Vanda M. Stepanek

PIK3CA mutation H1047R is associated with response to PI3K/AKT/mTOR signaling pathway inhibitors in early-phase clinical trials.

PIK3CA mutations may predict response to PI3K/AKT/mTOR inhibitors in patients with advanced cancers, but the relevance of mutation subtype has not been investigated. Patients with diverse cancers referred to the Clinical Center for Targeted Therapy were analyzed for PIK3CA and, if possible, KRAS mutations. Patients with PIK3CA mutations were treated, whenever possible, with agents targeting the PI3K/AKT/mTOR pathway. Overall, 105 (10%) of 1,012 patients tested harbored PIK3CA mutations. Sixty-six (median 3 prior therapies) of the 105 PIK3CA-mutant patients, including 16 individuals (of 55 PIK3CA-mutant patients tested) with simultaneous KRAS mutations, were treated on a protocol that included a PI3K/AKT/mTOR pathway inhibitor; 17% (11/66) achieved a partial response (PR). Patients with a PIK3CA H1047R mutation compared with patients who had other PIK3CA mutations or patients with wild-type PIK3CA treated on the same protocols had a higher PR rate (6/16, 38% vs. 5/50; 10% vs. 23/174, 13%, respectively; all P ≤ 0.02). None of the 16 patients with coexisting PIK3CA and KRAS mutations in codon 12 or 13 attained a PR (0/16, 0%). Patients treated with combination therapy versus single-agent therapies had a higher PR rate (11/38, 29% vs. 0/28, 0%; P = 0.002). Multivariate analysis showed that H1047R was the only independent factor predicting response [OR 6.6, 95% confidence interval (CI), 1.02-43.0, P = 0.047). Our data suggest that interaction between PIK3CA mutation H1047R versus other aberrations and response to PI3K/AKT/mTOR axis inhibitors warrants further exploration.

Objective Detection and Delineation of Oral Neoplasia Using Autofluorescence Imaging

Although the oral cavity is easily accessible to inspection, patients with oral cancer most often present at a late stage, leading to high morbidity and mortality. Autofluorescence imaging has emerged as a promising technology to aid clinicians in screening for oral neoplasia and as an aid to resection, but current approaches rely on subjective interpretation. We present a new method to objectively delineate neoplastic oral mucosa using autofluorescence imaging. Autofluorescence images were obtained from 56 patients with oral lesions and 11 normal volunteers. From these images, 276 measurements from 159 unique regions of interest (ROI) sites corresponding to normal and confirmed neoplastic areas were identified. Data from ROIs in the first 46 subjects were used to develop a simple classification algorithm based on the ratio of red-to-green fluorescence; performance of this algorithm was then validated using data from the ROIs in the last 21 subjects. This algorithm was applied to patient images to create visual disease probability maps across the field of view. Histologic sections of resected tissue were used to validate the disease probability maps. The best discrimination between neoplastic and nonneoplastic areas was obtained at 405 nm excitation; normal tissue could be discriminated from dysplasia and invasive cancer with a 95.9% sensitivity and 96.2% specificity in the training set, and with a 100% sensitivity and 91.4% specificity in the validation set. Disease probability maps qualitatively agreed with both clinical impression and histology. Autofluorescence imaging coupled with objective image analysis provided a sensitive and noninvasive tool for the detection of oral neoplasia.

Assessing PIK3CA and PTEN in early-phase trials with PI3K/AKT/mTOR inhibitors

Despite a wealth of preclinical studies, it is unclear whether PIK3CA or phosphatase and tensin homolog (PTEN) gene aberrations are actionable in the clinical setting. Of 1,656 patients with advanced, refractory cancers tested for PIK3CA or PTEN abnormalities, PIK3CA mutations were found in 9% (146/1,589), and PTEN loss and/or mutation was found in 13% (149/1,157). In multicovariable analysis, treatment with a phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) inhibitor was the only independent factor predicting response to therapy in individuals harboring a PIK3CA or PTEN aberration. The rate of stable disease ≥6 months/partial response reached 45% in a subgroup of individuals with H1047R PIK3CA mutations. Aberrations in the PI3K/AKT/mTOR pathway are common and potentially actionable in patients with diverse advanced cancers. This work provides further important clinical validation for continued and accelerated use of biomarker-driven trials incorporating rational drug combinations.

Noninvasive imaging of oral neoplasia with a high-resolution fiber-optic microendoscope.

The purpose of this study was to evaluate the ability of high‐resolution microendoscopy to image and quantify changes in cellular and architectural features seen in early oral neoplasia in vivo.

