Anthony John Iafrate

Genotypic and Histological Evolution of Lung Cancers Acquiring Resistance to EGFR Inhibitors

Lung cancers undergo dynamic genetic and histological changes upon developing resistance to EGFR inhibitors. The Shifting Sands of Lung Cancer Lung cancer is the leading cause of death globally and has proven very difficult to treat. The development almost a decade ago of tyrosine kinase inhibitors that specifically block the epidermal growth factor receptor (EGFR), which is switched on in many lung cancers, provided hope that targeted therapies would finally combat this deadly disease. However, only a certain subpopulation of lung cancer patients carrying specific activating mutations in EGFR responded clinically to EGFR inhibitors, and even among these patients, resistance to the inhibitor emerged within 12 months. To better understand how lung cancers develop drug resistance, Sequist and colleagues undertook a comprehensive genetic and histological analysis of 37 patients with non–small cell lung cancer (NSCLC), and they made some surprising discoveries. In an effort to understand the exact mechanism underscoring the acquisition of drug resistance in NSCLC patients treated with EGFR inhibitors, the investigators analyzed tumor biopsies from patients at the time they acquired resistance. All of the lung cancer patients retained their original activating EGFR mutations, but some patients had acquired another mutation in EGFR (T790M), which interferes with binding of the drug to the receptor, rendering the tumors resistant. Meanwhile, another group of patients became resistant because they developed amplification of a gene encoding the MET tyrosine kinase receptor, which, like EGFR, drives cell growth. Yet other patients acquired drug resistance mechanisms that had not been reported before including amplification of the EGFR gene itself and mutations in the PIK3CA gene (which encodes a subunit of the signaling molecule phosphatidylinositol 3-kinase). In addition, the authors observed that a few lung cancers transitioned from an epithelial cell morphology to a mesenchymal cell–like appearance, which is associated with a more aggressive type of tumor. In five patients, the authors discovered another type of transition that was even more surprising: the conversion of NSCLCs into small cell lung cancers (SCLCs), which are easier to treat. Indeed, these five patients responded well to the typical chemotherapy regimen used to treat SCLCs. To study the evolution of lung tumors in patients over the course of their disease, the investigators took serial biopsies from three lung cancer patients over 2 years. They found that when the patients acquired drug resistance and were then taken off the EGFR inhibitor, they lost the resistance mutations and their tumors once again became sensitive to treatment by either the same or a different EGFR inhibitor. The detailed genetic and histological analysis by Sequist and colleagues provides new insights into the shifting sands of drug resistance evolution in lung cancers and suggests that serial biopsies may be essential in the quest to reverse or even prevent the development of drug resistance. Lung cancers harboring mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors, but drug resistance invariably emerges. To elucidate mechanisms of acquired drug resistance, we performed systematic genetic and histological analyses of tumor biopsies from 37 patients with drug-resistant non–small cell lung cancers (NSCLCs) carrying EGFR mutations. All drug-resistant tumors retained their original activating EGFR mutations, and some acquired known mechanisms of resistance including the EGFR T790M mutation or MET gene amplification. Some resistant cancers showed unexpected genetic changes including EGFR amplification and mutations in the PIK3CA gene, whereas others underwent a pronounced epithelial-to-mesenchymal transition. Surprisingly, five resistant tumors (14%) transformed from NSCLC into small cell lung cancer (SCLC) and were sensitive to standard SCLC treatments. In three patients, serial biopsies revealed that genetic mechanisms of resistance were lost in the absence of the continued selective pressure of EGFR inhibitor treatment, and such cancers were sensitive to a second round of treatment with EGFR inhibitors. Collectively, these results deepen our understanding of resistance to EGFR inhibitors and underscore the importance of repeatedly assessing cancers throughout the course of the disease.

Unique Clinicopathologic Features Characterize ALK-Rearranged Lung Adenocarcinoma in the Western Population

Purpose: The anaplastic large cell kinase gene (ALK) is rearranged in ∼5% of lung adenocarcinomas within the Asian population. We evaluated the incidence and the characteristics of ALK-rearranged lung adenocarcinomas within the western population and the optimal diagnostic modality to detect ALK rearrangements in routine clinical practice. Experimental Design: We tested 358 lung adenocarcinomas from three institutions for ALK rearrangements by fluorescent in situ hybridization (FISH) and immunohistochemistry with and without tyramide amplification. The clinicopathologic characteristics of tumors with and without ALK rearrangements were compared. Results: We identified 20 (5.6%) lung adenocarcinomas with ALK rearrangements within our cohort of western patients. ALK rearrangement was associated with younger age (P = 0.0002), never smoking (P < 0.0001), advanced clinical stage (P = 0.0001), and a solid histology with signet-ring cells (P < 0.0001). ALK rearrangement was identified by FISH in 95% of cases and immunohistochemistry with and without tyramide amplification in 80% and 40% of cases, respectively, but neither FISH nor immunohistochemistry alone detected all cases with ALK rearrangement on initial screening. None of the ALK-rearranged tumors harbored coexisting EGFR mutations. Conclusions: Lung adenocarcinomas with ALK rearrangements are uncommon in the western population and represent a distinct entity of carcinomas with unique characteristics. For suspected cases, dual diagnostic testing, with FISH and immunohistochemistry, should be considered to accurately identify lung adenocarcinomas with ALK rearrangement. (Clin Cancer Res 2009;15(16):5216–23)

Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene represents a molecular target in a small subset of non-small cell lung cancers (NSCLCs). This fusion leads to constitutive ALK activation with potent transforming activity. In a pivotal phase 1 clinical trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib (PF-02341066) demonstrated impressive antitumor activity in the majority of patients with NSCLC harboring ALK fusions. However, despite these remarkable initial responses, cancers eventually develop resistance to crizotinib, usually within 1 y, thereby limiting the potential clinical benefit. To determine how cancers acquire resistance to ALK inhibitors, we established a model of acquired resistance to crizotinib by exposing a highly sensitive EML4-ALK–positive NSCLC cell line to increasing doses of crizotinib until resistance emerged. We found that cells resistant to intermediate doses of crizotinib developed amplification of the EML4-ALK gene. Cells resistant to higher doses (1 μM) also developed a gatekeeper mutation, L1196M, within the kinase domain, rendering EML4-ALK insensitive to crizotinib. This gatekeeper mutation was readily detected using a unique and highly sensitive allele-specific PCR assay. Although crizotinib was ineffectual against EML4-ALK harboring the gatekeeper mutation, we observed that two structurally different ALK inhibitors, NVP-TAE684 and AP26113, were highly active against the resistant cancer cells in vitro and in vivo. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitor 17-AAG. Thus, we have developed a model of acquired resistance to ALK inhibitors and have shown that second-generation ALK TKIs or Hsp90 inhibitors are effective in treating crizotinib-resistant tumors harboring secondary gatekeeper mutations.

A Novel, Highly Sensitive Antibody Allows for the Routine Detection of ALK-Rearranged Lung Adenocarcinomas by Standard Immunohistochemistry

Purpose: Approximately 5% of lung adenocarcinomas harbor an EML4-ALK gene fusion and define a unique tumor group that may be responsive to targeted therapy. However ALK-rearranged lung adenocarcinomas are difficult to detect by either standard fluorescence in situ hybridization or immunohistochemistry (IHC) assays. In the present study, we used novel antibodies to compare ALK protein expression in genetically defined lung cancers and anaplastic large cell lymphomas. Experimental Design: We analyzed 174 tumors with one standard and two novel monoclonal antibodies recognizing the ALK protein. Immunostained tissue sections were assessed for the level of tumor-specific ALK expression by objective quantitative image analysis and independently by three pathologists. Results: ALK protein is invariably and exclusively expressed in ALK-rearranged lung adenocarcinomas but at much lower levels than in the prototypic ALK-rearranged tumor, anaplastic large cell lymphoma, and as a result, is often not detected by conventional IHC. We further validate a novel IHC that shows excellent sensitivity and specificity (100% and 99%, respectively) for the detection of ALK-rearranged lung adenocarcinomas in biopsy specimens, with excellent interobserver agreement between pathologists (κ statistic, 0.94). Conclusions: Low levels of ALK protein expression is a characteristic feature of ALK-rearranged lung adenocarcinomas. However, a novel, highly sensitive IHC assay reliably detects lung adenocarcinomas with ALK rearrangements and obviates the need for fluorescence in situ hybridization analysis for the majority of cases, and therefore could be routinely applicable in clinical practice to detect lung cancers that may be responsive to ALK inhibitors. Clin Cancer Res; 16(5); 1561–71

Identification of genotype-correlated sensitivity to selective kinase inhibitors by using high-throughput tumor cell line profiling

Kinase inhibitors constitute an important new class of cancer drugs, whose selective efficacy is largely determined by underlying tumor cell genetics. We established a high-throughput platform to profile 500 cell lines derived from diverse epithelial cancers for sensitivity to 14 kinase inhibitors. Most inhibitors were ineffective against unselected cell lines but exhibited dramatic cell killing of small nonoverlapping subsets. Cells with exquisite sensitivity to EGFR, HER2, MET, or BRAF kinase inhibitors were marked by activating mutations or amplification of the drug target. Although most cell lines recapitulated known tumor-associated genotypes, the screen revealed low-frequency drug-sensitizing genotypes in tumor types not previously associated with drug susceptibility. Furthermore, comparing drugs thought to target the same kinase revealed striking differences, predictive of clinical efficacy. Genetically defined cancer subsets, irrespective of tissue type, predict response to kinase inhibitors, and provide an important preclinical model to guide early clinical applications of novel targeted inhibitors.

