MYB mediates downregulation of the colorectal cancer metastasis suppressor heterogeneous nuclear ribonucleoprotein L-like during epithelial-mesenchymal transition.

Heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial-mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from -273 to -10 base pairs upstream of the transcription start site identified by 5'-RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was reduced on EMT while that of SP1 and SP3 was unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB-downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression.

Selectin-Binding Assay by Flow Cytometry.

Cell-cell interactions mediated by selectins and their ligand glycans play pivotal roles in a variety of biological processes represented by leukocyte recruitment to inflammatory sites, lymphocyte homing, and extravasation of cancer cells. The interactions are enhanced at least partly through the upregulation of the selectin-ligand glycan expression, which is observed, for instance, during the activation of leukocytes or epithelial-mesenchymal transition of cancer cells. Selectin-binding assays such as cell adhesion assay or rolling assay have long been used to directly evaluate the activity of these cells in the selectin-mediated processes. In this chapter, we introduce a highly quantitative assay by flow cytometry using recombinant selectin-Ig(Fc) chimera proteins, showing our procedure and tips for E-selectin-binding assay of colon cancer cells undergoing epithelial-mesenchymal transition.

Clinical and in vitro studies of the correlation between MGMT and the effect of streptozocin in pancreatic NET.

PURPOSE: This study aimed to determine the correlation between DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) status and the response to streptozocin in advanced well-differentiated pancreatic neuroendocrine tumors (WD panNETs). METHODS: To test the hypothesis that MGMT deficiency was required for an alkylating drug response, we retrospectively reviewed the response of 13 patients with WD panNETs to alkylating agents in relation to MGMT status. We also studied MGMT expression in streptozocin resistance using panNET cell lines. RESULTS: The cohort included 54% of patients with and 46% without MGMT expression. Among these, 83.3% (5/6) of MGMT-negative cases showed a partial response to streptozocin. In contrast, only 14.2% (1/7) of MGMT-positive cases showed a partial response (P = 0.013). Induced expression of MGMT in BON1 cells (a panNET cell line with undetectable endogenous MGMT) produced streptozocin resistance. Knockdown of MGMT in QGP1 cells, which express MGMT endogenously, did not alter the response to streptozocin. CONCLUSIONS: We observed a relationship between MGMT status and streptozocin response in both patients and cell culture. Despite limited cases examined, high concordance of negative expression of MGMT and response to streptozocin treatment suggest that MGMT expression can be a potential biomarker for this treatment.

Gangliosides and Tumors.

Tumor-associated gangliosides play important roles in regulation of signal transduction induced by growth-factor receptors including EGFR, FGFR, HGF and PDGFR in a specific microdomain called glycosynapse in the cancer cell membranes, and in interaction with glycan recognition molecules involved in cell adhesion and immune regulation including selectins and siglecs. As the genes involved in the synthesis and degradation of tumor-associated gangliosides were identified, biological functions became clearer from the experimental results employing forced overexpression and/or knockdown/knockout of the genes. Studies on the regulatory mechanisms for their expression also achieved great advancements. Epigenetic silencing of glycan-related genes is a dominant mechanism in glycan alteration at early stages of carcinogenesis. Development of hypoxia resistance involving activation of a transcription factor HIF, and acquisition of cancer stem cell-like characteristics through epithelial-mesenchymal transition are important mechanisms for glycan modulations in the later stages of cancer progression. In the initial stages of studies, the gangliosides which specifically appear in cancers attracted attention under the name of tumor-associated gangliosides. However, it became apparent that not only the cancer-associated gangliosides but also the normal gangliosides present in nonmalignant cells and tissues perform important biological functions, and some of them tend to disappear in cancer cells resulting in the loss of the physiological functions, and this sometimes facilitates progression of cancers.

HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells.

Heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL), an RNA-binding protein that regulates alternative splicing of pre-mRNA, has been shown to regulate differentiation of lymphocytes, as well as metastasis of colorectal cancer cells. Here, we show that HNRNPLL promotes cell cycle progression and, hence, proliferation of colorectal cancer cells. Functional annotation analysis of those genes whose expression levels were changed threefold or more in RNA sequencing analysis between SW480 cells overexpressing HNRNPLL and those knocked down for HNRNPLL revealed enrichment of DNA replication-related genes by HNRNPLL overexpression. Among 13 genes detected in the DNA replication pathway, PCNA, RFC3 and FEN1 showed reproducible upregulation by HNRNPLL overexpression both at mRNA and at protein levels in SW480 and HT29 cells. Importantly, knockdown of any of these genes alone suppressed the proliferation-promoting effect induced by HNRNPLL overexpression. RNA-immunoprecipitation assay presented a binding of FLAG-tagged HNRNPLL to mRNA of these genes, and HNRNPLL overexpression significantly suppressed the downregulation of these genes during 12 h of actinomycin D treatment, suggesting a role of HNRNPLL in mRNA stability. Finally, analysis of a public RNA sequencing dataset of clinical samples suggested a link between overexpression of HNRNPLL and that of PCNA, RFC3 and FEN1. This link was further supported by immunohistochemistry of colorectal cancer clinical samples, whereas expression of CDKN1A, which is known to inhibit the cooperative function of PCNA, RFC3 and FEN1, was negatively associated with HNRNPLL expression. These results indicate that HNRNPLL stabilizes mRNA encoding regulators of DNA replication and promotes colorectal cancer cell proliferation.

HNRNPLL, a newly identified colorectal cancer metastasis suppressor, modulates alternative splicing of CD44 during epithelial-mesenchymal transition.

OBJECTIVE: Despite the recent advances in treatment of colon cancer, the prognosis is unfavourable for patients with distant metastases. The aim of this study was to identify targets for prevention and/or therapy of colon cancer metastasis. DESIGN: CMT93 cells, a murine rectal cancer cell line with poor metastasising activity, were transduced with lentiviral shRNA library and transplanted into the rectum of syngeneic C57BL/6 mice. Genomic DNA was collected from metastatic lesions, and the integrated shRNA were retrieved by PCR for sequencing, followed by identification of the candidate genes targeted by the shRNA. RESULTS: The genome-wide shRNA library screen identified Hnrnpll (heterogeneous nuclear ribonucleoprotein L-like) encoding a pre-mRNA splicing factor as a candidate metastasis suppressor gene. Knockdown of Hnrnpll enhanced matrigel invasion activity of colon cancer cells in vitro, as well as their metastatic ability in vivo. An RNA-immunoprecipitation analysis showed Hnrnpll-binding to Cd44 pre-mRNAs, and the level of Cd44 variable exon 6 (Cd44v6), a poor prognosis marker of colorectal cancer, was increased by knocking down Hnrnpll. A neutralising Cd44v6 antibody suppressed the matrigel invasion ability induced by Hnrnpll knockdown. HNRNPLL expression was downregulated when colon cancer cells were induced to undergo epithelial-mesenchymal transition (EMT). Immunohistochemistry of clinical samples indicated that colorectal cancer cells with low E-cadherin expression at the invasion front exhibited decreased HNRNPLL expression. CONCLUSIONS: HNRNPLL is a novel metastasis suppressor of colorectal cancer, and modulates alternative splicing of CD44 during EMT.

KSGal6ST generates galactose-6-O-sulfate in high endothelial venules but does not contribute to L-selectin-dependent lymphocyte homing.

The addition of sulfate to glycan structures can regulate their ability to serve as ligands for glycan-binding proteins. Although sulfate groups present on the monosaccharides glucosamine, uronate, N-acetylglucosamine and N-acetylgalactosamine are recognized by defined receptors that mediate important functions, the functional significance of galactose-6-O-sulfate (Gal6S) is not known. However, in vitro studies using synthetic glycans and sulfotransferase overexpression implicate Gal6S as a binding determinant for the lymphocyte homing receptor, L-selectin. Only two sulfotransferases have been shown to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase-1 (C6ST-1). In the present study, we use mice deficient in KSGal6ST and C6ST-1 to test whether Gal6S contributes to ligand recognition by L-selectin in vivo. First, we establish that KSGal6ST is selectively expressed in high endothelial venules (HEVs) in lymph nodes and Peyer's patches. We also determine by mass spectrometry that KSGal6ST generates Gal6S on several classes of O-glycans in peripheral lymph nodes. Furthermore, KSGal6ST, but not C6ST-1, is required for the generation of the Gal6S-containing glycan, 6,6'-disulfo-3'sLN (Siaα2→3[6S]Galß1→4[6S]GlcNAc) or a closely related structure in lymph node HEVs. Nevertheless, L-selectin-dependent short-term homing of lymphocytes is normal in KSGal6ST-deficient mice, indicating that the Gal6S-containing structures we detected do not contribute to L-selectin ligand recognition in this setting. These results refine our understanding of the biological ligands for L-selectin and introduce a mouse model for investigating the functions of Gal6S in other contexts.