Target-Based Therapeutic Matching in Early-Phase Clinical Trials in Patients with Advanced Colorectal Cancer and PIK3CA Mutations

Target-matched treatment with PI3K/AKT/mTOR pathway inhibitors in patients with diverse advanced cancers with PIK3CA mutations have shown promise. Tumors from patients with colorectal cancer were analyzed for PIK3CA, KRAS, and BRAF mutations. PIK3CA-mutated tumors were treated, whenever feasible, with agents targeting the PI3K/AKT/mTOR pathway. Of 194 patients analyzed, 31 (16%) had PIK3CA mutations and 189 (97%) were assessed for KRAS mutations. Patients with PIK3CA mutations had a higher prevalence of simultaneous KRAS mutations than patients with wild-type PIK3CA (71%, 22/31 vs. 43%, 68/158; P = 0.006). Of 31 patients with PIK3CA mutations, 17 (55%) were treated with protocols containing PI3K/AKT/mTOR pathway inhibitors [median age, 57 years; median number of prior therapies, 4; mTORC1 inhibitors (11), phosphoinositide 3-kinase (PI3K) inhibitors (5), or an AKT inhibitor (1)]. None (0/17) had a partial or complete response (PR/CR) and only 1 [6%, 95% confidence interval (CI), 0.01–0.27] had stable disease 6 months or more, which was not significantly different from a stable disease ≥6 month/PR/CR rate of 16% (11/67; 95% CI, 0.09–0.27) in patients with colorectal cancer without PIK3CA mutations treated with PI3K/AKT/mTOR pathway inhibitors (P = 0.44). Median progression-free survival was 1.9 months (95% CI, 1.5–2.3). In conclusion, our data provide preliminary evidence that in heavily pretreated patients with PIK3CA-mutant advanced colorectal cancer, protocols incorporating PI3K/AKT/mTOR inhibitors have minimal activity. PIK3CA mutations are associated with simultaneous KRAS mutations, possibly accounting for therapeutic resistance. Mol Cancer Ther; 12(12); 2857–63. ©2013 AACR.

Evaluation of a low-cost, portable imaging system for early detection of oral cancer

BackgroundThere is an important global need to improve early detection of oral cancer. Recent reports suggest that optical imaging technologies can aid in the identification of neoplastic lesions in the oral cavity; however, there is little data evaluating the use of optical imaging modalities in resource limited settings where oral cancer impacts patients disproportionately. In this article, we evaluate a simple, low-cost optical imaging system that is designed for early detection of oral cancer in resource limited settings. We report results of a clinical study conducted at Tata Memorial Hospital (TMH) in Mumbai, India using this system as a tool to improve detection of oral cancer and its precursors.MethodsReflectance images with white light illumination and fluorescence images with 455 nm excitation were obtained from 261 sites in the oral cavity from 76 patients and 90 sites in the oral cavity from 33 normal volunteers. Quantitative image features were used to develop classification algorithms to identify neoplastic tissue, using clinical diagnosis of expert observers as the gold standard.ResultsUsing the ratio of red to green autofluorescence, the algorithm identified tissues judged clinically to be cancer or clinically suspicious for neoplasia with a sensitivity of 90% and a specificity of 87%.ConclusionsResults suggest that the performance of this simple, objective low-cost system has potential to improve oral screening efforts, especially in low-resource settings.

BRAF mutation testing with a rapid, fully integrated molecular diagnostics system

Fast and accurate diagnostic systems are needed for further implementation of precision therapy of BRAF-mutant and other cancers. The novel IdyllaTM BRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those obtained by a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory employing polymerase chain reaction–based sequencing, mass spectrometric detection, or next-generation sequencing. In one set of 60 FFPE tumor samples (15 with BRAF mutations per Idylla), the Idylla and cobas results had an agreement of 97%. Idylla detected BRAF V600 mutations in two additional samples. The Idylla and MiSeq results had 100% concordance. In a separate set of 100 FFPE tumor samples (64 with BRAF mutation per Idylla), the Idylla and CLIA-certified laboratory results demonstrated an agreement of 96% even though the tests were not performed simultaneously and different FFPE blocks had to be used for 9 cases. The IdyllaTM BRAF Mutation Test produced results quickly (sample to results time was about 90 minutes with about 2 minutes of hands on time) and the closed nature of the cartridge eliminates the risk of PCR contamination. In conclusion, our observations demonstrate that the Idylla test is rapid and has high concordance with other routinely used but more complex BRAF mutation–detecting tests.

