Abstract Background and purpose When treated by radiotherapy, patients with squamous cell carcinomas of the head and neck (HNSCC) positive for HPV and p16INK4a possess a clearly favorable prognosis ...as compared to those with HPV-negative HNSCC. The aim of this work was to study whether the better outcomes might be caused by an enhanced cellular radiosensitivity. Materials and methods The radiation response of five HPV/p16INK4a -positive and five HPV-negative cell lines was characterized with regard to cellular radiosensitivity by colony formation assay. Furthermore G1- and G2-arrest, apoptosis and residual DNA double-strand breaks (DSB) were analyzed by the colcemid-based G1-efflux assay, propidium iodide staining, the detection of PARP cleavage, the fluorescence-based detection of caspase activity and the immunofluorescence staining of γH2AX and 53BP1 foci. Results On average, the cellular radiosensitivity of the HNSCC cell lines positive for HPV and p16INK4a was higher as compared to the sensitivity of a panel of five HPV-negative HNSCC cell lines (SF3 = 0.2827 vs. 0.4455). The higher sensitivity does not result from increased apoptosis or the execution of a permanent G1-arrest, but is rather associated with both, elevated levels of residual DSBs and extensive G2-arrest. Conclusions Increased cellular radiosensitivity due to compromised DNA repair capacity is likely to contribute to the improved outcome of patients with HPV/p16INK4a -positive tumors when treated by radiotherapy.
Transcriptional regulation of the
TERT
gene is a major cause of the cancer-specific increase in telomerase activity. Recently, frequent somatic mutations in the
TERT
promoter have been described in ...several tumor entities such as melanoma, glioblastoma, bladder cancer, and hepatocellular carcinoma. By generating a putative consensus binding site for ETS transcription factors within the
TERT
promoter, these mutations are predicted to increase promoter activity and
TERT
transcription. In order to improve the understanding of the role of
TERT
promoter mutation in liver tumorigenesis, the mutational status of the
TERT
promoter was analyzed in 78 hepatocellular carcinomas, 15 hepatocellular adenomas, and 52 intrahepatic cholangiocarciomas. The promoter region of
TERT
was screened for the two hotspot mutations using PCR and restriction fragment length analysis, utilizing the introduction of novel restriction sites by the somatic mutations.
TERT
promoter mutation was found in 37 of 78 hepatocellular carcinomas (47 %) and was restricted to the -124C>T mutation. Frequency of mutations was associated with grade of differentiation ranging from 39 % in well-differentiated tumors to 73 % in high-grade hepatocellular carcinomas.
TERT
promoter mutations were not found in 15 hepatocellular adenomas and 52 intrahepatic cholangiocarcinomas. These data show that
TERT
promoter mutation is the most frequent genetic alteration in hepatocellular carcinoma known at this time. The striking predominance of the -124C>T mutation compared with other tumor entities suggest a biological difference of the two hotspot mutations. Analysis of
TERT
promoter mutation might become a diagnostic tool distinguishing hepatocellular adenoma from well-differentiated hepatocellular carcinoma.
Abstract Purpose How EGF receptor (EGFR) inhibition induces cellular radiosensitization and with that increase in tumor control is still a matter of discussion. Since EGFR predominantly regulates ...cell cycle and proliferation, we studied whether a G1-arrest caused by EGFR inhibition may contribute to these effects. Materials and methods We analyzed human non-small cell lung cancer (NSCLC) cell lines either wild type (wt) or mutated in p53 (A549, H460, vs. H1299, H3122) and HCT116 cells (p21 wt and negative). EGFR was inhibited by BIBX1382BS, erlotinib or cetuximab; p21 was knocked down by siRNA. Functional endpoints analyzed were cell signaling, proliferation, G1-arrest, cell survival as well as tumor control using an A549 tumor model. Results When combined with IR, EGFR inhibition enhances the radiation-induced permanent G1 arrest, though solely in cells with intact p53/p21 signaling. This increase in G1-arrest was always associated with enhanced cellular radiosensitivity. Strikingly, this effect was abrogated when cells were re-stimulated, suggesting the initiation of dormancy. In line with this, only a small non-significant increase in tumor control was observed for A549 tumors treated with fractionated RT and EGFR inhibition. Conclusion For NSCLC cells increase in radiosensitivity by EGFR inhibition results from enhanced G1-arrest. However, this effect does not lead to improved tumor control because cells can be released from this arrest by re-stimulation.
