FEN1 Inhibition as a Potential Novel Targeted Therapy against Breast Cancer and the Prognostic Relevance of FEN1.

To improve breast cancer treatment and to enable new strategies for therapeutic resistance, therapeutic targets are constantly being studied. Potential targets are proteins of DNA repair and replication and genomic integrity, such as Flap Endonuclease 1 (FEN1). This study investigated the effects of FEN1 inhibitor FEN1-IN-4 in combination with ionizing radiation on cell death, clonogenic survival, the cell cycle, senescence, doubling time, DNA double-strand breaks and micronuclei in breast cancer cells, breast cells and healthy skin fibroblasts. Furthermore, the variation in the baseline FEN1 level and its influence on treatment prognosis was investigated. The cell lines show specific response patterns in the aspects studied and have heterogeneous baseline FEN1 levels. FEN1-IN-4 has cytotoxic, cytostatic and radiosensitizing effects, expressed through increasing cell death by apoptosis and necrosis, G2M share, senescence, double-strand breaks and a reduced survival fraction. Nevertheless, some cells are less affected by the cytotoxicity and fibroblasts show a rather limited response. In vivo, high FEN1 mRNA expression worsens the prognosis of breast cancer patients. Due to the increased expression in breast cancer tissue, FEN1 could represent a new tumor and prognosis marker and FEN1-IN-4 may serve as a new potent agent in personalized medicine and targeted breast cancer therapy.

Different Impacts of DNA-PK and mTOR Kinase Inhibitors in Combination with Ionizing Radiation on HNSCC and Normal Tissue Cells.

Despite substantial advancements in understanding the pathomechanisms of head and neck squamous cell carcinoma (HNSCC), effective therapy remains challenging. The application of kinase inhibitors (KIs) in HNSCC, specifically mTOR and DNA-PK inhibitors, can increase radiosensitivity and therefore presents a promising strategy when used simultaneously with ionizing radiation (IR) in cancer treatment. Our study focused on the selective DNA-PK-inhibitor AZD7648; the selective mTOR-inhibitor Sapanisertib; and CC-115, a dual inhibitor targeting both mTOR and DNA-PK. The impact of these KIs on HNSCC and normal tissue cells was assessed using various analytical methods including cell death studies, cell cycle analysis, real-time microscopy, colony-forming assays and immunohistochemical staining for γH2AX and downstream mTOR protein p-S6. We detected a strong inhibition of IR-induced DNA double-strand break (DSB) repair, particularly in AZD7648-treated HNSCC, whereas normal tissue cells repaired DNA DSB more efficiently. Additionally, AZD7648 + IR treatment showed a synergistic decline in cell proliferation and clonogenicity, along with an elevated G2/M arrest and cell death in the majority of HNSCC cell lines. CC-115 + IR treatment led to an elevation in G2/M arrest, increased cell death, and a synergistic reduction in cell proliferation, though the effect was notably lower compared to the AZD7648 + IR- treated group. Sapanisertib led to a high cellular toxicity in both HNSCC and normal tissue cells, even in non-irradiated cells. Regarding cell proliferation and the induction of apoptosis and necrosis, Sapanisertib + IR was beneficial only in HPV+ HNSCC. Overall, this study highlights the potential of AZD7648 as a radiosensitizing agent in advanced-stage HPV-positive and negative HNSCC, offering a promising therapeutic strategy. However, the dual mTOR/DNA-PK-I CC-115 did not provide a distinct advantage over the use of selective KIs in our investigations, suggesting limited benefits for its application in KI + IR therapy. Notably, the selective mTOR-inhibitor Sapanisertib was only beneficial in HPV+ HNSCC and should not be applied in HPV- cases.

Neutrophils in HNSCC Can Be Associated with Both a Worse or Favorable Prognosis.

