V600 Mutation Research Articles

The potential for BRAF V600 inhibitors in advanced cutaneous melanoma: rationale and latest evidence.

Historically, patients with advanced cutaneous melanoma have a poor prognosis and limited treatment options. The discovery of selective v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600 mutation as an oncogenic mutation in cutaneous melanoma and the importance of the mitogen-activated protein kinase (MAPK) pathway in its tumourigenesis have changed the treatment paradigm for melanoma. Selective BRAF inhibitors and now MEK inhibitors have demonstrated response rates far higher than standard chemotherapeutic options and we review the phase I-III results for these agents in this article. The understanding of mechanisms of resistance that may occur upstream, downstream, at the BRAF level or bypassing the MAPK pathway provides a platform for rational drug development and combination therapies.

Challenges Ahead for Companion Diagnostics

T he U.S. Food and Drug Administration ’ s (FDA) approval of vemurafenib (Plexxikon/Roche) last August was greeted as a milestone not only for melanoma treatment but also for personalized medicine. The drug is indicated for patients who harbor a BRAF V600 → E mutation. Vemurafenib was developed along with a companion diagnostic, in a test case for how the FDA plans to handle personalized drugs from now on. In a draft guidance issued last July 14, the FDA defined companion diagnostics as devices that provide essential information for the safe and effective use of a corresponding therapeutic product. It stated that it would review personalized drugs only in tandem with the diagnostic that was used to select patients for clinical trials, adding that personalized drug labels should refer specifically to approved diagnostics for use in selecting patients for treatment. FDA offi cials emphasize that the approach is needed to ensure that personalized drugs are given only to likely responders. But as the experience with vemurafenib shows, the new method encroaches on entrenched diagnostic turf. Hospitals, drug companies, and professional medical societies have all reacted against the guidance, claiming that it locks researchers and clinicians into infl exible protocols that stifl e innovation and limit access to treatment. They say that laboratory-developed tests (LDTs) that individual institutions create and administer can adequately determine patient eligibility for personalized care. Vemurafenib ’ s creators at Plexxikon, a Berkeley, Calif. – based biotechnology company, rank among the guidance ’ s most outspoken critics; they claim it imposes excessive burdens on early-stage drug development. However, Plexxikon also anticipated diagnostic scrutiny from the FDA even before their BRAF inhibitor, then known as PLX-4032, had shown human proof-of-concept antitumor effects. So in 2005, to accelerate the drug ’ s development, Plexxikon teamed with Roche Molecular Diagnostics (RMD), in Pleasanton, Calif., on the development of an error-free system for detecting BRAF mutations. Producing the test took several years — partly because RMD had to devise a way to work with paraffi n-embedded, formalin-fi xed tumor samples, in which DNA is often damaged. From these efforts emerged the Cobas 4800 V600 Mutation Assay, a closed, PCRbased system that fl ags V600 → E mutations with a “yes” or “no” fl uorescence reaction. Plexxikon credits the test with propelling vemurafenib from discovery to approval in just 5 years, specifi cally by reducing the size of the trial population to V600 → E mutation carriers only. Vemurafenib ’ s label now indicates that because it was tested only in patients selected with the Cobas assay, doctors should avoid using it in BRAF-mutated patients selected using some other method. FDA spokesperson Erica Jefferson emphasized that the label doesn ’ t constitute regulation. “Product labels are intended to guide treatment decisions and provide clinical information,” she said. “The language is worded to allow for future approved tests for use in selecting patients for treatment.”

Abstract A229: BRAF inhibitors upregulate EGFR ligands: A molecular link to RAF inhibitor-induced cutaneous squamous cell carcinomas.

