Search

Hypoxia is now established as a key factor influencing the pathophysiology of malignant growth. Among other effects, hypoxia modulates the expression of a multitude of genes through the induction of hypoxia-inducible transcription factors. This differential gene expression favors angiogenesis, cell survival, an invasive/metastatic phenotype, and resistance to anticancer therapies. Because benign tumors do not exhibit these traits, one might expect these entities to be neither hypoxic nor to induce the genetic hypoxia response program. To test this hypothesis, an investigation of the oxygenation status of 17 leiomyomas and 1 leiomyosarcoma of the uterus using polarographic needle electrodes (Eppendorf pO2 sensor) and the expression of hypoxia-related markers in biopsy specimens of the same tumors was carried out. Marker expression in eight additional archival leiomyosarcomas was also assessed. Leiomyoma tissue was generally found to be severely hypoxic, with median oxygen (O2) partial pressure values ranging from 1 to 5 mm Hg. In contrast, none of the hypoxia-related markers hypoxia-inducible factor (HIF)–1α, HIF-2α, glucose transporter-1, or carbonic anhydrase IX were expressed in any leiomyoma. Larger intercapillary distances were correlated with a poorer oxygenation status. Conversely, the expression of hypoxia-related markers was abundant in the leiomyosarcomas and they also exhibited a high-turnover phenotype (significantly increased proliferation and apoptosis). Uterine leiomyoma might therefore represent a state of oxygen-limited proliferation. Malignancy in the same organ system is associated with growth and metabolism beyond tissue-inherent limitations leading to the induction of hypoxia-related markers, thereby contributing to a self-perpetuating aggressive phenotype. [Cancer Res 2008;68(12):4719–26]

Supplementary Table 1 from Lack of Hypoxic Response in Uterine Leiomyomas despite Severe Tissue Hypoxia

Background and purposeTo evaluate the tolerability and outcomes of chemoradiation in elderly patients with locally advanced esophageal squamous cell carcinoma (ESCC).Materials and methodsThis multi-center retrospective analysis included 161 patients with SCC of the esophagus with a median age of 73 years (range 65-89 years) treated with definitive or neoadjuvant (chemo)radiotherapy between 2010 and 2019 at 3 large comprehensive cancer centers in Germany. Locoregional control (LRC), progression-free survival (PFS), distant metastasis-free survival (DMFS), overall survival (OS), and treatment-associated toxicities were analyzed, and parameters determining patient outcomes and treatment tolerance were assessed.ResultsThe delivery of radiotherapy without dose reduction was possible in 149 patients (93%). In 134 patients (83%), concomitant chemotherapy was initially prescribed; however, during the course of therapy, 41% of these patients (n = 55) required chemotherapy de-escalation due to treatment-related toxicities. Fifty-two patients (32%) experienced higher-grade acute toxicities, and 22 patients (14%) higher-grade late toxicities. The 2-year LRC, DMFS, PFS, and OS rates amounted to 67.5%, 33.8%, 31.4%, and 40.4%, respectively. Upon multivariate analysis, full-dose concomitant chemotherapy (vs. no or modified chemotherapy) was associated with significantly better DMFS (p=0.005), PFS (p=0.005) and OS (p=0.001). Furthermore, neoadjuvant chemoradiotherapy followed by tumor resection (vs. definitive chemoradiotherapy or definitive radiotherapy alone) significantly improved PFS (p=0.043) and OS (p=0.049). We could not identify any clinico-pathological factor that was significantly associated with LRC. Furthermore, definitive (chemo)radiotherapy, brachytherapy boost and stent implantation were significantly associated with higher-grade acute toxicities (pConclusionsChemoradiation with full-dose and unmodified concurrent chemotherapy has a favorable prognostic impact in elderly ESCC patients; however, about half of the analyzed patients required omission or adjustment of chemotherapy due to comorbidities or toxicities. Therefore, the identification of potential predictive factors for safe administration of concurrent chemotherapy in elderly ESCC patients requires further exploration to optimize treatment in this vulnerable patient cohort.

ImportanceThe number of older adults with head and neck squamous cell carcinoma (HNSCC) is increasing, and these patients are underrepresented in clinical trials. It is unclear whether the addition of chemotherapy or cetuximab to radiotherapy is associated with improved survival in older adults with HNSCC.ObjectiveTo examine whether the addition of chemotherapy or cetuximab to definitive radiotherapy is associated with improved survival in patients with locoregionally advanced (LA) HNSCC.Design, Setting, and ParticipantsThe Special Care Patterns for Elderly HNSCC Patients Undergoing Radiotherapy (SENIOR) study is an international, multicenter cohort study including older adults (≥65 years) with LA-HNSCCs of the oral cavity, oropharynx/hypopharynx, or larynx treated with definitive radiotherapy, either alone or with concomitant systemic treatment, between January 2005 and December 2019 at 12 academic centers in the US and Europe. Data analysis was conducted from June 4 to August 10, 2022.InterventionsAll patients underwent definitive radiotherapy alone or with concomitant systemic treatment.Main Outcomes and MeasuresThe primary outcome was overall survival. Secondary outcomes included progression-free survival and locoregional failure rate.ResultsAmong the 1044 patients (734 men [70.3%]; median [IQR] age, 73 [69-78] years) included in this study, 234 patients (22.4%) were treated with radiotherapy alone and 810 patients (77.6%) received concomitant systemic treatment with chemotherapy (677 [64.8%]) or cetuximab (133 [12.7%]). Using inverse probability weighting to attribute for selection bias, chemoradiation was associated with longer overall survival than radiotherapy alone (hazard ratio [HR], 0.61; 95% CI, 0.48-0.77; P P = .70). Progression-free survival was also longer after the addition of chemotherapy (HR, 0.65; 95% CI, 0.52-0.81; P P = .19). The survival benefit of the chemoradiation group was present in patients up to age 80 years (65-69 years: HR, 0.52; 95% CI, 0.33-0.82; 70-79 years: HR, 0.60; 95% CI, 0.43-0.85), but was absent in patients aged 80 years or older (HR, 0.89; 95% CI, 0.56-1.41).Conclusions and RelevanceIn this cohort study of older adults with LA- HNSCC, chemoradiation, but not cetuximab-based bioradiotherapy, was associated with longer survival compared with radiotherapy alone.

Background and PurposeTo compare two validated spinal instability scores regarding the stabilizing effects and skeletal-related events (SREs) of palliative radiotherapy (RT) in patients with spinal bone metastases (SBM).Materials and MethodsTwo hundred eighty-two osteolytic SBM of lung or breast cancer patients were analyzed for stability before and following RT based on the Spinal Instability Neoplastic Score (SINS) or the Taneichi score. Score concordance was quantified by absolute agreement and Cohen’s kappa coefficient. SREs were defined as fractures or local progression after RT. OS was quantified as the time between the start of RT and death from any cause.ResultsAt 3 and 6 months after RT, 35 and 50% of initially unstable SBM were re-stabilized according to SINS in patients still alive. Corresponding Taneichi score-based stabilization proportions were 25 and 46%, respectively. Comparison of both stability scores showed high absolute agreement for all time-points (range 71–78%, kappa range 0.35–0.44). SRE occurred more frequently in initially unstable SBM compared to stable SBM according to SINS (14 vs. 5%), but no such association could be shown for the Taneichi-based instability criterion. Poor general condition of patients was negatively associated with SINS-measured re-stabilization after 6 months, but no predictive factor for re-stabilization could be found for the Taneichi score.ConclusionsDespite the relatively high agreement between both stabilization scores, the SINS should be considered the standard for future studies on the stabilization effects of RT in SBM.

