Additional Tumor Types Research Articles

Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology.

- In 2013, an evidence-based guideline was published by the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology to set standards for the molecular analysis of lung cancers to guide treatment decisions with targeted inhibitors. New evidence has prompted an evaluation of additional laboratory technologies, targetable genes, patient populations, and tumor types for testing. - To systematically review and update the 2013 guideline to affirm its validity; to assess the evidence of new genetic discoveries, technologies, and therapies; and to issue an evidence-based update. - The College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology convened an expert panel to develop an evidence-based guideline to help define the key questions and literature search terms, review abstracts and full articles, and draft recommendations. - Eighteen new recommendations were drafted. The panel also updated 3 recommendations from the 2013 guideline. - The 2013 guideline was largely reaffirmed with updated recommendations to allow testing of cytology samples, require improved assay sensitivity, and recommend against the use of immunohistochemistry for EGFR testing. Key new recommendations include ROS1 testing for all adenocarcinoma patients; the inclusion of additional genes ( ERBB2, MET, BRAF, KRAS, and RET) for laboratories that perform next-generation sequencing panels; immunohistochemistry as an alternative to fluorescence in situ hybridization for ALK and/or ROS1 testing; use of 5% sensitivity assays for EGFR T790M mutations in patients with secondary resistance to EGFR inhibitors; and the use of cell-free DNA to "rule in" targetable mutations when tissue is limited or hard to obtain.

Lapatinib.

The human epidermal growth factor receptor (HER) family of receptor tyrosine kinases plays an important role in the biology of many cancers. In breast and gastrointestinal cancer, and at lower rates also in additional tumor types, HER2 and its homo- or heterodimerization with HER1 or HER3 are essential for cancer cell growth and survival. Breast cancer patients overexpressing HER2 have a more aggressive course of their disease. The poor prognosis associated with HER2 overexpression can be substantially improved by adding HER2-targeted therapy to standard of care using the monoclonal antibody trastuzumab. Lapatinib, an oral dual tyrosine kinase inhibitor, blocks HER1 and HER2 tyrosine kinase activity by binding to the ATP-binding site of the receptor's intracellular domain, resulting in inhibition of tumor cell growth. Lapatinib is generally well tolerated with diarrhea being the most common adverse effect. However, although being mainly of mild to moderate severity, interruption or discontinuation of treatment has been reported in a substantial proportion of patients in clinical trials. In 2007, lapatinib has been approved in combination with capecitabine in patients with advanced HER2-positive breast cancer upon progressive disease following standard therapy with anthracyclines, taxanes, and trastuzumab. In 2013, the approval was extended to a chemotherapy-free combination with trastuzumab for patients with metastatic HER2-positive, hormone receptor-negative breast cancer progressing on prior trastuzumab and chemotherapy. Since 2010, lapatinib is approved in combination with letrozole in the treatment of postmenopausal women with advanced HER2- and hormone receptor-positive breast cancer. In contrast, in first-line cytotoxic-based therapy of both early and advanced HER2-positive breast cancer, data from clinical trials did not provide evidence of additional benefit of lapatinib compared to trastuzumab. Moreover, over the past few years, novel HER2-targeted drugs, either alone or as a combined anti-HER2 approach, have been extensively evaluated, demonstrating a more favorable outcome. Also, neither in first- nor second-line treatment of advanced gastric cancer, lapatinib has been proven to be superior compared to trastuzumab as hitherto standard of care HER2 blockade. Therefore, lapatinib has become somewhat less important in patients with HER2-positive breast cancer during the past 10years since its first introduction. Nevertheless, consideration of treatment with lapatinib appears to be reasonable in selected patients not only in the approved applications but also beyond, and further indications such as HER2-positive refractory metastatic colorectal cancer may arise in future. Also, lapatinib may have distinct advantages over antibodies in targeting truncated HER2 and crossing the blood-brain barrier. Finally, the favorable cardiac toxicity profile of lapatinib makes it an attractive alternative to trastuzumab-based regimens in patients at risk for cardiac events.

Abstract A043: Identification of a patient enrichment strategy supporting development of a potent and selective dual TTK/CLK2 inhibitor CC-671

