Samir N. Khleif

Cancer classification using the Immunoscore: a worldwide task force

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the ‘Immunoscore’ into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

AACR-FDA-NCI Cancer Biomarkers Collaborative Consensus Report: Advancing the Use of Biomarkers in Cancer Drug Development

Recent discoveries in cancer biology have greatly increased our understanding of cancer at the molecular and cellular level, but translating this knowledge into safe and effective therapies for cancer patients has proved to be challenging. There is a growing imperative to modernize the drug development process by incorporating new techniques that can predict the safety and effectiveness of new drugs faster, with more certainty, and at lower cost. Biomarkers are central to accelerating the identification and adoption of new therapies, but currently, many barriers impede their use in drug development and clinical practice. In 2007, the AACR-FDA-NCI Cancer Biomarkers Collaborative stepped into the national effort to bring together disparate stakeholders to clearly delineate these barriers, to develop recommendations for integrating biomarkers into the cancer drug development enterprise, and to set in motion the necessary action plans and collaborations to see the promise of biomarkers come to fruition, efficiently delivering quality cancer care to patients. Clin Cancer Res; 16(13); 3299–318. ©2010 AACR.

Clinical Trial Designs for the Early Clinical Development of Therapeutic Cancer Vaccines

There are major differences between therapeutic tumor vaccines and chemotherapeutic agents that have important implications for the design of early clinical trials. Many vaccines are inherently safe and do not require phase I dose finding trials. Patients with advanced cancers and compromised immune systems are not good candidates for assessing either the toxicity or efficacy of therapeutic cancer vaccines. The rapid pace of development of new vaccine candidates and the variety of possible adjuvants and modifications in method of administration makes it important to use efficient designs for clinical screening and evaluation of vaccine regimens. We review the potential advantages of a wide range of clinical trial designs for the development of tumor vaccines. We address the role of immunological endpoints in early clinical trials of tumor vaccines, investigate the design implications of attempting to use disease stabilization as an end point and discuss the difficulties of reliably utilizing historical control data. Several conclusions for expediting the clinical development of effective cancer vaccines are proposed.

A phase I vaccine trial with peptides reflecting ras oncogene mutations of solid tumors.

Mutations in the ras genes occur in 20% of all human cancers. These genes, in turn, produce mutated proteins that are unique to cancer cells, rendering them distinguishable from normal cells by the immune system. Thus, mutated Ras proteins may form potential targets for immune therapy. We conducted a phase I/pilot clinical trial in patients with advanced cancers to test the toxicity and the ability to induce an immune response by vaccination with 13-mer mutated Ras peptides reflecting codon 12 mutations. These peptides corresponded to each of the patients own tumor Ras mutation. Patients were vaccinated monthly x3 subcutaneously with the specific Ras peptide along with Detox adjuvant (RiBi ImmunoChem Research, Inc., Hamilton, MT, U.S.A.) at one of five different peptide dose levels (100, 500, 1,000, 1,500, and 5,000 micrograms). Three out of 10 evaluable patients generated a mutant Ras specific CD4+ and/or CD8+ T-cell immune response. The CD8+ cytotoxic cells specific for Gly to Val mutation at codon 12 were capable of lysing an HLA-A2-matched tumor cell line carrying the corresponding mutant but not the wild-type ras gene. The treatment has been well tolerated with no evidence of serious acute or delayed systemic side effects on any of the five dose levels. We demonstrated that we can generate in cancer patients specific T-lymphocyte responses that detect single amino acid differences in Ras oncoproteins. Neither the immune responses nor the minor side effects seen were found to be dose dependent. This approach may provide a unique opportunity for generating a tumor-directed therapy. Also, in vitro stimulation of these cells with the corresponding peptide generated specific T-cell lines that could be used for adoptive immune therapy.

Recommendations from the iSBTc-SITC/FDA/NCI Workshop on Immunotherapy Biomarkers

Purpose: To facilitate development of innovative immunotherapy approaches, especially for treatment concepts exploiting the potential benefits of personalized therapy, there is a need to develop and validate tools to identify patients who can benefit from immunotherapy. Despite substantial effort, we do not yet know which parameters of antitumor immunity to measure and which assays are optimal for those measurements. Experimental Design: The iSBTc-SITC (International Society for Biological Therapy of Cancer-Society for Immunotherapy of Cancer), FDA (Food and Drug Administration), and NCI (National Cancer Institute) partnered to address these issues for immunotherapy of cancer. Here, we review the major challenges, give examples of approaches and solutions, and present our recommendations. Results and Conclusions: Although specific immune parameters and assays are not yet validated, we recommend following standardized (accurate, precise, and reproducible) protocols and use of functional assays for the primary immunologic readouts of a trial; consideration of central laboratories for immune monitoring of large, multi-institutional trials; and standardized testing of several phenotypic and functional potential potency assays specific to any cellular product. When reporting results, the full QA (quality assessment)/QC (quality control) should be conducted and selected examples of truly representative raw data and assay performance characteristics should be included. Finally, to promote broader analysis of multiple aspects of immunity, and gather data on variability, we recommend that in addition to cells and serum, RNA and DNA samples be banked (under standardized conditions) for later testing. We also recommend that sufficient blood be drawn to allow for planned testing of the primary hypothesis being addressed in the trial, and that additional baseline and posttreatment blood is banked for testing novel hypotheses (or generating new hypotheses) that arise in the field. Clin Cancer Res; 17(10); 3064–76. ©2011 AACR.