Purpose About one fifth of patients with head and neck cancer are aged 70 years and older at the time of diagnosis. In these patients, risk factors (R1 status or extracapsular extension of lymph node metastases, ECE) often lead to a need for combined chemoradiotherapy (CRT) in the postoperative setting. However, there is considerable concern about the toxicity of such therapy in this age group. Methods Retrospective evaluation of the data of 53 patients ≥ 70 years of age who underwent surgery in our hospital between 1999 and 2015 for tumors of the oral cavity, the oropharynx, the hypopharynx, or the larynx, who subsequently received adjuvant radiation therapy. Two younger patients ( Results Univariate analyses revealed a statistically significant influence of many factors on overall survival (OS) and progression-free survival (PFS), including Karnofsky performance score (KPS), alcohol consumption, smoking, R status, ECE, chemotherapy, and discontinuation of RT. Younger patients had better OS and PFS compared to the elderly (p = 0.013 and 0.012, respectively). In a multivariate Cox regression, no independent influence of age on OS and PFS was found. Survival was primarily dependent on the addition of chemotherapy to radiotherapy (RT), application of the full course of RT, continued alcohol abuse, KPS, and the presence of ECE. Toxicity analysis showed a higher incidence of chronic renal failure but, generally, side effects for elderly patients were not substantially greater. Conclusion Performance status and behavioral risk factors but not chronological age are crucial for the prognosis of patients who require adjuvant chemoradiation.

This retrospective study aimed to evaluate the stability and fracture rates of osteolytic spinal bone metastases (SBM) in elderly patients following palliative radiotherapy (RT) and to derive prognostic factors for stability and survival. A total of 322 patients aged at least 70 years received palliative RT at two major German academic medical centers or at the German Cancer Research Center. Stability assessment was based on the validated Taneichi score prior to RT and at 3 and 6 months after RT. The survival time following RT was assessed, and prognostic factors for stability and survival were analyzed. Prior to RT, 183 patients (57%) exhibited unstable SBM and 68 patients (21%) pathological fractures. At 3 and 6 months after RT, significant recalcification and stabilization were evident in 19% (23/118) and 40% (31/78) of surviving patients, respectively. Only 17 patients (5%) experienced new pathological fractures following RT. Tumor histology was found to significantly influence stabilization rates with only breast cancer patients exhibiting increased stabilization compared to patients with other histologies. The median survival time and 6‑month survival rates following RT were 5.4 months (95% confidence interval 4.4–7.2 months) and 48%, respectively. The patients’ performance status was found to be the strongest predictor for survival after RT in this patient cohort; further factors demonstrating a significant association with survival were the application of systemic treatment, the number of SBM and the primary tumor histology. To analyze the influence of age on survival after RT, study patients were stratified into 3 age groups (i.e., 70–74 years, 75–79 years, and ≥80 years). The subgroup of patients aged at least 80 years showed a strong trend towards a worse survival time following RT compared to younger patients (i.e., 6‑month survival rate 39% vs. 51%; p = 0.06, log-rank test). Prognostic factors influencing overall survival such as performance status and histology should guide the choice for palliative RT for SBM. Strongly hypofractionated RT regimes may be advisable for most elderly patients considering the overall poor prognosis in order to reduce hospitalization times.

Simple Summary Progress in the field of in situ proteomics allows for the simultaneous detection of multiple biomarkers within one cancer tissue specimen. As a result, biological hypotheses previously only assessable ex vivo can now be studied in human cancer tissue. However, methods for objective analysis have so far been lacking behind. In this study, we established a free, objective, and entirely open-source-based method for the analysis of multiplexed immunofluorescence specimens. This will gain further importance with the availability of more advanced multiplexing methods in the future. Abstract (1) Background: The immune system has physiological antitumor activity, which is partially mediated by cytotoxic T lymphocytes (CTL). Tumor hypoxia, which is highly prevalent in cancers of the head and neck region, has been hypothesized to inhibit the infiltration of tumors by CTL. In situ data validating this concept have so far been based solely upon the visual assessment of the distribution of CTL. Here, we have established a set of spatial statistical tools to address this problem mathematically and tested their performance. (2) Patients and Methods: We have analyzed regions of interest (ROI) of 22 specimens of cancers of the head and neck region after 4-plex immunofluorescence staining and whole-slide scanning. Single cell-based segmentation was carried out in QuPath. Specimens were analyzed with the endpoints clustering and interactions between CTL, normoxic, and hypoxic tumor areas, both visually and using spatial statistical tools implemented in the R package Spatstat. (3) Results: Visual assessment suggested clustering of CTL in all instances. The visual analysis also suggested an inhibitory effect between hypoxic tumor areas and CTL in a minority of the whole-slide scans (9 of 22, 41%). Conversely, the objective mathematical analysis in Spatstat demonstrated statistically significant inhibitory interactions between hypoxia and CTL accumulation in a substantially higher number of specimens (16 of 22, 73%). It showed a similar trend in all but one of the remaining samples. (4) Conclusion: Our findings provide non-obvious but statistically rigorous evidence of inhibition of CTL infiltration into hypoxic tumor subregions of cancers of the head and neck. Importantly, these shielded sites may be the origin of tumor recurrences. We provide the methodology for the transfer of our statistical approach to similar questions. We discuss why versions of the Kcross and pcf.cross functions may be the methods of choice among the repertoire of statistical tests in Spatstat for this type of analysis.

Simple Summary The introduction of immune-checkpoint inhibitors improved the therapeutic landscape for patients with advanced malignant melanoma. However, many patients, including patients with melanoma brain metastases, do not derive benefit from immune-checkpoint blockade. Hence, biomarkers are needed to identify potential mechanisms of resistance and optimize patient selection. This study aimed to explore the role of hypoxia-mediated immunosuppression within the tumor microenvironment of patients with metastatic melanoma using multiplex immunofluorescence. We analyzed the prognostic relevance of the hypoxia surrogate marker GLUT-1, the adenosine-synthesizing ectoenzymes CD73/CD39, and the infiltration by CD8 positive lymphocytes, and evaluated their spatial interaction within the tumor microenvironment (TME). Finally, we outlined the role of the melanoma immune phenotype for the patient’s prognosis and discussed the importance of tumor hypoxia and the adenosine system in shaping the tumor immune phenotype. Abstract Following the introduction of immune checkpoint inhibitors, a substantial prolongation of the overall survival has been achieved for many patients with multiple brain metastases from melanoma. However, heterogeneity between individual tumor responses is incompletely understood. In order to determine the impact of the individual tumor phenotype on the prognosis of melanoma patients, we examined surgical sections from 33 patients who were treated with radiotherapy (whole-brain radiotherapy, WBRT, stereotactic radiotherapy, STX, or both) and Ipilimumab. We analyzed multiplex staining of the hypoxia marker GLUT-1, the adenosine (ADO)-associated enzymes CD73 and CD39, and CD8, a marker of cytotoxic T lymphocytes (CTL) on a single-cell basis using QuPath. Additionally, the MOSAIC interaction analysis algorithm was used to explore the hypothesis that CTL systematically avoid GLUT-1high tumor areas. Our results revealed, that a strong GLUT-1 expression, low numbers of CTL, or exclusion of CTL from the tumor were correlated with significant prognostic detriment. Hypoxic tumors overall have smaller amounts of CTL, and spatial analysis revealed a repellent effect of hypoxia on CTL. In contrast to in vitro studies, specific upregulation of ADO-related enzymes CD73 and CD39 in GLUT-1high tumor regions was never observed. In this study, we could show direct in vivo evidence for hypoxia-mediated immunosuppression in melanoma. Moreover, this study suggests a significant prognostic relevance of the tumor immune phenotype, the strength of CD8 infiltration in the tumor, and the expression of hypoxia marker GLUT-1 on melanoma cells. Last, our results suggest a temporal stability of the microenvironment-mediated immunosuppressive phenotype in melanoma.

