Background
Pulmonary metastasectomy is an integral part of the interdisciplinary treatment of patients with pulmonary metastases (PMs) from colorectal carcinoma (CRC). Although alterations in the ...epidermal growth factor receptor (EGFR) pathway are common in CRC, there is still insufficient data regarding PM. We hypothesized that EGFR expression and Kirsten rat sarcoma viral oncogene homolog (
KRAS
)/
BRAF
mutations (Mts) might be associated with clinicopathological variables and the outcome in patients undergoing pulmonary metastasectomy.
Methods
In this single-center study, 44 patients undergoing pulmonary metastasectomy from primary CRC were included and prospectively followed up. Tissue specimens of resected PMs were assessed. Restriction fragment length analysis was used for
BRAF V600E
and
KRAS
codons 12 and 13 Mt analyses. EGFR expression was evaluated by immunohistochemistry. Patients were followed up in 3–6-month intervals.
Results
EGFR expression was evident in 49 % of the PMs, whereas Mts in
KRAS
and
BRAF
were detected in 48 and 0 %, respectively. Time to lung-specific recurrence after metastasectomy was significantly decreased in patients with
KRAS
mutated PMs in univariate (
p
= 0.013) and multivariate analysis (
p
= 0.035), whereas EGFR expression had no impact on recurrence free survival. Moreover, KRAS Mts were associated with the number of PMs (
p
= 0.037) and with the lung as first site of recurrence after metastasectomy (
p
= 0.047).
Discussion
This is the first evaluation of EGFR pathway alterations in the setting of pulmonary metastasectomy. Our data suggest that patients with
KRAS
Mts are at high risk for early pulmonary recurrence and have a more diffuse pattern of metastasis. These findings may have impact on the therapeutic management of CRC patients with pulmonary spreading.
Summary The significance of epidermal growth factor receptor (EGFR) signaling is well studied in a number of different tumors, but limited data is available with regard to head and neck squamous cell ...carcinoma (HNSCC). Since anti-EGFR therapies are currently under investigation in these malignancies as well, comprehensive information about the alteration of EGFR in HNSCC is necessary to design the most appropriate therapeutic protocols. We examined retrospectively the gene copy number of EGFR by FISH and the protein expression by immunohistochemistry using different epitope-specific antibodies in paraffin-embedded primary tumors of five different regions, from 71 HNSCC patients who had not been treated with anti-EGFR therapy. In seven cases corresponding lymph node metastases were also available for comparative analyses. We also determined the mutational status of tyrosine kinase (TK) domain (exon 19 and 21) and the extracellular deletion mutation (vIII) of EGFR, the KRAS mutation at codon 12 and the presence of HPV infection. Eight of the 71 cases (11.3%) showed EGFR gene amplification (most of them localized into the hypopharyngeal region) and the increased gene copy number (amplification + polysomy) was 43.7%. Despite pronounced intratumoral heterogeneity of EGFR protein expression being found, the high EGFR expression correlated with poor prognosis. On the other hand, the phosphorylation of EGFR was associated with prolonged survival. No mutations in the TK domain of EGFR were found in any of the HNSCC patients and only two cases were KRAS mutant at codon 12. We detected vIII deletion mutation of EGFR in 21% of the samples, but there was no statistically significant correlation between the presence of vIII mutant form and patient survival. EGFR vIII mutation was, however, associated with increased gene copy number. Fourteen of 71 cases (19.7%) were HPV-positive and the incidence of infection showed a decreasing tendency from the oral cavity towards the larynx. Interestingly, in contrast to previous findings, we could not observe improved survival in HPV-positive patients compared to non-infected patients, most probably due to the fact that the majority of these HNSCC patients were smokers and alcohol consumers. In conclusion, we found that increased EGFR protein levels and gene copy numbers (not gene amplification alone) have prognostic significance in the investigated HNSCC patient population. However, the relatively high incidence of the EGFR-vIII mutant form warrants careful therapeutic decision-making when choosing between different anti-EGFR treatment options.