Implementing multiplexed genotyping of non-small-cell lung cancers into routine clinical practice

BACKGROUND Personalizing non-small-cell lung cancer (NSCLC) therapy toward oncogene addicted pathway inhibition is effective. Hence, the ability to determine a more comprehensive genotype for each case is becoming essential to optimal cancer care. METHODS We developed a multiplexed PCR-based assay (SNaPshot) to simultaneously identify >50 mutations in several key NSCLC genes. SNaPshot and FISH for ALK translocations were integrated into routine practice as Clinical Laboratory Improvement Amendments-certified tests. Here, we present analyses of the first 589 patients referred for genotyping. RESULTS Pathologic prescreening identified 552 (95%) tumors with sufficient tissue for SNaPshot; 51% had ≥1 mutation identified, most commonly in KRAS (24%), EGFR (13%), PIK3CA (4%) and translocations involving ALK (5%). Unanticipated mutations were observed at lower frequencies in IDH and β-catenin. We observed several associations between genotypes and clinical characteristics, including increased PIK3CA mutations in squamous cell cancers. Genotyping distinguished multiple primary cancers from metastatic disease and steered 78 (22%) of the 353 patients with advanced disease toward a genotype-directed targeted therapy. CONCLUSIONS Broad genotyping can be efficiently incorporated into an NSCLC clinic and has great utility in influencing treatment decisions and directing patients toward relevant clinical trials. As more targeted therapies are developed, such multiplexed molecular testing will become a standard part of practice.

Loss of the Mismatch Repair Protein MSH6 in Human Glioblastomas Is Associated with Tumor Progression during Temozolomide Treatment

Purpose: Glioblastomas are treated by surgical resection followed by radiotherapy [X-ray therapy (XRT)] and the alkylating chemotherapeutic agent temozolomide. Recently, inactivating mutations in the mismatch repair gene MSH6 were identified in two glioblastomas recurrent post-temozolomide. Because mismatch repair pathway inactivation is a known mediator of alkylator resistance in vitro, these findings suggested that MSH6 inactivation was causally linked to these two recurrences. However, the extent of involvement of MSH6 in glioblastoma is unknown. We sought to determine the overall frequency and clinical relevance of MSH6 alterations in glioblastomas. Experimental Design: The MSH6 gene was sequenced in 54 glioblastomas. MSH6 and O6-methylguanine methyltransferase (MGMT) immunohistochemistry was systematically scored in a panel of 46 clinically well-characterized glioblastomas, and the corresponding patient response to treatment evaluated. Results:MSH6 mutation was not observed in any pretreatment glioblastoma (0 of 40), whereas 3 of 14 recurrent cases had somatic mutations (P = 0.015). MSH6 protein expression was detected in all pretreatment (17 of 17) cases examined but, notably, expression was lost in 7 of 17 (41%) recurrences from matched post–XRT + temozolomide cases (P = 0.016). Loss of MSH6 was not associated with O6-methylguanine methyltransferase status. Measurements of in vivo tumor growth using three-dimensional reconstructed magnetic resonance imaging showed that MSH6-negative glioblastomas had a markedly increased rate of growth while under temozolomide treatment (3.17 versus 0.04 cc/mo for MSH6-positive tumors; P = 0.020). Conclusions: Loss of MSH6 occurs in a subset of post–XRT + temozolomide glioblastoma recurrences and is associated with tumor progression during temozolomide treatment, mirroring the alkylator resistance conferred by MSH6 inactivation in vitro. MSH6 deficiency may therefore contribute to the emergence of recurrent glioblastomas during temozolomide treatment.

ALK Rearrangements Are Mutually Exclusive with Mutations in EGFR or KRAS: An Analysis of 1,683 Patients with Non–Small Cell Lung Cancer