Abundance and signaling of the epidermal growth factor receptor (EGFR) and programmed cell death protein ligand 1 (PD-L1) in head and neck squamous cell carcinoma (HNSCC) are not only genetically determined but are also subject to the traits of the tumor microenvironment, which has hitherto not been clarified completely. We investigated the impact of hypoxia on the EGFR system and on PD-L1 in six HPV negative HNSCC cell lines in vitro and in FaDu xenografts in vivo. Protein levels of EGFR, AKT, pAKT, ERK1/2, pERK1/2, CA IX, cleaved PARP (apoptosis), LC3B (autophagy), and PD-L1 were quantified by western blot after oxygen deprivation or CoCl2, staurosporine, and erlotinib treatment. In FaDu xenograft tumors the expression of EGFR, CA IX andCD34 staining were analyzed. Reduced oxygen supply strongly downregulated EGFR protein levels and signaling in FaDu cells in vitro and in vivo, and a transient downregulation of EGFR signaling was found in three other HNSCC cell lines. PD-L1 was affected by oxygen deprivation in only one HNSCC cell line showing increased protein amounts. The results of this study indicate a significant impact of the traits of the tumor microenvironment on crucial molecular targets of cancer therapies with high clinical relevance for therapy resistance and response in HNSCC.

Background To date, only limited magnetic resonance imaging (MRI) data are available concerning tumor regression during neoadjuvant radiochemotherapy (RCT) of rectal cancer patients, which is a prerequisite for adaptive radiotherapy (RT) concepts. This exploratory study prospectively evaluated daily fractional MRI during neoadjuvant treatment to analyze the predictive value of MR biomarkers for treatment response. Methods Locally advanced rectal cancer patients were examined with daily MRI during neoadjuvant RCT. Contouring of the tumor volume was performed for each MRI scan by using T2- and diffusion-weighted-imaging (DWI)-sequences. The daily apparent-diffusion coefficient (ADC) was calculated. Volumetric and functional tumor changes during RCT were analyzed and correlated with the pathological response after surgical resection. Results In total, 171 MRI scans of eight patients were analyzed regarding anatomical and functional dynamics during RCT. Pathological complete response (pCR) could be achieved in four patients, and four patients had a pathological partial response (pPR) following neoadjuvant treatment. T2- and DWI-based volumetry proved to be statistically significant in terms of therapeutic response, and volumetric thresholds at week two and week four during RCT were defined for the prediction of pCR. In contrast, the average tumor ADC values widely overlapped between both response groups during RCT and appeared inadequate to predict treatment response in our patient cohort. Conclusion This prospective exploratory study supports the hypothesis that MRI may be able to predict pCR of rectal cancers early during neoadjuvant RCT. Our data therefore provide a useful template to tailor future MR-guided adaptive treatment concepts.

This retrospective multi-center analysis aimed to assess the clinical response and stabilizing effects of palliative radiotherapy (RT) for spinal bone metastases (SBM) in head and neck cancer (HNC), and to establish potential predictive factors for stability and overall survival (OS). Patients included in this analysis were treated at the University Hospitals of Mainz, Freiburg, and Heidelberg between 2001 and 2019. Clinical information was taken from the medical records. The stability of affected vertebral bodies was assessed according to the validated spine instability neoplastic score (SINS) based on CT-imaging before RT, as well as 3 and 6 months after RT. OS was quantified as the time between the start of palliative RT and death from any cause or last follow-up. Potential predictive factors for stability and OS were analyzed using generalized estimating equations and Cox regression for time-varying covariates to take into account multiple observations per patient. The mean follow-up time of 66 included patients after the first palliative RT was 8.1 months (range 0.3&ndash, 85.0 months). The majority of patients (70%, n = 46) had squamous cell carcinomas (SCC) originating from the pharynx, larynx and oral cavity, while most of the remaining patients (26%, n = 17) suffered from salivary glands tumors. A total of 95 target volumes including 178 SBM were evaluated that received a total of 81 irradiation series. In patients with more than one metastasis per irradiated region, only the most critical bone metastasis was analyzed according to the SINS system. Prior to RT, pain and neurologic deficits were present in 76% (n = 72) and 22% (n = 21) of irradiated lesions, respectively, and 68% of the irradiated lesions (n = 65) were assessed as unstable or potentially unstable prior to RT. SBM-related pain symptoms and neurologic deficits responded to RT in 63% and 47% of the treated lesions, respectively. Among patients still alive at 3 and 6 months after RT with potentially unstable or unstable SBM, a shift to a better stability class according to the SINS was observed in 20% and 33% of the irradiated SBM, respectively. Pathological fractures of SBM were frequently detected before the start of irradiation (43%, n = 41), but after RT, new fractures or increasing vertebral body sintering within the irradiated region occurred rarely (8%, n = 8). A pathological fracture before RT was negatively associated with stabilization 6 months after RT (OR 0.1, 95% CI 0.02&ndash, 0.49, p = 0.004), while a Karnofsky performance score (KPS) &ge, 70% was associated positively with a stabilization effect through irradiation (OR 6.09, 95% CI 1.68&ndash, 22.05, p = 0.006). Mean OS following first palliative RT was 10.7 months, and the KPS (&ge, 70% vs. &lt, 70%) was shown to be a strong predictive factor for OS after RT (HR 0.197, 95% CI 0.11&ndash, 0.35, p &lt, 0.001). There was no significant difference in OS between patients with SCC and non-SCC. Palliative RT in symptomatic SBM of HNC provides sufficient symptom relief in the majority of patients, while only about one third of initially unstable SBM show re-stabilization after RT. Since patients in our multi-center cohort exhibited very limited OS, fractionation schemes should be determined depending on the patients&rsquo, performance status.