The prognostic significance of tumor-infiltrating neutrophils in head and neck squamous cell carcinoma (HNSCC) is poorly understood. It is unclear how the presence of neutrophils affects prognosis due to their polarization into cytotoxic N1 or immunosuppressive N2. Therefore, we determined the number of CD66b+ neutrophil granulocytes separately in the stromal and epithelial compartments in cancer tissues from 397 patients with HNSCC. Tumor samples from six historical patient groups were processed into tissue microarrays and stained immunohistochemically. In total, 21.9% were HPV positive (p16+). Neutrophil counts were much lower in the stromal compartment (372 ± 812) than in the epithelial cancer compartment (1040 ± 1477) (p < 0.001), with large differences between groups. In three groups with high neutrophil infiltration, high rates were associated with a favorable prognosis, whereas in two groups, high rates were a negative prognostic factor. In p16- oropharyngeal and hypopharyngeal cancer high infiltration was associated with a favorable prognosis. Cancers with an exclusion of neutrophils in the epithelial compartment were associated with improved prognosis. In oropharyngeal and hypopharyngeal HPV-negative cancer high neutrophil infiltration rates were clearly associated with prolonged survival. Neutrophil granulocytes in HNSCC may contribute to a favorable or unfavorable prognosis.

Acriflavine-Functionalized Silica@Manganese Ferrite Nanostructures for Synergistic Radiation and Hypoxia Therapies.

Mesoporous and nonmesoporous SiO2@MnFe2O4 nanostructures were loaded with the hypoxia-inducible factor-1 inhibitor acriflavine for combined radiation and hypoxia therapies. The X-ray irradiation of the drug-loaded nanostructures not only triggered the release of the acriflavine inside the cells but also initiated an energy transfer from the nanostructures to surface-adsorbed oxygen to generate singlet oxygen. While the drug-loaded mesoporous nanostructures showed an initial drug release before the irradiation, the drug was primarily released upon X-ray radiation in the case of the nonmesoporous nanostructures. However, the drug loading capacity was less efficient for the nonmesoporous nanostructures. Both drug-loaded nanostructures proved to be very efficient in irradiated MCF-7 multicellular tumor spheroids. The damage of these nanostructures toward the nontumorigenic MCF-10A multicellular spheroids was very limited because of the small number of nanostructures that entered the MCF-10A spheroids, while similar concentrations of acriflavine without nanostructures were toxic for the MCF-10A spheroids.

The Effect of Xevinapant Combined with Ionizing Radiation on HNSCC and Normal Tissue Cells and the Impact of Xevinapant on Its Targeted Proteins cIAP1 and XIAP.

The poor prognosis of HNSCC is partly due to treatment resistance. The SMAC mimetic Xevinapant is a promising new approach to targeted cancer therapy. Xevinapant inhibits cIAP1/2 and XIAP, leading to apoptosis, necroptosis and inhibition of prosurvival signaling. Combining Xevinapant with IR could improve therapeutic potential. The effect of Xevinapant in combination with IR on HNSCC and healthy tissue cells was investigated. Cell growth, cell death, clonogenic survival and DNA double-strand breaks (DSBs) were studied, and intracellular cIAP1 and XIAP levels were evaluated. Xevinapant had cytostatic and cytotoxic, as well as radiosensitizing, effects on the malignant cells, while healthy tissue cells were less affected. Apoptotic and necrotic cell death was particularly affected, but the increase in residual DSBs and the reduced survival implied an additional effect of Xevinapant on DNA damage repair and other cell inactivation mechanisms. cIAP1 and XIAP levels varied for each cell line and were affected by Xevinapant and IR treatment. There was an association between higher IAP levels and increased cell death. Xevinapant appears to be a potent new drug for HNSCC therapy, especially in combination with IR. IAP levels could be an indicator for impaired DNA damage repair and increased susceptibility to cellular stress.

Increased Radiation Sensitivity in Patients with Phelan-McDermid Syndrome.

Phelan-McDermid syndrome is an inherited global developmental disorder commonly associated with autism spectrum disorder. Due to a significantly increased radiosensitivity, measured before the start of radiotherapy of a rhabdoid tumor in a child with Phelan-McDermid syndrome, the question arose whether other patients with this syndrome also have increased radiosensitivity. For this purpose, the radiation sensitivity of blood lymphocytes after irradiation with 2Gray was examined using the G0 three-color fluorescence in situ hybridization assay in a cohort of 20 patients with Phelan-McDermid syndrome from blood samples. The results were compared to healthy volunteers, breast cancer patients and rectal cancer patients. Independent of age and gender, all but two patients with Phelan-McDermid syndrome showed significantly increased radiosensitivity, with an average of 0.653 breaks per metaphase. These results correlated neither with the individual genetic findings nor with the individual clinical course, nor with the respective clinical severity of the disease. In our pilot study, we saw a significantly increased radiosensitivity in lymphocytes from patients with Phelan-McDermid syndrome, so pronounced that a dose reduction would be recommended if radiotherapy had to be performed. Ultimately, the question arises as to the interpretation of these data. There does not appear to be an increased risk of tumors in these patients, since tumors are rare overall. The question, therefore, arose as to whether our results could possibly be the basis for processes, such as aging/preaging, or, in this context, neurodegeneration. There are no data on this so far, but this issue should be pursued in further fundamentally based studies in order to better understand the pathophysiology of the syndrome.