Comparison of multispectral wide-field optical imaging modalities to maximize image contrast for objective discrimination of oral neoplasia

Multispectral widefield optical imaging has the potential to improve early detection of oral cancer. The appropriate selection of illumination and collection conditions is required to maximize diagnostic ability. The goals of this study were to (i) evaluate image contrast between oral cancer∕precancer and non-neoplastic mucosa for a variety of imaging modalities and illumination∕collection conditions, and (ii) use classification algorithms to evaluate and compare the diagnostic utility of these modalities to discriminate cancers and precancers from normal tissue. Narrowband reflectance, autofluorescence, and polarized reflectance images were obtained from 61 patients and 11 normal volunteers. Image contrast was compared to identify modalities and conditions yielding greatest contrast. Image features were extracted and used to train and evaluate classification algorithms to discriminate tissue as non-neoplastic, dysplastic, or cancer; results were compared to histologic diagnosis. Autofluorescence imaging at 405-nm excitation provided the greatest image contrast, and the ratio of red-to-green fluorescence intensity computed from these images provided the best classification of dysplasia∕cancer versus non-neoplastic tissue. A sensitivity of 100% and a specificity of 85% were achieved in the validation set. Multispectral widefield images can accurately distinguish neoplastic and non-neoplastic tissue; however, the ability to separate precancerous lesions from cancers with this technique was limited.

Prospective evaluation of a portable depth-sensitive optical spectroscopy device to identify oral neoplasia.

A portable, depth-sensitive clinical spectroscopy device for noninvasive early diagnosis of oral cancer is described. We carried out a pilot study to evaluate the ability of the device to identify oral neoplasia using a previously developed diagnostic algorithm. A total of 79 oral sites in 33 subjects, including 28 patients with oral lesions and 5 healthy volunteers, were measured and analyzed. Measurements of 54 nonkeratinized oral sites yielded an area under the receiver operating characteristic curve of 0.90. Measurements of 25 keratinized oral sites yielded an area under the receiver operating characteristic curve of 0.83.

Abstract 5607: BRAF and KRAS mutation testing in plasma cell-free DNA with ICE COLD-PCR in patients with advanced cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable, low-risk, inexpensive, and repeatedly available source of biologic material for mutation analysis and monitoring of molecular changes throughout cancer therapy. Methods: DNA in plasma from patients with advanced cancers who progressed on systemic therapy was tested for BRAF V600 and KRAS G12 and G13 mutations using the ICE COLD-PCR platform. ICE COLD-PCR, “Improved and Complete Enrichment COamplification at Lower Denaturation,” selectively amplifies mutant DNA by exploiting differences in denaturation temperatures between mutant DNA duplexes and normal “wild-type” DNA duplexes. KRAS Exon 2 and BRAF Exon 15 ICE COLD-PCR was performed on plasma samples. Amplicons were analyzed using Sanger sequencing and results were compared to the mutation status of archival primary or metastatic tumor tissue as determined in a CLIA-certified laboratory during routine clinical care. Results: Plasma samples from 77 patients with advanced cancers and known tumor tissue BRAF and/or KRAS mutation status (colorectal cancer, n=38; melanoma, n=17; non-small cell lung cancer, n=7; other cancers, n=15) were obtained before treatment and, if possible, sequentially during therapy and tested for BRAF (42), KRAS (34) or BRAF and KRAS (1) mutations in cfDNA. BRAF mutations were detected in 93% (40/43) of archival tumor samples compared to 70% (30/43) of plasma cfDNA samples (agreement 77%). In addition, 20 patients treated with systemic therapy had serial plasma samples collected and the change in relative abundance of BRAF-mutant compared to wild-type cfDNA corresponded with the clinical course of 15 patients and was discrepant for 1 patient; in 5 patients no BRAF mutated cfDNA was detected at any time point. KRAS mutations were detected in 83% (29/35) of archival tumor samples compared to 74% (26/35) of plasma cfDNA samples (agreement 80%). In addition, 12 patients treated with systemic therapy had serial plasma collected and the change in relative abundance of KRAS-mutant compared to wild-type cfDNA corresponded with clinical course in 10 patients; in 2 patients no KRAS mutated cfDNA was detected at any time point. Conclusions: Detection of BRAF and KRAS mutations in cfDNA can provide a fast and noninvasive alternative to mutation testing in tumor tissue with a potential to be used for monitoring response to cancer therapy. Citation Format: Filip Janku, Ben Legendre, Katherine Richardson, Gerald S. Falchook, Aung Naing, Veronica R. Holley, Siqing Fu, David S. Hong, Sarina A. Piha-Paul, Jennifer J. Wheler, Ralph G. Zinner, Vivek Subbiah, Apostolia M. Tsimberidou, Daniel D. Karp, Vanda M. Stepanek, Goran Cabrilo, Rajyalakshmi Luthra, Funda Meric-Bernstam, Agop Y. Bedikian, Bryan K. Kee, Cathy Eng, Michael J. Overman, Kevin B. Kim, Amy Kruempel, Jaclyn Pope, Courtney Cubrich, Grant Wu, Marcia Lewis, Razelle Kurzrock. BRAF and KRAS mutation testing in plasma cell-free DNA with ICE COLD-PCR in patients with advanced cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5607. doi:10.1158/1538-7445.AM2014-5607