theorem was traditionally regarded as a critical test of one’s mathematical acumen, of whether one could cross from elementary to advanced solutions. The phrase is now taken more generally, encompassing make-or-break transitions of all kinds. For example, the transition from genomic knowledge to diagnostic application may be regarded as a pons asinorum. [Wikicommons/Oliver Byrne’s Elements of Euclid, 1847]

Stephen C. Peiper, M.D. ([email protected]), is Peter A. Herbut Professor & Chair, Department of Pathology, Anatomy & Cell Biology and Zi-xuan Wang, Ph.D., is Scientific Director, Genomic Pathology Laboratory, Assistant Professor, Departments of Surgery and Pathology, Anatomy & Cell Biology, Thomas Jefferson University.

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