BackgroundThis study aimed to analyze the oncological long-term results and late toxicity of carbon ion-based radiotherapy (RT) of patients with sacral chordoma and to identify potential prognostic factors for local control (LC) and overall survival (OS). MethodsA total of 68 patients with sacral chordoma (median age 61 years, range 34 - 84 years) treated at the Heidelberg Ion Beam Therapy Center were included in this study. Of these 52 patients (77%) received a primary RT and 16 patients (23%) received a RT in a recurrent situation. All patients were treated with carbon ion RT, either in combination with photons or as a monotherapy, with a median radiation dose of 66 Gy RBE (range 60 - 74 Gy). In 40 patients (59%), RT was performed in the postoperative situation. Postoperative care included regular MRI scans, which were performed in 3-month intervals in the first year and in 6-month intervals in the following years. Local progression was defined as an enlargement of the maximum tumor diameter by 10% or a new tumor growth within the planning target volume (PTV). LC and OS were determined using the Kaplan-Meier method. Furthermore, the relevance of various prognostic factors for LC and OS was assessed by univariate and multivariate analysis.Results:The median follow-up period was 60 months (range 1 - 96 months). The 5-year rates for LC, metastasis-free and disease-free survival and OS were 43%, 82%, 44% and 82%, respectively. Local recurrence was observed in 31 patients (46%), occurring after a median follow-up time of 24 months (range 2 – 72 months). Only 10% of local recurrences occurred later than 5 years after RT. The univariate analysis showed a statistical significance for the initial macroscopic tumor volume (GTV) and a strong trend for the therapy situation (primary vs. recurrence situation) to predict a local tumor recurrence. In the log-rank test and univariate analysis, the age of patients, size of the GTV and PTV were identified as strong predictors of OS after RT of sacral chordoma. In the multivariate testing, borderline significance was evident for the therapeutic situation (primary vs. recurrence situation) to predict local tumor recurrence, while none of the tested factors were significant for the prediction of OS after RT.The incidence of late toxicity ≥ III° according to CTCAE v5.0 was 21%. Sacral insufficiency fractures occurred in 49% of patients (maximum III°: 16%) and were thus by far the most frequent late side effect in our analysis. Radiogenic damage to the peripheral nerves, intestinal tract and skin was observed in only 9% (≥ III°: 5%), 3% (all II°) and 9% (all I°) of patients.Conclusion Our analysis showed only moderate long-term local control rates after carbon ion-based RT, with sacral chordomas having a particularly poor prognosis in the recurrent situation. A recent study (ISAC) evaluates the safety and effectiveness of further dose escalation and hypofractionation of proton and carbon ion therapy in sacral chordoma. Here, the potential benefits of dose escalation must be weighed against the possible side effects.

Purpose Since the introduction of ipilimumab (IPI) for the treatment of patients with metastatic malignant melanoma, we have observed remarkable responses after hypofractionated whole brain irradiation (WBRT) or stereotactic radiotherapy (STX) for brain metastases of malignant melanoma. We sought to investigate the impact of the sequence of these treatment modalities. Methods We retrospectively evaluated the survival of melanoma patients with brain metastases who were treated with WBRT or STX and received IPI in close temporal relation between October 2010 and March 2015. Follow-up was obtained until November 2016. A total of 27 patients with advanced melanoma and brain metastases who were treated with WBRT before 2010, and who had not received IPI, served as historical controls. Results We identified a total of 41 patients of whom 15 were treated with STX, 7 with a combination of STX and WBRT and 19 with WBRT alone. All patients received at least 2 doses of IPI. The median time interval between radiotherapy and IPI was 2 months. Patients treated with IPI after radiotherapy had a censored median survival of 11 months, compared with 3 months for the patients who received IPI prior to radiotherapy. Patients who received IPI before radiotherapy showed a similar survival as historical controls, who had not received IPI. We observed long-term survivors after radiotherapy of brain metastases followed by IPI. Conclusions These data suggest that the sequence of RT and immune checkpoint inhibition with IPI may be crucial for the success of combined modality treatment of melanoma brain metastases.

Background Radioresistance is a common feature of head and neck squamous cell carcinoma (HNSCC). We previously showed that the irradiation- activated vascular endothelial growth factor (VEGF)-extracellular signal-regulated kinase (ERK)-axis is fundamental for the survival of resistant tumors. In this study, we examined if treatment with potent multikinase (MK) inhibitors, sorafenib and sunitinib, could radiosensitize tumor cells. Methods Cultured HNSCC cell lines were treated with inhibitors and subsequently irradiated. Radiosensitizing effects were functionally assessed by annexin-V apoptosis and clonogenic assays and confirmed by Western blot. Additionally, we surveyed human HNSCC tissue microarrays (TMAs) for activated ERK expression. Results Based on combination indexes, we found that combining irradiation with both inhibitors exerted strong and supra-additive antitumor effects on clonogenic survival. Kinase inhibition enhanced irradiation-induced apoptotic rates and inhibited postradiogenic phospho-ERK-expression. Patients with recurrent HNSCC displayed significantly lower extracellular signal-regulated kinase phosphorylation (pERK) levels than relapse-free patients. Conclusion We propose further evaluation of sorafenib and sunitinib as potential radiosensitizing agents in HNSCC treatment. © 2017 Wiley Periodicals, Inc. Head Neck 39: 623–632, 2017

Background: The receptor for the epidermal growth factor (EGFR) is widely considered to be one of the central drivers of oncogenesis in squamous cell carcinomas of the head and neck region (HNSCC). Inhibition of EGFR using monoclonal antibodies is established both in the curative and the palliative setting in this disease. HNSCCs are known to contain abundant hypoxic tissue areas and hypoxia has been shown to be involved in the (down)regulation of EGFR membrane expression. Methods: A novel method for multiplex immunofluorescence and single-cell segmentation (via DAPI-stained nuclei) was established, to study the expression of EGFR, the endogenous hypoxia marker CA IX, and intratumoural diffusion distances from microvessels (using CD34 staining) in 58 human HNSCCs and 9 normal/cancer adjacent tissues. Results: EGFR was found to be significantly downregulated with increasing distance from tumour microvessels, whereas the opposite was true for CA IX. Larger diffusion-limited areas were correlated with higher expression of CA IX. Conclusions: The hypoxic tumour microenvironment may have a major role in mediating resistance against anti-EGFR strategies by downregulating EGFR molecules on tumour cells.

In the early 1920s, Warburg published experimental data on the enhanced conversion of glucose to pyruvate (followed by lactate formation) even in the presence of abundant oxygen (aerobic glycolysis, Warburg effect). He attributed this metabolic trait to a respiratory injury and considered this a universal metabolic alteration in carcinogenesis. This interpretation of the data was questioned since the early 1950s. Realistic causative mechanisms and consequences of the Warburg effect were described only during the past 15 years and are summarized in this article. There is clear evidence that mitochondria are not defective in most cancers. Aerobic glycolysis, a key metabolic feature of the Warburg phenotype, is caused by active metabolic reprogramming required to support sustained cancer cell proliferation and malignant progression. This metabolic switch is directed by altered growth factor signaling, hypoxic or normoxic activation of HIF-1α- transcription, oncogene activation or loss-of-function of suppressor genes, and is implemented in the hostile tumor microenvironment. The 'selfish' reprogramming includes (a) overexpression of glucose transporters and of key glycolytic enzymes, and an accelerated glycolytic flux with subsequent accumulation and diversion of glycolytic intermediates for cancer biomass synthesis, (b) high-speed ATP production that meets the energy demand, and (c) accumulation of lactate which drives tumor progression and largely contributes to tumor acidosis, which in turn synergistically favors tumor progression and resistance to certain antitumor therapies, and compromises antitumor immunity. Altogether, the Warburg effect is the central contributor to the cancer progression machinery.