Preoperative Radiochemotherapy in Rectal Cancer: Is There an Impact of Oxaliplatin on Pathologic Complete Response and Survival Rates under "Real World" Conditions?

This study aimed to evaluate the benefit of additional administration of oxaliplatin during fluorouracil-based neoadjuvant radiochemotherapy (nRCT) in terms of pathologic complete remission (pCR), disease-free survival (DFS), and overall survival (OS) in patients with advanced rectal cancer. Between 2006 and 2021, 669 patients (pts) were diagnosed with locally advanced rectal cancer, of whom a total of 414 pts with nRCT were identified and included in the study. A total of 283 pts were treated by nRCT using concurrent chemotherapy with fluorouracil or capecitabine; 131 pts were treated using a combination of fluorouracil or capecitabine and oxaliplatin. Propensity score matching analyses (PSM) with 114 pts in each group were used to balance the patients' characteristics. OS, DFS, pCR-rate, and potential prognostic factors were compared between the two groups. The median follow-up time was 59.5 weeks in the fluorouracil-group and 43 weeks in the fluorouracil/oxaliplatin group (p = 0.003). After PSM, the pCR-rate (including sustained clinical complete remission) was 27% (31/114 pts) in the fluorouracil/oxaliplatin group and 16% (18/114 pts) in the fluorouracil-group (p = 0.033). There was no difference between these two groups for both 10-year OS and DFS neither before nor after PSM, respectively (OS: 72.6% vs. 55.4%, p = 0.066, and 67.8% vs. 55.1%, p = 0.703, and DFS: 44.8% vs. 46.8%, p = 0.134, and 44.7% vs. 42.3%, p = 0.184). Multivariate analysis identified regression grading according to Dworak grade 4 (HR: 0.659; CI: 0.471-0.921; p = 0.015) and age over 60 years (HR: 2.231; CI: 1.245-4.001; p = 0.007) as independent predictors for OS. In conclusion, the addition of oxaliplatin to fluorouracil during nRCT significantly improved pCR-rate without having an impact on survival.

In Vitro Analysis of Superparamagnetic Iron Oxide Nanoparticles Coated with APTES as Possible Radiosensitizers for HNSCC Cells.

Superparamagnetic iron oxide nanoparticles (SPION) are being investigated for many purposes, e.g., for the amplification of ionizing radiation and for the targeted application of therapeutics. Therefore, we investigated SPIONs coated with (3-Aminopropyle)-Triethoxysilane (SPION-APTES) for their influence on different head and neck squamous cell carcinoma (HNSCC) cell lines, as well as for their suitability as a radiosensitizer. We used 24-well microscopy and immunofluorescence microscopy for cell observation, growth curves to determine cytostatic effects, and colony formation assays to determine cytotoxicity. We found that the APTES-SPIONs were very well taken up by the HNSCC cells. They generally have a low cytotoxic effect, showing no significant difference in clonogenic survival between the control group and cells treated with 20 µg Fe/mL (p > 0.25) for all cell lines. They have a cytostatic effect on some cell lines cells (e.g., Cal33) that is visible across different radiation doses (1, 2, 8 Gy, p = 0.05). In Cal33, e.g., SPION-APTES raised the doubling time at 2 Gy from 24.53 h to 41.64 h. Importantly, these findings vary notably between the cell lines. However, they do not significantly alter the radiation effect: only one out of eight cell lines treated with SPION-APTES showed a significantly reduced clonogenic survival after ionizing radiation with 2 Gy, and only two showed significantly reduced doubling times. Thus, although the APTES-SPIONs do not qualify as a radiosensitizer, we were still able to vividly demonstrate and analyze the effect that the APTES-SPIONs have on various cell lines as a contribution to further functionalization.

Tumor-specific radiosensitizing effect of the ATM inhibitor AZD0156 in melanoma cells with low toxicity to healthy fibroblasts.

