Refined high-content imaging-based phenotypic drug screening in zebrafish xenografts.

Zebrafish xenotransplantation models are increasingly applied for phenotypic drug screening to identify small compounds for precision oncology. Larval zebrafish xenografts offer the opportunity to perform drug screens at high-throughput in a complex in vivo environment. However, the full potential of the larval zebrafish xenograft model has not yet been realized and several steps of the drug screening workflow still await automation to increase throughput. Here, we present a robust workflow for drug screening in zebrafish xenografts using high-content imaging. We established embedding methods for high-content imaging of xenografts in 96-well format over consecutive days. In addition, we provide strategies for automated imaging and analysis of zebrafish xenografts including automated tumor cell detection and tumor size analysis over time. We also compared commonly used injection sites and cell labeling dyes and show specific site requirements for tumor cells from different entities. We demonstrate that our setup allows us to investigate proliferation and response to small compounds in several zebrafish xenografts ranging from pediatric sarcomas and neuroblastoma to glioblastoma and leukemia. This fast and cost-efficient assay enables the quantification of anti-tumor efficacy of small compounds in large cohorts of a vertebrate model system in vivo. Our assay may aid in prioritizing compounds or compound combinations for further preclinical and clinical investigations.

Primary tumor side is associated with prognosis of colorectal cancer patients with brain metastases.

BACKGROUND: Brain metastases (BM) are a rare complication in colorectal cancer (CRC) patients and associated with an unfavorable survival prognosis. Primary tumor side (PTS) was shown to act as a prognostic and predictive biomarker in several trials including metastatic CRC (mCRC) patients. Here, we aim to investigate whether PTS is also associated with the outcome of CRC patients with BM. METHODS: Patients treated for CRC BM between 1988 and 2017 at an academic care center were included. Right-sided CRC was defined as located in the appendix, cecum and ascending colon and left-sided CRC was defined as located in the descending colon, sigma and rectum. RESULTS: Two hundred and eighty-one CRC BM patients were available for this analysis with 239/281 patients (85.1%) presenting with a left-sided and 42/281 patients (14.9%) with a right-sided primary CRC. BM-free survival (BMFS) was significantly longer in left-sided compared with right-sided CRC patients (33 versus 20 months, P = 0.009). Overall survival from CRC diagnosis as well as from diagnosis of BM was significantly longer in patients with a left-sided primary (42 versus 25 months, P = 0.002 and 5 versus 4 months, P = 0.005, respectively). In a multivariate analysis including graded prognostic assessment, PTS remained significantly associated with prognosis after BM (hazard ratio 0.65; 95% confidence interval: 0.46-0.92 months, P = 0.0016). CONCLUSIONS: PTS was associated with survival times after the rare event of BM development in CRC patients. Therefore, its prognostic value remains significant even thereafter.

Tumor mutational burden and immune infiltrates in renal cell carcinoma and matched brain metastases.

BACKGROUND: Tumor mutational burden (TMB) and density of tumor-infiltrating lymphocytes (TIL) have been postulated as predictive biomarkers for immunotherapy. Therefore, we investigated the concordance of TMB and TIL of primary/extracranial renal cell carcinoma (RCC) specimens and matched brain metastases (BM). PATIENTS AND METHODS: Twenty specimens from 10 patients were retrieved from the Vienna Brain Metastasis Registry (6/10 primary tumor, 4/10 lung metastasis, 10/10 matched BM). TMB was assessed using the TruSight Oncology 500 gene panel with libraries sequenced on a NextSeq instrument. TIL subsets (CD3+, CD8+, CD45RO+, FOXP3+, PD-L1+) were investigated using immunohistochemistry (Ventana Benchmark Ultra system) and automated tissue analysis (Definiens software). RESULTS: No significant difference in TMB, CD3+, CD8+, CD45RO+, FOXP3+ or PD-L1+ expression was observed between extracranial and matched intracranial specimens (P > 0.05). Higher CD8+ TIL (P = 0.053) and CD45RO+ TIL (P = 0.030) densities in the primary tumor compared with the intracranial samples were observed in specimens collected after exposure to systemic treatment. Neither extracranial sample origin (lung metastasis versus primary RCC) nor extracranial disease status at BM diagnosis (progressive versus stable disease) were significantly associated with TMB or TIL densities in extracranial and intracranial samples (P > 0.05). No significant correlation was found between the median differences of TMB or TIL densities from extracranial to intracranial samples and BM-free survival. CONCLUSION: The comparable immunological microenvironment of extra- and intracranial tumor samples in our study underscores the immunological activation also in BM from RCC, and therefore, supports the development of immune modulatory treatments also in patients with brain metastatic RCC.