Abstract Selective inhibitors of BRAF such as vemurafenib (PLX4032) and GSK2118436 show remarkable efficacy in melanoma patients with tumors harboring BRAF V600 mutations. However these compounds paradoxically activate the MAPK pathway in RAS-mutant cells and are associated clinically with appearance of skin tumors such as cutaneous squamous cell carcinomas (cuSCC), particularly of the keratoacanthoma subtype. Here we further investigated the molecular links between BRAF inhibitor treatment and skin neoplasm. In soft agar, BRAF inhibitors induced growth of HRAS-mutant cuSCC B9 cells via upregulation of several EGFR ligands. The EGFR inhibitor Tarceva antagonized this transforming effect. The “Paradox Breakers,” a class of BRAF inhibitors recently discovered at Plexxikon, did not activate the MAPK pathway in cells with RAS mutation or EGFR family kinase activation and failed to upregulate EGFR ligands or induce soft agar growth of B9 cells. Also when tested in vivo, subcutaneous B9-tumor growth was accelerated by a structural analog (PLX4720) of vemurafenib but not by an equally effective BRAF inhibitor of the Paradox Breaker class. Taken together, a novel mechanism responsible for BRAF inhibitor-induced cuSCC was uncovered. Our data suggest that combination treatment of EGFR inhibitors with BRAF inhibitors might prevent skin tumor growth and that Paradox Breakers represent a new generation of BRAF inhibitors that have fewer unwanted side effects and the potential for greater clinical efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A229.

Phase III randomized, open-label, multicenter trial (BRIM3) comparing BRAF inhibitor vemurafenib with dacarbazine (DTIC) in patients with V600EBRAF-mutated melanoma.

LBA4 Background: About 50% of melanomas have an activating V600EBRAF mutation which led to the hypothesis that inhibition of the mutated BRAF kinase may be of clinical benefit. Phase I and II trials with vemurafenib (previously PLX4032/RO5185426), an orally available inhibitor of oncogenic BRAF kinase, showed response rates (RR; CR+PR) >50% in V600EBRAF- mutated melanoma patients (pts). We conducted a phase III trial to determine if vemurafenib improved overall survival (OS) and progression-free survival (PFS) in melanoma pts with V600EBRAF mutation. Methods: Pts with previously untreated, unresectable stage IIIC or stage IV melanoma that tested positive for V600EBRAF mutation by the cobas 4800 BRAF V600 Mutation Test were randomized (1:1) to vemurafenib (960 mg po bid) or DTIC (1,000 mg/m2, IV, q3w). Randomization was stratified by PS, stage, LDH, and geographic region. Pts were assessed for tumor responses after weeks 6, 12, and then q9 weeks. Co-primary endpoints were OS and PFS on the intent-to-treat population; secondary endpoints included RR, response duration, and safety. Final analysis was planned at 196 deaths. Results: 675 pts were enrolled at 103 centers worldwide between Jan and Dec 2010. Treatment cohorts were well-balanced. At the pre-planned interim analysis (50% of deaths needed for final analysis), the hazard ratios for OS and PFS were 0.37 (95% CI 0.26 to 0.55; p<0.0001) and 0.26 (95% CI 0.20 to 0.33; p<0.0001), respectively, both in favor of vemurafenib. The confirmed RR was 48.4% and 5.5% to vemurafenib and DTIC, respectively, among the 65% of pts evaluable for RR to date. Benefit in OS, PFS, and RR was seen in all subgroups examined. Due to these data, the DTIC cohort has been allowed to cross over to vemurafenib. At the time of data analysis, 66% of vemurafenib pts and 25% DTIC pts were still on treatment. The most common toxicities of vemurafenib were: diarrhea, rash, alopecia, photosensitivity, fatigue, arthralgia, and keratoacanthoma/skin squamous cell carcinoma. Conclusions: Vemurafenib is associated with significantly improved OS and PFS compared to DTIC in pts with previously untreated, V600EBRAF-mutated metastatic melanoma.

Phase I/II study to assess safety, pharmacokinetics, and efficacy of the oral MEK 1/2 inhibitor GSK1120212 (GSK212) dosed in combination with the oral BRAF inhibitor GSK2118436 (GSK436).