The first representatives of the new fluorescent boro-β-carboline family were synthesized by the insertion of the difluoroboranyl group into the oxaza or diaza core. The resulting compounds showed ...good photophysical properties with fine Stokes-shifts in the range of 38-85 nm with blue and green emission. The energetics of the excitation states and molecular orbitals of two members were investigated by quantum chemical computations suggesting effects for the improved properties of diazaborinino-carbolines over oxazaborolo-carbolines. These properties nominated this chemotype as a new fluorophore for the development of fluorescent probes. As an example, diazaborinino-carbolines were used for the specific labeling of anti-Her2 antibody trastuzumab. The fluorescent conjugate showed a high fluorophore-antibody ratio and was confirmed as a useful tool for labeling and confocal microscopy imaging of tumour cells
in vitro
together with the
ex vivo
two-photon microscopy imaging of tumour slices.
The first representatives of fluorescent boro-β-carbolines were applied for labeling trastuzumab. The antibody fluorophore conjugate was confirmed as a useful tool for labeling and imaging tumour cells in confocal and two-photon microscopy.
Head and neck squamous cell carcinomas (HNSCCs) are among the most abundant malignancies worldwide. Patients with recurrent/metastatic disease undergo combination chemotherapy containing cetuximab, ...the monoclonal antibody used against the epidermal growth factor receptor (EGFR). Cetuximab augments the effect of chemotherapy; however, a significant number of patients show therapy resistance. The mechanism of resistance is yet to be unveiled, although extracellular alterations of the receptor have been reported, and their role in cetuximab failure has been proposed.
Here, we investigate possible effects of the multi-exon deletion variant (EGFRvIII), and the single nucleotide polymorphism EGFR R521K on cetuximab efficacy.
Our results show that in HNSCC patients, the EGFRvIII allele frequency is under 1%; therefore, it cannot lead to common resistance. EGFR R521K, present in 42% of the patients, is investigated in vitro in four HNSCC cell lines (two wild-type and two heterozygous for EGFR R521K). While no direct effect is found to be related to the EGFR status, cells harboring R521K show a reduced sensitivity in ADCC experiments and in vivo xenograft experiments. However, this preclinical difference is not reflected in the progression-free or overall survival of HNSCC patients. Furthermore, NK cell and macrophage presence in tumors is not related to EGFR R521K.
Our results suggest that EGFR R521K, unlike reported previously, is unable to cause cetuximab resistance in HNSCC patients; therefore, its screening before therapy selection is not justifiable.
Tumor hypoxia promotes neoangiogenesis and contributes to the radio- and chemotherapy resistant and aggressive phenotype of cancer cells. However, the migratory response of tumor cells and the role ...of small GTPases regulating the organization of cytoskeleton under hypoxic conditions have yet to be established. Accordingly, we measured the proliferation, migration, RhoA activation, the mRNA and protein levels of hypoxia inducible factor-1alpha (HIF-1α) and three small G-proteins, Rac1, cdc42 and RhoA in a panel of five human tumor cell lines under normoxic and hypoxic conditions. Importantly, HT168-M1 human melanoma cells with high baseline migration capacity showed increased HIF-1α and small GTPases expression, RhoA activation and migration under hypoxia. These activities were blocked by anti- HIF-1α shRNA. Moreover, the in vivo metastatic potential was promoted by hypoxia mimicking CoCl2 treatment and reduced upon inhibition of HIF-1α in a spleen to liver colonization experiment. In contrast, HT29 human colon cancer cells with low migration capacity showed limited response to in vitro hypoxia. The expression of the small G-proteins decreased both at mRNA and protein levels and the RhoA activation was reduced. Nevertheless, the number of lung or liver metastatic colonies disseminating from orthotopic HT29 grafts did not change upon CoCl2 or chetomin treatment. Our data demonstrates that the hypoxic environment induces cell-type dependent changes in the levels and activation of small GTPases and results in varying migratory and metastasis promoting responses in different human tumor cell lines.
