NTCP model validation method for DAHANCA patient selection of protons versus photons in head and neck cancer radiotherapy.

Introduction: Prediction models using logistic regression may perform poorly in external patient cohorts. However, there is a need to standardize and validate models for clinical use. The purpose of this project was to describe a method for validation of external NTCP models used for patient selection in the randomized trial of protons versus photons in head and neck cancer radiotherapy, DAHANCA 35. Material and methods: Organs at risk of 588 patients treated primarily with IMRT in the randomized controlled DAHANCA19 trial were retrospectively contoured according to recent international recommendations. Dose metrics were extracted using MatLab and all clinical parameters were retrieved from the DAHANCA database. The model proposed by Christianen et al. to predict physician-rated dysphagia was validated through the closed testing, where change of the model intercept, slope and individual beta's were tested for significant prediction improvements. Results: Six months prevalence of dysphagia in the validation cohort was 33%. The closed testing procedure for physician-rated dysphagia showed that the Christianen et al. model needed an intercept refitting for the best match for the Danish patients. The intercept update increased the risk of dysphagia for the validation cohort by 7.9 ± 2.5% point. For the raw model performance, the Brier score (mean squared residual) was 0.467, which improved significantly with a new intercept to 0.415. Conclusions: The previously published Dutch dysphagia model needed an intercept update to match the Danish patient cohort. The implementation of a closed testing procedure on the current validation cohort allows quick and efficient validation of external NTCP models for patient selection in the future.

Prognostic factors for survival after salvage total laryngectomy following radiotherapy or chemoradiation failure: a 10-year retrospective longitudinal study in eastern Denmark.

OBJECTIVE: The primary aims were to determine the rates of and prognostic factors for overall survival, disease-specific survival and disease-free survival following salvage total laryngectomy. DESIGN: Retrospective longitudinal study. SETTING: Tertiary medical centres. PARTICIPANTS: A total of 142 patients in eastern Denmark undergoing salvage total laryngectomy for squamous cell carcinoma of the larynx or hypopharynx. MAIN OUTCOME MEASURES: 5-year overall survival, 5-year disease-specific survival, 5-year disease-free survival and prognostic factors for these outcomes. RESULTS: 5-year overall survival, disease-specific survival and disease-free survival were 37.7%, 54.9% and 55.3%, respectively. N classification at primary diagnosis, lymph node excision and postoperative complications within 1 year after salvage total laryngectomy were prognostic factors for shorter overall survival, disease-specific survival and disease-free survival. Residual tumour/recurrence was negatively associated with overall survival, close or involved resection margins with disease-specific survival, and second primary cancer was associated with longer disease-specific survival and disease-free survival. Nine per cent of all patients had residual tumour and 33.8% developed a recurrence. CONCLUSION: Our overall survival, disease-specific survival and disease-free survival findings are in accordance with previous studies. With the purpose of identifying recurrent tumour, we suggest extra attention being given to patients with higher N classification and need for lymph node excision during salvage total laryngectomy along with use of frozen sections. The high number of patients with recurrence within 1 year after salvage total laryngectomy occurred although thorough and regular follow-up visits were performed.

Risk factors for postoperative complications after total laryngectomy following radiotherapy or chemoradiation: a 10-year retrospective longitudinal study in Eastern Denmark.

OBJECTIVES: To determine the rates of and risk factors for postoperative complications following total laryngectomy in patients treated with radiotherapy or chemoradiation. DESIGN: Retrospective longitudinal study. SETTING: Tertiary medical centres. PARTICIPANTS: A total of 143 patients undergoing total laryngectomy for squamous cell carcinoma of the larynx or hypopharynx. MAIN OUTCOME MEASURES: Overall postoperative complications and fistula formation. RESULTS: Overall postoperative complications, fistula formation, wound infection, bleeding and wound necrosis within one year after total laryngectomy occurred in 56.6%, 42.3%, 31.0%, 11.3% and 9.2% of patients, respectively. Stenosis of the pharynx/oesophagus and stoma shrinkage within five years after surgery were each seen in 18.2% of cases. In 66.7% of cases, conservative treatment of the fistulas was chosen. Rehospitalisations within five years occurred for 44.8% with a median rate of 1.5 (range 1-11). Smoking status (P = 0.005 and 0.013) and chronic obstructive pulmonary disease (COPD) (P = 0.013 and 0.011) were significant risk factors for both overall postoperative complications and fistula formation in uni- and multivariate analysis. Tumour localisation in the hypopharynx was associated with overall postoperative complications (P = 0.036). Residual tumour or cancer recurrence was associated with late-onset fistulas (P < 0.001). CONCLUSION: The frequencies of postoperative complications after total laryngectomy were comparable with similar international studies, although fistula formation rate is increasing in Denmark. We suggest optimising treatment of COPD and to further encourage to smoking cessation. We propose that development of fistulas >2 months after surgery prompts immediate biopsies. Additionally, we suggest standardised registration of surgical techniques to identify variables affecting the frequency of postoperative complications.

