Treatment outcome of atypical EGFR mutations in the German National Network Genomic Medicine Lung Cancer (nNGM).

BACKGROUND: Atypical EGFR mutations occur in 10%-30% of non-small-cell lung cancer (NSCLC) patients with EGFR mutations and their sensitivity to classical epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI) is highly heterogeneous. Patients harboring one group of uncommon, recurrent EGFR mutations (G719X, S768I, L861Q) respond to EGFR-TKI. Exon 20 insertions are mostly insensitive to EGFR-TKI but display sensitivity to exon 20 inhibitors. Clinical outcome data of patients with very rare point and compound mutations upon systemic treatments are still sparse to date. PATIENTS AND METHODS: In this retrospective, multicenter study of the national Network Genomic Medicine (nNGM) in Germany, 856 NSCLC cases with atypical EGFR mutations including co-occurring mutations were reported from 12 centers. Clinical follow-up data after treatment with different EGFR-TKIs, chemotherapy and immune checkpoint inhibitors were available from 260 patients. Response to treatment was analyzed in three major groups: (i) uncommon mutations (G719X, S7681, L861Q and combinations), (ii) exon 20 insertions and (iii) very rare EGFR mutations (very rare single point mutations, compound mutations, exon 18 deletions, exon 19 insertions). RESULTS: Our study comprises the largest thus far reported real-world cohort of very rare EGFR single point and compound mutations treated with different systemic treatments. We validated higher efficacy of EGFR-TKI in comparison to chemotherapy in group 1 (uncommon), while most exon 20 insertions (group 2) were not EGFR-TKI responsive. In addition, we found TKI sensitivity of very rare point mutations (group 3) and of complex EGFR mutations containing exon 19 deletions or L858R mutations independent of the combination partner. Notably, treatment responses in group 3 (very rare) were highly heterogeneous. Co-occurring TP53 mutations exerted a non-significant trend for a detrimental effect on outcome in EGFR-TKI-treated patients in groups 2 and 3 but not in group 1. CONCLUSIONS: Based on our findings, we propose a novel nNGM classification of atypical EGFR mutations.

Natural IgM antibodies: from parias to parvenus.

Over the years, natural IgM antibodies were considered as the parias among the immune competent molecules. Their characteristic properties, like low affinity, cross-reactivity and pentameric structure, were assessed as difficult and nebulous. Today, mainly based on the persistent work of a few researchers and the key discoveries on innate immunity, natural IgM antibodies are "back on stage". Their important role in the immune response against invasive particles, modified self-components and altered cells is accepted. All the so far negatively judged features have to be seen in a different light, e.g. low affinity seems to be good for function and does not exclude specificity, cross-reactivity is no longer judged as unspecific, but instead as a very economic way of immune recognition and the pentameric structure is important for binding capacity and functional activities. In addition, with the use of natural IgM antibodies, a new field of tumour-specific targets has been encountered, the carbo-neo-epitopes, which are commonly found on post-transscriptionally modified membrane receptors. Having understood the typical features of natural IgM antibodies, their renaissance opens a new area of cancer therapeutics and diagnostics.

The "early birds": natural IgM antibodies and immune surveillance.

Precancerous epithelial lesions are sites of uncontrolled cellular proliferation generated by irreversible genetic alterations. Not all of those lesions progress to invasive cancer, some may even regress, but the early detection of abnormal cells can be crucial for patient survival. Immune surveillance mechanisms are responsible for the removal of transformed cells and antibodies play an important role in these immune processes. In the past, analysis of the immunoglobuline repertoire has focused mainly on xenoimmunizations or the investigation of cancer patient immunity. The human hybridoma technology (Trioma technique) offers the unique possibility to study the humoral immunity of healthy people. Using this technique a series of tumor-binding antibodies could be isolated which all have several features in common: they are germ-line coded IgM antibodies, they predominantly bind to carbohydrates on post-transcriptionally modified antigens, they induce apoptosis and, most importantly, they detect not only malignant cells but also precursor stages. These data demonstrate that the body has a comprehensive defense system against malignant cells based on the production of natural antibodies.

Death by stress: natural IgM-induced apoptosis.

Stress kills and hence should be avoided. On the other hand, stress induction can be used to remove malignant cells by inducing cellular suicide. Natural IgM antibodies act as first-line defense in immune surveillance. These antibodies selectively kill aberrant cells by using different apoptotic stress mechanisms. They can be isolated from patients but also from healthy donors by using the human hybridoma technology. They are components of the innate immunity, and, on the basis of specific screening methods, should also be detectable in any other individual. The three tumor-specific, apoptosis-inducing natural IgM antibodies described in this review are good examples for stress-induced apoptosis and nature's resourceful ways to fight malignant growth.

