Cetuximab, irinotecan and fluorouracile in fiRst-line treatment of immunologically-selected advanced colorectal cancer patients: the CIFRA study protocol

Abstract Background Combination of chemotherapies (fluoropirimidines, oxaliplatin and irinotecan) with biologic drugs (bevacizumab, panitumumab, cetuximab) have improved clinical responses and survival of metastatic colorectal cancer (mCRC). However, patients’ selection thorough the identification of predictive factors still represent a challange. Cetuximab (Erbitux®), a chimeric monoclonal antibody binding to the Epidermal Growth Factor Receptor (EGFR), belongs to the Immunoglobulins (Ig) grade 1 subclass able to elicite both in vitro and in vivo the Antibody-Dependent Cell-mediated Cytotoxicity (ADCC). ADCC is the cytotoxic killing of antibody-coated target cells by immunologic effectors. The effector cells express a receptor for the Fc portion of these antibodies (FcγR); genetic polymorphisms of FcγR modify the binding affinity with the Fc of IgG1. Interestingly, the high-affinity FcγRIIIa V/V is associated with increased ADCC in vitro and in vivo. Thus, ADCC could partially account for cetuximab activity. Methods/design CIFRA is a single arm, open-label, phase II study assessing the activity of cetuximab in combination with irinotecan and fluorouracile in FcγRIIIa V/V patients with KRAS, NRAS, BRAF wild type mCRC. The study is designed with a two-stage Simon model based on a hypothetical higher response rate (+ 10%) of FcγRIIIa V/V patients as compared to previous trials (about 60%) assuming ADCC as one of the possible mechanisms of cetuximab action. The test power is 95%, the alpha value of the I-type error is 5%. With these assumptions the sample for passing the first stage is 14 patients with > 6 responses and the final sample is 34 patients with > 18 responses to draw positive conclusions. Secondary objectives include toxicity, responses’ duration, progression-free and overall survival. Furthermore, an associated translational study will assess the patients’ cetuximab-mediated ADCC and characterize the tumor microenvironment. Discussion The CIFRA study will determine whether ADCC contributes to cetuximab activity in mCRC patients selected on an innovative immunological screening. Data from the translational study will support results’ interpretation as well as provide new insights in host-tumor interactions and cetuximab activity. Trial registration The CIFRA trial (version 0.0, June 21, 2018) has been registered into the NIH-US National Library of Medicine, ClinicalTrials.gov database with the identifier number (NCT03874062).

Download Full-text

Chemosensitivity of Patient-Derived Cancer Stem Cells Identifies Colorectal Cancer Patients with Potential Benefit from FGFR Inhibitor Therapy

Cancers ◽

10.3390/cancers12082010 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2010

Author(s):

Takehito Yamamoto ◽

Hiroyuki Miyoshi ◽

Fumihiko Kakizaki ◽

Hisatsugu Maekawa ◽

Tadayoshi Yamaura ◽

...

Keyword(s):

Colorectal Cancer ◽

Stem Cells ◽

Cancer Patients ◽

Growth Factor Receptor ◽

Genetic Alterations ◽

Inhibitor Therapy ◽

Epithelial Stem Cells ◽

Colorectal Cancer Patients

Some colorectal cancer patients harboring FGFR (fibroblast growth factor receptor) genetic alterations, such as copy number gain, mutation, and/or mRNA overexpression, were selected for enrollment in several recent clinical trials of FGFR inhibitor, because these genetic alterations were preclinically reported to be associated with FGFR inhibitor sensitivity as well as poor prognosis, invasiveness, and/or metastatic potential. However, few enrolled patients were responsive to FGFR inhibitors. Thus, practical strategies are eagerly awaited that can stratify patients for the subset that potentially responds to FGFR inhibitor chemotherapy. In the present study, we evaluated the sensitivity to FGFR inhibitor erdafitinib on 25 patient-derived tumor-initiating cell (TIC) spheroid lines carrying wild-type RAS and RAF genes, both in vitro and in vivo. Then, we assessed possible correlations between the sensitivity and the genetic/genomic data of the spheroid lines tested. Upon their exposure to erdafitinib, seven lines (7/25, 28%) responded significantly. Normal colonic epithelial stem cells were unaffected by the inhibitors. Moreover, the combination of erdafitinib with EGFR inhibitor erlotinib showed stronger growth inhibition than either drug alone, as efficacy was observed in 21 lines (84%) including 14 (56%) that were insensitive to erdafitinib alone. The in vitro erdafitinib response was accurately reflected on mouse xenografts of TIC spheroid lines. However, we found little correlation between their genetic/genomic alterations of TIC spheroids and the sensitivity to the FGFR inhibitor. Accordingly, we propose that direct testing of the patient-derived spheroids in vitro is one of the most reliable personalized methods in FGFR-inhibitor therapy of colorectal cancer patients.