Oncogenic HPV types are major human carcinogens. Under hypoxia, HPV-positive cancer cells can repress the viral E6/E7 oncogenes and induce a reversible growth arrest. This response could contribute to therapy resistance, immune evasion, and tumor recurrence upon reoxygenation. Here, we uncover evidence that HPV oncogene repression is mediated by hypoxia-induced activation of canonical PI3K/mTORC2/AKT signaling. AKT-dependent downregulation of E6/E7 is only observed under hypoxia and occurs, at least in part, at the transcriptional level. Quantitative proteome analyses identify additional factors as candidates to be involved in AKT-dependent E6/E7 repression and/or hypoxic PI3K/mTORC2/AKT activation. These results connect PI3K/mTORC2/AKT signaling with HPV oncogene regulation, providing new mechanistic insights into the cross talk between oncogenic HPVs and their host cells., Hypoxia is linked to therapeutic resistance and poor clinical prognosis for many tumor entities, including human papillomavirus (HPV)-positive cancers. Notably, HPV-positive cancer cells can induce a dormant state under hypoxia, characterized by a reversible growth arrest and strong repression of viral E6/E7 oncogene expression, which could contribute to therapy resistance, immune evasion and tumor recurrence. The present work aimed to gain mechanistic insights into the pathway(s) underlying HPV oncogene repression under hypoxia. We show that E6/E7 downregulation is mediated by hypoxia-induced stimulation of AKT signaling. Ablating AKT function in hypoxic HPV-positive cancer cells by using chemical inhibitors efficiently counteracts E6/E7 repression. Isoform-specific activation or downregulation of AKT1 and AKT2 reveals that both AKT isoforms contribute to hypoxic E6/E7 repression and act in a functionally redundant manner. Hypoxic AKT activation and consecutive E6/E7 repression is dependent on the activities of the canonical upstream AKT regulators phosphoinositide 3-kinase (PI3K) and mechanistic target of rapamycin (mTOR) complex 2 (mTORC2). Hypoxic downregulation of E6/E7 occurs, at least in part, at the transcriptional level. Modulation of E6/E7 expression by the PI3K/mTORC2/AKT cascade is hypoxia specific and not observed in normoxic HPV-positive cancer cells. Quantitative proteome analyses identify additional factors as candidates to be involved in hypoxia-induced activation of the PI3K/mTORC2/AKT signaling cascade and in the AKT-dependent repression of the E6/E7 oncogenes under hypoxia. Collectively, these data uncover a functional key role of the PI3K/mTORC2/AKT signaling cascade for viral oncogene repression in hypoxic HPV-positive cancer cells and provide new insights into the poorly understood cross talk between oncogenic HPVs and their host cells under hypoxia.

Purpose This retrospective analysis aimed to evaluate the stability of spinal metastases in malignant melanoma patients following radiotherapy (RT), and to assess prognostic factors for survival. Methods Forty-one patients with malignant melanoma and osteolytic spinal bone metastases were irradiated at the university clinics of Heidelberg and Mainz between July 2003 and October 2013. Three and six months after palliative RT, only 20 and 15 patients, respectively, were still alive and were therefore assessed for spinal stability using the Taneichi score based on CT imaging. Additionally, overall survival (OS) and bone survival (BS) rates as well as prognostic factors for BS were evaluated for all study patients. Results Before RT, 19 patients (46.3%) were rated unstable. In the surviving patients, none of the unstable metastases were classified as stable 6 months after RT. Five-year OS was 23.3% and median BS was 4 months (range 0.5-29.8). Accordingly, only 36.6% of the patients were still alive 6 months after RT. Karnofsky performance score (KPS) Conclusions Our study population was characterized by poor BS and a lack of benefit with regard to stabilization of initially unstable spinal bone metastases 3 and 6 months after RT. This applies in particular to patients with a KPS

It is currently unclear whether adjuvant therapy for WHO grade III anaplastic astrocytomas (AA) should be carried out as combined chemoradiotherapy with temozolomide (TMZ)—analogous to the approach for glioblastoma multiforme—or as radiotherapy (RT) alone. A retrospective analysis of data from 90 patients with AA, who were treated between November 1997 and February 2014. Assessment of overall (OS) and progression-free survival (PFS) was performed according to treatment categories: (1) 50 %, RT + TMZ according to protocol, (2) 11 %, RT + TMZ with dose reduction, (3) 26 %, RT alone, and (4) 13 %, individualized, primarily palliative therapy. No dose reduction was necessary in the RT alone group. Median OS was 85, 69, and 43 months for treatment categories 1/2, 3, and 4, respectively. These differences were not statistically significant. PFS was 35, 29, 48, and 33 months for categories 1, 2, 3, and 4, respectively; again without significant differences between categories. In a subgroup of 39 patients with known IDH1 R132H status, the presence of this mutation correlated with significantly longer OS (p = 0.01) and PFS (p = 0.002). Complete or partial tumor resection and younger age also correlated with a significantly better prognosis, and this influence persisted in multivariate analysis. In the IDH1 R132H subgroup analysis, only this marker retained an independent prognostic value. A general superiority of combined chemoradiotherapy compared to RT alone could not be demonstrated. Biomarkers for predicting the benefits of combination therapy using RT and TMZ are needed for patients with AA.

In this chapter we allude to a series of facts and fallacies often encountered in the description of tumor hypoxia, a relevant trait of the tumor microenvironment and a paramount driver of tumor aggressiveness and treatment resistance. The critical role of diffusion distances, terminological inconsistencies considering O

Radiotherapy (RT) in combination with chemoimmunotherapy is highly efficient in the treatment of diffuse large B‑cell lymphoma (DLBCL). This retrospective analysis evaluated the efficacy of the treatment volume and the dose concept of involved-site RT (ISRT). We identified 60 histologically confirmed stage I–IV DLBCL patients treated with multimodal cytotoxic chemoimmunotherapy and followed by consolidative ISRT from 2005–2015. Progression-free survival (PFS) and overall survival (OS) were estimated by Kaplan–Meier method. Univariate analyses were performed by log-rank test and Mann–Whitney U‑test. After initial chemoimmunotherapy (mostly R‑CHOP; rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone), 19 (36%) patients achieved complete response (CR), 34 (64%) partial response (PR) or less. Excluded were 7 (12%) patients with progressive disease after chemoimmunotherapy. All patients underwent ISRT with a dose of 40 Gy. After a median follow-up of 44 months, 79% of the patients remained disease free, while 21% presented with failure, progressive systemic disease, or death. All patients who achieved CR after chemoimmunotherapy remained in CR. Of the patients achieving PR after chemotherapy only 2 failed at the initial site within the ISRT volume. No marginal relapse was observed. Ann Arbor clinical stage I/II showed significantly improved PFS compared to stage III/IV (93% vs 65%; p ≤ 0.021). International Prognostic Index (IPI) score of 0 or 1 compared to 2–5 has been associated with significantly increased PFS (100% vs 70%; p ≤ 0.031). Postchemoimmunotherapy status of CR compared to PR was associated with significantly increased PFS (100% vs 68%; p ≤ 0.004) and OS (100% vs 82%; p ≤ 0.026). Only 3 of 53 patients developed grade II late side effects, whereas grade III or IV side effects have not been observed. These data suggest that a reduction of the RT treatment volume from involved-field (IF) to involved-site (IS) is sufficient because no marginal failures occurred. The concept of IS will likely reduce the risk for late sequelae of RT.

Oncogenic human papillomaviruses (HPVs) are closely linked to major human malignancies, including cervical and head and neck cancers. It is widely assumed that HPV-positive cancer cells are under selection pressure to continuously express the viral E6/E7 oncogenes, that their intracellular p53 levels are reconstituted on E6/E7 repression, and that E6/E7 inhibition phenotypically results in cellular senescence. Here we show that hypoxic conditions, as are often found in subregions of cervical and head and neck cancers, enable HPV-positive cancer cells to escape from these regulatory principles: E6/E7 is efficiently repressed, yet, p53 levels do not increase. Moreover, E6/E7 repression under hypoxia does not result in cellular senescence, owing to hypoxia-associated impaired mechanistic target of rapamycin (mTOR) signaling via the inhibitory REDD1/TSC2 axis. Instead, a reversible growth arrest is induced that can be overcome by reoxygenation. Impairment of mTOR signaling also interfered with the senescence response of hypoxic HPV-positive cancer cells toward prosenescent chemotherapy in vitro. Collectively, these findings indicate that hypoxic HPV-positive cancer cells can induce a reversible state of dormancy, with decreased viral antigen synthesis and increased therapeutic resistance, and may serve as reservoirs for tumor recurrence on reoxygenation.