CRA8503 Background: In preclinical models, the BRAF/MEK inhibitor (i) combination GSK436/GSK212 has demonstrated enhanced activity against BRAF-mutant cancer cells compared to either drug alone, delayed emergence of GSK436 resistance, and prevented proliferative skin lesions attributable to BRAFi exposure. Methods: Eligible patients (pts) had BRAF V600 mutation positive solid tumors. Part 1: pharmacokinetic (PK) drug-drug interaction (DDI) study. Part 2: Dose escalation of continuous daily dosing of the combination followed by expansion cohorts; Part 3: Randomized phase II trial in untreated stage IV melanoma. Results: 45 pts have received ≥ 1 dose of GSK212 + GSK436, including 43 melanoma (all BRAFi naïve), 1 NSCLC and 1 salivary duct carcinoma. PK results of 7 pts in Part 1 showed no effect of GSK212 on single dose of GSK436. There was no clinically meaningful DDI between GSK436 and GSK212 after repeat dosing of the combination (Part 2). GSK436 was dosed 75-150 mg BID in combination with GSK212 1.0, 1.5, 2.0 mg QD. The recommended dose was 2 mg QD GSK212 in combination with 150 mg BID GSK436. At 1.5 mg GSK212, there was one DLT, a recurrent grade (G) 2 neutrophilic panniculitis. The only G4 adverse event (AE) was a sepsis-like syndrome with fever/hypotension. G3 AEs included generalized rash (n=2, 4%) and neutropenia (n=2, 4%). Skin toxicity ≥ G2 occurred in 9 (20%) pts; of these, G2 rash (n=4, 8%) and G2 macular rash (n=1, 2%). No cutaneous squamous cell carcinoma (SCC) or hyperproliferative skin lesions have occurred at any dose level. Other common G2 toxicities were pyrexia (n=5, 11%), vomiting (n=2, 4%) and fatigue (n=2, 4%). Of 16 evaluable pts in Part 2, 13 pts had PR and 3 SD for an ORR of 81% (95% CI 54.4%-96.0%) and all but 2 pts remain on study. In 10 evaluable pts who received 150 mg BID GSK436 + ≥1 mg QD GSK212, 9 pts had PR and 1 SD. Conclusions: GSK212 at 2 mg QD combines safely with GSK436 150 mg BID, no SCC thus far and decreased frequency of rash compared to previous trials of single agent GSK436 and GSK212, respectively. The preliminary anti-tumor activity warrants further investigation; the randomized phase II trial (Part 3) is accruing.

Frequency of mutations associated with targeted therapy in malignant melanoma patients.

8597 Background: BRAF, RAS, KIT, and TP53 genes are frequently mutated in primary malignant melanomas, and activating mutations involving RAS and BRAF V600, as well as imatinib-sensitive KIT mutations, have been exploited for targeted therapy. Here we studied the prevalence of mutations in these four genes in primary malignant melanoma samples to establish a strategy to identify patients who might benefit from targeted gene therapy. In addition, results were cross-referenced with pathological and histogenetic indicators. Methods: 198 confirmed formalin-fixed, paraffin embedded (FFPE) primary melanoma tissue samples were screened for mutations in BRAF (exons 11, 12 and 15), RAS (H-, K-, N-, exons 1 and 2), KIT (exons 8, 9, 11, 13, and 17), and TP53 (exons 4-9) using direct DNA sequencing. Results: Overall, 140 (70.7%) of the 198 melanoma samples had mutations in one or more of the genes analyzed. BRAF mutations were the most prevalent, with the majority involving codon V600 (Table). Among the BRAF V600 mutations, 66% were V600E and 30% were V600K. NRAS and KIT had similar mutation frequencies. Most NRAS mutations occurred at position Q61, of which 50% were Q61L and 33% were Q61R. About one-third of the KIT mutations were those likely to be sensitive to imatinib. BRAF V600, NRAS Q61, and imatinib-sensitive CKIT mutations were mutually exclusive. TP53 mutations were observed in approximately one-third of samples, often concurrent with other mutations. Conclusions: BRAF, NRAS, CKIT, and TP53 genes are frequently mutated in primary malignant melanomas. The predominance of BRAF V600, NRAS Q61, and imatinib-sensitive KIT mutations signifies the potential for targeted therapeutic strategies in melanoma patients. The high percentage of BRAF V600K mutation illustrates the importance of expanding clinical trials of BRAF inhibitors to include patients with V600K and possibly other V600 mutations. The mutually exclusive pattern of mutations also provides a hierarchical screening strategy for samples that have limited tissue availability. Gene Prevalence, % Predominant mutations BRAF 44.9 V600 (88%) NRAS 18.7 Q61 (89%) CKIT 17.1 Imatinib sensitive (33%) TP53 34.6 None

An open-label pilot study of vemurafenib in previously treated metastatic melanoma patients with brain metastases.