Download Full-text

The roles and prognostic significance of ABI1-TSV-11 expression in patients with left-sided colorectal cancer

Scientific Reports ◽

10.1038/s41598-021-90220-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yu Zhang ◽

Zhaohui Zhong ◽

Mei Li ◽

Jingyi Chen ◽

Tingru Lin ◽

...

Keyword(s):

Colorectal Cancer ◽

Prognostic Significance ◽

Growth Factor Receptor ◽

The Cancer Genome Atlas ◽

Actin Dynamics ◽

Elevated Expression ◽

Sw480 Cells ◽

And Migration

AbstractAbnormally expressed and/or phosphorylated Abelson interactor 1 (ABI1) participates in the metastasis and progression of colorectal cancer (CRC). ABI1 presents as at least 12 transcript variants (TSVs) by mRNA alternative splicing, but it is unknown which of them is involved in CRC metastasis and prognosis. Here, we firstly identified ABI1-TSV-11 as a key TSV affecting the metastasis and prognosis of left-sided colorectal cancer (LsCC) and its elevated expression is related to lymph node metastasis and shorter overall survival (OS) in LsCC by analyzing data from The Cancer Genome Atlas and TSVdb. Secondly, ABI1-TSV-11 overexpression promoted LoVo and SW480 cells adhesion and migration in vitro, and accelerated LoVo and SW480 cells lung metastasis in vivo. Finally, mechanism investigations revealed that ABI1-isoform-11 interacted with epidermal growth factor receptor pathway substrate 8 (ESP8) and regulated actin dynamics to affect LoVo and SW480 cells biological behaviors. Taken together, our data demonstrated that ABI1-TSV-11 plays an oncogenic role in LsCC, it is an independent risk factor of prognosis and may be a potential molecular marker and therapeutic target in LsCC.

Download Full-text

Necroptosis regulates tumor repopulation after radiotherapy via RIP1/RIP3/MLKL/JNK/IL8 pathway

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-019-1423-5 ◽

2019 ◽

Vol 38 (1) ◽

Cited By ~ 5

Author(s):

Yiwei Wang ◽

Minghui Zhao ◽

Sijia He ◽

Yuntao Luo ◽

Yucui Zhao ◽

...

Keyword(s):

Colorectal Cancer ◽

Growth Stimulation ◽

Underlying Mechanism ◽

Promoting Effect ◽

Clinical Prognosis ◽

Associated Proteins ◽

Radiation Induced ◽

Colorectal Cancer Patients

Abstract Background Tumor cell repopulation after radiotherapy is a major cause for the tumor radioresistance and recurrence. This study aims to investigate the underlying mechanism of tumor repopulation after radiotherapy, with focus on whether and how necroptosis takes part in this process. Methods Necroptosis after irradiation were examined in vitro and in vivo. And the growth-promoting effect of necroptotic cells was investigated by chemical inhibitors or shRNA against necroptosis associated proteins and genes in in vitro and in vivo tumor repopulation models. Downstream relevance factors of necroptosis were identified by western blot and chemiluminescent immunoassays. Finally, the immunohistochemistry staining of identified necroptosis association growth stimulation factor was conducted in human colorectal tumor specimens to verify the relationship with clinical outcome. Results Radiation-induced necroptosis depended on activation of RIP1/RIP3/MLKL pathway, and the evidence in vitro and in vivo demonstrated that the inhibition of necroptosis attenuated growth-stimulating effects of irradiated tumor cells on living tumor reporter cells. The JNK/IL-8 were identified as downstream molecules of pMLKL during necroptosis, and inhibition of JNK, IL-8 or IL-8 receptor significantly reduced tumor repopulation after radiotherapy. Moreover, the high expression of IL-8 was associated with poor clinical prognosis in colorectal cancer patients. Conclusions Necroptosis associated tumor repopulation after radiotherapy depended on activation of RIP1/RIP3/MLKL/JNK/IL-8 pathway. This novel pathway provided new insight into understanding the mechanism of tumor radioresistance and repopulation, and MLKL/JNK/IL-8 could be developed as promising targets for blocking tumor repopulation to enhance the efficacy of colorectal cancer radiotherapy.