In this chapter we allude to a series of facts and fallacies often encountered in the description of tumor hypoxia, a relevant trait of the tumor microenvironment and a paramount driver of tumor aggressiveness and treatment resistance. The critical role of diffusion distances, terminological inconsistencies considering O2 partial pressures vs. O2 concentrations and with it the use of inept units, the impact of O2 depletion on proliferation and cell viability, the switch in the Warburg dogma, the distribution of hypoxic subvolumes within a tumor, the involvement of O2 diffusion shunts in the development of chronic hypoxia, and the role of endogenous biomarkers as surrogates for the assessment of hypoxia are discussed in more detail. Special emphasis is put on the clinical relevance of these misconceptions and misinterpretations and their impact on the assessment of hypoxia as well as hypoxia-targeted treatment planning.

We have established a novel in situ protein analysis pipeline, which is built upon highly sensitive, multichannel immunofluorescent staining of paraffin sections of human and xenografted tumor tissue. Specimens are digitized using slide scanners equipped with suitable light sources and fluorescence filter combinations. Resulting digital images are subsequently subjected to quantitative image analysis using a primarily object-based approach, which comprises segmentation of single cells or higher-order structures (e.g., blood vessels), cell shape approximation, measurement of signal intensities in individual fluorescent channels and correlation of these data with positional information for each object. Our approach could be particularly useful for the study of the hypoxic tumor microenvironment as it can be utilized to systematically explore the influence of spatial factors on cell phenotypes, e.g., the distance of a given cell type from the nearest blood vessel on the cellular expression of hypoxia-associated biomarkers and other proteins reflecting their specific state of activation or function. In this report, we outline the basic methodology and provide an outlook on possible use cases.

PET-CT is widely used for both the staging and planning of primary or neoadjuvant chemoradiotherapy for esophageal cancer. Inclusion of PET-CT information into radiotherapy planning often leads to substantial modifications of the target volume. In the case of detection of distant metastases, it may also result in a switch to a palliative treatment approach. This spares patients from therapy-related toxicities that provide no clinical benefit. However, due to a lack of studies, it is currently unclear whether the advantages of PET-CT also translate into a measurable improvement in patient survival. A retrospective analysis assessed the survival data of 145 patients with esophageal carcinoma stages I (eight patients; 5%), II (45; 31%), III (79; 55%), IV (8; 5%) and unknown (5; 4%). Patients were treated between 1999 and 2014 either with primary chemoradiation (n = 101) or neoadjuvant chemoradiation at the Department of Radiation Oncology, University Medical Center Mainz, followed by transabdominal or transthoracic tumor resection (n = 44). Of the 145 patients, 64 (44%) had undergone PET-CT. Univariate analysis showed the use of PET-CT to be associated with significantly longer local recurrence-free survival (p = 0.006) and tended to translate into a measurable improvement of overall survival (p = 0.071). Since more patients underwent surgery in the group planned using PET-CT (20% vs. 44%; p = 0.002), we carried out a multivariate Cox regression analysis to adjust for this possible confounding factor. Surgery (p = 0.042; HR 0.55; 95% confidence interval: 0.31–0.98) as well as the use of PET-CT (p = 0.048; HR 0.60; 95% confidence interval: 0.36–0.99) nearly halved the risk of local recurrence. It was only in the group of patients with PET-CT that a trend towards a shorter overall survival was evident in lymph node-positive patients (p = 0.16), whereas nodal stage did not impact on survival in patients staged without PET-CT (p = 0.97). To the best of our knowledge these data suggest for the first time that the use of PET-CT in the framework of staging and planning of primary or neoadjuvant chemoradiotherapy for esophageal cancer has a favorable impact on patient survival.

Background: Experimental studies have established a causal connection between tumour hypoxia, hypoxia-associated proteome changes and downregulation of E-cadherin, the final common pathway of epithelial-to-mesenchymal transition (EMT). Our study aimed at elucidating the interrelationship of these processes in cancers of the uterine cervix in vivo. Methods: Tumour oxygenation was assessed in 48 squamous cell carcinomas (SCC) of the uterine cervix using polarographic needle electrodes. The expression pattern of E-cadherin was investigated by immunohistochemistry and western blotting, and was compared with that of the hypoxia-inducible proteins glucose transporter (GLUT)-1 and carbonic anhydrase (CA) IX in biopsy specimens of the oxygenation measurement tracks. Results: The majority of cervical cancers (52%) were E-cadherin positive, with a complete absence of the antigen in only 10% of the tumours. No correlation was found between the level of E-cadherin expression and the oxygenation status (mean pO2, median pO2 and hypoxic fractions). In patients showing partial expression of E-cadherin (38%), staining was not preferentially diminished in GLUT-1- or CA IX-positive areas, and loss of E-cadherin occurred independently of tumour cell scattering. Conclusion: Our data provide no evidence in favour of a hypoxia-induced EMT as a mechanistic basis of cervical cancer invasiveness.

Tumor hypoxia is a hallmark of solid malignant tumor growth, profoundly influences malignant progression and contributes to the development of therapeutic resistance. Pathogenesis of tumor hypoxia is multifactorial, with contributions from both acute and chronic factors. Spatial distribution of hypoxia within tumors is markedly heterogeneous and often changes over time, e.g., during a course of radiotherapy. Substantial changes in the oxygenation status can occur within the distance of a few cell layers, explaining the inability of currently used molecular imaging techniques to adequately assess this crucial trait. Due to the possible importance of tumor hypoxia for clinical decision-making, there is a great demand for molecular tools which may provide the necessary resolution down to the single cell level. Exogenous and endogenous markers of tumor hypoxia have been investigated for this purpose. Their potential use may be greatly enhanced by multiparametric in situ methods in experimental and human tumor tissue.

Hypoxia-associated proteome changes have been shown to be associated with resistance to chemo- and radiotherapy. Our study evaluated the role of the hypoxia-inducible (HIF)-1 target gene carbonic anhydrase (CA) IX in the prediction of the response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer (stages II and III). A total of 29 pretreatment biopsy specimens were stained for CA IX by immunohistochemistry, converted to digital images and evaluated in a quantitative fashion using image analysis software. Contrary to our expectations, a trend towards a correlation between better tumor regression (50%) and higher expression of CA IX (p=0.056) was found. CA IX was also present more frequently in pathological tumor stage T1 (pT1) tumors (p=0.048). Conversely, no association with lymph node metastasis was identified. In conclusion, as a single marker, CA IX expression is not able to identify a hypoxia-related treatment resistant phenotype in rectal cancer.

Systematic studies on the oxygenation status of solid tumors have shown that the development of hypoxic/anoxic tissue subvolumes is a pathophysiological trait in a wide range of human malignancies. As a result of this characteristic property, adenosine (ADO) accumulation (range: 50-100 μM) occurs caused by intra- and extracellular generation of ADO. Extracellular nucleotide catabolism by hypoxia-/HIF-1α-sensitive, membrane-associated ecto-5'-nucleotidases most probably is the major source of ADO in the halo of cancer cells upon specific genetic alterations taking place during tumor growth. Extracellular ADO can act through autocrine and paracrine pathways following receptor-binding and involving different intracellular signalling cascades. Hypoxia-driven receptor activation can lead to a broad spectrum of strong immune-suppressive properties facilitating tumor escape from immune control (e.g., inhibition of CD4+, CD8+, NK and dendritic cells, stimulation of Treg cells). In addition, tumor growth and progression is promoted by ADO-driven direct stimulation of tumor cell proliferation, migration, invasion, metastatic dissemination, and an increase in the production of molecules stimulating tumor angiogenesis. Hypoxia- driven ADO accumulation in the tumor microenvironment thus plays a critical role in tumor growth and progression at multiple pathophysiological levels.