Anti-EGFR antibody therapy is still one of the clinical choices in head and neck squamous cell carcinoma (HNSCC) patients, but the emergence of cetuximab resistance questioned its effectiveness and ...reduced its applicability. Although several possible reasons of resistance against the antibody treatment and alternative therapeutic proposals have been described (EGFR alterations, activation of other signaling pathways), there is no method to predict the effectiveness of anti-EGFR antibody treatments and to suggest novel therapeutics. Our study investigated the effect of EGFR R521K alteration on efficiency of cetuximab therapy of HNSCC cell lines and tried to find alternative therapeutic approaches against the resistant cells.
After genetic characterization of HNSCC cells, we chose one wild type and one R521K+ cell line for
proliferation and apoptosis tests, and
animal models using different therapeutic agents.
Although the cetuximab treatment affected EGFR signalization in both cells, it did not alter
cell proliferation or apoptosis.
cetuximab therapy was also ineffective on R521K harboring tumor xenografts, while blocked the tumor growth of EGFR-wild type xenografts. Interestingly, the cetuximab-resistant R521K tumors were successfully treated with c-MET tyrosine kinase inhibitor SU11274.
Our results suggest that HNSCC cell line expressing the R521K mutant form of EGFR does not respond well to cetuximab treatment
or
, but hopefully might be targeted by c-MET tyrosine kinase inhibitor treatment.
Ependymomas are common pediatric brain tumors that originate from the ependyma and characterized by poor prognosis due to frequent recurrence. However, the current WHO grading system fails to ...accurately predict outcome. In a retrospective study, we analyzed 54 intracranial pediatric ependymomas and found a significantly higher overall survival in supratentorial cases when compared to infratentorial tumors. Next we performed region-specific immunohistochemical analysis of the ependyma in neonatal and adult ependyma from the central canal of spinal cord to the choroid plexus of lateral ventricles for components of cell-cell junctions including cadherins, claudins and occludin. We found robust claudin-5 expression in the choroid plexus epithelia but not in other compartments of the ependyma. Ultrastructural studies demonstrated distinct regional differences in cell-cell junction organization. Surprisingly, we found that 9 out of 20 supratentorial but not infratentorial ependymomas expressed high levels of the brain endothelial tight junction component claudin-5 in tumor cells. Importantly, we observed an increased overall survival in claudin-5 expressing supratentorial ependymoma. Our data indicates that claudin-5 expressing ependymomas may follow a distinct course of disease. The assessment of claudin-5 expression in ependymoma has the potential to become a useful prognostic marker in this pediatric malignancy.
Some Vinca alkaloids (eg, vinblastine, vincristine) have been widely used as antitumor drugs for a long time. Unfortunately, vindoline, a main alkaloid component of Catharanthus roseus (L.) G. Don, ...itself, has no antitumor activity. In our novel research program, we have prepared and identified new vindoline derivatives with moderate cytostatic activity.
Here, we describe the effect of conjugation of vindoline derivative with oligoarginine (tetra‐, hexa‐, or octapeptides) cell‐penetrating peptides on the cytostatic activity in vitro and in vivo. Br‐Vindoline‐(l)‐Trp‐OH attached to the N‐terminus of octaarginine was the most effective compound in vitro on HL‐60 cell line. Analysis of the in vitro activity of two isomer conjugates (Br‐vindoline‐(l)‐Trp‐Arg8 and Br‐vindoline‐(d)‐Trp‐Arg8 suggests the covalent attachment of the vindoline derivatives to octaarginine increased the antitumor activity significantly against P388 and C26 tumour cells in vitro. The cytostatic effect was dependent on the presence and configuration of Trp in the conjugate as well as on the cell line studied. The configuration of Trp notably influenced the activity on C26 and P388 cells: conjugate with (l)‐Trp was more active than conjugate with the (d)‐isomer. In contrast, conjugates had very similar effect on both the HL‐60 and MDA‐MB‐231 cells. In preliminary experiments, conjugate Br‐vindoline‐(l)‐Trp‐Arg8 exhibited some inhibitory effect on the tumor growth in P388 mouse leukemia tumor‐bearing mice. Our results indicate that the conjugation of modified vindoline could result in an effective compound even with in vivo antitumor activity.