The chemokine receptor CCR7 is expressed on epithelium of non-inflamed gastric mucosa, Helicobacter pylori gastritis, gastric carcinoma and its precursor lesions and up-regulated by H. pylori.

CCR7 chemokine-receptor expression on tumour cells of gastric carcinoma has been associated with lymph-node metastasis and is thought to play an important role in metastasis. However, so far it is unknown whether CCR7 is newly up-regulated on gastric carcinoma or already expressed in non-neoplastic gastric epithelium. Therefore, epithelial CCR7 expression was investigated in the process of gastric carcinogenesis: non-inflamed mucosa --Helicobacter pylori gastritis -- intestinal metaplasia/dysplasia -- gastric carcinoma. CCR7 was expressed by gastric epithelium in non-inflamed gastric mucosa (n = 5), H. pylori gastritis (n = 17), intestinal metaplasia (n = 10), dysplasia (n = 3) and on tumour cells in 20 of 24 patients with gastric carcinoma (13/14 intestinal-type; 7/10 diffuse-type) as tested by immunohistochemistry. As CCR7 expression by gastric epithelium was significantly stronger in H. pylori gastritis than in non-infected mucosa, the influence of H. pylori on CCR7 receptor expression of gastric epithelial cells was investigated by fluorescence activated cell sorter analysis. H. pylori strains up-regulated the CCR7 chemokine-receptor in CCR7-positive cell lines. No difference in CCR7 up-regulation between cag(+) and cag(-)H. pylori strains was found. Epithelial CCR7 up-regulation by H. pylori may alter the metastatic fate of gastric carcinoma. Additionally, CCR7 expression not only on gastric carcinoma, but also on non-neoplastic gastric epithelium, suggests a novel biological function.

Natural IgM antibodies, the ignored weapons in tumour immunity.

During its lifetime each multi-cellular organism is permanently exposed to infectious agents and transformed cells. Without an early recognition and a rapid elimination system, there would be no development and no life. The innate or natural immunity, seems to be more important for the detection of "foreign" cells and particles than has been thought. Even if not every transformed cell has the ability and potency for malignant behaviour, the important question is not, why malignant cells arise, but instead, why malignancy occurs so infrequently. We have shown in a recent paper, by using the human hybridoma technology, that tumour immunity is not induced by malignant cells, but instead the result of innate immunity and that natural IgM antibodies play an important role in immunosurveillance mechanisms against transformed cells in humans (Brändlein et al., 2003b). In this review typical features of natural IgM antibodies are discussed and tumour-specific reactivities and different apoptotic functions on epithelial cancer cells are illustrated.

Pleiotropic effects of CXC chemokines in gastric carcinoma: differences in CXCL8 and CXCL1 expression between diffuse and intestinal types of gastric carcinoma.

CXC chemokines modulate host immunity, neovascularization, growth and invasive behaviour of tumours. Despite their relevance in tumour biology, chemokine expression in intestinal- and diffuse-type gastric carcinoma, which exhibit a completely different growth pattern, has not been investigated in detail. In this study, expression of the CXC chemokines CXCL8 [interleukin (IL)-8], CXCL1 [growth-related oncogene alpha (Gro alpha)], CXCL9 [monokine induced by interferon (IFN)-gamma] and CXCL10 [IFN-gamma-inducible protein-10 (IP-10)] and the corresponding chemokine receptors CXCR1-3 was investigated by immunohistochemistry in intestinal- and diffuse-type gastric carcinoma. Tumour cells of all patients expressed CXCL8. CXCL8 expression was significantly stronger in tumour cells of diffuse- rather than intestinal-type gastric carcinoma (P < 0.01) as determined by a semiquantitative score. CXCL1 was expressed almost exclusively by diffuse- but not intestinal-type carcinoma cells. The corresponding chemokine receptors, CXCR1 and CXCR2, were found on carcinoma cells. Furthermore, CXCL8 expression correlated with number of tumour vessels (P < 0.01), suggesting an angiogenetic function in gastric carcinoma not only in vitro but also in vivo. CXCL10 and CXCL9, attractants for T cells, were expressed by peritumorous macrophages in close proximity to IFN-gamma-producing CXCR3-positive T cells in both tumour types. These chemokines may attract gastric carcinoma-infiltrating T cells via an IFN-gamma-mediated pathway and enhance host immunity against the tumour. In gastric carcinoma a complex interplay between CXC-chemokine signals derived from both tumour cells and tumour-infiltrating immune cells may exhibit pleiotropic effects in tumour biology that go far beyond their originally described functions as leucocyte chemoattractants. Because CXCL8 and CXCL1, which are known to increase growth and invasive behaviour of malignant tumours, are significantly stronger expressed in diffuse- than intestinal-type gastric carcinoma, one may speculate that these chemokines influence the different growth pattern of gastric carcinoma types.

IgVH-genes analysis from psoriatic arthritis shows involvement of antigen-activated synovial B-lymphocytes.