Nonalcoholic steatohepatitis (NASH)-induced hepatocellular carcinoma (HCC) and its precursor, nonalcoholic fatty liver disease (NAFLD) are an unmet health issue due to widespread obesity. We assessed ...copy number changes of genes associated with hepatocarcinogenesis and oxidative pathways at a single-cell level. Eleven patients with NASH-HCC and 11 patients with NAFLD were included. Eight probes were analyzed using multiplex interphase fluorescence in situ hybridization (miFISH), single-cell imaging and phylogenetic tree modelling: Telomerase reverse transcriptase (TERT), C-Myc (MYC), hepatocyte growth factor receptor tyrosine kinase (MET), tumor protein 53 (TP53), cyclin D1 (CCND1), human epidermal growth factor receptor 2 (HER2), the fragile histidine triad gene (FHIT) and FRA16D oxidoreductase (WWOX). Each NASH-HCC tumor had up to 14 distinct clonal signal patterns indicating multiclonality, which correlated with high tumor grade. Changes frequently observed were TP53 losses, 45%; MYC gains, 36%; WWOX losses, 36%; and HER2 gains, 18%. Whole-genome duplications were frequent (82%) with aberrant tetraploid cells evolving from diploid ancestors. Non-tumorous NAFLD/NASH biopsies did not harbor clonal copy number changes. Fine mapping of NASH-HCC using single-cell multiplex FISH shows that branched tumor evolution involves genome duplication and that multiclonality increases with tumor grade. The loss of oxidoreductase WWOX and HER2 gains could be potentially associated with NASH-induced hepatocellular carcinoma.
In patients with non-small cell lung cancer (NSCLC) harboring a fusion of the neurotrophic receptor kinase (NTRK) gene 1 or 3, treatment with tropomyosin kinase (TRK) inhibitors have shown promising ...results, however so far no data on efficacy of these agents in patients with NSCLC and NTRK2 fusion are available. We present a case of a female patient with NTRK2-positive NSCLC with a complete ongoing response on therapy with larotrectinib, suggesting efficacy of first-generation TRK inhibitors also in NTRK2-positive NSCLC.
Abstract Background and purpose There is a great need to improve the outcome of locoregionally advanced squamous cell carcinomas of the head and neck (HNSCC). Standard treatment includes a ...combination of surgery, radio- and chemotherapy. The addition of molecular targeting agents to conventional treatment may improve outcomes. In this study the Raf inhibitor sorafenib was used to increase the radiosensitivity of HNSCC cell lines. Material and methods In a panel of six cell lines (A549, FaDu, UTSCC 60A, UTSCC 42A, UTSCC 42B, UTSCC 29) radiosensitivity was measured by colony formation assay and apoptosis and cell cycle analysis were performed by flow cytometry. DNA repair was analyzed by 53BP1 immunohistochemistry. Results Sorafenib added prior to irradiation resulted in an increased cellular radiosensitivity (DEF0.5 = 1.11–1.84). Radiosensitization was not caused by an enhanced rate of apoptosis or cell cycle effects. In contrast, sorafenib was shown for the first time to block the repair of DNA double-strand breaks (DSB). Conclusion Our data suggest that sorafenib may be used to overcome the radioresistance of HNSCC through the inhibition of DSB repair.
Core needle biopsies (CNBs) are widely used to determine human epidermal growth factor receptor 2 (HER2) status in breast cancer. Recent publications reported up to 20% false-positive results on CNBs ...if immunohistochemistry (IHC) is compared with fluorescent in situ hybridization (FISH). To clarify, if confirmation of IHC positivity by FISH is generally required, we analyzed the reliability of IHC positivity on CNBs versus surgical specimens in a multi-institutional study.