Our results indicate that the conjugation of modified vindoline could result in an effective compound even with in vivo antitumor activity.
Abstract
Squamous cell carcinomas of the Head and Neck region (HNSCCs) have about 600.000 new cases every year. Advanced disease is treated by radio-chemotherapy, which has ambiguous success. ...Cetuximab, a monoclonal antibody against EGF receptor, is proven to be useful in combination therapy, thus it is in clinical use in HNSCC. However, a large number of patients show therapy resistance and do not benefit from combined therapy. EGF receptor is a widely used target in the therapy against cancer cells. Antibodies (as cetuximab) and inhibitors (TKI) are in use. However, genetic variations of the receptor are described to influence therapy efficacy. The extracellular missense polymorphism R521K of the receptor is reported to be abundant in HNSCC. However, its role in therapy outcome is yet to be cleared. In the present work, we mapped the possible effects of cetuximab therapy in wild type and mutant genotype HNSCCs. We selected two wild type (Pe/Ca-PJ41, Cal27) and two R521K (Pe/Ca-PJ15, FaDu) cell lines, performed in vitro proliferation tests and antibody binding assays, measured in vitro Antigen-Dependent Cellular Cytotoxicity (ADCC), and quantified receptor phosphorylation inhibition by cetuximb. In our in vivo xenograft experiments, we analyzed subcutaneous tumor growth by cetuximab, in combination with inhibition of NK cell and macrophage activity. Importantly, we analyzed clinical samples in the National Institute of Oncology, originated from 95 relapsed HNSCC patients treated with cetuximab to reveal any connections between EGFR R521K single nucleotide polymorphism and clinical outcome. In cell lines, cetuximab did not show significant cytotoxic effects, but was effective in the ADCC assay. Significantly, it enhanced the killing of cancer cells more effectively in wild type cell line models than on R521K harboring cells. Although antibody binding assay and EGF receptor phosphorylation was not different among the cell lines used, in vivo xenograft models showed strong antitumor effect of cetuximab in wild type models, and only moderate or no response in EGFR R521K models. Our clinical study, showed significantly lower progression-free survival among patients with R521K tumors. Overall survival of the two genotype groups showed only a trend of similar pattern, without statistically significant difference. Immune cell quantification showed no difference between the NK cell and macrophage infiltration in the two groups. Our data suggests the possible effect of EGFR 521 genotype on cetuximab therapy efficacy, but per se is insufficient to explain the dramatic difference between patient responses. Further data collection and analysis might be needed to reassure if R521K should play role in therapy selection for HNSCC, while clinical samples collected after cetuximab treatment would be exceptionally useful in order to track immune cell changes in patients with wild type and EGFR R521K tumors. This work was funded by the National Research, Development and Innovation Office grant K/116295 (2015). Financial support from the 2019 Thematic Excellence Program (TUDFO/51757/2019-ITM) is greatly acknowledged.
Citation Format: Mihaly T. Cserepes, Zita Hegedus, Ivan Ranđelović, Istvan Kenessey, Mónika Meilinger-Dobra, Kristóf G. Csikó, Andrea Ladanyi, Éva Remenár, Jozsef Tovari. Correlations of genetic variation R521K of EGF receptor and the in vitro, in vivo and clinical phenotypes of head and neck cancers after cetuximab treatment abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1863.
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