Sialic acid cyclization of human Th homing receptor glycan associated with recurrent exacerbations of atopic dermatitis.

BACKGROUND: The molecular pathogenesis underlying recurrent exacerbations of atopic dermatitis (AD) is unclear. Some peripheral CCR4(+) and CCR7(+) helper memory T cells express the specific homing receptor, sialyl 6-sulfo Lewis X (G152 glycan). This glycan loses receptor activity via cyclization of its sialic acid moiety, thus becoming cyclic sialyl 6-sulfo Lewis X (G159 glycan). These findings suggest that the disordered expression of G152 and G159 glycans may be associated with recurrent exacerbations of AD. OBJECTIVE: To assess the possible association of G152 and G159 glycans, which are expressed on peripheral helper T (Th) cells, with frequency of exacerbations. METHODS: The percentage of glycan-expressing cells among peripheral blood CD4(+)CD45RO(+) lymphocytes was determined by flow cytometry. The association of glycans with the frequency of exacerbations determined by recurrence scores as well as with current disease activity was statistically tested. RESULTS: Current disease activity was significantly associated with CCR4(+)CCR7(-) memory Th cells expressing CSLEX-1 glycan, the conventional skin-trafficking receptor without sialic-acid-cyclization activity. In contrast, the frequency of exacerbations was positively and negatively associated with CCR4(+)CCR7(+) memory Th cells expressing G152 and G159 glycans, respectively. Receiver operating characteristics analyses indicated that the ratio of the G152(+)/G159(+) cell percentages discriminated patients with highly recurrent AD with the best accuracy. CONCLUSION: Flow cytometric determination of G159 and G152 glycans on peripheral helper memory T cells may be clinically useful for identifying patients with highly recurrent AD. Disordered sialic acid cyclization of G152 glycan may underlie highly recurrent AD, which may provide a novel therapeutic approach.

Transcription factors c-Myc and CDX2 mediate E-selectin ligand expression in colon cancer cells undergoing EGF/bFGF-induced epithelial-mesenchymal transition.

Sialyl Lewis x (sLe(x)) and sialyl Lewis a (sLe(a)) glycans are expressed on highly metastatic colon cancer cells. They promote extravasation of cancer cells and tumor angiogenesis via interacting with E-selectin on endothelial cells. Recently, epithelial-mesenchymal transition (EMT) has been noted as a critical phenotypic alteration in metastatic cancer cells. To address the association between sLe(x/a) expression and EMT, we assessed whether sLe(x/a) are highly expressed on colon cancer cells undergoing EMT. Treatment of HT29 and DLD-1 cells with EGF and/or basic FGF (bFGF) induced EMT and significantly increased sLe(x/a) expression resulting in enhanced E-selectin binding activity. The transcript levels of the glycosyltransferase genes ST3GAL1/3/4 and FUT3 were significantly elevated and that of FUT2 was significantly suppressed by the treatment. We provide evidence that ST3GAL1/3/4 and FUT3 are transcriptionally up-regulated by c-Myc with probable involvement of Ser62 phosphorylation, and that FUT2 is transcriptionally down-regulated through the attenuation of CDX2. The contribution of c-Myc and CDX2 to the sLe(x/a) induction was proved to be significant by knockdown or forced expression experiments. Interestingly, the cells undergoing EMT exhibited significantly increased VEGF secretion, which can promote tumor angiogenesis in cooperation with sLe(x/a). Finally, immunohistological study indicated high E-selectin ligand expression on cancer cells undergoing EMT in vivo, supporting their coexistence observed in vitro. These results suggest a significant link between sLe(x/a) expression and EMT in colon cancer cells and a pivotal role of c-Myc and CDX2 in regulating sLe(x/a) expression during EMT.