8548 Background: Melanoma is a common cause of cerebral metastases, and indicates a poor prognosis. The BRAF inhibitor vemurafenib (V; RG7204, PLX4032) has shown dramatic activity in Phase I and II studies in metastatic melanoma harboring V600-mutated BRAF. These studies excluded patients with active brain metastasis. The activity of V for brain metastases is unknown. Methods: Open label, single arm trial of V to evaluate the safety and efficacy of V in patients with metastatic melanoma with BRAF V600 mutations (by the cobas 4800 BRAF V600 Mutation Test) and non-resectable-brain mets, pretreated by radiotherapy and/or chemotherapy. Patients may have symptoms related to their brain mets and be on a stable dose of steroids. V is administered continuously at the dose of 960 mg BID. Twenty patients are planned to be include in this study. Staging (MRI brain and CT thorax/abdomen) is scheduled every 4 weeks in the first 2 months and then every 8 weeks. In addition, the serum S100 levels are determined. Results: Four patients with V600 BRAF positive melanoma metastatic to the brain and pretreated with temozolamide and/or total brain radiotherapy V are on treatment between 2 and 10 weeks to date. All patients are young (between 24 and 48 years), have 3 to more than 10 brain mets, ECOG PS 0-2 and are on dexamethasone. Two patients were taking morphine to control pain and neurological symptoms. There was no dose reduction for V. AEs included rash, fatigue in two patients and disseminated Herpes zoster and mild transient paresis of nervus facialis in one patient. Two patients’ staging reports are currently available. Patient 1 presented an confirmed partial response in brain and body (RECIST) associated with a reduction of S-100 from 141.6µg/l (pre-treatment) to 4.4µg/l (day 43). Dexamethasone and morphine doses were reduced. Patient 2 showed a minor response in brain and body at week 4 associated with a reduction of S-100 from 35.6µg/l (day 1) to 3.26µg/l (day 15). Conclusions: V has been well tolerated in symptomatic patients with melanoma metastatic to the brain to date. There are early but strong indications for activity in brain mets.

Tumor genetic analyses of patients with metastatic melanoma treated with the BRAF inhibitor GSK2118436 (GSK436).

8501 Background: GSK436 is a highly potent, selective ATP-competitive BRAF inhibitor. BRF112680, a first in human study, assessed safety, pharmacokinetics, pharmacodynamics, and efficacy. Efficacy of single-agent GSK436 was demonstrated in BRAF V600 mutation-positive (mut+) metastatic melanoma patients (pts), with an unconfirmed response rate (RR) of 77% in V600E mut+ pts and 44% in V600K mut+ pts (based on October 2010 data cut). This study evaluates the relationship of genetic alterations in target genes to RR. Methods: To date, samples from 38 pts have been evaluated; 5 pts had samples at pre-treatment (preTx) and progression, 32 pts had a preTx sample only, and 1 pt had a progression sample only. Tumor DNA was evaluated by Sequenom (SEQ) for a targeted panel of mutations in 11 genes (AKT1, BRAF, CDK4, CDKN2A, CTNNB1, GNAQ, GNA11, KIT, MEK1, MEK2, NRAS). PTEN mutation and copy number were derived by sequencing and array CGH (Agilent 1M) or MPLA, respectively. Results: BRAF mutation status was confirmed in all pt samples via SEQ. At preTx, 30 tumors were BRAF V600E, and 7 were V600K. The BRAF mut was retained in the 5 pts with both preTx and progression samples; in 3/5 the ratio of mut:wt decreased in the progression sample. To date, a PTEN alteration (mutation, hemizygous or homozygous deletion) was observed in 9/37 preTx sample pts (24%); 8 of which were V600E+. Pts with a PTEN alteration had lower RR (1/9=11%) compared with pts in which no PTEN alteration was observed (15/28=54%). In addition to the BRAF mutation, we observed in separate preTx samples mutations in MEK2 and CDK4; both patients had SD. An additional pt with a BRAF and β-catenin mutation had PR. Conclusions: These data support genetic characterization of melanomas for mutations outside of BRAF as predictors of RR to BRAF inhibition; the V600 mutation type itself is strongly predictive. Our data suggest that alterations in PTEN may be associated with a decrease in RR to GSK436. Mutations in the MAPK signaling pathway (MEK2) and outside it (CDK4) have been identified, which may also be associated with RR. Additional analyses are being performed to identify further predictors of response to BRAF inhibition.

BRIM-2: An open-label, multicenter phase II study of vemurafenib in previously treated patients with BRAF V600E mutation-positive metastatic melanoma.