Download Full-text

CD30/CD16A Tandab AFM13-Induced Target Cell Lysis By NK-Cells Is Enhanced By CD137 Co-Stimulation and Blocking PD-1

Blood ◽

10.1182/blood.v126.23.2747.2747 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2747-2747 ◽

Cited By ~ 1

Author(s):

Xing Zhao ◽

Narendiran Rajasekaran ◽

Uwe Reusch ◽

Jens-Peter Marschner ◽

Martin Treder ◽

...

Keyword(s):

Tumor Growth ◽

Nk Cells ◽

Target Cell ◽

Tumor Size ◽

Target Cells ◽

In Vivo Models ◽

Effector Cells ◽

Cytotoxicity Assays

Abstract Introduction: AFM13 is a CD30/CD16A bispecific tetravalent TandAb antibody that recruits and activates NK-cells by specific binding to CD16A for targeted lysis of CD30+ tumor cells. Given promising clinical activity and safety profile of AFM13 and proof-of-mechanism demonstrating dependence on the immune response, potential synergy of AFM13 and checkpoint modulators was evaluated. Methods: Efficacy of AFM13 alone or in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies was assessed by in vitro cytotoxicity assays with human PBMCs or enriched NK-cells and CD30+ target cells as well as patient-derived xenograft in vivo models with autologous PBMC. To evaluate NK-cell-mediated lysis of CD30+ lymphoma cell lines, 4 hour cytotoxicity assays were performed with PBMCs or enriched NK-cells as effector cells in the presence of suboptimal concentrations of AFM13 alone, and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies. For the in vivo model tumor fragments derived from surgical specimens of newly diagnosed patients with CD30+ Hodgkin Lymphoma were xenografted (PDX) in immuno-deficient mice. After 28 days mice were reconstituted with autologous patient-derived PBMC and treated with AFM13 alone and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies weekly for a total of three weeks. Tumor size, tumor-infiltrating human lymphocytes and intra-tumoral cytokines were evaluated on day 58. Results: AFM13 as a single agent at suboptimal concentrations induced effector-to-target cell-dependent lysis of CD30+ lymphoma cells up to 40% using enriched NK-cells as effector cells in a 4 hour in vitro assay. Immune-modulating antibodies alone mediated substantially lower lysis (<25%). However, the addition of anti-PD-1 or anti-CD137 to AFM13 strongly enhanced specific lysis up to 70%, whereas the addition of anti-CTLA-4 to AFM13 showed no beneficial effect. The most impressive increase of efficacy was observed when AFM13 was applied together with a combination of anti-PD-1 and anti-CD137. In vivo, reduction of tumor growth was observed when AFM13 and anti-PD-1 were used as single agents or when AFM13 was combined with anti-CD137. Synergy was most impressive in these PDX models for the combination of AFM13 and anti-PD-1 which led to a very strong reduction of tumor size. Of note, reduction of tumor growth was strongly correlated with infiltrating NK- and T-cells and intra-tumoral cytokines. Conclusions: The combination trials performed with companion intra-tumoral assessment of lymphocytes and cytokines may enhance the efficacy of AFM13 in patients. This may be explained by a potential cross-talk between NK-cells and T-cell which was enhanced when AFM13 was used in combination with checkpoint modulators. Disclosures No relevant conflicts of interest to declare.