Colonic epithelial cells express specific ligands for mucosal macrophage immunosuppressive receptors siglec-7 and -9.

Immune cells are known to express specific recognition molecules for cell surface glycans. However, mechanisms involved in glycan-mediated cell-cell interactions in mucosal immunity have largely been left unaccounted for. We found that several glycans preferentially expressed in nonmalignant colonic epithelial cells serve as ligands for sialic acid-binding Ig-like lectins (siglecs), the immunosuppressive carbohydrate-recognition receptors carried by immune cells. The siglec ligand glycans in normal colonic epithelial cells included disialyl Lewis(a), which was found to have binding activity to both siglec-7 and -9, and sialyl 6-sulfo Lewis(x), which exhibited significant binding to siglec-7. Expression of these siglec-7/-9 ligands was impaired upon carcinogenesis, and they were replaced by cancer-associated glycans sialyl Lewis(a) and sialyl Lewis(x), which have no siglec ligand activity. When we characterized immune cells expressing siglecs in colonic lamina propriae by flow cytometry and confocal microscopy, the majority of colonic stromal immune cells expressing siglec-7/-9 turned out to be resident macrophages characterized by low expression of CD14/CD89 and high expression of CD68/CD163. A minor subpopulation of CD8(+) T lymphocytes also expressed siglec-7/-9. Siglec-7/-9 ligation suppressed LPS-induced cyclooxygenase-2 expression and PGE(2) production by macrophages. These results suggest that normal glycans of epithelial cells exert a suppressive effect on cyclooxygenase-2 expression by resident macrophages, thus maintaining immunological homeostasis in colonic mucosal membranes. Our results also imply that loss of immunosuppressive glycans by impaired glycosylation during colonic carcinogenesis enhances inflammatory mediator production.

Induction of 6-sulfated glycans with cell adhesion activity via T-bet and GATA-3 in human helper T cells.

BACKGROUND: Cell surface 6-sulfated glycans play important roles in various immunological events through cell-to-cell interactions. The 6-sulfation process is mediated by 6-sulfotransferase family isoenzymes. We previously demonstrated that GlcNAc6ST-1, one of the isoenzyme genes, is induced by GATA-3 and NF-κB in human helper T (Th) cells. However, transcriptional regulation of HEC-GlcNAc6ST, another isoenzyme important in Th cells, remains unclear. METHODS: 5'-RACE analysis, chromatin immunoprecipitation, and reporter assays were performed to reveal transcriptional regulation of HEC-GlcNAc6ST. RNA-knockdown and forced expression experiments were performed to demonstrate the contribution of HEC-GlcNAc6ST to the 6-sulfated glycan expression. RESULTS: We identified potential binding sites of Sp1, T-bet, and GATA-3 in the HEC-GlcNAc6ST promoter. Reporter assays indicated that transfection of Sp1 enhanced the activity, whereas mithramycin A, an Sp1-specific inhibitor, repressed it. Transfection of T-bet increased the activity, which was inhibited by introducing a mutation into the potential T-bet binding site. GATA-3 alone could not elevate the activity, although co-transfection of protein kinase A, which is known to enhance IL-5 transcription in Th2 cells through phosphorylation of GATA-3, caused elevation. RNA-knockdown and forced expression of HEC-GlcNAc6ST in Jurkat cells down- and up-regulated α2,6-sialylated 6-sulfo N-acetyllactosamine, a preferential ligand for B-cell-specific CD22 antigen, respectively. From these results, we concluded that T-bet and GATA-3 as well as Sp1 control the expression of glycan with cell-adhesion activity by regulating HEC-GlcNAc6ST transcription in Th cells. GENERAL SIGNIFICANCE: These results may provide a clue to biological regulation of Th-cell interaction with selectins and other carbohydrate-recognition molecules by T-bet and GATA-3.

Altered expression of glycan genes in cancers induced by epigenetic silencing and tumor hypoxia: clues in the ongoing search for new tumor markers.