PURPOSE: Despite new treatment options, melanoma continues to have an unfavorable prognosis. DNA damage response (DDR) inhibitors are a promising drug class, especially in combination with chemotherapy (CT) or radiotherapy (RT). Manipulating DNA damage repair during RT is an opportunity to exploit the genomic instability of cancer cells and may lead to radiosensitizing effects in tumors that could improve cancer therapy. METHODS: A panel of melanoma-derived cell lines of different origin were used to investigate toxicity-related clonogenic survival, cell death, and cell cycle distribution after treatment with a kinase inhibitor (KI) against ATM (AZD0156) or ATR (VE-822, berzosertib), irradiation with 2 Gy, or a combination of KI plus ionizing radiation (IR). Two fibroblast cell lines generated from healthy skin tissue were used as controls. RESULTS: Clonogenic survival indicated a clear radiosensitizing effect of the ATM inhibitor (ATMi) AZD0156 in all melanoma cells in a synergistic manner, but not in healthy tissue fibroblasts. In contrast, the ATR inhibitor (ATRi) VE-822 led to additive enhancement of IR-related toxicity in most of the melanoma cells. Both inhibitors mainly increased cell death induction in combination with IR. In healthy fibroblasts, VE-822 plus IR led to higher cell death rates compared to AZD0156. A significant G2/M block was particularly induced in cancer cells when combining AZD0156 with IR. CONCLUSION: ATMi, in contrast to ATRi, resulted in synergistic radiosensitization regarding colony formation in melanoma cancer cells, while healthy tissue fibroblasts were merely affected with respect to cell death induction. In connection with an increased number of melanoma cells in the G2/M phase after ATMi plus IR treatment, ATMi seems to be superior to ATRi in melanoma cancer cell treatments when combined with RT.

Effect of Citrate- and Gold-Stabilized Superparamagnetic Iron Oxide Nanoparticles on Head and Neck Tumor Cell Lines during Combination Therapy with Ionizing Radiation.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. They are associated with alcohol and tobacco consumption, as well as infection with human papillomaviruses (HPV). Therapeutic options include radiochemotherapy, surgery or chemotherapy. Nanoparticles are becoming more and more important in medicine. They can be used diagnostically, but also therapeutically. In order to provide therapeutic alternatives in the treatment of HNSCC, the effect of citrate-coated superparamagnetic iron oxide nanoparticles (Citrate-SPIONs) and gold-coated superparamagnetic iron oxide nanoparticles (Au-SPIONs) in combination with ionizing irradiation (IR) on two HPV positive and two HPV negative HNSCC and healthy fibroblasts and keratinocytes cell lines were tested. Effects on apoptosis and necrosis were analyzed by using flow cytometry. Cell survival studies were performed with a colony formation assay. To better understand where the SPIONs interact, light microscopy images and immunofluorescence studies were performed. The HNSCC and healthy cell lines showed different responses to the investigated SPIONs. The cytotoxic effects of SPIONs, in combination with IR, are dependent on the type of SPIONs, the dose administered and the cell type treated. They are independent of HPV status. Reasons for the different cytotoxic effect are probably the different compositions of the SPIONs and the related different interaction of the SPIONs intracellularly and paramembranously, which lead to different strong formations of double strand breaks.

An In Vitro Approach for Investigating the Safety of Lipotransfer after Breast-Conserving Therapy.

The application of lipotransfer after breast-conserving therapy (BCT) and irradiation in breast cancer patients is an already widespread procedure for reconstructing volume deficits of the diseased breast. Nevertheless, the safety of lipotransfer has still not been clarified yet due to contradictory data. The goal of this in vitro study was to further elucidate the potential effects of lipotransfer on the irradiated remaining breast tissue. The mammary epithelial cell line MCF-10A was co-cultured with the fibroblast cell line MRC-5 and irradiated with 2 and 5 Gy. Afterwards, cells were treated with conditioned medium (CM) from adipose-derived stem cells (ADSC), and the effects on the cellular functions of MCF-10A cells and on gene expression at the mRNA level in MCF-10A and MRC-5 cells were analyzed. Treatment with ADSC CM stimulated transmigration and invasion and decreased the surviving fraction of MCF-10A cells. Further, the expression of cytokines, extracellular, and mesenchymal markers was enhanced in mammary epithelial cells. Only an effect of ADSC CM on irradiated fibroblasts could be observed. The present data suggest epithelial-mesenchymal transition-like changes in the epithelial mammary breast cell line. Thus, the benefits of lipotransfer after BCT should be critically weighed against its possible risks for the affected patients.