Methods for estimating the site of origin of locoregional recurrence in head and neck squamous cell carcinoma.

PURPOSE: Methods to estimate the likely origin of recurrences after radiation therapy for head and neck squamous cell carcinoma are compared. METHODS AND MATERIALS: A total of 25 patients meeting the following inclusion criteria were randomly selected: curatively intended intensity-modulated radiotherapy planned on a positron emission tomography-computed tomography (PET/CT) scan during the period 2005-2009; squamous cell carcinoma in the oral cavity, pharynx or larynx; complete clinical response followed by locoregional recurrence; and a CT scan at recurrence before any salvage therapy. Exclusion criteria were previous cancer in the area, surgery prior to radiotherapy, or a synchronous cancer. Three methods of estimating focal points of recurrence origin and two volume overlap methods assigning the recurrences to the most central target volumes encompassing at least 50% or 95% of the recurrence volumes were tested. Treatment planning and recurrence scans were rigid and deformable co-registered in order to transfer focal points to the treatment planning scan. Double determinations of all volumes, points, and co-registrations were made. RESULTS: The volume overlap methods assigned the recurrences to significantly more peripheral target volumes than focal methods (p < 0.0001 for all comparisons of 95% overlap vs. focal methods, p < 0.028 for all comparisons of 50% overlap vs. focal methods). Repeated registrations of the same point had higher reproducibility with deformable registration than with rigid registration (median distance 0.31 vs. 0.35 cm, p = 0.015). No significant differences were observed among the focal methods. CONCLUSION: Significant differences between methods were found which may affect strategies to improve radiotherapy based on pattern of failure analyses.

Early Mortality after Radical Radiotherapy in Head and Neck Cancer - A Nationwide Analysis from the Danish Head and Neck Cancer Group (DAHANCA) Database.

AIMS: Curative-intent radiotherapy (RT) or chemoradiation (CRT) of squamous cell carcinoma of the head and neck (HNSCC) produces high survival rates, but is associated with substantial toxicity. However, there are no commonly accepted quality metrics for early mortality in radiation oncology. To assess the applicability of early mortality as a clinical quality indicator, this study investigated the temporal distribution, risk factors and trends of 90- and 180-day overall and non-cancer mortality in a nationwide cohort of HNSCC patients treated with RT/CRT. MATERIALS AND METHODS: Information on all HNSCC patients treated with curative-intent RT/CRT in Denmark between 2000 and 2017 was obtained from the national Danish Head and Neck Cancer Group clinical database. Deaths in patients with residual or recurrent disease after RT/CRT were classified as cancer-related. Possible risk factors were investigated using logistic regression analysis. RESULTS: Data from 11 419 patients were extracted. In total, 90- and 180-day mortality risks were 3.1% and 7.1%, respectively. There was a uniform temporal distribution of 180-day mortality. In multivariable analysis, increasing age, stage, performance status, earlier treatment year and hypopharyngeal cancer were significantly associated with an increased risk (P < 0.05). Risk factor estimates were comparable for 90- versus 180-day mortality as well as for overall versus non-cancer mortality. Between 2000 and 2017 there was a significant decrease in 180-day mortality, which was driven by a reduction in cancer-related events. CONCLUSION: The distribution of 180-day overall and non-cancer mortality did not indicate a well-defined early high-risk period. Moreover, risk factor estimates were highly similar across risk periods and groups. Taken together, our findings question the applicability of early mortality as a standard metric for treatment-associated toxicity.