Abstract CC-671 is a potent and selective TTK1 (Mps1) and CLK2 inhibitor. In support of a phase I proof of concept, we used an image-based, multiplex cell assay to evaluate the antiproliferative impact of CC-671 across a broad panel of cancer cell lines representing tumors from various anatomic origins. CC-671 potently and selectively inhibited proliferation and induced apoptosis. Cell lines representing leukemia, lymphoma, colorectal cancer (CRC), head and neck (H&N), and bladder cancers were uniformly sensitive to CC-671 while the cell lines from other tumor types, such as breast and lung cancers, were differentially sensitive to CC-671. Cell cycle analysis using phospho-histone H3 (pHH3) as a mitosis biomarker suggested that CC-671 had cell line-specific mitotic effects. CC-671 accelerated mitotic exit in 120 cell lines while causing mitotic block in 37 cell lines at concentrations < 1 μM. Custom gene expression signatures correlating with sensitivity or resistance were generated. Mapping these signatures to a gene expression database of clinical tumor samples identified additional CC-671 sensitive tumor types including Wilms tumor, superficial bladder cancer, triple-negative, and luminal Btype breast cancer, and several squamous cell carcinomas (lung, esophageal, cervical, H&N). To identify a breast cancer patient selection profile, the CC-671 antiproliferative activity was assessed in a panel of 49 breast cancer cell lines. CC-671 potently inhibited the growth of breast cancer cell lines with 50% inhibitory concentrations (IC50 values) < 100 nM in 14 lines and > 10 μM in 21 lines. Triple-negative breast cancer (TNBC) cell lines were significantly more sensitive to CC-671 than non-TNBC (ER+/PR+ and/or HER2+) lines. Within TNBC, the mesenchymal (M) and basal-like 1 (BL1) subtypes were more sensitive to CC-671. In addition, TNBC lines with cancer stem cell (CSC)-like properties were also sensitive, indicating a potential role of TTK1 and CLK2 in cancer stem cells. Interestingly, mutations in several genes (phosphatidylinositol-4,5-bisphosphate 3kinase 110 kDa catalytic subunit alpha [PIK3CA], serine/threonine kinase 11 [STK11], and phosphatase and tensin homolog [PTEN]) in the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway tend to associate with CC-671 TNBC sensitivity. These mutational correlates are being confirmed in additional TNBC lines. Citation Format: Shuichan Xu, Tam Tran, Tao Shi, Ning Jiang, Dan Zhu, Jennifer R. Riggs, John Boylan. Identification of a patient enrichment strategy supporting development of a potent and selective dual TTK/CLK2 inhibitor CC-671 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A043.

Abstract 3093: Expression of DLL3 in metastatic melanoma, glioblastoma and high-grade extrapulmonary neuroendocrine carcinomas as potential indications for rovalpituzumab tesirine (Rova-T; SC16LD6.5), a delta-like protein 3 (DLL3)-targeted antibody drug conjugate (ADC)

Abstract Expression of DLL3 was examined in additional tumor types, as it was found to be highly expressed in tumor-initiating cells (TIC) in small cell lung cancer (SCLC), where a DLL3-targeted antibody drug conjugate (ADC), rovalpituzumab tesirine (Rova-T; SC16LD6.5; Saunders et al. 2015 Sci Transl Med 7:302ra136)) exerted clinically meaningful anti-tumor effects in a phase I trial (Spigel et al. 2016 Lancet Oncology; In Press). DLL3 expression was profiled by qRT-PCR, ELISA and immunohistochemistry in multiple tumor types. Patient-derived xenografts (PDX) from melanoma and ovarian small cell carcinoma were established and used for efficacy studies to determine the ability of Rova-T to impact tumor growth and TIC frequency. DLL3 expression was seen in metastatic melanoma (55%), low grade gliomas (90%), glioblastoma (70%), medullary thyroid cancer (65%), carcinoids (33%), dispersed neuroendocrine tumors in the pancreas (9%), bladder (57%) and prostate (24%), testicular cancer (90%), and lung adenocarcinomas with neuroendocrine features (80%). Unlike SCLC, where DLL3 does not predict clinical outcome on standard therapies, DLL3 expression negatively correlates with overall survival in melanoma and small cell bladder cancer. In mice bearing DLL3 positive melanoma PDX, treatment with a single dose of Rova-T resulted in effective and durable responses (>100 days), which correlated with a significant impact on TIC frequency. Similarly, in mice bearing DLL3-positive ovarian small cell PDX, a single dose of Rova-T resulted in effective and durable responses (>100 days). Our results show that DLL3 is expressed in many neuroendocrine tumors (lung, ovarian, prostate, bladder, etc), metastatic melanoma, medullary thyroid cancer, low-grade gliomas and glioblastoma. Given pre-clinical results showing efficacy of Rova-T in melanoma and ovarian small cell carcinoma, as well as encouraging clinical data with Rova-T in patients with recurrent/refractory SCLC, clinical evaluation of Rova-T in DLL3-positive melanoma, glioblastoma, medullary thyroid cancer and other high-grade neuroendocrine carcinomas is warranted. A “basket” trial enrolling patients with DLL3-positive solid tumors is now recruiting patients (NCT02709889). Citation Format: Laura R. Saunders, Samuel A. Williams, Sheila Bheddah, Kumiko Isse, Sarah Fong, Marybeth A. Pysz, Himisha Beltran, Loredana Puca, Verena Sailer, Juan M. Mosquera, Yu Yin, Jiaoti Huang, Andrew J. Armstrong, Jorge Garcia, Cristina Magi-Galluzzi, Vadim Koshkin, Petros Grivas, Farhad Kosari, John Cheville, Justin C. Moser, Thomas J. Flotte, Thorvardur Halfdanarson, Aaron Mansfield, Konstantinos N. Leventakos, Julian R. Molina, Douglas W. Ball, Barry D. Nelkin, Jill E. Shea, Courtney L. Scaife, Scott J. Dylla. Expression of DLL3 in metastatic melanoma, glioblastoma and high-grade extrapulmonary neuroendocrine carcinomas as potential indications for rovalpituzumab tesirine (Rova-T; SC16LD6.5), a delta-like protein 3 (DLL3)-targeted antibody drug conjugate (ADC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3093. doi:10.1158/1538-7445.AM2017-3093