Five pathologic laboratories contributed to this study by performing IHC on 500 CNBs and the corresponding surgical specimens overall. If IHC revealed score 2+ or 3+, HER2 status was confirmed by FISH in a central laboratory. We compared evaluation according to US Food and Drug Administration-approved scoring criteria and recently published American Society of Clinical Oncology (ASCO)-College of American Pathologists (CAP) guidelines.
CNBs scored 3+ revealed five false-positive results if scoring followed the US Food and Drug Administration criteria (five of 40; 12.5%) and two false-positives in terms of the ASCO-CAP criteria (two of 33; 6.1%). IHC was false negative in one CNB only. By contrast, IHC on surgical specimens revealed five false-negative results, but only one false-positive result (one of 35; 2.9%) if scored following US Food and Drug Administration-approved criteria. With the aid of the ASCO-CAP criteria, false-positive IHC results were obtained in only one of the five participating institutions.
IHC 3+ scores on CNBs proved to be reliable in four of the five participating institutions if scoring followed the ASCO-CAP criteria. Therefore, accurate determination of HER2 status in breast cancer is possible on CNB using the common strategy to screen all cases by IHC and retest only 2+ scores by FISH. Prerequisites are quality assurance and the application of the new ASCO-CAP criteria.
Mismatch repair-deficient (dMMR) prostate cancer (PCa) is a rare (1–5%) but highly actionable molecular subgroup of PCa, vulnerable to immune checkpoint inhibitors. Our case of sporadic dMMR PCa due ...to large monoallelic co-deletion of EPCAM, MSH2, and MSH6 features a clinically aggressive disease presentation and a major response to pembrolizumab. We report a 65-year-old patient with primary metastatic PCa, Gleason score 5 + 5 = 10, with penile and lymph node metastases at diagnosis. Patient showed rapid progression on first-line ADT and enzalutamide. Tumor next-generation sequencing (NGS) revealed microsatellite instability and a tumor mutational burden of 40.8 mutations/megabase. Immunohistochemistry showed co-loss of MSH2 and MSH6. Review of NGS row data confirmed large monoallelic deletion in chromosome 2p, including EPCAM, MSH2, and MSH6. No germline alterations in mismatch repair genes were detected. Patient showed excellent response to pembrolizumab, which is still ongoing. We conclude that early molecular tumor profiling is essential to enable personalized management of advanced PCa, especially in patients with aggressive or atypical disease course.
Women with triple-negative breast cancer (TNBC) do not benefit from endocrine therapy or trastuzumab. Chemotherapy is the only systemic therapy currently available. To reduce the elevated risk of ...disease progression in these patients, better treatment options are needed, which are less toxic and more targeted to this patient population. We performed a comprehensive analysis of potential targetable genetic aberrations affecting the receptor tyrosine kinase/RAS/MAPK pathway, which are observed at higher frequencies in adenocarcinomas of other organs. Sixty-five individual TNBCs were studied by sequence analysis for
HER2
(exon 18–23),
EGFR
(exon 18–21),
KRAS
(exon 2), and
BRAF
(exon 15) mutations. In addition, a tissue microarray was constructed to screen for
EGFR
gene copy gain and
EML4
-
ALK
fusion by FISH. Triple-negative status was confirmed by immunohistochemistry and FISH on tissue microarray sections. EGFR and CK5/6 immunohistochemical analyses were performed for identification of the basal-like phenotype. In addition, mutation analysis of
TP53
(exon 5–8) was included. Sequence analysis revealed
HER2
gene mutation in only one patient (heterozygous missense mutation in exon 19: p.L755S). No mutations were found in
EGFR
,
KRAS,
and
BRAF
. High polysomy of
EGFR
was detected in 5 of the 62 informative cases by FISH. True
EGFR
gene amplification accompanied by strong membranous EGFR protein expression was observed in only one case. No rearrangement of the
ALK
gene was detected. Basal-like phenotype was identified in 38 of the 65 TNBCs (58.5 %).
TP53
gene mutation was found in 36/63 (57.1 %) tumors. We conclude that targetable genetic aberrations in the receptor tyrosine kinase/RAS/MAPK pathway occur rarely in TNBC.
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