Download Full-text

MACC1 expression as a candidate prognostic biomarker in colorectal cancer patients receiving adjuvant oxaliplatin-based therapy.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.4_suppl.567 ◽

2019 ◽

Vol 37 (4_suppl) ◽

pp. 567-567 ◽

Cited By ~ 1

Author(s):

Lynn Katherine Symonds ◽

Kelsey K. Baker ◽

Mary Weber Redman ◽

Lisa Koch ◽

Kelly Carter ◽

...

Keyword(s):

Colorectal Cancer ◽

Cox Regression ◽

Prognostic Biomarker ◽

Patient Characteristics ◽

Hazard Ratios ◽

Platinum Based Chemotherapy ◽

Tumor Mutational Burden ◽

Colorectal Cancer Patients

567 Background: MACC1, part of the HGF-MET pathway, drives proliferation and regulation of MET expression in vitro. In vivo, MACC1 is associated with tumor progression and studies suggest greater MACC1 expression is associated with resistance to platinum-based chemotherapy. We hypothesized that MACC1 may be a prognostic biomarker in colorectal cancer (CRC). Methods: MACC1 expression was evaluated by immunohistochemistry on tumor microarrays (N = 428). Patients were stage I-III CRC who received an oxaliplatin-based regimen (either with 5-FU (FOLFOX) or capecitabine (XELOX)) within the HeCOG 6C/08 observational study. MACC1 expression was assessed by a blinded GI pathologist using a scale ranging from 0 (no staining) to 3+ (strong expression). Each patient had at least 3 samples and the strongest result was used for the final score. MACC1 positivity was defined as ≥2+ expression and 0-1+ as MACC1-. Cox regression models were used to estimate hazard ratios (HR) for the association of MACC1 expression with patient characteristics, disease-free (DFS), and overall survival (OS). Results: 400/428 CRC tumors were evaluable: 322 (80.5%) were MACC1+ and 78 (19.5%) MACC1-. Mucinous features were less likely in MACC1+ patients (24% vs. 38%, p = 0.02). Other unfavorable features including grade, lymphovascular invasion, perineural invasion, and tumor mutational burden were not significantly different. There was no difference for stage, microsatellite instability, BRAF, KRAS, or NRASstatus between MACC1+/- cancers. There was a trend towards worse survival in MACC1+ patients regardless of treatment (DFS HR 1.55 [95% CI: 0.87, 2.76], OS HR 1.59 [95% CI 0.74, 3.4]). This difference was not statistically significant for OS (p = 0.26) or DFS (p = 0.08) even when stratified by clinicopathologic variables. Conclusions: Patients with MACC1+ CRC tumors who received adjuvant oxaliplatin-based therapy were less likely to have mucinous histology. They had a trend toward independently worse survival that was not significant when accounting for stage and clinicopathologic variables. Studies focused on the predictive role of MACC1 and oxaliplatin in stage III CRC are in progress.

Download Full-text

In Vitro and In Vivo Characterization of CD19/CD3 Tandab AFM11 and CD19/CD16A Tandab AFM12 Targeting NHL

Blood ◽

10.1182/blood.v126.23.2763.2763 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2763-2763

Author(s):

Xing Zhao ◽

Narendiran Rajasekaran ◽

Uwe Reusch ◽

Michael Weichel ◽

Kristina Ellwanger ◽

...

Keyword(s):