Effect of estrogen withdrawal on energy-rich phosphates and prediction of estrogen dependence monitored by in vivo 31P magnetic resonance spectroscopy of four human breast cancer xenografts.

The effect of estrogen withdrawal on energy metabolism was studied in four human breast cancer xenografts: the estrogen-dependent MCF-7 and ZR75-1 and the estrogen-independent ZR75/LCC-3 and MDA-MB-231. The tumors were grown in ovariectomized nude mice with a s.c. implanted estrogen pellet. After Gompertzian growth was verified, the estrogen pellet was removed from half of the animals. In vivo 31P magnetic resonance spectroscopy of the tumors was performed 1 day before and on days 2, 6, and 14 after estrogen removal. Estrogen withdrawal induced a significant increase in the nucleoside triphosphate:Pi ratio in the two estrogen-dependent xenografts, whereas this ratio remained unchanged in the estrogen-independent tumors. In ZR75/LCC-3 tumors a slight decrease in nucleoside triphosphate:Pi was observed following onset of estrogen stimulation after initial growth without estrogen. Extracts of freeze-clamped tumors prepared 14 days after estrogen removal were analyzed for ATP and phosphocreatine content. Our findings suggest a correlation between estrogen withdrawal and the steady-state concentrations of ATP, phosphocreatine, and Pi in human breast cancer xenografts. Discrimination analysis of the pretherapeutic spectra enabled us to identify the tumor line and the estrogen dependence of the tumors in 80-90% of all cases.

Growth inhibition in response to estrogen withdrawal and tamoxifen therapy of human breast cancer xenografts evaluated by in vivo 31P magnetic resonance spectroscopy, creatine kinase activity, and apoptotic index.

Estrogen withdrawal versus tamoxifen (TAM) treatment was compared in two human breast cancer xenografts, the estrogen-dependent ZR75-1 and its estrogen-independent subline ZR75/LCC-3. The following parameters were determined: tumor growth, NTP:P(i) by 31P magnetic resonance spectroscopy, apoptotic index, and creatine kinase (CK) activity. Tumors of each line were grown in ovariectomized nude mice during stimulation from a s.c. 17 beta-estradiol pellet. At a tumor size of approximately 350 mm3, the pellet was removed from one-half of the animals. The remaining one-half served as controls. In parallel experiments, injections of TAM were initiated instead of estrogen withdrawal. Estrogen withdrawal as well as TAM induced growth inhibition of ZR75-1 tumors, whereas ZR75/LCC-3 was resistant to both types of therapy. Growth inhibition of ZR75-1 by estrogen withdrawal, but not by TAM, was accompanied by an 80% increase of the NTP:P(i) ratio (P < 0.01) and a significantly decreased cytosolic CK activity (P < 0.01). No significant change in pH was observed. These changes seemed not to be related to changes in apoptotic index. None of the described changes occurred in ZR75/LCC-3. The present data indicate: (a) ZR75-1 and ZR75/LCC-3 xenografts respond differently to estrogen withdrawal and TAM with regard to growth inhibition, 31P magnetic resonance spectroscopy, and CK activity; (b) estrogen withdrawal, but not TAM, induced a decrease in the CK activity of estrogen-dependent tumor tissue, and (c) increased apoptosis did not explain the growth inhibition and the increase in NTP:P(i) induced by estrogen withdrawal. The results indicate other growth inhibitory mechanisms of TAM in addition to competitive inhibition of the estrogen receptor.

Systemic chemotherapy of brain metastases from small-cell lung cancer: a review.

PURPOSE: For decades the treatment of choice in small-cell lung cancer (SCLC) with brain metastases has been corticosteroids and radiotherapy (RT) because of a presumed lack of penetrance of cytostatic agents into parenchymatous brain metastases. In recent years, several reports have appeared on radiologic and clinical responses to systemic chemotherapy without additional RT in patients with metastatic SCLC in the brain. We reviewed the literature and focused on the methodologic aspects in comparison with RT data. DESIGN: We reviewed 12 patient series that included 116 patients and were published between 1981 and 1990. RESULTS: The overall brain response to chemotherapy without irradiation in patients with intracranial metastases at diagnosis was 76%, whereas the response rate of brain relapses was 43%. CONCLUSIONS: We conclude that intracranial metastases from SCLC seem to respond to chemotherapy as readily as other metastatic locations of SCLC do. Thus first-line cranial irradiation probably should be applied routinely only in cases of delayed brain metastases. Whether consolidating cranial RT should be given after a few courses of initial chemotherapy in SCLC patients with brain metastases at diagnosis is unclear and warrants a randomized evaluation.