Abstract 3345: Selective killing of SMARCA2- and SMARCA4-deficient tumors by inhibition of EZH2: In vitro and in vivo preclinical models

Abstract The histone methyltransferase EZH2 is the enzymatic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes the methylation of H3K27 thereby repressing target gene transcription. EZH2 is amplified, overexpressed, or mutated in multiple cancer types, most notably Follicular Lymphoma (FL) and germinal center Diffuse Large B-cell Lymphoma (GCB-DLBCL). We previously reported that preclinical models of malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SNF5, SMARCB1), are selectively killed by potent and selective inhibitors of EZH2. Here we report another class of SWI/SNF-altered cancers named small cell carcinoma of the ovary hypercalcemic type (SCCOHT) that is dependent on EZH2 activity. SCCOHT is a very aggressive form of cancer that responds poorly to conventional therapy with a one-year overall survival rate of only 50%. Very few novel agents have been approved for this indication; thus there is a need for targeted therapeutics in SCCOHT. SMARCA4 and SMARCA2 are co-inactivated in this tumor type that has many rhabdoid features. We demonstrate that tazemetostat, an EZH2 inhibitor currently in phase 2 clinical trials, induces potent and selective killing in SMARCA2 and SMARCA4-deficient ovarian cell lines. In addition to small molecule inhibitor data, we conducted functional genomics studies with CRISPR pooled screening, and confirmed that SCCOHT is also sensitive to CRISPR-mediated EZH2 gene ablation. Dose-dependent anti-tumor effects were observed upon tazemetostat treatment in SCCOHT xenografts deficient in both SMARCA2 and SMARCA4. We also report on additional non-ovarian tumor types with dual SMARCA2/SMARCA4 loss including NSCLC that exhibit EZH2 dependence representing additional potential therapeutic indications for tazemetostat treatment. Citation Format: Elayne Chan-Penebre, Kelli Armstrong, Allison Drew, Alexandra R. Grassian, Igor Feldman, Maria Roche, Peter Ho, Dorothy Brach, Alejandra Raimondi, Robert A. Copeland, Richard Chesworth, Jesse J. Smith, Scott A. Ribich. Selective killing of SMARCA2- and SMARCA4-deficient tumors by inhibition of EZH2: In vitro and in vivo preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3345. doi:10.1158/1538-7445.AM2017-3345

Abstract 3814: A pan cancer analysis of the tumor inflammation signature

Abstract Introduction: The Tumor Inflammation Signature (TIS) is an 18 gene signature in development for the detection of adaptive immune response within tumors by measuring expression of genes associated with cytotoxic cells, antigen presentation, and IFNγ activity. The TIS has previously been shown to enrich for a population of patients who respond to the anti-PD1 therapy pembrolizumab. We characterized the behavior of the TIS across a range of tumor gene expression data downloaded from the TCGA in order to understand the distribution of TIS within and between tumor types, with special emphasis on those tumors for which anti-PD1 therapy is approved for use. Methods: The TIS was trained to predict response to pembrolizumab using gene expression profiles from 289 biopsies from 11 different tumor types. Its association with clinical response was retrospectively assessed in an independent set of 200 samples from 14 additional tumor types. We calculated TIS scores in over 9000 samples from 31 TCGA RNASeq datasets. We contrasted TIS with mutational load, with overall survival, and with other gene expression signatures. Results: While TIS scores are higher in classically immunogenic tumor types, they display a significant amount of intersample variability within most tumor types, and a subset of patients can be identified that possess elevated TIS scores consistent with responsiveness to pembrolizumab. TIS genes have highly conserved co-expression patterns across tumor types, consistent with a model in which the genes measure immune-intrinsic transcriptomic activity with minimal contribution of tumor-intrinsic gene expression. Notably, ranking tumors by median TIS score is superior to mutational load ranking at predicting clinical response to anti-PD1. TIS was minimally correlated with mutational load in most tumor types, except in tumor types known for hypermutation-driven by mismatch repair deficiency. TIS and mutation burden had their greatest variability in melanoma; in most other tumors, TIS retains more variability than mutation burden, possibly reflecting patients with anti-tumor immune responses driven by one or a few mutation-derived neoantigens. Average mutation burden and average TIS are positively correlated within most tumor types. Notable exceptions to this trend include two tumors for which anti-PD1 is currently approved. Bladder urothelial carcinoma has lower average TIS than its mutation burden would predict, and kidney renal clear cell carcinoma has much higher average TIS than its mutation burden would predict. Conclusion: The TIS has the potential to enrich for anti-PD1 responders independent of tumor type. It may identify rare potential responders in tumors with low average response rates. These results support further research into the efficacy of anti-PD1 in patients with high TIS scores, regardless of tumor type. Citation Format: Patrick Danaher, Sarah Warren, Alessandra Cesano. A pan cancer analysis of the tumor inflammation signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3814. doi:10.1158/1538-7445.AM2017-3814

Development of a clinical trial immunohistochemistry (IHC) assay using a novel antibody to CD38.