Fisetin induces DNA double-strand break and interferes with the repair of radiation-induced damage to radiosensitize triple negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is associated with aggressiveness and a poor prognosis. Besides surgery, radiotherapy serves as the major treatment modality for TNBC. However, response to radiotherapy is limited in many patients, most likely because of DNA damage response (DDR) signaling mediated radioresistance. Y-box binding protein-1 (YB-1) is a multifunctional protein that regulates the cancer hallmarks among them resisting to radiotherapy-induced cell death. Fisetin, is a plant flavonol of the flavonoid family of plant polyphenols that has anticancer properties, partially through inhibition of p90 ribosomal S6 kinase (RSK)-mediated YB-1 phosphorylation. The combination of fisetin with radiotherapy has not yet been investigated. METHODS: Activation status of the RSK signaling pathway in total cell lysate and in the subcellular fractions was analyzed by Western blotting. Standard clonogenic assay was applied to test post-irradiation cell survival. γH2AX foci assay and 3 color fluorescence in situ hybridization analyses were performed to study frequency of double-strand breaks (DSB) and chromosomal aberrations, respectively. The underlying repair pathways targeted by fisetin were studied in cells expressing genomically integrated reporter constructs for the DSB repair pathways via quantifying the expression of green fluorescence protein by flow cytometry. Flow cytometric quantification of sub-G1 cells and the protein expression of LC3-II were employed to measure apoptosis and autophagy, respectively. Kinase array and phosphoproteomics were performed to study the effect of fisetin on DDR response signaling. RESULTS: We showed that the effect of fisetin on YB-1 phosphorylation in TNBC cells is comparable to the effect of the RSK pharmacological inhibitors. Similar to ionizing radiation (IR), fisetin induces DSB. Additionally, fisetin impairs repair of IR-induced DSB through suppressing the classical non-homologous end-joining and homologous recombination repair pathways, leading to chromosomal aberration as tested by metaphase analysis. Effect of fisetin on DSB repair was partially dependent on YB-1 expression. Phosphoproteomic analysis revealed that fisetin inhibits DDR signaling, which leads to radiosensitization in TNBC cells, as shown in combination with single dose or fractionated doses irradiation. CONCLUSION: Fisetin acts as a DSB-inducing agent and simultaneously inhibits repair of IR-induced DSB. Thus, fisetin may serve as an effective therapeutic strategy to improve TNBC radiotherapy outcome.

Deep learning for brain metastasis detection and segmentation in longitudinal MRI data.

PURPOSE: Brain metastases (BM) occur frequently in patients with metastatic cancer. Early and accurate detection of BM is essential for treatment planning and prognosis in radiation therapy. Due to their tiny sizes and relatively low contrast, small BM are very difficult to detect manually. With the recent development of deep learning technologies, several res earchers have reported promising results in automated brain metastasis detection. However, the detection sensitivity is still not high enough for tiny BM, and integration into clinical practice in regard to differentiating true metastases from false positives (FPs) is challenging. METHODS: The DeepMedic network with the binary cross-entropy (BCE) loss is used as our baseline method. To improve brain metastasis detection performance, a custom detection loss called volume-level sensitivity-specificity (VSS) is proposed, which rates metastasis detection sensitivity and specificity at a (sub)volume level. As sensitivity and precision are always a trade-off, either a high sensitivity or a high precision can be achieved for brain metastasis detection by adjusting the weights in the VSS loss without decline in dice score coefficient for segmented metastases. To reduce metastasis-like structures being detected as FP metastases, a temporal prior volume is proposed as an additional input of DeepMedic. The modified network is called DeepMedic+ for distinction. Combining a high-sensitivity VSS loss and a high specificity loss for DeepMedic+, the majority of true positive metastases are confirmed with high specificity, while additional metastases candidates in each patient are marked with high sensitivity for detailed expert evaluation. RESULTS: Our proposed VSS loss improves the sensitivity of brain metastasis detection, increasing the sensitivity from 85.3% for DeepMedic with BCE to 97.5% for DeepMedic with VSS. Alternatively, the precision is improved from 69.1% for DeepMedic with BCE to 98.7% for DeepMedic with VSS. Comparing DeepMedic+ with DeepMedic with the same VSS loss, 44.4% of the FP metastases are reduced in the high-sensitivity model and the precision reaches 99.6% for the high-specificity model. The mean dice coefficient for all metastases is about 0.81. With the ensemble of the high-sensitivity and high-specificity models, on average only 1.5 FP metastases per patient need further check, while the majority of true positive metastases are confirmed. CONCLUSIONS: Our proposed VSS loss and temporal prior improve brain metastasis detection sensitivity and precision. The ensemble learning is able to distinguish high confidence true positive metastases from metastases candidates that require special expert review or further follow-up, being particularly well-fit to the requirements of expert support in real clinical practice. This facilitates metastasis detection and segmentation for neuroradiologists in diagnostic and radiation oncologists in therapeutic clinical applications.