Effect of tumor necrosis factor alpha on vascular resistance, nitric oxide production, and glucose and oxygen consumption in perfused tissue-isolated human melanoma xenografts.

The effect of tumor necrosis factor alpha (TNF-alpha) on vascular resistance, nitric oxide production, and consumption of oxygen and glucose was examined in a perfused tissue-isolated tumor model in nude mice. One experimental group was perfused with heparinized Krebs-Henseleit buffer, a second one was perfused with TNF-alpha (500 microgram/kg) 5 h before perfusion. The vascular resistance increased significantly 5 h after TNF-alpha injection. The increase in vascular resistance did not seem to be mediated by a decrease in tumor nitric oxide production, as determined by perfusate nitrate/nitrite concentrations, but may be due to aggregation of leukocytes, platelets, and erythrocytes and/or endothelial consumption among the three experimental groups. The oxygen consumption was linearly dependent on the amount of available oxygen in the perfusate, whereas the glucose consumption was constant and independent of the glucose delivery rate. The present experiments provide new insights into physiological and metabolic mechanisms of action of TNF- alpha for optimization of future treatment schedules involving TNF-alpha.

Changes in vascularization of human breast cancer xenografts responding to antiestrogen therapy.

To elucidate the previously suggested vascular effect(s) of antiestrogen therapy, we studied the effect of estrogen withdrawal and tamoxifen on 1) vascular resistance, 2) glucose and oxygen consumption, and 3) vascular density in a perfused breast cancer line (ZR75-1). Furthermore, we examined ZR75-1 tumors by functional CT-scanning (fCT) to determine changes in parameters related to tumor capillary transfer constants and vascular volume fraction in response to antiestrogenic manipulations. The vascular resistance decreased significantly from 42.7 to 20.8 mmHg x min x g x ml(-1) (P< .03) on day 9 after estrogen withdrawal, but not after 9 days of tamoxifen treatment. The estrogen-depleted tumors were significantly smaller than controls on day 9. There was no difference in nutrient consumption or vascular density in any of the experimental groups compared to controls. fCT showed an increase (P < .03) in vascular volume fraction during tumor growth, and this parameter was significantly lower after estrogen withdrawal when compared to controls (P < .05). Vascular resistance correlated with tumor size (R = 0.7, P < .0001), indicating that vascular resistance increases during tumor growth. The changes in vascular parameters after estrogen withdrawal indicate a vascular remodeling effect. This inhibition of vascular development by hormone deprivation may have important implications for future planning of multimodal treatment regimens.

Modulation of A-NK cell rigidity: In vitro characterization and in vivo implications for cell delivery.

The delivery of cells to specific regions of the vasculature is a critical step in many therapeutic strategies. These include the packaging of DNA or RNA in cell "vehicles" for delivery to tissues, the reconstitution of differentiated cells to an organ using embryonic stem cells, and the enhancement of the immune response using effector lymphocytes. In most cases, these cells must be injected systemically. Unfortunately, ex vivo manipulation or activation can affect cell visco-elastic properties, making it difficult for the injected cells to traverse capillary beds. Compounding the problem is the fact that common agents used in the laboratory for increasing cell deformability generally have adverse side effects on the therapeutic potential of the cells. Using micropipet aspiration techniques, cytotoxicity assays and in vivo trafficking studies we show that: (1) the rigidity of injected effector cells directly affects resistance to passage through tissue; (2) modulation of cytoskeletal organization can be used to decrease cell rigidity, but can also compromise therapeutic efficacy; and (3) thioglycollate, an agent which does not influence effector lymphocyte cytotoxic activity, reduces cell rigidity and entrapment in the lungs.

Waiting times for diagnosis and treatment of head and neck cancer in Denmark in 2010 compared to 1992 and 2002.