7561 Background: CD38 is a type II transmembrane glycoprotein expressed on normal lymphoid and myeloid cells and can be highly expressed in hematologic malignancies. An IHC prototype assay was developed to detect CD38 expression in formalin-fixed, paraffin-embedded (FFPE) tissue specimens from three relapsed or refractory non-Hodgkin’s lymphoma (NHL) subtypes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL) for selection of patients for treatment with daratumumab in a Phase 2 clinical trial (NCT02413489). Methods: This assay is based on EnVision FLEX IHC technology using CD38, clone DAK-CD38, primary antibody that has been developed and manufactured by Agilent Technologies. The assay staining protocol was developed for Dako PT Link and Autostainer Link 48. Specificity of DAK-CD38 staining was demonstrated by Western blot analysis of cancer cell lysates, as well as IHC on both normal and cancer tissue. Assay precision and robustness were evaluated using commercially procured FFPE DLBCL, FL and MCL specimens. Results: CD38 IHC DAK-CD38 detected a broad range of CD38 expression in DLBCL, FL, and MCL specimens. FFPE specimens derived from cancer cell lines exhibited a range of IHC staining intensity and confirmed the reported expression of CD38 in the scientific literature. All precision and robustness results met acceptance criteria for both IHC intensity and percent positive cells staining. Conclusions: Our studies demonstrate that the Dako CD38 IHC DAK-CD38 assay is sensitive, specific, precise, and robust for the detection of CD38 expression in DLBCL, FL and MCL. This assay may also have the potential to be used for the detection of CD38 in additional tumor types.

Endocrinological side-effects of immune checkpoint inhibitors.

Three mAbs targeting immune checkpoint proteins are available for the treatment of patients with melanoma, lung, and kidney cancer, and their use will likely expand in the future to additional tumor types. We here update the literature on the incidence and pathophysiology of endocrine toxicities induced by these agents, and discuss management guidance. Immune checkpoint inhibition may trigger autoimmune syndromes involving different organs, including several endocrine glands (pituitary, thyroid, adrenals, and endocrine pancreas). Hypophysitis is more frequently associated with ipilimumab, whereas the incidence of thyroid dysfunction is higher with nivolumab/pembrolizumab. Primary adrenal insufficiency can rarely occur with either treatment. Autoimmune diabetes is very rare. As hypophysitis and adrenalitis may be life-threatening, endocrinological evaluation is essential particularly in patients developing fatigue and other symptoms consistent with adrenal insufficiency. Corticosteroids should be promptly used when hypophysitis-induced adrenal insufficiency or adrenalitis are diagnosed, but not in thyroiditis or diabetes. No impact of corticosteroids on the efficacy/activity of immune checkpoint-inhibiting drugs is reported. Hormonal deficiencies are often permanent. In absence of predicting factors, accurate information to patients provided by the oncology care team is essential for early diagnosis and to limit the consequences of checkpoint inhibition-related endocrine toxicity.

Application of immunomodulatory therapeutic antibodies - a new perspective in oncology

In the past decade major advances in tumor immunology, a better understanding of antigen recognition by T-cells likewise discovering the regulatory inhibitory signals resulted in the development of new immunotherapies with promising durable responses in various solid tumor types and in hematologic malignancies. This review focuses on immunomodulatory antibodies, namely immune checkpoint inhibitor therapy. The prototype of this new class of immune stimulating agents was cytotoxic T-lymphocyte antigen-4 (CTLA-4) antagonists. After demonstrating enhanced survival, ipilimumab was approved first in the United States in 2011, further on in the European Union for second-line (2011) and for first-line therapy (2013) of metastatic melanoma. Additional T-cell intrinsic pathways were identified and targeted for clinical development. Antibodies blocking the PD-1 pathway also showed promising clinical activity and objective tumor response in several types of tumors, including metastatic melanoma, non-small- cell lung cancer. On the other hand antitumor activity is frequently accompanied by significant reversible immune-related adverse events. To explore potential new immune checkpoint targets bring forth several challanges. Future clinical development will involve identifying potential biomarkers anticipating responsiveness to pathway blockade and additional tumor types likely to respond to the therapy. Furthermore, combination strategies, immune checkpoint inhibitors combined with cancer vaccines, targeted inhibitors and traditional chemotherapies are being evaluated in pre-clinical studies. Orv. Hetil., 2016, 157(Suppl. 2), 9-16.