Anti‐PD‐1 synergizes with cyclophosphamide to induce potent anti‐tumor vaccine effects through novel mechanisms

Programmed death‐1 receptor (PD‐1) is expressed on T cells following TCR activation. Binding of this receptor to its cognate ligands, programmed death ligand (PDL)‐1 and PDL‐2, down‐regulates signals by the TCR, promoting T‐cell anergy and apoptosis, thus leading to immune suppression. Here, we find that using an anti‐PD‐1 antibody (CT‐011) with Treg‐cell depletion by low‐dose cyclophosphamide (CPM), combined with a tumor vaccine, induces synergistic antigen‐specific immune responses and reveals novel activities of each agent in this combination. This strategy led to complete regression of established tumors in a significant percentage of treated animals, with survival prolongation. We show for the first time that combining CT‐011 and CPM significantly increases the number of vaccine‐induced tumor‐infiltrating CD8+ T cells, with simultaneous decrease in infiltrating Treg cells. Interestingly, we find that CT‐011 prolongs Treg‐cell inhibition induced by CPM, leading to a sustainable significant synergistic decrease of splenic and tumor‐infiltrated Treg cells. Surprisingly, we find that the anti‐tumor effect elicited by the combination of CT‐011 and CPM is dependent on both CD8+ and CD4+ T‐cell responses, although the antigen we used is a class I MHC‐restricted peptide. Thus, we describe a novel and effective therapeutic approach by combining multiple strategies to target several tumor‐mediated immune inhibitory mechanisms.

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

ABSTRACT A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.

Apoptosis is associated with cleavage of a 5 kDa fragment from RB which mimics dephosphorylation and modulates E2F binding.

Dephosphorylation of the RB protein has been reported to be associated with apoptosis. In contrast, we show that treatment of HL60 cells with etoposide or cytosine arabinoside or treatment of breast epithelial cells with α-FAS is associated with the cleavage of a 5 kDa fragment from the C-terminus of RB, resulting in a truncated product that we have designated as p100cl. This cleavage event coincides with the activation of cysteine proteases at the onset of apoptosis, is blocked by the addition of iodoacetamide to cells prior to the onset of apoptosis, and results in the expression of faster migrating protein species which can mimic dephosphorylated RB. The free 5 kDa fragment is detected only during apoptosis, predicts a cleavage site that we have mapped to a unique CPP32-like recognition sequence which is present at the C-terminus of all reported RB homologues, and results in a truncated RB protein with enhanced E2F binding affinity. While the causality for this cleavage event in the apoptotic process is still under investigation, our findings suggest distinct post-translational pathways for the RB product between cells examined during growth arrest (p105 hypophosphorylated RB) or apoptosis (p100cl).

Human herpesvirus 6 (HHV-6) ORF-1 transactivating gene exhibits malignant transforming activity and its protein binds to p53

The 357 amino acid open reading frame 1 (ORF-1), also designated DR7, within the SalI-L fragment of human herpesvirus 6 (HHV-6) exhibited transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter and increased HIV-1 replication (Kashanchi et al., Virology, 201, 95 – 106, 1994). In the current study, the SalI-L transforming region was localized to the SalI-L-SH subfragment. Several ORFs identified in SalI-L-SH by sequence analysis were cloned into a selectable mammalian expression vector, pBK-CMV. Only pBK/ORF1 transformed NIH3T3 cells. Furthermore, cells expressing ORF-1 protein produced fibrosarcomas when injected into nude mice, whereas control cells, expressing either no ORF-1 protein or C-terminal truncated (after residue 172) ORF-1 protein, were not tumorigenic. Western blot analysis of proteins extracted from the tumors revealed ORF-1 protein. Additional studies indicated that ORF-1 was expressed in HHV-6-infected human T-cells by 18 h. Co-immunoprecipitation experiments showed that ORF-1 protein bound to tumor suppressor protein p53, and the ORF-1 binding domain on p53 was located between residues 28 and 187 of p53, overlapping with the specific DNA binding domain. Functional studies showed that p53-activated transcription was inhibited in ORF-1, but not in truncated ORF-1, expressing cells. Importantly, the truncated ORF-1 mutant also failed to cause transformation. Analysis of several human tumors by PCR revealed ORF-1 DNA sequences in some angioimmunoblastic lymphadenopathies, Hodgkins and non-Hodgkins lymphomas and glioblastomas. The detection of ORF-1 sequences in human tumors, while not proof per se, is a prerequisite for establishing its role in tumor development. Taken together, the results demonstrate that ORF-1 is an HHV-6 oncogene that binds to and affects p53. The identification of both transforming and transactivating activities within ORF-1 is a characteristic of other viral oncogenes and is the first reported for HHV-6.

A systematic approach to biomarker discovery; Preamble to "the iSBTc-FDA taskforce on immunotherapy biomarkers"

The International Society for the Biological Therapy of Cancer (iSBTc) has initiated in collaboration with the United States Food and Drug Administration (FDA) a programmatic look at innovative avenues for the identification of relevant parameters to assist clinical and basic scientists who study the natural course of host/tumor interactions or their response to immune manipulation. The task force has two primary goals: 1) identify best practices of standardized and validated immune monitoring procedures and assays to promote inter-trial comparisons and 2) develop strategies for the identification of novel biomarkers that may enhance our understating of principles governing human cancer immune biology and, consequently, implement their clinical application. Two working groups were created that will report the developed best practices at an NCI/FDA/iSBTc sponsored workshop tied to the annual meeting of the iSBTc to be held in Washington DC in the Fall of 2009. This foreword provides an overview of the task force and invites feedback from readers that might be incorporated in the discussions and in the final document.