David R. Hout

Successful treatment of hepatic oligometastases with stereotactic ablative radiotherapy and radiofrequency ablation in an anaplastic lymphoma kinase fusion‐positive lung cancer patient

Local ablative therapy with stereotactic ablative radiotherapy may improve survival in oncogene‐addicted lung cancer patients with extracranial oligometastatic disease treated with targeted therapies. There is limited data on the use of radiofrequency ablation (RFA) in this same setting. We present a case of an anaplastic lymphoma kinase (ALK)‐positive lung cancer patient with hepatic oligometastatic progression who was successfully treated with both stereotactic ablative radiation and RFA while continuing with an ALK inhibitor.

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer

Patients with lung cancers harboring an activating anaplastic lymphoma kinase (ALK) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off ΔCt of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.

Detection of a TRAF1-ALK fusion in an anaplastic large cell lymphoma patient with chemotherapy and ALK inhibitor-resistant disease

BackgroundThe anaplastic lymphoma kinase (ALK) gene encodes a receptor tyrosine kinase, which was first identified as the fusion partner of the nucleophosmin (NPM1) gene in the recurrent t(2;5)(p23;q35) found in a subset of anaplastic large cell lymphoma (ALCL). Several distinct, non-NPM1, ALK fusions have subsequently been described in lymphomas and other tumor types. All of these fusions result in the constitutive expression and activation of ALK and ALK signaling pathways, ultimately leading to the malignant phenotype.Case reportA non-NPM1 fusion partner of ALK was identified in a 32-year-old Caucasian male ALCL patient whose disease was refractory to standard chemotherapy and autologous stem cell transplantation, and exhibited a poor response to a first-generation ALK inhibitor. Non-allele-specific ALK RT-qPCR revealed ALK overexpression and 5′ RACE PCR revealed that the patient’s lymphoma expressed a TRAF1-ALK fusion.ConclusionsWe report the case of an ALCL patient whose tumor harbored the newly recognized TRAF1-ALK fusion and describe the clinical outcome after treatment with an ALK inhibitor. The short survival of our patient may reflect a propensity toward aggressive behavior in lymphomas that express this ALK fusion.

Abstract 1930: The unknown piece of the pie: Molecular markers in triple-negative lung cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Lung cancer is the main cause of cancer mortality worldwide. Approximately ∼80% of lung cancer cases are non-small cell lung cancer (NSCLC) in type and >50% of NSCLC are adenocarcinoma in histopathology. A shifting paradigm in the field of pulmonary oncology is the identification of molecular markers that are therapeutic targets and/or provide prognostic information. The recent recognition of the role biomarkers such as EGFR, KRAS, and ALK play in NSCLC adenocarcinoma patients demonstrates this trend. Although these three highly characterized molecular markers make up one-quarter to two-thirds of NSCLC adenocarcinoma patients, the remaining population, termed “Triple-Negative Lung Cancer” (TNLC), has yet to be fully defined. The complete absence of therapeutic targets for triple-negative lung cancer patients undeniably supports the need to identify up-regulated and mutated genes that better define this poor prognostic cohort. Recent genome-wide expression profiles subdivided TNLC patients into a poor prognostic group based on the overexpression of the DEP domain containing 1 gene (DEPDC1) (Okayama et al., 2011). We hypothesized that overexpression of DEPDC1 correlates with a specific sub-population of adenocarcinoma patients. Our initial studies indicate that DEPDC1 is constitutively expressed in both normal lung as well as lung cancer specimens. DEPDC1 transcripts are present as two variants (V1 and V2), with constitutive expression of the V1 variant in normal lung. We hypothesized that expression of the V2 transcript could be unique to NSCLC adenocarcinomas. Screening of FFPE NSCLC adenocarcinoma specimens using a proprietary qPCR approach demonstrated the presence of the DEPDC1 V2 variant but at levels that were not sufficient to serve as a sole biomarker. We therefore investigated whether the ratio of DEPDC1 V2 expression relative to V1 could serve as a biomarker in TNLC. Our preliminary data suggest that the ratio of V2 to V1 expression of DEPDC1 does in fact segregate TNLC into specific sub-populations. These studies have exciting diagnostic as well as therapeutic implications as Phase I/II studies using novel peptide vaccines derived against DEPDC1 have proven tolerable and efficacious for patients with bladder cancer and could potentially be extended to target these newly identified TNLC sub-populations. Citation Format: Brock L. Schweitzer, Kasey D. Lawrence, John Handshoe, Rachel Skelton, Lindsay Chatfield, Liquan Xue, Stephan W. Morris, David R. Hout. The unknown piece of the pie: Molecular markers in triple-negative lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1930. doi:10.1158/1538-7445.AM2013-1930