Kinase inhibitors increase individual radiation sensitivity in normal cells of cancer patients.

PURPOSE: Kinase inhibitors (KI) are known to increase radiosensitivity, which can lead to increased risk of side effects. Data about interactions of commonly used KI with ionizing radiation on healthy tissue are rare. PATIENTS AND METHODS: Freshly drawn blood samples were analyzed using three-color FISH (fluorescence in situ hybridization) to measure individual radiosensitivity via chromosomal aberrations after irradiation (2 Gy). Thresholds of 0.5 and 0.6 breaks/metaphase (B/M) indicate moderate or clearly increased radiosensitivity. RESULTS: The cohorts consisted of healthy individuals (NEG, n = 219), radiosensitive patients (POS, n = 24), cancer patients (n = 452) and cancer patients during KI therapy (n = 49). In healthy individuals radiosensitivity (≥ 0.6 B/M) was clearly increased in 5% of all cases, while in the radiosensitive cohort 79% were elevated. KI therapy increased the rate of sensitive patients (≥ 0.6 B/M) to 35% significantly compared to 19% in cancer patients without KI (p = 0.014). Increased radiosensitivity of peripheral blood mononuclear cells (PBMCs) among patients occurred in six of seven KI subgroups. The mean B/M values significantly increased during KI therapy (0.47 ± 0.20 B/M without compared to 0.50 ± 0.19 B/M with KI, p = 0.047). CONCLUSIONS: Kinase inhibitors can intensify individual radiosensitivity of PBMCs distinctly in 85% of tested drugs.

PD-1 and PD-L1 expression predict regression and prognosis following neoadjuvant radiochemotherapy of oesophageal adenocarcinoma.

Background and purpose: PD-1 and PD-L1 are involved in anticancer immunosurveillance, and their expression may be predictive for therapeutic effectiveness of specific antibodies. Their influence on response to neoadjuvant radiochemotherapy (RCT) and prognosis in patients with oesophageal adenocarcinoma (OAC) remains to be defined. Materials and methods: Between 10/2004 and 06/2018, complete pre-RCT biopsy-specimens were available from 76 patients with locally advanced, non-metastatic OAC scheduled for trimodality therapy. We evaluated intra- and peritumoural expression of CD8, PD-1 and PD-L1 in pre-treatment specimens to determine their influence on tumour regression grade and survival. PD-1 and PD-L1 expression were considered positive (+) if ≥1% of all cells were stained positive, otherwise negative (-); densities of CD8+ cells were categorized as being high (Hi) or low (Lo) according to the median. Results: A negative PD-L1 expression in peritumoural cells predicted a poor tumour regression (RD 0.24 [95% CI 0.03-0.44], p = 0.023). A positive PD-1 expression in intra- as well as peritumoural cells was identified as an unfavourable prognostic factor (HR 0.52 [95% CI 0.29-0.93], p = 0.028; HR 0.50 [0.25-0.99], p = 0.047, respectively). With respect to CD8+ infiltration, positive PD-1 and PD-L1 expressions attenuated its favourable prognostic effect in intratumoural area (LoCD8/PD1 + vs. HiCD8/PD1-: HR 0.25 [0.09-0.69], p = 0.007; LoCD8/PDL1+ vs. HiCD8/PDL1-: HR 0.32 [0.12-0.89], p = 0.028) and were associated with negative outcome when seen in peritumoural area (HiCD8/PD1+ vs. LoCD8/PD1-: HR 0.29 [0.11-0.74], p = 0.010); HiCD8/PDL1+ vs. LoCD8/PDL1-: HR 0.33 [0.12-0.90], p = 0.031). Conclusions: PD-1 and PD-L1 expression were identified to be of predictive and prognostic value in patients with OAC, particularly when considering CD8+ infiltration. Further validation by a large size dataset is required.