The glycan molecules that preferentially appear in cancers are clinically utilized as serum tumor markers. The exact reason, however, why glycans are useful as tumor markers remain elusive. Here, we will summarize lessons learned from well-established cancer-associated glycans, and propose strategies to develop new cancer markers. Our recent results on cancer-associated glycans, sialyl Lewis A and sialyl Lewis X, indicated that the repressed transcription of some glycan genes by epigenetic silencing during early carcinogenesis, and the transcriptional induction of some other glycan genes by tumor hypoxia accompanying cancer progression at locally advanced stages, are two major factors determining cancer-associated glycan expression. Multiple genes are involved in glycan synthesis, and epigenetic silencing of a part of such genes leads to accumulation of glycans having truncated incomplete structures, which are readily detected by specific antibodies. Glycans are very unique and advantageous as marker molecules because they are capable of reflecting epigenetic silencing in their structures. Transcriptional induction of some glycan genes by tumor hypoxia at the later stages produces further glycan modifications, such as an unusual increase of the N-glycolyl sialic acid residues in the glycan molecules. The entire process of malignant transformation thus creates abnormal glycans, whose structures reveal the effects of both epigenetic silencing and tumor hypoxia. The second advantage of a glycan marker over a proteinous marker is that they can reflect the plurality of genetic anomalies in a singular molecule, as it is synthesized by the cooperative action of multiple genes. Glycans are sometimes covalently bound to well-known cancer-associated proteins, such as CD44v, and this eventually contributes to a high cancer specificity and functional relevancy in cancer progression.

[Case of cancer-associated retinopathy in which immediate treatment succeeded].

PURPOSE: Cancer-associated retinopathy (CAR) leads to progressive loss of visual functions and a small number of reports have described effective treatment for the disease. We herein report a case of a CAR patient whose visual acuity improved with corticosteroid and a radical treatment of the tumor. CASE: A 70-year-old man with rapidly progressive loss of visual acuity and visual field was referred to our clinic. The visual acuity of the patient had decreased to finger count OD and 20/30 OS at the initial visit, though visual acuity three weeks before the initial visit was 20/16 in both eyes. The patient suffered from night blindness, Goldmann perimetry revealed ring-like scotoma, and the electroretinogram was negative. Fundus examination showed only attenuation of the vessels but no apparent sign of retinal degeneration. Chest computed tomography (CT) revealed a small cell carcinoma in the lung of 1.2 cm diameter. One week after the initial visit, as the visual acuity in both eyes decreased to hand motion, the patient was treated with chemotherapy, radiation, and corticosteroid pulse therapy. After the treatment, the visual acuity of the patient improved to 20/50 OD and 20/67 OS, but the visual field of the patient did not improve. The tumor in the lung was disappeared on CT. CONCLUSION: Corticosteroid treatment for CAR patients may be effective when performed rapidly after clinical symptoms appear.

Accuracy of epidermal growth factor receptor gene mutation analysis by direct sequencing method based on small biopsy specimens from patients with non-small cell lung cancer: analysis of results in 19 patients.

BACKGROUND: The importance of an epidermal growth factor receptor (EGFR) gene mutation has been recognized in patients with non-small cell lung cancer (NSCLC), and many reports have indicated that the presence of somatic mutations in the EGFR gene is a strong predictor of both clinical and in vitro sensitivity to EGFR tyrosine kinase inhibitors; thus necessitating the standardization of a mutation screening system based on the sources of tissue samples. METHODS: In this study, we compared the results of EGFR mutation analyses in 19 small biopsy specimens with results obtained in surgical materials from the same patients with NSCLC. We used a laser microdissection method and a direct sequencing method, and we confirmed the accuracy of EGFR mutation analysis with small biopsy specimens. RESULTS: The results obtained from the biopsy specimens were identical to those obtained from the surgical materials in 18 of the 19 patients analyzed. For the 1 patient in whom the results obtained from the two sets of materials were not identical, the number of cancer cells in one bronchoscopic specimen was insufficient to perform analyses of all three exons of interest (i.e., exons 18, 19, and 21), and so only exon 19 was sequenced, and no mutation was demonstrated. CONCLUSION: We conclude that satisfactory accuracy can be achieved by the genomic analysis of a small biopsy specimen from a patient with NSCLC and we note that it is possible to conduct prospective clinical trials that include patient assignment for treatment based on the results obtained.

Significance of NF-kappaB/GATA axis in tumor necrosis factor-alpha-induced expression of 6-sulfated cell recognition glycans in human T-lymphocytes.