Rates of immune cell infiltration in patients with triple-negative breast cancer by molecular subtype

In patients with triple-negative breast cancer (TNBC), tumor-infiltrating lymphocytes (TILs) are associated with improved survival. Lehmann et al. identified 4 molecular subtypes of TNBC [basal-like (BL) 1, BL2, mesenchymal (M), and luminal androgen receptor (LAR)], and an immunomodulatory (IM) gene expression signature indicates the presence of TILs and modifies these subtypes. The association between TNBC subtype and TILs is not known. Also, the association between inflammatory breast cancer (IBC) and the presence of TILs is not known. Therefore, we studied the IM subtype distribution among different TNBC subtypes. We retrospectively analyzed patients with TNBC from the World IBC Consortium dataset. The molecular subtype and the IM signature [positive (IM+) or negative (IM-)] were analyzed. Fisher’s exact test was used to analyze the distribution of positivity for the IM signature according to the TNBC molecular subtype and IBC status. There were 88 patients with TNBC in the dataset, and among them 39 patients (44%) had IBC and 49 (56%) had non-IBC. The frequency of IM+ cases differed by TNBC subtype (p = 0.001). The frequency of IM+ cases by subtype was as follows: BL1, 48% (14/29); BL2, 30% (3/10); LAR, 18% (3/17); and M, 0% (0/21) (in 11 patients, the subtype could not be determined). The frequency of IM+ cases did not differ between patients with IBC and non-IBC (23% and 33%, respectively; p = 0.35). In conclusion, the IM signature representing the underlying molecular correlate of TILs in the tumor may differ by TNBC subtype but not by IBC status.

Abstract 2220: Insight ALK ScreenTM, a highly sensitive and specific RT-qPCR first-line screening assay for comprehensive detection of all oncogenic anaplastic lymphoma kinase (ALK) fusions: clinical validation

More than 15 different ALK fusions (e.g., CLTC-ALK, multiple EML4-ALK variants, KIF5B-ALK, NPM-ALK, RanBP2-ALK, TFG-ALK, TPM4-ALK, others) cause malignancies including NSCLC, NHL, and inflammatory myofibroblastic tumors. Preliminary studies suggest several other cancer subsets may also express ALK fusions (e.g., breast, colorectal and esophageal cancers). Several ALK small-molecule inhibitors are in development, and patients treated with one clinical stage inhibitor have had marked antitumor responses. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for ALK have been used to select patients likely to benefit from ALK inhibitor therapy; however, these methods are problematic. Both methods have suboptimal sensitivity for certain ALK fusions (e.g., EML4-ALK), and FISH is technically demanding and has a prolonged turn-around time (5-7 days). To address the unmet need for a highly sensitive, practical assay for clinical ALK detection, we developed Insight ALK Screen™, a real-time PCR test for ALK fusions and wild-type ALK upregulation. The test operates by qPCR of two regions corresponding to the ALK extracellular and intracellular kinase domains; for example, EML4-ALK or other ALK fusion events result in a ΔCt (change in threshold cycle) increase in transcripts encoding the ALK kinase domain over wild-type ALK expression levels.This assay design enables Insight ALK Screen to detect any ALK fusion regardless of the ALK fusion partner. We are validating Insight ALK Screen in comparison to ALK IHC and FISH using NSCLC FFPE specimens from the Clarient tumor bank. Eighty-two NSCLCs were screened using Insight ALK Screen, and 17 of the 82 (20.7%) showed either an ALK fusion or wild-type ALK upregulation. All 17 ALK-expressing NSCLCs were next tested using an EML4-ALK variant-specific RT-PCR assay; 6 (35.3%) of the 17 cases (7.3% of the original 82 cases) were shown to be EML4-ALK-positive. Ongoing studies are assessing the genetic mechanisms (e.g., other EML4-ALK variants not tested, other ALK fusions, ALK gene amplification, chromosome 2 polysomy, etc.) underlying the aberrant ALK expression in the other 11 of 17 NSCLCs; these data will be presented at the meeting together with complete IHC, FISH and clinical-molecular correlative information. Together with its ease-of-use, small tissue requirements, multiplex compatibility, quantitation of ALK expression levels, lack of bias for specific fusions, and quick turn-around (24-48 hrs), our preliminary results support Insight ALK Screen as the first-line ALK diagnostic method of choice. 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 2220. doi:10.1158/1538-7445.AM2011-2220