Influence of alectinib and crizotinib on ionizing radiation - in vitro analysis of ALK/ROS1-wildtype lung tissue cells.

(1) Background: Just little is known about the interaction of ALK/ROS1-targeting kinase inhibitors with ionizing radiation (IR), particularly regarding side effects. We investigated the toxicity in two different lung cell lines both ALK/ROS1 wildtype (healthy and tumor origin) as representatives for normal lung tissue; (2) Methods: Human lung cell line BEAS-2B and malignant A549 lung cancer cells (ALK/ROS1 wt) were treated with alectinib or crizotinib, 2 Gy irradiation or a combination of KI and IR. Cell toxicity was analyzed by cell death (Annexin, 7AAD), colony forming, migration assay and live-cell imaging (TMRM, DRAQ7, Caspase3/7). Cell cycle (Hoechst) were analyzed by flow cytometry; (3) Results: Crizotinib led to higher cell death rates than alectinib, when cells were treated with 10 µM KI. Alectinib induced a more intense growth inhibition of colonies. Both inhibitors showed additive effects in combination with irradiation. Combination treatment (IR + KI) does not lead to synergistic effect on neither cell death nor colony forming; (4) Conclusions: The influence of simultaneous KI and IR was studied in non-mutated ALK/ROS1 cell lines. Both KIs seems to be well tolerated in combination with thoracic radiotherapy and lacked synergistic reinforcement in cellular toxicity. This supports the feasibility of ALK/ROS1 inhibition in combination with thoracic irradiation in future clinical trials.

Baseline Quality of Life of Physical Function Is Highly Relevant for Overall Survival in Advanced Rectal Cancer.

In advanced rectal cancer, neoadjuvant radiochemotherapy and total mesorectal excision lead to long overall survival. The quality of life (QOL) of the patients is clearly related to the prognosis. Our question was whether the prognosis can be represented with only one question or one score from the QOL questionnaires. 360 consecutively recruited patients diagnosed with advanced rectal cancer were questioned during radiochemotherapy and a follow-up of 8 years. The questionnaires QLQ-C30 and QLQ-CR38 were used; 10 functional and 17 symptom scores were calculated. The functional score "physical function" and the symptom scores "fatigue", "nausea and vomiting", "pain" and "appetite loss" were highly prognostic (p < 0.001) for overall survival. "Physical function" was highly prognostic at all time points up to 1 year after starting therapy (p ≤ 0.001). The baseline "physical function" score divided the cohort into a favorable group with an 8-year overall survival rate of 70.4% versus an unfavorable group with 47.5%. In the multivariable analysis, baseline "physical function", age and distant metastases were independent predictors of overall survival. The score "physical function" is a powerful unrelated risk factor for overall survival in patients with rectal cancer. Future analyses should study whether increased "physical function" after diagnosis could improve survival.

Palbociclib Induces Senescence in Melanoma and Breast Cancer Cells and Leads to Additive Growth Arrest in Combination With Irradiation.

INTRODUCTION: Several kinase inhibitors (KI) bear the potential to act as radiosensitizers. Little is known of the radiosensitizing effects of a wide range of other KI like palbociclib, which is approved in ER+/HER2- metastatic breast cancer. METHOD: In our study, we used healthy donor fibroblasts and breast cancer and skin cancer cells to investigate the influence of a concomitant KI + radiation therapy. Cell death and cell cycle distribution were studied by flow cytometry after Annexin-V/7-AAD and Hoechst staining. Cellular growth arrest was studied in colony-forming assays. Furthermore, we used C12-FDG staining (senescence) and mRNA expression analysis (qPCR) to clarify cellular mechanisms. RESULTS: The CDK4/6 inhibitor palbociclib induced a cell cycle arrest in the G0/G1 phase. Cellular toxicity (cell death) was only slightly increased by palbociclib and not enhanced by additional radiotherapy. As the main outcome of the colony formation assays, we found that cellular growth arrest was induced by palbociclib and improved by radiotherapy in an additive manner. Noticeably, palbociclib treatment clearly induced senescence not only in breast cancer and partly in melanoma cells, but also in healthy fibroblasts. According to these findings, the downregulation of senescence-related FOXM1 might be an involved mechanism of the senescence-induction potential of palbociclib. CONCLUSION: The effect on cellular growth arrest of palbociclib and radiotherapy is additive. Palbociclib induces permanent G0/G1 cell cycle arrest by inducing senescence in fibroblasts, breast cancer, and melanoma cells. Direct cell death induction is only a minor secondary mechanism of action. Concomitant KI and radiotherapy is a strategy worth studying in clinical trials.