Abstract IA02: Human ES cells for modeling brain cancer

Abstract Diffuse Intrinsic Pontine Gliomas (DIPG) are a highly aggressive tumor of the brainstem occurring primarily in children. The outlook of patients with this disease is particularly dire due to the tumor's inaccessible location and its lethal course over one or two years. Recent sequencing data identified, for the first time in human cancer, heterozygous mutations in the histone H3 that create a lysine to methionine amino acid substitution. Using a human ES-based platform, we demonstrate the exquisitely cell-context specific oncogenic role of the H3K27M mutation. The model recapitulated the histological features and in vivo progression of the human tumor, as well as its transcriptomal and epigenetic profile. Genome wide analyses indicate a resetting of the transformed precursors to a developmentally more primitive state, with evidence of major modifications of histone marks at master regulatory genes. Drug screens in the ES model identified a compound that targets menin, a protein with multiple partners the mixed leukemia lineage protein MLL. The compounds have been validated in patient derived DIPG cell lines and are undergoing intense preclinical assessments. The ES model, coupled with more recent gene editing technology, provides a very powerful platform for the dissection of the requirements of neoplastic transformation in specific tumors, the specification of cells of origin, as well insight into the downstream effectors of individual and combined mutations. Human pluripotent stem cells have an excellent potential as platform for modeling cancer. Work to extend these findings to additional mutations and tumor types is ongoing. Ref. Funato et al. Science. 2014 Dec 19;346 (6216) Citation Format: Viviane Tabar. Human ES cells for modeling brain cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA02.

Successes and Challenges of PARP Inhibitors in Cancer Therapy.

Successes and Challenges of PARP Inhibitors in Cancer Therapy.

Systematic transcriptome analysis reveals tumor-specific isoforms for ovarian cancer diagnosis and therapy

Tumor-specific molecules are needed across diverse areas of oncology for use in early detection, diagnosis, prognosis and therapy. Large and growing public databases of transcriptome sequencing data (RNA-seq) derived from tumors and normal tissues hold the potential of yielding tumor-specific molecules, but because the data are new they have not been fully explored for this purpose. We have developed custom bioinformatic algorithms and used them with 296 high-grade serous ovarian (HGS-OvCa) tumor and 1,839 normal RNA-seq datasets to identify mRNA isoforms with tumor-specific expression. We rank prioritized isoforms by likelihood of being expressed in HGS-OvCa tumors and not in normal tissues and analyzed 671 top-ranked isoforms by high-throughput RT-qPCR. Six of these isoforms were expressed in a majority of the 12 tumors examined but not in 18 normal tissues. An additional 11 were expressed in most tumors and only one normal tissue, which in most cases was fallopian or colon. Of the 671 isoforms, the topmost 5% (n = 33) ranked based on having tumor-specific or highly restricted normal tissue expression by RT-qPCR analysis are enriched for oncogenic, stem cell/cancer stem cell, and early development loci--including ETV4, FOXM1, LSR, CD9, RAB11FIP4, and FGFRL1. Many of the 33 isoforms are predicted to encode proteins with unique amino acid sequences, which would allow them to be specifically targeted for one or more therapeutic strategies--including monoclonal antibodies and T-cell-based vaccines. The systematic process described herein is readily and rapidly applicable to the more than 30 additional tumor types for which sufficient amounts of RNA-seq already exist.

IS5-4 - Checkpoint Inhibitors as a Foundation for Anti-Cancer Immune Therapy

Substantial experimental data demonstrate that patients with advanced cancer can generate T lymphocyte immune responses to tumor specific mutations, re-expressed developmental proteins and tissue differentiation antigens. In normal immune function, T lymphocyte responses are tightly regulated by inhibitory checkpoints to prevent both self-reactivity and excessive tissue damage in response to infection. Similar regulatory checkpoints act to limit tumor-specific immune responses. Antagonist antibodies targeting CTLA-4, PD-1 or PD-1 ligand (B7-H1/PD-L1) were brought into clinical development to test the hypothesis that blockade of T-cell inhibitory ligand-receptor interactions and/or inhibition of T-regulatory cells could activate/expand/restore anti-tumor T-lymphocyte responses and produce meaningful clinical benefit in advanced cancer. Indeed, anti-CTLA-4 (ipilimumab) prolonged survival in 2 randomized trials conducted in metastatic melanoma, and demonstrated clinical activity in a small subset of patients with other malignancies through limited phase 2 evaluation. Several antibody PD1/PD-L1 pathway antagonists are now in clinical trials and overall demonstrated objective response rates of 17-41% in metastatic melanoma, 10-22% in NSCLC, and 12- 29% in metastatic renal carcinoma. Responses were also observed in colon (2/37), gastric, ovarian (1/20), and head and neck cancer. Phase 2 studies in additional tumor types are indicated based on the association between tumor PD-L1 expression and response to PD1/PD-L1 pathway antagonists. A substantial proportion of responders to PD-1/PD-L1 blockade remained progression-free for long periods without maintenance therapy. Autoimmune type toxicity can be observed with both ipilimumab and the PD1/PD-L1 pathway antagonists; treatment algorithms for toxicity management to include steroids and other immune suppressants as necessary have been instituted successfully. The first combination of an anti-CTLA- 4 plus an anti-PD-1 produced greater toxicity than expected for either single agent, but toxicity was manageable, and activity in melanoma appeared to surpass reported data for either single agent. Multiple other immune checkpoints are known, and based on current data, it is reasonable to predict that inhibition of one or more checkpoints will likely become the foundation on which successful and broadly active combination immunotherapies are built.