8509 Background: Most agents tested in large phase II trials in patients (pts) with metastatic melanoma have response rates of 10-20%. Approximately 50% of melanomas harbor a V600E activating mutation in the BRAF gene, which can now be targeted with specific inhibitors. In a Phase I study, treatment with vemurafenib (V), an orally available inhibitor of mutant BRAF, led to a high incidence of tumor regressions in BRAF-mutant melanoma (Flaherty et al. NEJM 2010). Here we report the results of BRIM-2, a pivotal Phase II trial of V in previously treated pts with BRAF-mutant melanoma. Methods: The primary endpoint was best overall response rate (BORR), with a target of 30% and a lower boundary of the exact 95% CI of at least 20% by independent review committee (IRC). BRAF mutation status was determined using a PCR-based investigational, companion diagnostic assay (cobas 4800 BRAF V600 Mutation Test, Roche Molecular Systems). Pts without active CNS metastasis who had received at least one prior line of therapy for metastatic melanoma received V (960 mg po twice daily) until progressive disease, unacceptable toxicity, or death. Results: 132 pts were enrolled (Oct 2009-Mar 2010), median age 51.5 yr, 54% ECOG PS 1, 61% M1c, 49% elevated LDH, and 49% >1 line of prior therapy. At the time of analysis (Sept 27, 2010, median follow-up 7 mo), BORR was 52.3% (95% CI: 43–61%; 2.3% CR, 50% PR) by IRC, with 29.5% SD and only 13.6% PD. Median duration of response was 6.8 mo (95% CI: 5.6-NR), and median PFS was 6.2 mo (95% CI: 5.6–6.8%). Median OS had not been reached. Most common adverse events (AEs; ≥25% pts, mostly Grade 1-2) were arthralgia, rash, photosensitivity, fatigue, alopecia, pruritis, and skin papilloma. The most common Grade 3 AE was cutaneous squamous cell carcinoma (24.2%), the majority centrally reviewed as keratoacanthoma-type. AEs were generally reversible with dose modification or interruption. 42% of pts required dose reductions, most commonly for rash, arthralgia and LFT abnormalities. Conclusions: BRIM2 met its primary endpoint based on BORR, confirming that V is an active agent in pts with BRAF mutation-positive melanoma who have completed prior first-line therapy.

Molecular testing for BRAF V600 mutations in the BRIM-2 trial of the BRAF inhibitor vemurafenib in metastatic melanoma.

10523 Background: The BRAF inhibitor vemurafenib (V) has shown dramatic activity in early clinical trials in patients with BRAFV600 mutation-positive melanoma. Preclinical and Phase 1 clinical data show the drug has minimal activity in tumors lacking BRAF mutations, creating a need for an accurate diagnostic test to identify appropriate patients for therapy. Methods: To assess eligibility for BRIM-2, a Phase 2 trial of V in patients with BRAFV600 mutation-positive metastatic melanoma, tumor samples were screened with an investigational PCR assay (cobas 4800 BRAF V600 Mutation Test) designed to detect the canonical V600E (1799T>A) mutation. 328 samples were tested of which 327 samples gave valid results. BRAF mutations were detected in 184 (56.3 %); 132 patients with BRAF-mutation positive melanoma were enrolled into the trial based on clinical eligibility criteria. Sanger sequencing was performed retrospectively on DNA samples from all enrolled patients. Results: Fifteen samples (11.4%) gave invalid Sanger results. Sanger detected a V600E mutation in 97 samples (73.5%), V600K mutation in 9 samples (6.8%) and a wild-type (WT) result in 11 cases (8.3%). Samples were also subjected to “deep” sequencing with a quantitative picotiter plate pyrosequencing method (454 GS-Titanium) to resolve differences between PCR and Sanger results. Using 454 sequencing of samples with invalid Sanger results revealed a V600E mutation in 14 cases and a V600K mutation in 1 case. Of 11 samples with a WT Sanger result, 454 sequencing detected a V600E mutation in all cases, with a median percentage of mutant alleles of 8%; ten of the 11 samples had a percentage < 25%. The presence of a V600K mutation was confirmed by 454 sequencing in all 9 samples identified as having V600K mutations by Sanger. Conclusions: 1) the investigational PCR test had a low failure rate; 2) the PCR test was more sensitive in the detection of V600E mutations than Sanger sequencing, which may miss mutations in cases with low % mutant alleles; 3) the PCR test detects V600K mutations, and 4) in all 132 cases, a codon 600 mutation was confirmed by Sanger and/or 454 sequencing. Robust, rapid and accurate molecular testing was achieved in this multi-center clinical trial.