Radioresistance is a common feature of head and neck squamous cell carcinoma (HNSCC). We previously showed that the irradiation- activated vascular endothelial growth factor (VEGF)-extracellular signal-regulated kinase (ERK)-axis is fundamental for the survival of resistant tumors. In this study, we examined if treatment with potent multikinase (MK) inhibitors, sorafenib and sunitinib, could radiosensitize tumor cells.Cultured HNSCC cell lines were treated with inhibitors and subsequently irradiated. Radiosensitizing effects were functionally assessed by annexin-V apoptosis and clonogenic assays and confirmed by Western blot. Additionally, we surveyed human HNSCC tissue microarrays (TMAs) for activated ERK expression.Based on combination indexes, we found that combining irradiation with both inhibitors exerted strong and supra-additive antitumor effects on clonogenic survival. Kinase inhibition enhanced irradiation-induced apoptotic rates and inhibited postradiogenic phospho-ERK-expression. Patients with recurrent HNSCC displayed significantly lower extracellular signal-regulated kinase phosphorylation (pERK) levels than relapse-free patients.We propose further evaluation of sorafenib and sunitinib as potential radiosensitizing agents in HNSCC treatment. © 2017 Wiley Periodicals, Inc. Head Neck 39: 623-632, 2017.

Systematic studies on the oxygenation status of solid tumors have shown that the development of hypoxic/anoxic tissue subvolumes is a pathophysiological trait in a wide range of human malignancies. As a result of this characteristic property, adenosine (ADO) accumulation (range: 50-100 μM) occurs caused by intra- and extracellular generation of ADO. Extracellular nucleotide catabolism by hypoxia-/HIF-1α-sensitive, membrane-associated ecto-5'-nucleotidases most probably is the major source of ADO in the halo of cancer cells upon specific genetic alterations taking place during tumor growth. Extracellular ADO can act through autocrine and paracrine pathways following receptor-binding and involving different intracellular signalling cascades. Hypoxia-driven receptor activation can lead to a broad spectrum of strong immune-suppressive properties facilitating tumor escape from immune control (e.g., inhibition of CD4+, CD8+, NK and dendritic cells, stimulation of Treg cells). In addition, tumor growth and progression is promoted by ADO-driven direct stimulation of tumor cell proliferation, migration, invasion, metastatic dissemination, and an increase in the production of molecules stimulating tumor angiogenesis. Hypoxia- driven ADO accumulation in the tumor microenvironment thus plays a critical role in tumor growth and progression at multiple pathophysiological levels.

Tumor hypoxia is a hallmark of solid malignant tumor growth, profoundly influences malignant progression and contributes to the development of therapeutic resistance. Pathogenesis of tumor hypoxia is multifactorial, with contributions from both acute and chronic factors. Spatial distribution of hypoxia within tumors is markedly heterogeneous and often changes over time, e.g., during a course of radiotherapy. Substantial changes in the oxygenation status can occur within the distance of a few cell layers, explaining the inability of currently used molecular imaging techniques to adequately assess this crucial trait. Due to the possible importance of tumor hypoxia for clinical decision-making, there is a great demand for molecular tools which may provide the necessary resolution down to the single cell level. Exogenous and endogenous markers of tumor hypoxia have been investigated for this purpose. Their potential use may be greatly enhanced by multiparametric in situ methods in experimental and human tumor tissue.

Hypoxia-associated proteome changes have been shown to be associated with resistance to chemo- and radiotherapy. Our study evaluated the role of the hypoxia-inducible (HIF)-1 target gene carbonic anhydrase (CA) IX in the prediction of the response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer (stages II and III). A total of 29 pretreatment biopsy specimens were stained for CA IX by immunohistochemistry, converted to digital images and evaluated in a quantitative fashion using image analysis software. Contrary to our expectations, a trend towards a correlation between better tumor regression (>50 %) and higher expression of CA IX (p = 0.056) was found. CA IX was also present more frequently in pathological tumor stage T1 (pT1) tumors (p = 0.048). Conversely, no association with lymph node metastasis was identified. In conclusion, as a single marker, CA IX expression is not able to identify a hypoxia-related treatment resistant phenotype in rectal cancer.

Hypoxia is an important factor mediating tumor progression and therapeutic resistance, in part through proteome changes mediated by the transcription factor hypoxia-inducible factor (HIF)-1. Since glioblastoma multiforme is the epitome of a highly aggressive tumor entity, while lower-grade astrocytomas often show a prolonged clinical course, a profound difference in the extent of hypoxic tissue areas and corresponding magnitude of HIF-1 activity may exist between these entities. In this study, to address this question, serial sections of 11 glioblastomas and 10 anaplastic astrocytomas were immunostained for HIF-1α, glucose transporter (GLUT)-1, carbonic anhydrase (CA) IX (i.e., hypoxia-related markers), Ki67 (proliferation), phosphorylated ribosomal protein S6 [p-rpS6; mammalian target of rapamycin (mTOR) activity] and CD34 (microvascular endothelium). Digital scans of whole tumor sections were registered to achieve geometric correspondence for subsequent morphometric operations. HIF-1α-, GLUT-1- and CA IX-positive staining was found in all 11 glioblastomas, showing a preferential expression in tissue areas adjacent to necroses. A considerable spatial overlap between GLUT-1 and CA IX, and a colocalization of these proteins with areas of enlarged mean diffusion distances were observed. Conversely, 8 of the 10 anaplastic astrocytomas were completely negative for hypoxia-related markers. The glioblastomas also showed significantly greater heterogeneity of intercapillary distances, larger diffusion-limited tissue fractions, significantly higher mTOR activity and a trend for higher proliferation rates. Microregionally, mTOR and proliferation showed a significant spatial overlap with areas of shorter mean diffusion distances. In conclusion, diffusion-limited hypoxia, leading to the expression of hypoxia-related markers is a pivotal element of the glioblastoma phenotype and may be driven by dysregulated growth and proliferation in normoxic subregions.

The introduction of small molecule BRAFV600 kinase inhibitors represents a milestone in the targeted therapy of patients with metastatic melanoma by a significant increase in therapeutic efficacy in terms of overall and progression-free survival compared with conventional chemotherapy. Beside BRAFV600 inhibitor treatment, radiotherapy is a further mainstay for the therapy of metastatic melanoma and thus a concomitant or sequential application of BRAFV600 inhibitors and radiotherapy is inevitable. Recent reports show a significant radiosensitization of the irradiated healthy tissue in patients with melanoma after the combination of radiotherapy and BRAFV600 inhibitors, evoking concern in clinical practice. We review interactions of BRAFV600 inhibitors and radiation with regard to antitumor effects and an increased radiotoxicity in the healthy tissue.