BACKGROUND AND AIM: Significant tumour progression was observed during waiting time for treatment of head and neck cancer. To reduce waiting times, a Danish national policy of fast track accelerated clinical pathways was introduced in 2007. This study describes changes in waiting time and the potential influence of fast track by comparing waiting times in 2010 to 2002 and 1992. METHODS: Charts of all new patients diagnosed with squamous cell carcinoma of the oral cavity, pharynx and larynx at the five Danish head and neck oncology centres from January to April 2010 (n=253) were reviewed and compared to similar data from 2002 (n=211) and 1992 (n=168). RESULTS: The median time to diagnosis was 13 days (2010) versus 17 days (2002; p<0.001) and 20 days (1992; p<0.001). Median days from diagnosis to treatment start were 25 (2010) versus 47 (2002; p<0.001) and 31 (1992; p<0.001). Total pre-treatment time was median 41 days in 2010 versus 69 days (2002) (p<0.001) and 50 days (1992; p<0.001). Significantly more diagnostic imaging was done in 2010 compared to 2002 and 1992. When compared to current fast track standards the adherence to diagnosis improved slightly from 47% (1992) to 51% (2002) and 64% (2010); waiting time for radiotherapy was within standards for 7%, 1% and 22% of cases, respectively; waiting time for surgery was within standards for 17%, 22% and 48%, respectively. CONCLUSION: The study showed a significant reduction in delay of diagnosis and treatment of head and neck cancer in 2010, but still less than half of all patients start treatment within the current standards.

Reduction of interstitial fluid pressure after TNF-alpha treatment of three human melanoma xenografts.

Tumour necrosis factor-alpha (TNF-alpha) reduced the interstitial fluid pressure (IFP) to 54-64% (P < 0.05) and the mean arterial blood pressure (MABP) to 70% (P < 0.01) of control values after 5 h in three human melanoma tumour lines transplanted to nude mice.

Creatine and cyclocreatine treatment of human colon adenocarcinoma xenografts: 31P and 1H magnetic resonance spectroscopic studies.

Creatine (Cr) and cyclocreatine (cyCr) have been shown to inhibit the growth of a variety of human and murine tumours. The purpose of this study was to evaluate the anti-tumour effect of these molecules in relation to drug accumulation, energy metabolism, tumour water accumulation and toxicity. Nude mice carrying a human colon adenocarcinoma (LS174T) with a creatine kinase (CK) activity of 2.12 units mg(-1) protein were fed Cr (2.5% or 5%) or cyCr (0.025%, 0.1% or 0.5%) for 2 weeks and compared with controls fed standard diet. Cr concentrations of 2.5% and 5% significantly inhibited tumour growth, as did 0.1% and 0.5% cyCr. In vivo 31P magnetic resonance spectroscopy (MRS) after 2 weeks of treatment showed an increase in [phosphocreatine (PCr)+phosphocyclocreatine (PcyCr)]/nucleoside triphosphate (NTP) with increasing concentrations of dietary Cr and cyCr, without changes in absolute NTP contents. The antiproliferative effect of the substrates of CK was not related to energy deficiency but was associated with acidosis. Intratumoral substrate concentrations (measured by 1H-MRS) of 4.8 micromol g(-1) wet weight Cr (mice fed 2.5% Cr) and 6.2 micromol g(-1) cyCr (mice fed 0.1% cyCr) induced a similar decrease in growth rate, indicating that both substrates were equally potent in tumour growth inhibition. The best correlant of growth inhibition was the total Cr or (cyCr+Cr) concentrations in the tissue. In vivo, these agents did not induce excessive water accumulation and had no systemic effects on the mice (weight loss, hypoglycaemia) that may have caused growth inhibition.

Cells shed from tumours show reduced clonogenicity, resistance to apoptosis, and in vivo tumorigenicity.

The goal of this study was to compare growth characteristics of cells shed from a tumour with the native tumour cells. The human colon adenocarcinoma LS174T and its highly metastatic subline LS LiM 6 were grown as tissue-isolated tumours in nude mice and perfused to collect shed cells. The tumours were then excised and prepared into single-cell suspensions. Clonogenicity in 0.3-0.9% agarose, apoptotic fraction, and in vivo tumorigenicity were determined for each population. In both tumour lines, shed cells were less clonogenic, more apoptotic and less tumorigenic than cells isolated directly from their native tissue. These findings suggest that shed cells have a low metastatic potential compared to native tumour cells, most likely because they represent an apoptotic population.