T Cell ◽

Target Cell ◽

Binding Sites ◽

Effector Cell ◽

Nk Cell ◽

Target Cells ◽

Tumor Target ◽

Effector Cells

Abstract Introduction: CD19 is expressed by B cells from early development through differentiation into plasma cells, and represents a validated target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Different CD19-targeting T-cell engagers are investigated in clinical studies for the treatment of NHL or ALL, including Affimed's AFM11, a bispecific CD19/CD3 TandAb antibody, which is currently investigated in a phase 1 dose escalation study. Indeed, Affimed's bispecific tetravalent platform comprises not only T-cell engaging TandAbs with two binding sites for CD3, but also NK-cell recruiting TandAbs with two binding sites for CD16A. In the present study, Affimed's AFM11, was characterized and compared in in vitro and in vivo studies with the CD19/CD16A TandAb AFM12. Methods: Analogous to the CD19/CD3 TandAb AFM11, a bispecific tetravalent TandAb AFM12 was constructed with two binding sites for CD19 and two sites for CD16A. Both TandAbs were characterized side by side for their biophysical properties, binding affinities to CD19+ tumor target cells and to their respective effector cells by flow cytometry. Kinetics and dose-response characteristics were evaluated in in vitro cytotoxicity assays. Potency and efficacy of both TandAbs were compared on different CD19+ tumor target cell lines using primary human effector cells. To compare the efficacy of AFM11 and AFM12 a patient-derived tumor xenograft model was developed. Results: AFM12 mediated efficacious target cell lysis with a very fast on-set in vitro. Lysis induced by AFM11 was less efficacious (lower specific lysis than AFM12) but reproducibly more potent (lower EC50 value). In addition to the potency and efficacy of AFM11 and AFM12, different aspects of safety, such as effector cell activation in the presence and absence of target cells were investigated and will be described. Conclusions: Affimed's CD19/CD3 and CD19/CD16A TandAbs with identical anti-CD19 tumor-targeting domains but different effector cell-recruiting domains represent interesting molecules to study T-cell- or NK-cell-based immunotherapeutic approaches. The comparison of AFM11 and AFM12 demonstrated that AFM12-mediated lysis was fast and efficacious, whereas AFM11 showed a higher potency. In summary, the NK-cell recruiting TandAb AFM12 represents an alternative to T-cell recruiting molecules, as it may offer a different side effect profile, comparable to that of AFM13, the first NK-cell TandAb clinically investigated. Disclosures No relevant conflicts of interest to declare.

Download Full-text

A specific Upregulated lncRNA in Colorectal Cancer Promotes Cancer Progression by Modulating miR-185-5p/CCND2 Axis

10.21203/rs.3.rs-923878/v1 ◽

2021 ◽

Author(s):

Junshu Li ◽

Yanhong Ji ◽

Na Chen ◽

Huiling Wang ◽

Chao Fang ◽

...

Keyword(s):

Colorectal Cancer ◽

Network Analysis ◽

Tumor Growth ◽

Cancer Progression ◽

Gene Mutations ◽

Disease Free Survival ◽

Luciferase Reporter ◽

Colorectal Cancer Patients

Abstract BackgroundAdenomatous polyposis coli (APC) gene mutations were found in most colorectal cancer patients and functioned as an important inducer of tumorigenesis. Long non-coding RNA (lncRNA) plays a crucial role in the pathogenesis of various diseases, including colorectal cancer (CRC). Here we investigated the role of SURC which is specific upregulated in CRC progression. MethodsBased on the previous microarray results, weighted correlation network analysis (WGCNA) and lncRNA-mRNA co-expression network analysis were used to identify a lncRNA (SURC) and found it was specific up-regulated in CRC patients by qPCR and FISH staining. Chromatin immunoprecipitation (ChIP) assay was used to demonstrate the regulatory effect and mechanism of APC mutation on SURC expression. The effects of SURC on proliferation and cell cycle were determined by in vitro and in vivo experiments. Chromatin Isolation by RNA Purification (CHIRP) and luciferase reporter assay were carried out to illustrate the interaction between SURC, miR-185-5p and CCND2.ResultsWe found that SURC was specific up-regulated in CRC, but not in other solid tumor, when compared with normal adjacent tissues. High expression of SURC correlates with poorer disease-free survival and overall survival of CRC patients. Mutated APC protein resulted in stabilization of β-catenin in CRC, which promotes the transcription of SURC via binding to its promoter. Knockdown of SURC impaired CRC cell proliferation, colony formation and CRC tumor growth. Mechanistically, after transcription, SURC was transferred to cytoplasm and inhibits miR-185-5p expression via binding to miR-185-5p and inhibiting the synthesis of miR-185-5p from pri-miR-185-5p, which results in CCND2 expression.ConclusionCollectively, these results indicated that SURC promoted CRC tumor growth via interacting with miR-185-5p and regulating the activity of miR-185-5p/CCND2 axis which would be a novel diagnosis and prognosis prediction target for CRC.