Musing on Microarrays and Genomic Analysis

Copyright: © 2014 Halper J. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The paper on molecular profiling of human chondrosarcoma cells by Desiderio et al. [1] in this issue confirms several important findings by other investigators, such as that cells cultured in 3-D structures approximate their natural environment more closely than monolayer cultures and, as a result, these 3-D cultures are quite suitable for drug discovery and screening using gene expression microarrays. The expression of stemness markers by chondrospheres described in this report also supports the use of 3-D cultures in regenerative medicine. This is not the first report of gene expression in chondrosarcoma, e.g., Yoshitaka et al. used microRNA microarrays to analyze 20 chondrosarcoma samples obtained at surgery and 2 chondrosarcoma cell lines [2]. However, there appears to be little or no overlap between these two studies though both employed microarrays from the same manufacturer. The reasons are multiple: Desiderio et al. used primary tumor-derived chondrospheres, a type of 3-D culture whereas Yoshitaka et al. analyzed microRNAs from two commercially available chondrosarcoma cell lines maintained in traditional 2-D configuration, and microRNAs from 20 surgically obtained chondrosarcomas. Authors of both studies observed changes in expression of many genes, but emphasized changes in those deemed of importance to their particular investigation, and, as a result analyzed only a very selected few changes. As Desiderio et al. were mostly interested in drug resistance, metastasis and “stemness” they directed their attention to genes likely to be involved in regulation of these three processes. In contrast, Yoshitaka et al. concentrated on genes whose changes in expression can be utilized in diagnosis. Genomic analysis is being developed for other types of malignancies, such as ovarian cancer [3], acute leukemia [4] and follicular lymphoma [5] to name just a few. All of this helps to drive an important point home: there are many many genes involved in carcinogenesis, some already known, and many more to be still discovered as pointed out by Lawrence et al. [6]. In their just published report the authors conclude that many new candidate cancer genes remain to be discovered and that the number of additional cancer genes will go up as more tumors within the same (histopathological) group will be examined and as additional tumor types will undergo genomic analysis [6]. This will enhance the current capacity of current genomic collection, however, it will take quite a bit of effort to understand which mutations, be it somatic point mutations, substitutions, small insertion, and/or deletions contribute to carcinogenesis, and can be utilized as diagnostic and/or prognostic markers. Such analysis will also contribute to our knowledge of genes and their functions, such as in cell proliferation, apoptosis, genome stability, chromatin regulation, immune evasion, RNA processing and protein homeostasis [6]. Integrated genomic analyses carry the promise to revolutionize diagnosis and determination of proper treatment modalities for a variety of malignancies, and, indeed, other diseases as well. It also remains to be seen to what extent this new technology will transform pathology, esp., diagnostic surgical pathology, or whether, genomic analysis will even supersede this venerable old discipline!

Ongoing comparison of confocal laser endomicroscopy (PCLE) pulmonary images with surgical biopsies for lung cancer diagnosis

<h3>Background</h3> Probe-based confocal laser endomicroscopy (pCLE) is a new FDA approved tool used for in vivo microscopic imaging of proximal and distal airways including bronchial and alveolar walls, microvessels, and inflammatory cells. Light at 488 nm wavelength is focused via a pinhole-sized aperture incorporated into a flexible probe that fits into the working channel of a standard bronchoscope and reflected back through that aperture to produce an image that can aid in the identification of neoplastic processes. Studies have demonstrated elastin and collagen fibers as having endogenous fluorescence by pCLE. No studies have correlated in vivo imaging by pCLE to pathologic samples obtained during bronchoscopy. This combination could prove diagnostically advantageous to both clinician and pathologist. <h3>Design</h3> Selected patients undergoing bronchoscopy from 7/17/ 12 to present are subjected to pCLE followed by biopsy. pCLE and bronchoscopy are performed to help determine abnormal bronchial or alveolar changes that warrant a biopsy. We correlate pCLE images with biopsies obtained during bronchoscopy. Patients ranged from 47 to 82 years of age, and biopsies include adeno, squamous cell, poorly differentiated, small cell and metastatic carcinomas. When necessary, immunohistochemistry (IHC) and electron microscopy (EM) are performed for further tumor classification. Studies are ongoing to add additional tumor types. <h3>Results/findings</h3> By pCLE, differences between normal, inflammation and neoplasia can be recognized. Normal findings consist of organized connective tissue fibers. Inflammatory cells, mostly macrophages, can be recognized within tissue spaces. In neoplastic tissue, a ragged surface can be observed along with disorganization of the collagen/elastin fibers within the bronchi/ alveoli. Differences between squamous (SCC) and adenocarcinomas (ADC) can be suggested on the basis of imaging patterns. SCC shows friability and dense areas of auto fluorescence with dark spots representing tumor groups. ADC shows less fluorescence and gives a mottled appearance to the tissue. ADC with bronchioloalveolar differentiation and a lepidic pattern of growth can be suspected when a studded appearance is noted by pCLE. Disarray of fibers increases with more poorly differentiated carcinomas. Biopsies and pCLE images can be correlated by comparing tissue patterns with images. The use of IHC and ultrastructural evaluation (EM) further support our pCLE and microscopic findings. <h3>Conclusions</h3> pCLE allows the distinction between benign and malignant tissues, assists in obtaining diagnostic tissue, and recognizes tumor growth patterns. Correlation between biopsies and pCLE can help aid sampling and diagnostic techniques. Larger studies can enhance our understanding of how pCLE can help differentiate tumor types.