Cell-in-cell phenomenon: leukocyte engulfment by non-tumorigenic cells and cancer cell lines.

BACKGROUND: Research on cell-in-cell (CIC) phenomena, including entosis, emperipolesis and cannibalism, and their biological implications has increased in recent years. Homotypic and heterotypic engulfment of various target cells by numerous types of host cells has been studied in vitro and in tissue sections. This work has identified proteins involved in the mechanism and uncovered evidence for CIC as a potential histopathologic predictive and prognostic marker in cancer. Our experimental study focused on non-professional phagocytosis of leukocytes. RESULTS: We studied the engulfment of peripheral blood mononuclear cells isolated from healthy donors by counting CIC structures. Two non-tumorigenic cell lines (BEAS-2B, SBLF-9) and two tumour cell lines (BxPC3, ICNI) served as host cells. Immune cells were live-stained and either directly co-incubated or treated with irradiation or with conventional or microwave hyperthermia. Prior to co-incubation, we determined leukocyte viability for each batch via Annexin V-FITC/propidium iodide staining. All host cells engulfed their targets, with uptake rates ranging from 1.0% ± 0.5% in BxPC3 to 8.1% ± 5.0% in BEAS-2B. Engulfment rates of the cancer cell lines BxPC3 and ICNI (1.6% ± 0.2%) were similar to those of the primary fibroblasts SBLF-9 (1.4% ± 0.2%). We found a significant negative correlation between leukocyte viability and cell-in-cell formation rates. The engulfment rate rose when we increased the dose of radiotherapy and prolonged the impact time. Further, microwave hyperthermia induced higher leukocyte uptake than conventional hyperthermia. Using fluorescent immunocytochemistry to descriptively study the proteins involved, we detected ring-like formations of diverse proteins around the leukocytes, consisting, among others, of α-tubulin, integrin, myosin, F-actin, and vinculin. These results suggest the involvement of actomyosin contraction, cell-cell adhesion, and the α-tubulin cytoskeleton in the engulfment process. CONCLUSIONS: Both non-tumorigenic and cancer cells can form heterotypic CIC structures by engulfing leukocytes. Decreased viability and changes caused by microwave and X-ray irradiation trigger non-professional phagocytosis.

Kinase Inhibitors of DNA-PK, ATM and ATR in Combination with Ionizing Radiation Can Increase Tumor Cell Death in HNSCC Cells While Sparing Normal Tissue Cells.

(1) Kinase inhibitors (KI) targeting components of the DNA damage repair pathway are a promising new type of drug. Combining them with ionizing radiation therapy (IR), which is commonly used for treatment of head and neck tumors, could improve tumor control, but could also increase negative side effects on surrounding normal tissue. (2) The effect of KI of the DDR (ATMi: AZD0156; ATRi: VE-822, dual DNA-PKi/mTORi: CC-115) in combination with IR on HPV-positive and HPV-negative HNSCC and healthy skin cells was analyzed. Cell death and cell cycle arrest were determined using flow cytometry. Additionally, clonogenic survival and migration were analyzed. (3) Studied HNSCC cell lines reacted differently to DDRi. An increase in cell death for all of the malignant cells could be observed when combining IR and KI. Healthy fibroblasts were not affected by simultaneous treatment. Migration was partially impaired. Influence on the cell cycle varied between the cell lines and inhibitors; (4) In conclusion, a combination of DDRi with IR could be feasible for patients with HNSCC. Side effects on healthy cells are expected to be limited to normal radiation-induced response. Formation of metastases could be decreased because cell migration is impaired partially. The treatment outcome for HPV-negative tumors tends to be improved by combined treatment.