Download Full-text

Anti-Metastatic Activity of an Anti-EGFR Monoclonal Antibody against Metastatic Colorectal Cancer with KRAS p.G13D Mutation

International Journal of Molecular Sciences ◽

10.3390/ijms21176037 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6037

Author(s):

Tomokazu Ohishi ◽

Yukinari Kato ◽

Mika K. Kaneko ◽

Shun-ichi Ohba ◽

Hiroyuki Inoue ◽

...

Keyword(s):

Colorectal Cancer ◽

Monoclonal Antibody ◽

Metastatic Colorectal Cancer ◽

Growth Factor Receptor ◽

High Sensitivity ◽

Kras Mutations ◽

Cytotoxicity Activities ◽

Hct 116 Cells

The now clinically-used anti-epidermal growth factor receptor (EGFR) monoclonal antibodies have demonstrated significant efficacy only in patients with metastatic colorectal cancer (mCRC), with wild-type Kirsten rat sarcoma viral oncogene homolog (KRAS). However, no effective treatments for patients with mCRC with KRAS mutated tumors have been approved yet. Therefore, a new strategy for targeting mCRC with KRAS mutated tumors is desired. In the present study, we examined the anti-tumor activities of a novel anti-EGFR monoclonal antibody, EMab-17 (mouse IgG2a, kappa), in colorectal cancer (CRC) cells with the KRAS p.G13D mutation. This antibody recognized endogenous EGRF in CRC cells with or without KRAS mutations, and showed a high sensitivity for CRC cells in flow cytometry, indicating that EMab-17 possesses a high binding affinity to the endogenous EGFR. In vitro experiments showed that EMab-17 exhibited antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities against CRC cells. In vivo analysis revealed that EMab-17 inhibited the metastases of HCT-15 and HCT-116 cells in the livers of nude mouse metastatic models, unlike the anti-EGFR monoclonal antibody EMab-51 of subtype mouse IgG1. In conclusion, EMab-17 may be useful in an antibody-based therapy against mCRC with the KRAS p.G13D mutation.

Download Full-text

O7 A bispecific VHH approach to leverage the potent and widely applicable tumor cytolytic capacity of Vγ9Vδ2 T cells

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.12 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A6.2-A7

Author(s):

LA King ◽

R Lameris ◽

RC Roovers ◽

P Parren ◽

TD de Gruijl ◽

...

Keyword(s):

Colorectal Cancer ◽

T Cells ◽

T Cell ◽

Xenograft Model ◽

Cell Receptor ◽

Target Cells ◽

Mouse Xenograft Model ◽

Vγ9vδ2 T Cells

Vγ9Vδ2-T cells include a unique and potent subset of T cells which play an important role in tumor defense. Vγ9Vδ2-T cells recognize and can lyse butyrophilin 3A1-expressing target cells with elevated levels of non-peptide phosphoantigens (pAg), induced by cell stress or malignancy. To date, Vγ9Vδ2-T cell based cancer immunotherapeutic approaches were well tolerated and in some cases capable of inducing relevant clinical responses. In an effort to improve the efficacy and consistency of Vγ9Vδ2-T cell based cancer immunotherapy, we designed a bispecific VHH that binds to both Vγ9Vδ2-T cells and EGFR expressed by tumor cells and results in the target-specific activation of Vγ9Vδ2-T cells and subsequent lysis of colorectal cancer cell lines and primary colorectal cancer samples both in vitro and in an in vivo mouse xenograft model. Of note, tumor cell lysis was independent of mutations in KRAS and BRAF that are known to impair the efficacy of clinically registered anti-EGFR monoclonal antibodies as well as common Vγ9Vδ2-T cell receptor sequence variations. In combination with the conserved monomorphic nature of the Vγ9Vδ2-TCR and the facile replacement of the tumor-specific VHH, this immunotherapeutic approach can in principle be applied to a large group of cancer types.Disclosure InformationL.A. King: None. R. Lameris: None. R.C. Roovers: None. P. Parren: None. T.D. de Gruijl: None. H.J. van der Vliet: None.

Download Full-text