HER-2/neu vaccine-primed autologous T-cell infusions for the treatment of advanced stage HER-2/neu expressing cancers

This phase I study evaluated the feasibility of expanding HER-2/neu (HER2) vaccine-primed peripheral blood T-cells ex vivo and assessed the safety of T-cell infusions. Eight patients with HER2(+) treatment refractory metastatic cancers were enrolled. T-cells could be expanded to predefined parameters in seven patients (88%). Ninety-two percent of adverse events were grade 1 or 2. Three of seven patients developed infusion-related inflammatory reactions at their disease sites. HER2-specific T-cells significantly increased in vivo compared to pre-infusion levels (p = 0.010) and persisted in 4/6 patients (66%) over 70 days after the first infusion. Partial clinical responses were observed in 43% of patients. Levels of T-regulatory cells in peripheral blood prior to infusion (p < 0.001), the level of HER2-specific T-cells in vivo (p = 0.030), and development of diverse clonal T-cell populations (p < 0.001) were associated with response. The generation of HER2 vaccine-primed autologous T-cells for therapeutic infusion is feasible and well tolerated. This approach provides a foundation for the application of T-cell therapy to additional solid tumor types.

Observational study of real world clinical performance of microrna molecular profiling for cancer of unknown primary (CUP).

e22173 Background: Molecular profiling of CUP may effectively identify the underlying tumor type and potentially influence treatment decisions and outcomes. A microRNA (miR) array has been shown in concordance studies at leading academic centers to perform well in correctly identifying the tumor of origin in CUP. This series of consecutive referrals tested in a CLIA-laboratory extends these findings to the real world clinical setting. Methods: MicroRNA was isolated from 258 consecutive specimens referred to Rosetta Genomics, analyzed on a 64 miR-based array, and interpreted for any of 42 specific tumor types as previously described. The laboratory medical director routinely collected follow-up information on correlation of test prediction with other clinical information, pathology findings, supplemental IHC, treatment response, and clinical course as well as physician perception of relevance and utility of test result. This information was scored categorically into 3 classes of concordance depending on agreement with clinical and/or pathological data. The composite data was de-identified and analyzed for assessment of overall test performance in this real world clinical setting. Results: Of 258 specimens submitted 90% were paraffin embedded tissue (FFPE). Sufficient tumor material was received in 217 (85%) cases, of which 192 (88%) were successfully processed and reported with a mean TAT of 7 days. The most common result was colorectal (12%), followed by breast (10.4%), upper body squamous cell (7.3%), ovarian (6.8%), and biliary tract/pancreatic (6.8%); and the remaining 109 cases (57%) represented 30 additional tumor types. Overall the concordance with the clinical and/or pathological best final diagnosis was 86%, and when the reporting algorithm yielded a single diagnosis (51% of cases) the concordance was 89%. Clinical utility as measured by consideration of a shift in therapy or significantly increased decisional certainty for therapy was observed in approximately 70% of the cases. Conclusions: MicroRNA analysis in CUP cases performs comparably in the real world setting to that previously reported in academic studies and adds information of value to the management of patients.

Abstract 2217: A systems pharmacogenomic approach to identify synergistic molecular mechanisms of combined mTOR/HDAC inhibition.