Sulfated glycans play critical roles in various cell recognition events among leukocytes. The 6-sulfated lactosamine glycans in particular have been widely noted for their importance because they are involved in cell recognition events mediated by cell-adhesion molecules such as selectins and sialic acid-recognizing molecules such as siglecs and also in the activation of CD44 in binding to extracellular matrix hyaluronate. A pro-inflammatory cytokine, tumor necrosis factor-alpha, induces expression of 6-sulfated glycans on human leukocytes. Here we report that the transcription of the GlcNAc6ST-1 gene, the gene encoding a sulfotransferase for 6-sulfated glycan synthesis, is induced in human T-lymphoid cells through tandem NF-kappaB and GATA motifs in its 5'-regulatory region. Results of our reporter assays, immunoprecipitation, and chromatin immunoprecipitation analyses indicated that GATA-3 and/or GATA-2, but not GATA-1, associates with NF-kappaB in a transcription factor complex on the 5'-regulatory region of the gene and acts synergistically with NF-kappaB in triggering GlcNAc6ST-1 transcription. Recently, a skin-homing subset of helper memory T cells exhibiting the Th2 marker CCR4 was shown to specifically express 6-sulfated glycans. The transactivation mechanism described here suggested that GlcNAc6ST-1 transcription is coordinated with the NF-kappaB/GATA-3 axis, which is known to figure heavily in Th2 cell differentiation. In line with this, in vitro differentiation of human T cells to Th2 cells was found to significantly induce GlcNAc6ST-1 transcription and 6-sulfated glycan expression.

Clinical significance of pretreatment serum amphiregulin and transforming growth factor-alpha, and an epidermal growth factor receptor somatic mutation in patients with advanced non-squamous, non-small cell lung cancer.

Circulating amphiregulin and transforming growth factor-alpha (TGF-alpha) have been found to be correlated with an unfavorable response to gefitinib based on the identification of patients with a higher probability of resistance to the drug. However, the association between an epidermal growth factor receptor (EGFR) somatic mutation and the overexpression of its ligands has not been determined. To verify the clinical significance of the two serum markers and EGFR mutation status, we determined serum amphiregulin and TGF-alpha levels by enzyme-linked immunosorbent assay in 93 patients with advanced non-squamous, non-small cell lung cancer and EGFR somatic mutation status using the peptic nucleic acid-locked nucleic acid clamp method in 46 cases. The relationship between each independent clinicopathological variable and the response to gefitinib therapy was examined. We also evaluated the risk factors associated with prognosis. Fourteen (41.0%) of 34 progressive disease cases were positive for amphiregulin (P = 0.007). Eleven (32.4%) of 34 progressive disease cases were positive for TGF-alpha (P = 0.005). The median survival time of patients with the EGFR somatic mutation was significantly longer (P = 0.01). The same was true of amphiregulin- (P = 0.046) and TGF-alpha-negative patients (P < 0.01). In multivariate analysis, serum TGF-alpha positivity (hazard ratio, 2.558; P = 0.005) and the wild type EGFR gene (hazard ratio, 1.894; P = 0.003) were significant independent prognostic factors. Our study demonstrates that the status of the serum EGFR ligand, in addition to EGFR activating mutation, is a predictive factor for response to gefitinib therapy.

Elevation of serum thioredoxin in patients with gefitinib-induced interstitial lung disease.

OBJECTIVE: Interstitial lung disease (ILD) is a severe adverse event of gefitinib therapy. However, the mechanism still remains unclear. The objective of this study was to examine whether or not oxidative stress, one of the common factors in drug-associated ILD, is involved in the pathogenesis of gefitinib-induced ILD. PATIENTS AND METHODS: Using an enzyme-linked immunosorbent assay (ELISA), we measured the concentration of serum thioredoxin (Trx), a redox-active protein with antioxidative effects, in 44 patients treated with gefitinib, including three patients who had ILD. RESULTS: In patients who had gefitinib-induced ILD, serum Trx levels were significantly elevated. They decreased after cessation of gefitinib therapy accompanying clinical improvement of ILD. CONCLUSION: It was suggested that oxidative stress may be involved as a part of mechanisms causing or worsening gefitinib-induced ILD.