Potential Therapeutic Strategies to Overcome Acquired Resistance to BRAF or MEK Inhibitors in BRAF Mutant Cancers

Recent clinical trials with selective inhibitors of the BRAF and MEK kinases have shown promising results in patients with tumors harboring BRAF V600 mutations. However, as has been observed previously with similarly successful targeted therapies, acquired resistance to these agents is an emerging problem that limits their clinical benefit. Several recent studies from our laboratory and others have investigated the causes of acquired resistance to BRAF and MEK inhibitors, and multiple resistance mechanisms have been identified. Here, we review these mechanisms and suggest that they can be broadly grouped into two main classes: ERK-dependent and ERK-independent. We also propose distinct therapeutic strategies that might be employed to overcome each class of acquired resistance..

Abstract 2212: The analytic performance of a real-time PCR-based assay for the BRAF V600E mutation used as the companion diagnostic test for the novel BRAF inhibitor RG7204 (PLX4032) in metastatic melanoma

Abstract Approximately 50% of human melanomas harbor activating mutations in the BRAF oncogene, and a large majority are of the V600E (1799T&amp;gt;A) type. RG7204 (PLX4032), a highly selective small molecule that targets V600E-mutated BRAF, has shown striking efficacy in Phase 1 and Phase 2 clinical trials in metastatic melanoma. These findings create a critical need for a robust well-validated companion diagnostic test that is both sensitive and specific for the mutation. Here we describe analytic performance characteristics of the cobas BRAF V600 Mutation Test, a real-time PCR assay, which was designed to detect the V600E (1799T&amp;gt;A) mutation in formalin-fixed paraffin-embedded (FFPE) samples of malignant melanoma, and has been used to select patients in clinical trials of RG7204. Analytic sensitivity was assessed using DNA blends from melanoma cell lines, DNA blends from FFPE tumor sections of melanoma, and individual FFPE tumor specimens with low levels of BRAF mutant alleles, as determined by 454 sequencing (a quantitative “deep” sequencing method). A &amp;gt; 96% hit rate was obtained across all specimen types with 5% mutant sequences at a DNA input of 125 ng per PCR reaction, a DNA amount readily obtained from a single 5-micron FFPE tissue section. The calculated PROBIT values (95% probability) for the cobas test ranged from 0.6 to 30 ng per PCR genomic DNA input for specimens with 5% mutant sequences. The cobas test was compared to bidirectional Sanger sequencing using a set of 219 FFPE melanoma samples. The cobas test detected 14 V600E mutations among the 120 samples (11.7%) that were called mutation-negative by Sanger sequencing, and in all 14 cases a V600E mutation was confirmed by 454 sequencing. Conversely the cobas test detected no V600E mutation in 5 of 99 samples (5%) that were called mutant-positive by Sanger, and in all 5 cases 454 sequencing detected no mutation. The cobas test showed no cross reactivity with BRAF homologs, including RAF1, ARAF and the BRAF pseudogene; however, some cross-reactivity with V600K and V600D was observed with the cobas test. When a panel of tumor samples (including samples with low levels of mutant alleles and low tumor content) was repeatedly tested by two operators, using different instruments and reagents lots, a correct mutation call was obtained in 98.75% of tests. These analytic studies demonstrate that the cobas BRAF V600 Mutation Test is a reproducible assay that is more sensitive and more specific than conventional Sanger sequencing for the detection of the V600E (1799T&amp;gt;A) mutation and detects some non-V600E mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2212. doi:10.1158/1538-7445.AM2011-2212

Abstract SY03-04: Novel imaging approaches for immunotherapy and targeted therapy of melanoma in animal models and humans