Abstract The necessity of combining targeted therapeutics to achieve optimal, lasting clinical benefit is clear, but standardized approaches for identifying the interactive effects of these combinations are not yet established. Discerning drug synergy at the molecular level has proven particularly challenging, yet identification of cooperatively responding, biologically-relevant targets could be useful for defining patient subsets for which the combination would be active. Here we used a transcriptional co-expression systems-level analysis to define the cooperative molecular response to the synergistic combination of mTOR/HDAC inhibitors in multiple myeloma (MM), and in other tumor types including triple negative breast cancer. Co-expression analysis of cells treated individually and in combination defined the contribution of each drug to the combination, and identified a distinct network of 126 genes cooperatively targeted by both drugs. We interrogated the cooperative network genes for differential expression between normal and malignant cells, as well as for correlation with survival in a large patient dataset. 37 of the cooperatively affected genes were both differentially expressed in MM and predictive of survival (p&amp;lt;0.01). Analysis of additional tumor types showed similar results. The pharmacodynamic response of the survival-linked signature to the drug combination was evaluated using the NanoString gene expression platform in a large number of cell lines from multiple tumor types and in ex vivo-treated primary patient samples before and after treatment. We found the expression change of signature genes to be highly specific for biological response to the drug combination across tumor types. Additionally, to link the response signature to a central molecular effect of combination treatment, Ingenuity transcription factor enrichment testing was performed. Based on these predictions, subsequent analysis of CHIP-Seq datasets was performed, and two oncogenic transcription factors (TFs) were found to bind nearly all genes of this signature. We then experimentally linked drug combination response to diminished expression of these TFs at the protein level ahead of cell cycle and apoptotic changes. Further experiments have been performed to establish a direct link between these TFs, our gene signature, and drug response. Thus, a systems-level genomic approach has identified a gene signature indicative of drug combination activity, mechanism, disease specificity, and clinical potential. Citation Format: John K. Simmons, Aleksandra M. Michalowski, Ben Gamache, Jyoti Patel, Adriana Zingone, Ke Zhang, Michael Kuehl, Jing Huang, Ola Landgren, Beverly A. Mock. A systems pharmacogenomic approach to identify synergistic molecular mechanisms of combined mTOR/HDAC inhibition. [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 2217. doi:10.1158/1538-7445.AM2013-2217

Abstract LB-227: Pilot clinical trials of FSPG (BAY 94-9392): An 18F-labeled glutamate derivative for PET imaging of system xC- activity in tumors

Abstract Visualizing adaptations of tumor metabolism by employing positron emission tomography (PET) with a specific imaging probe has the potential to better characterize tumors with higher sensitivity and specificity. The glutamate-cystine exchanger (system xC-, a SLC7A11/SLC3A2 heterodimer) is an important transporter in tumor cells and a promising new target for imaging. This exchanger transports the amino acids L-cystine and L-glutamate, thus providing a rate-limiting precursor for glutathione biosynthesis. This contributes to redox maintenance, which is especially important in tumor cells. Recently, it was reported that a splice variant of the stem cell marker CD44 stabilizes the xCT subunit of system xC- at the cell membrane and promotes its proper function. FSPG (BAY 94-9392) is an 18F-labeled glutamate derivative that provides specific visualization of system xC- activity with PET/CT. Through this multicenter pilot trial, FSPG has successfully been translated into cancer patients and shows favorable pharmacokinetics and an absence of adverse events. A favorable biodistribution pattern previously seen in mice was also confirmed in these patients with different tumor types. A high lesion detection rate (80-100%) was found in lung cancer (n=10), hepatocellular cancer (n=5), head and neck cancer (n=5), and colorectal cancer (n=5) patients who also had positive FDG-PET/CT scans. Importantly, very low background uptake in healthy brain parenchyma enabled the detection of high-grade brain cancers as well as small brain metastases and skull base lesions (n=4) not well seen with FDG-PET. Primary breast tumors (n=4) and a low-grade brain tumor, however, showed minimal uptake of FSPG. Immunohistochemical (IHC) analyses on these subjects’ pathology samples showed significant correlation between FSPG uptake and protein expression of both the xCT subunit and CD44. In breast tumor samples specifically, IHC showed that absence of CD44 results in low signal from FSPG-PET even if xCT is present, indicating the importance of CD44 co-expression for system xC- function. Future clinical studies will further explore the potential of this compound in additional tumor types and expand upon the initial findings in a larger number of subjects. Here we present the first clinical examples demonstrating the ability of the tracer to measure system xC- activity in patients. FSPG-PET/CT may provide improved characterization and sensitivity of tumor detection in patients and further improve the understanding of the role of system xC- in cancer biology. It could be a valuable tool for improved tumor classification in patients, linking uptake of the tracer with malignancy and cancer stem cell properties of tumors. Furthermore, the ability to noninvasively probe this important pathway in humans may allow for more rational treatment planning, patient stratification and better development of new drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-227. doi:1538-7445.AM2012-LB-227

INI1-Deficient Tumors

Significant progress has been made in understanding the molecular genetic alterations involved in sarcomagenesis. Cytogenetic and molecular studies have identified nonrandom genetic abnormalities, including tumor suppressor gene inactivation. Mutations, deletions, and other somatic alterations in the tumor suppressor gene INI1 (hSNF5; SMARCB1), which encodes a subunit of the SWI/SNF chromatin remodeling complex, were first described in the malignant rhabdoid tumor of infancy. Since then, INI1 has also been implicated in the pathogenesis of additional tumor types including renal medullary carcinomas and epithelioid sarcomas and a subset of epithelioid malignant peripheral nerve sheath tumors, myoepithelial carcinomas, and extraskeletal myxoid chondrosarcomas. As varied as this group appears, they all show loss of INI1 protein expression, a propensity for rhabdoid cytomorphology, and sometimes other overlapping immunohistochemical and histologic findings. We will review the clinicopathologic features of these tumor types and emphasize the clinical utility of INI1 immunohistochemistry in differential diagnosis.