Purpose: Anaplastic lymphoma kinase (ALK) gene rearrangements define a distinct molecular subset of non–small cell lung cancer (NSCLC). Recently, several case reports and small series have reported that ALK rearrangements can overlap with other oncogenic drivers in NSCLC in crizotinib-naïve and crizotinib-resistant cancers. Experimental Design: We reviewed clinical genotyping data from 1,683 patients with NSCLC and investigated the prevalence of concomitant EGFR or KRAS mutations among patients with ALK-positive NSCLC. We also examined biopsy specimens from 34 patients with ALK-positive NSCLC after the development of resistance to crizotinib. Results: Screening identified 301 (17.8%) EGFR mutations, 465 (27.6%) KRAS mutations, and 75 (4.4%) ALK rearrangements. EGFR mutations and ALK rearrangements were mutually exclusive. Four patients with KRAS mutations were found to have abnormal ALK FISH patterns, most commonly involving isolated 5′ green probes. Sufficient tissue was available for confirmatory ALK immunohistochemistry in 3 cases, all of which were negative for ALK expression. Among patients with ALK-positive NSCLC who acquired resistance to crizotinib, repeat biopsy specimens were ALK FISH positive in 29 of 29 (100%) cases. Secondary mutations in the ALK kinase domain and ALK gene amplification were observed in 7 of 34 (20.6%) and 3 of 29 (10.3%) cases, respectively. No EGFR or KRAS mutations were identified among any of the 25 crizotinib-resistant, ALK-positive patients with sufficient tissue for testing. Conclusions: Functional ALK rearrangements were mutually exclusive with EGFR and KRAS mutations in a large Western patient population. This lack of overlap was also observed in ALK-positive cancers with acquired resistance to crizotinib. Clin Cancer Res; 19(15); 4273–81. ©2013 AACR.

BRAF Gene Amplification Can Promote Acquired Resistance to MEK Inhibitors in Cancer Cells Harboring the BRAF V600E Mutation

Amplification of an upstream gene leads to resistance to cancer drugs targeted downstream. Making Resistance Futile One of the lures of research into signal transduction is the possibility that it could lead to the development of highly targeted therapies that specifically interfere with pathways that are perturbed under pathological conditions. For instance, the discovery that mutations in BRAF are associated with various cancers led to the investigation of pharmacological inhibitors of BRAF—or the downstream kinase MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase)—as therapies for individuals with BRAF-mutant tumors. All too often, however, initial promising clinical responses to such targeted therapies are followed by relapse, as cancer cells develop resistance to a particular therapeutic agent. Corcoran et al. explored the mechanisms whereby BRAF-mutant colorectal cancer cells became resistant to a MEK inhibitor and discovered that resistance involved amplification of the mutant BRAF gene. Furthermore, they found that cancer cells resistant to inhibition of either MEK or BRAF alone remained sensitive to the combined inhibition of BRAF and MEK, undergoing apoptosis at low concentrations of the inhibitors. They thus propose that, in individuals with BRAF-mutant tumors, combined inhibition of MEK and BRAF could overcome resistance to targeted therapy—and perhaps prevent it from arising. Oncogenic BRAF mutations are found in several tumor types, including melanomas and colorectal cancers. Tumors with BRAF mutations have increased mitogen-activated protein kinase pathway activity and heightened sensitivity to BRAF and MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase) inhibitors. To identify potential mechanisms of acquired drug resistance, we generated clones resistant to the allosteric MEK inhibitor AZD6244 from two BRAF V600E mutant colorectal cancer cell lines that are highly sensitive to MEK or BRAF inhibition. These AZD6244-resistant (AR) clones, which exhibited cross-resistance to BRAF inhibitors, acquired resistance through amplification of the BRAF gene. A small percentage of treatment-naïve parental cells showed preexisting BRAF amplification. We observed similar amplification in a subset of cells in a BRAF-mutant colorectal cancer. In cell lines, BRAF amplification increased the abundance of phosphorylated MEK and impaired the ability of AZD6244 to inhibit ERK (extracellular signal–regulated kinase) phosphorylation. The ability of AZD6244 to inhibit ERK phosphorylation in AR cells was restored by treatment with a BRAF inhibitor at low concentrations that reduced the abundance of phosphorylated MEK to amounts observed in parental cells. Combined MEK and BRAF inhibition fully overcame resistance to MEK or BRAF inhibitors alone and was also more effective in parental cells compared to treatment with either inhibitor alone. These findings implicate BRAF amplification as a mechanism of resistance to both MEK and BRAF inhibitors and suggest combined MEK and BRAF inhibition as a clinical strategy to overcome, or possibly prevent, this mechanism of resistance.

Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition

Although mechanisms of acquired resistance of epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here we observe that acquired resistance caused by the EGFRT790M gatekeeper mutation can occur either by selection of pre-existing EGFRT790M-positive clones or via genetic evolution of initially EGFRT790M-negative drug-tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug-tolerant cells had a diminished apoptotic response to third-generation EGFR inhibitors that target EGFRT790M; treatment with navitoclax, an inhibitor of the anti-apoptotic factors BCL-xL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor–resistant patient tumors. These findings provide evidence that clinically relevant drug-resistant cancer cells can both pre-exist and evolve from drug-tolerant cells, and they point to therapeutic opportunities to prevent or overcome resistance in the clinic.