Hypoxia is now established as a key factor influencing the pathophysiology of malignant growth. Among other effects, hypoxia modulates the expression of a multitude of genes through the induction of hypoxia-inducible transcription factors. This differential gene expression favors angiogenesis, cell survival, an invasive/metastatic phenotype, and resistance to anticancer therapies. Because benign tumors do not exhibit these traits, one might expect these entities to be neither hypoxic nor to induce the genetic hypoxia response program. To test this hypothesis, an investigation of the oxygenation status of 17 leiomyomas and 1 leiomyosarcoma of the uterus using polarographic needle electrodes (Eppendorf pO2 sensor) and the expression of hypoxia-related markers in biopsy specimens of the same tumors was carried out. Marker expression in eight additional archival leiomyosarcomas was also assessed. Leiomyoma tissue was generally found to be severely hypoxic, with median oxygen (O2) partial pressure values ranging from 1 to 5 mm Hg. In contrast, none of the hypoxia-related markers hypoxia-inducible factor (HIF)–1α, HIF-2α, glucose transporter-1, or carbonic anhydrase IX were expressed in any leiomyoma. Larger intercapillary distances were correlated with a poorer oxygenation status. Conversely, the expression of hypoxia-related markers was abundant in the leiomyosarcomas and they also exhibited a high-turnover phenotype (significantly increased proliferation and apoptosis). Uterine leiomyoma might therefore represent a state of oxygen-limited proliferation. Malignancy in the same organ system is associated with growth and metabolism beyond tissue-inherent limitations leading to the induction of hypoxia-related markers, thereby contributing to a self-perpetuating aggressive phenotype. [Cancer Res 2008;68(12):4719–26]

In locally advanced solid tumors, oxygen (O2) delivery is frequently reduced or even abolished. This is due to abnormalities of the tumor microvasculature, adverse diffusion geometries, and tumor-associated and/or therapy-induced anemia. Up to 50-60% of locally advanced solid tumors may exhibit hypoxic and/or anoxic tissue areas that are heterogeneously distributed within the tumor mass. In approximately 30% of pretreatment patients, a decreased O2 transport capacity of the blood as a result of tumor-associated anemia can greatly contribute to the development of tumor hypoxia. While normal tissues can compensate for this O2 deficiency status by a rise in blood flow rate, locally advanced tumors (or at least larger tumor areas) cannot adequately counteract the restriction in O2 supply and thus the development of hypoxia. Hypoxia-induced alteration in gene expression and thus in the proteome (< 1% O2, or < 7 mmHg), and/or genome changes (< 0.1% O2, or < 0.7 mmHg) may promote tumor progression via mechanisms enabling cells to overcome nutritive deprivation, to escape from the hostile metabolic microenvironment and to favor unrestricted growth. Sustained hypoxia may thus lead to cellular changes resulting in a more clinically aggressive phenotype. In addition, hypoxia is known to directly or indirectly confer resistance to X- and gamma-radiation, and some chemotherapies leading to treatment failures. Whereas strong evidence has accumulated that hypoxia plays a pivotal role in tumor progression and acquired treatment resistance, the mechanism(s) by which treatment efficacy and survival may be compromised by anemia (independent of hypoxia) are not fully understood.

Background Strongly increased uptake of glucose is a hallmark of solid malignant tumors. This phenotype can be triggered by hypoxia-induced gene expression changes or can occur independently of hypoxia as a consequence of malignant transformation itself, and is often referred to as the Warburg effect. The glycolytic phenotype has been associated with malignant progression and resistance to radio- and chemotherapy. Methods We have chosen squamous cell carcinomas of the vulva (SCC-V) as a representative solid tumor entity to study the central players of this pathway, namely glucose transporter (GLUT)-1, carbonic anhydrase (CA) IX, hexokinase (HK)-2 and pyruvate kinase (PK)-M2, and have investigated their relationships to tumor microvessels (CD34, αSMA) and proliferation (Ki67). Expression of these proteins was analyzed in 38 SCC-Vs, 5 vulvar dysplasias and 10 non-neoplastic squamous epithelia of the vulva using multiparametric immunohistochemistry in registered serial sections (MIRSS). Results Expression of GLUT-1 in invasive carcinomas was predominantly located in the outer layers of the tumor cell aggregates close to the vascularized tumor stroma, and only to a lesser extent colocalized with CA IX, which was repeatedly found at larger diffusion distances away from microvessels. CA IX expression was lower in invasive carcinomas compared to dysplasias and non-neoplastic tissue and higher in recurrent vs. primary tumors. Ki67-positive proliferating cells were partially colocalized with GLUT-1. However, HK-2 and PK-2 - proteins centrally involved in the Warburg phenotype - did not show such a correlation. Conclusions Consistent with prior studies, the pattern of GLUT-1 clearly indicated that a large part of its expression is presumably unrelated to hypoxia. However, there was also no association with HK-2 and PK-M2, suggesting that the functional background of this expression is also independent of aerobic glycolysis. CA IX may be worth consideration as a marker of biological hypoxia, as should its pathophysiological consequences in SCC-V. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-760) contains supplementary material, which is available to authorized users.

Hypoxia is a hallmark of tumors leading to (mal-)adaptive processes, development of aggressive phenotypes and treatment resistance. Based on underlying mechanisms and their duration, two main types of hypoxia have been identified, coexisting with complex spatial and temporal heterogeneities. Chronic hypoxia is mainly caused by diffusion limitations due to enlarged diffusion distances and adverse diffusion geometries (e.g., concurrent vs. countercurrent microvessels, Krogh- vs. Hill-type diffusion geometry) and, to a lesser extent, by hypoxemia (e.g., in anemic patients, HbCO formation in heavy smokers), and a compromised perfusion or flow stop (e.g., due to disturbed Starling forces or intratumor solid stress). Acute hypoxia mainly results from transient disruptions in perfusion (e.g., vascular occlusion by cell aggregates), fluctuating red blood cell fluxes or short-term contractions of the interstitial matrix. In each of these hypoxia subtypes oxygen supply is critically reduced, but perfusion-dependent nutrient supply, waste removal, delivery of anticancer or diagnostic agents, and repair competence can be impaired or may not be affected. This detailed differentiation of tumor hypoxia may impact on our understanding of tumor biology and may aid in the development of novel treatment strategies, tumor detection by imaging and tumor targeting, and is thus of great clinical relevance.

Hypoxia is a hallmark of tumors leading to (mal-)adaptive processes, development of aggressive phenotypes and treatment resistance. Based on underlying mechanisms and their duration, two main types of hypoxia have been identified, coexisting with complex spatial and temporal heterogeneities. Chronic hypoxia is mainly caused by diffusion limitations due to enlarged diffusion distances and adverse diffusion geometries (e.g., concurrent vs. countercurrent microvessels, Krogh- vs. Hill-type diffusion geometry) and, to a lesser extent, by hypoxemia (e.g., in anemic patients, HbCO formation in heavy smokers), and a compromised perfusion or flow stop (e.g., due to disturbed Starling forces or intratumor solid stress). Acute hypoxia mainly results from transient disruptions in perfusion (e.g., vascular occlusion by cell aggregates), fluctuating red blood cell fluxes or short-term contractions of the interstitial matrix. In each of these hypoxia subtypes oxygen supply is critically reduced, but perfusion-dependent nutrient supply, waste removal, delivery of anticancer or diagnostic agents, and repair competence can be impaired or may not be affected. This detailed differentiation of tumor hypoxia may impact on our understanding of tumor biology and may aid in the development of novel treatment strategies, tumor detection by imaging and tumor targeting, and is thus of great clinical relevance.

This chapter briefly summarizes the most important processes by which hypoxia, lactate accumulation, and acidosis may influence malignant progression and therapeutic resistance of solid malignant tumors. While these phenomena are often elements of an integrated reaction, they may occur independently of each other under certain circumstances. The latter information may be of interest with regard to possible “targeted” therapeutic interventions.