Heinz Zwierzina

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).

Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells

Introduction We describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. Methods Automation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and α-smooth muscle actin (α-SMA) was investigated by IHC. Results Lower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of α-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype. Conclusion We demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers.

Effect of inhibitors of Na+/H+‐exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes

Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N‐formyl‐Met‐Leu‐Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling. Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP‐induced cell swelling. Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP. H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP‐induced increase in CV. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP‐stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole. Western analysis with a monoclonal anti gastric H+/K+ATPase β‐subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+‐dependent H+‐extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.

Cloning and genomic characterization of LST1: a new gene in the human TNF region

The leucocyte specific transcript — 1 (LST1) represents the human homolog of the mouse B144 transcript, encoded within the tumor necrosis factor (TNF) region of the human major histocompatibility complex class III interval. The gene is localized about 4 kilobases upstream of the lymphotoxin ß gene. It spans a polymorphic genomic region encompassing the microsatellites TNFd and TNFe in intron 3 and a polymorphic Pvu II restriction site 260 base pairs downstream of the polyadenylation signal. Isolation of a full-length cDNA clone revealed that LST1 codes for IFN-γ-inducible 800 nt transcripts, which are present in lymphoid tissues, T cells, macrophages, and histiocyte cell lines. The cDNA contains three long open reading frames (ORF) with the most likely ORF encoding a transmembrane protein. Its close linkage to the TNF genes and pattern of expression point toward a possible role for LST1 in the immune response.

LST1: A Gene with Extensive Alternative Splicing and Immunomodulatory Function

The gene of the leukocyte-specific transcript (LST1) is encoded within the TNF region of the human MHC. The LST1 gene is constitutively expressed in leukocytes and dendritic cells, and it is characterized by extensive alternative splicing. We identified 7 different LST1 splice variants in PBMC; thus, 14 LST1 splice variants (LST1/A-LST1/N) have been detected in various cell types. These isoforms code for transmembrane as well as soluble LST1 proteins characterized by two alternative open reading frames at their 3′ end. We demonstrate the presence of the transmembrane variant LST1/C on the cell surface of the monocytic cell lines U937 and THP1. Recombinant expression of LST1/C permitted its profound inhibitory effect on lymphocyte proliferation to be observed. In contrast, the alternative transmembrane variant LST1/A, the extracellular domain of which shows no amino acid sequence homology to LST1/C exerted a weaker but similar inhibitory effect on PBMC. These data demonstrate the protein expression of LST1 on the cell surface of mononuclear cells, and they show an inhibitory effect on lymphocyte proliferation of two LST1 proteins although they have only a very short amino acid homology.

Identification of Endosomal Epidermal Growth Factor Receptor Signaling Targets by Functional Organelle Proteomics

Epidermal growth factor (EGF) receptor (EGFR) signal transduction is organized by scaffold and adaptor proteins, which have specific subcellular distribution. On a way from the plasma membrane to the lysosome EGFRs are still in their active state and can signal from distinct subcellular locations. To identify organelle-specific targets of EGF receptor signaling on endosomes a combination of subcellular fractionation, two-dimensional DIGE, fluorescence labeling of phosphoproteins, and MALDI-TOF/TOF mass spectrometry was applied. All together 23 EGF-regulated (phospho)proteins were identified as being differentially associated with endosomal fractions by functional organelle proteomics; among them were proteins known to be involved in endosomal trafficking and cytoskeleton rearrangement (Alix, myosin-9, myosin regulatory light chain, Trap1, moesin, cytokeratin 8, septins 2 and 11, and CapZβ). Interestingly R-Ras, a small GTPase of the Ras family that regulates cell survival and integrin activity, was associated with endosomes in a ligand-dependent manner. EGF-dependent association of R-Ras with late endosomes was confirmed by confocal laser scanning immunofluorescence microscopy and Western blotting of endosomal fractions. EGFR tyrosine kinase inhibitor gefitinib was used to confirm EGF-dependent regulation of all identified proteins. EGF-dependent association of signaling molecules, such as R-Ras, with late endosomes suggests signaling specification through intracellular organelles.

Improving the efficacy of cancer immunotherapy

A series of cancer vaccines have been evaluated in clinical trials with encouraging results, but the demonstration of clinical benefit in confirmatory studies has so far proven to be difficult. The development of cancer vaccines is hampered by a range of issues particular to this field of research. On 12th March 2008, the Biotherapy Development Association convened a workshop to discuss issues faced by scientists and clinicians involved in the development of cancer vaccines. This paper is a review of the field, based on discussions held at the BDA workshop, and describes biological barriers encountered in generating effective immune responses to tumours, methodological obstacles encountered in the improvement of immunological monitoring which aims to improve inter-laboratory and inter-trial comparisons, challenges in clinical trial design and problems posed by the lack of specific regulation for cancer vaccines and the impact on their development. Ultimately, a number of general solutions are posed: (1) better patient selection, (2) use of multi-modal treatments that affect several aspects of the immune system at once, (3) a requirement for the development of good biomarkers to stratify patients for selection prior to trial and as surrogates for clinical response and (4) harmonisation of SOPs for immunological monitoring of clinical trials.

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.

EGFR inhibition as a therapy for head and neck squamous cell carcinoma

Background: Improved understanding of disease biology of head and neck squamous cell carcinoma (HNSCC) with nearly universal expression of EGFR has led to the introduction of targeted therapies to interrupt signalling of this negative prognostic marker. Objective: We performed a literature review on the mechanisms and efficacy of anti-EGFR antibodies and EGFR tyrosine kinase inhibitors in patients with locally advanced or recurrent/metastatic HNSCC. Results/conclusion: Clinical trials in HNSCC have administered EGFR directed drugs as single agents, in combination with chemotherapy or radiotherapy and demonstrated a good safety profile with antitumour activity in a subgroup of patients. The biology of responsiveness is still unclear, although there is growing evidence of an association of skin toxicity or presence of shorter EGFR intron 1 cytosine–adenine repeats with positive outcome.

The preclinical and clinical activity of aviscumine: A potential anticancer drug

Extracts from the European mistletoe plant Viscumalbum have been studied for decades for their direct and indirect anticancer activity. Therefore, scientists were interested in identifying the active compound (mistletoe lectin) in these extracts and making it available as a highly purified molecule for drug development. Recombinant mistletoe lectin (INN: aviscumine) was produced in Escherichiacoli. It has been shown to have immunomodulatory and cytotoxic activity in invitro and in animal models and can target tumour cells. Clinical phase I studies also demonstrated immunomodulatory activity, which appears to have a positive effect on disease stabilisation. This review explores the current knowledge base for aviscumines mechanism of action, efficacy and side-effects in both preclinical studies and clinical trials, and it considers aviscumines potential as a cancer therapy.