Abstract Molecular imaging may allow gaining a better understanding of the pharmacodynamic (PD) effects of immunotherapy strategies and novel targeted therapies for cancer. Two general approaches for imaging can be used, the administration of radiolabeled small molecule tracers that specifically accumulate in cells modulated by the therapy or the genetic labeling of immune cells followed by the administration of a detection probe. The use of targeted agents that block the MAPK pathway is providing unprecedented response rates in melanoma with the BRAF V600 mutation. The positron emitting tomography (PET) probe [18F]fluorodeoxy-glucose ([18F]FDG) can noninvasively image an effect of the therapy in inhibiting glucose metabolism, and effects on cell replication can be studied with the PET probe 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT). In vitro studies in a panel of BRAF V600E mutant melanoma cell lines showed differential effects of the BRAF inhibitor PLX4032 with most cell lines being highly sensitive and a minority being resistant. Oncogenic analysis did not discern between these cell lines. Despite evidence of blocking the MAPK pathway by detecting cell cycle arrest both by flow cytometry or the incorporation of FLT in nearly all cell lines, some cell lines failed to lose viability when exposed to the BRAF inhibitor. The best way to discern between poorly responsive and highly responsive cells was the differential uptake of 2DG. In the clinic, PLX4032 has resulted in response rates up to 80% in patients with BRAF V600+ metastatic melanoma, which are preceded by dramatic decrease in [18F]FDG uptake in PET scans. Areas of progression due to acquired resistance to PLX4032 are visualized by reactivation of [18F]FDG uptake in PET scans. Therapy with anti-CTLA4 antibodies results in durable objective responses in approximately 10% of patients with metastatic melanoma, and the anti-CTLA4 antibody ipilimumab has demonstrated improvement in overall survival in a randomized clinical trial. A key issue is the location where lymphocyte proliferation occurs after the release of the CTLA4 cell cycle inhibitory checkpoint in lymphocytes. Immune monitoring assays had failed to detect a large lymphocytic expansion in blood, while tumors of patients responding to this therapy had markedly increased lymphocytic intratumoral infiltrates. We analyzed changes in glucose metabolism using the PET probe [18F]FDG, and cell replication with the PET probe [18F]FLT, after the administration of the CTLA4-blocking antibody tremelimuamb. In these studies, molecular imaging with [18F]FLT allowed mapping and noninvasive imaging of cell proliferation in the spleen, the largest secondary lymphoid organ, after CTLA4 blockade in patients with metastatic melanoma. A better understanding of the distribution and tumor targeting of lymphocytes may allow advancing adoptive cell transfer (ACT) immunotherapy for cancer. We have used T cell receptor (TCR) engineering together with molecular imaging to study the antitumor activity in an animal model of TCR-engineered ACT therapy. Splenocytes were activated ex vivo and genetically modified with a retroviral vector expressing a TCR for tyrosinase and either luciferase for bioluminescence imaging (BLI) or sr39tk for positron emitting tomography (PET) imaging. Genetic labeling with the BLI and PET reporter genes allowed visualization of the distribution and antigen-specific tumor homing of TCR transgenic T cells, with trafficking correlated with antitumor efficacy. After an initial brief stage of systemic distribution, TCR redirected and genetically labeled T cells demonstrated an early pattern of specific distribution to antigen-matched tumors and locoregional lymph nodes, followed by a more promiscuous distribution one week later with additional accumulation in antigen-mismatched tumors. Therefore, the molecular imaging reporter gene labeling approach allowed the non-invasive imaging of the in vivo dynamics of the TCR transgenic cells. The approach of TCR engineering can result in responses in patients with advanced cancer. In an ongoing clinical trial we have treated patients with metastatic melanoma with the ACT of lymphocytes genetically modified to express a high affinity TCR for the melanosomal antigen MART-1. This approach has led to high initial antitumor responses followed by tumor progression once the T cells lost their immunological potency in vivo. PET imaging with [18F]FDG demonstrated profound effects on tumor lesions beyond the changes in size criteria provided by CT scans, allowing the study of T cell responses to cancer. In conclusion, the use of molecular imaging in preclinical models and in the clinic allows longitudinal analysis of biological processes without altering their course, thereby providing important information to advance immunotherapy and targeted therapy strategies for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY03-04. doi:10.1158/1538-7445.AM2011-SY03-04

Could BRAF Mutations in Melanocytic Lesions Arise from DNA Damage Induced by Ultraviolet Radiation?

Although BRAF V600 mutations in melanocytic tumors are not UV-signature mutations, it is plausible that they could still arise from error-prone replication of UV-damaged DNA. We propose a mechanism for their origin, taking into consideration melanocytic-specific BRAF tandem mutations, nearby potential pyrimidine dimer sites, the properties of specialized DNA polymerases, and biological selection.

Bovine serum albumin reverses inhibition of RT-PCR by melanin.

Bovine serum albumin reverses inhibition of RT-PCR by melanin.