The pathogenesis of psoriatic arthritis (PA), which occurs in 5-7% of patients with psoriasis vulgaris, is enigmatic. There are no molecular data about synovial B-cells of chronic synovitis of PA. In order to understand the B-cell response in PA, we analysed IgVH genes and specified replacement to silent mutation (R/S) ratios of the complementarity determining regions (CDR) and framework regions (FR) as well as VH families. To prove the existence of a common pattern we additionally analysed the IgVH genes at the amino-acid level. From 5 PA patients we took cryo-tissue sections with somatically mutated IgVH genes (sum R/sum S in the CDRs: 2.5-6.8), 62 of which were analysed. In one patient two cases of clonally related IgVH genes were observed. Identical amino-acid replacements in IgVH1 and IgVH4 were found at the same mutational "cold spot". These data indicate that antigen-activated B-cells participate in the formation of chronic synovitis of PA. Since, neither histopathologically nor immunohistochemically, no germinal centres could be detected, the clonally related IgVH genes may be interpreted as residues of a germinal centre reaction that occurred before synovectomy. The existence of identical amino-acid replacement mutations in IgVH1 and IgVH4 genes suggests that a limited number of antigens are common to all PA patients analysed. The recombinant expression of the IgVH could help to define B-cell specificities underlying the pathogenesis of chronic synovitis and dermatitis of PA.

Regulation of the new coexpressed CD55 (decay-accelerating factor) receptor on stomach carcinoma cells involved in antibody SC-1-induced apoptosis.

The human monoclonal antibody SC-1 was isolated from a patient with a diffuse-type adenocarcinoma of the stomach using somatic cell hybridization. The immunoglobulin (Ig)M antibody reacts specifically with diffuse- (70%) and intestinal-type (25%) gastric adenocarcinoma and induces apoptosis in vitro and in vivo. When used in clinical trials with stomach carcinoma patients, significant apoptotic and regressive effects in primary tumors have been observed with the antibody SC-1. The SC-1 receptor is a new 82 kd membrane-bound isoform of glycosylphosphatidylinositol (GPI)-linked CD55 (decay-accelerating factor, DAF). CD55 is known to protect cells from lysis through autologous complement and is coexpressed with the ubiquitously distributed 70 kd isoform. The SC-1-specific CD55 isoform is up-regulated shortly after antibody binding, followed by an internalization of the antibody/receptor-complex, whereas the membranous expression of wild-type CD55 remains unchanged. The apoptotic process is marked by cleavage of cytokeratin 18, indicating the involvement of caspase-6 in the apoptotic process. In contrast to other apoptotic pathways, a cleavage of poly(ADP-ribose)polymerase (PARP) is not observed. The expression of the cell-cycle regulator c-myc becomes up-regulated, whereas expression of topoisomerase IIalpha is down-regulated. Induction of apoptosis leads to an increase in the internal Ca(2+) concentration, which is not necessary for the apoptotic process but for the transport of newly synthesized SC-1-specific CD55 isoform to the membrane.

Deactivation of regulatory proteins hnRNP A1 and A2 during SC-1 induced apoptosis.

Phosphorylation and activation of caspases play an important role in the induction of apoptosis. During tumor specific apoptosis, induced by the human monoclonal antibody SC-1, tyrosine phosphorylation and serine dephosphorylation of several proteins is observed. In this paper we describe the identification of two dephosphorylated proteins as heterogeneous nuclear ribonucleoproteins A1 and A2 (hnRNP A1, hnRNP A2). The dephosphorylation of these proteins is important for apoptosis since the amount of apoptotic cell death can be decreased by the specific serine/threonine phosphatase inhibitor okadaic acid. We also investigated the effect of serine kinase inhibitor H7 on SC-1 induced apoptosis, which leads to a dose dependent increase in apoptosis. We could also show that 24 hours after the induction of apoptosis the hnRNP A1 protein is cleaved into different cleavage products. Further, we found a decreased expression of caspase-2 in early apoptosis signalling and an overexpression 24 hours after induction of apoptosis. Our results show that the phosphorylation status of the hnRNP A1 and A2 plays a significant role in early SC-1 induced apoptosis signalling and further indicate the role of caspase activation during the apoptotic process.

A novel proliferation-associated variant of CFR-1 defined by a human monoclonal antibody.

The germline coded human monoclonal IgM antibody 103/51 was isolated from a gastric carcinoma patient. This antibody binds to a 130-kd membrane molecule and has a mitotic effect on tumor cells in vitro. To characterize the target, we sequenced the protein and showed that the antibody binds to the cysteine-rich fibroblast growth factor receptor (CFR)-1, which is highly homologous to MG-160 and the E-selectin-ligand (ESL)-1. The epitope was determined by glycosidase-digestion experiments to be an N-linked carbohydrate side chain. Immunohistochemistry was used to investigate the tissue distribution of CFR-1. Different healthy tissues were tested and only the collecting tubes of the kidney, the Golgi apparatus, and the glomerular and fascicular zones of the adrenal gland stained positive. However, on malignant tissue the receptor is overexpressed in nearly all tested stomach cancers (12 of 15) and other tested carcinomas (13 of 15). Most interestingly, the receptor is also present in Helicobacter pylori gastritis and gastric dysplasia, but absent on uninflamed stomach mucosa. This restricted tissue pattern indicates that antibody 103/51 reacts with a membrane-bound variant of CFR-1, which is mainly expressed on transformed cells and precursor lesions and is essential for proliferation processes. The possible activity of antibody 103/51 as an activating ligand in these proliferative changes of gastric epithelial mucosa is discussed.

The two subunits of the interleukin-4 receptor mediate independent and distinct patterns of ligand endocytosis.

Interleukin-4 (IL-4) triggers cellular responses by interaction with the bipartite interleukin-4 receptor (IL-4R). IL-4-responsive cells specifically endocytose IL-4. We studied the ligand internalization properties of the human IL-4R and analyzed the specific functions of its two subunits IL-4Ralpha and gammac in this process. IL-4 mutant RY, which binds to IL-4Ralpha but does not recruit gammac into the receptor complex was used as a tool to show that IL-4Ralpha can promote independent ligand uptake in human T cells. Internalization was limited, however, by rapid IL-4 dissociation, suggesting that one important function of gammac in IL-4 endocytosis is to retain the ligand sufficiently long within the ternary receptor complex. We then measured IL-4 internalization by murine Ba/F3 cells that were stably transfected with various human IL-4R constructs. Efficient IL-4 uptake required the cytoplasmic section of the receptor. The intracellular domains of IL-4Ralpha and gammac were responsible for independent endocytosis processes with distinct kinetics. IL-4Ralpha-mediated internalization resulted in long-term intracellular maintainance of IL-4, whereas gammac directed the associated radioligand to intracellular breakdown and rapid release in the form of degraded protein. Mutants of either IL-4R subunit deficient in Janus kinase activation were not impaired in internalization, indicating that IL-4 endocytosis is not functionally connected to signal transduction.

Activation of STAT5 by IL-4 relies on Janus kinase function but not on receptor tyrosine phosphorylation, and can contribute to both cell proliferation and gene regulation.

We have investigated mechanisms and consequences of STAT5 activation through the human IL-4 receptor (IL-4R). By functionally expressing receptor mutants in the murine pro-B cell line Ba/F3, we could show that phosphorylated tyrosine residues within the IL-4R alpha chain are dispensable for IL-4-induced STAT5 activity. However, disruption of a membrane-proximal proline-rich sequence motif ('box1') in either subunit of the bipartite IL-4R abolished not only ligand-induced tyrosine phosphorylation of Janus kinases JAK1 and JAK3, but also IL-4-triggered activation of STAT5 and concomitant cell proliferation. A dominant-negative version of STAT5b, but not of STAT5a, interfered with IL-4-induced DNA synthesis in Ba/F3 cells, suggesting an involvement of STAT5b in the control of cell proliferation through IL-4R. Reporter gene experiments finally showed that transcription from promoters of STAT5 target genes can be specifically induced by challenging cells with IL-4, and that both STAT5a and STAT5b can contribute to IL-4-triggered transcriptional control.

The interleukin-4 receptor activates STAT5 by a mechanism that relies upon common gamma-chain.

Interleukin (IL)-4 signaling proceeds via cytoplasmic activation of the Janus kinases JAK1 and JAK3 and the signal transducer and activator of transcription STAT6. We show that the IL-4 receptor, like other cytokine receptor systems utilizing the common receptor gamma-chain (gammac), is also connected to a signaling pathway that involves STAT5. Both STAT5a and STAT5b become tyrosine-phosphorylated and acquire specific DNA-binding properties in response to IL-4 receptor stimulation in the murine pro-B cell line Ba/F3. In preactivated human T cells, STAT5 became activated in an IL-4-dependent fashion as assayed by IL-4-induced STAT5 translocation from the cytoplasm to the cell nucleus and by binding to cognate DNA. Moreover, stimulation of preactivated human T cells by IL-4 led to specific transcriptional up-regulation of STAT5 target genes. IL-4 receptor-mediated STAT5 activation is dependent on the presence of gammac and JAK3 within the receptor complex. In COS-7 cells, the JAK/STAT pathway leading from the IL-4 receptor to STAT5-dependent regulation of a reporter gene relied largely on coexpression of JAK3. In Ba/F3 cells, studies on signal transduction evoked by directed specific receptor homo- or heterodimerization revealed that STAT5 activation can be triggered exclusively by IL-4R heterodimers containing gammac.