Brain metastases as first manifestation of advanced cancer: exploratory analysis of 459 patients at a tertiary care center.

Symptomatic brain metastases (BM) are a frequent and late complication in cancer patients. However, a subgroup of cancer patients presents with BM as the first symptom of metastatic cancer. Here we aimed to analyze the clinical course and prognostic factors of this particular BM patient population. Patients presenting with newly diagnosed BM without a history of metastatic cancer were identified from the Vienna Brain Metastasis Registry. Clinical characteristics and overall survival were retrieved by chart review. 459/2419 (19.0%) BM patients presented with BM as first symptom of advanced cancer. In 374/459 (81.5%) patients, an extracranial primary tumor, most commonly lung cancer, could be identified within 3 months after BM diagnosis. In 85/459 (18.5%) patients no extracranial primary tumor could be identified despite comprehensive diagnostic workup within the first 3 months after diagnosis of BM. Survival of patients with identified extracranial tumor differed only numerically from patients with cancer of unknown primary (CUP), however patients receiving targeted therapy after molecular workup showed significantly enhanced survival (20 months vs. 7 months; p = 0.003; log rank test). The GPA score showed a statistically significant association with median overall survival times in the CUP BM patients (class I: 46 months; class II: 7 months; class III: 4 months; class IV: 2 months; p < 0.001; log rank test). The GPA score has a strong prognostic value in patients with CUP BM and may be useful for patient stratification in the clinical setting. Comprehensive diagnostic workup including advanced imaging techniques and molecular tissue analyses appears to benefit patients by directing specific molecular targeted therapies.

The course of quality of life and neurocognition in newly diagnosed patients with glioblastoma.

BACKGROUND: The importance of QoL and neurocognitive functions in patients with glioblastoma (GB) is above controversy by now. We followed newly diagnosed GB patients treated with radio-chemotherapy during their course of disease by continuously evaluating their quality of life (QoL) and cognitive functions. METHODS: We included consecutive patients with newly diagnosed GB from 2010 to 2013 at the Medical University of Vienna. To assess QoL the EORTC QLQ C30 and BN20 questionnaire were used. Neurocognition was measured with the NeuroCog FX. The evaluations were done 6 times every three months, beginning at the beginning of radio-chemotherapy. RESULTS: 42 patients participated in this study. We also recorded QoL and neurocognition in 23 patients after the first disease progression. Patients maintained their cognitive summary score until relapse. Patients with left-sided tumors showed significant lower scores in the subscale verbal fluency than patients with right-sided tumors. The global health score of QoL decreased after the fifth evaluation (13months after diagnosis) whereas a peak of fatigue symptoms was obtained at the third evaluation. Furthermore, fatigue symptoms increased strongly 7months after diagnosis and patients' financial difficulties were mentioned more frequently by younger patients and in patients with lower education levels. CONCLUSIONS: QoL and cognitive long-term assessments are feasible also in some patients with GB after a symptomatic progression. Our study demonstrates maintenance of QoL and cognitive summary scales before tumor progression. Moreover, it highlights subgroups according to tumor location and socioeconomic factors.

Decreased body mass index is associated with impaired survival in lung cancer patients with brain metastases: A retrospective analysis of 624 patients.

Body mass index (BMI) is a prognostic factor in several cancer types. We investigated the prognostic role of BMI in a large patient cohort with newly diagnosed lung cancer brain metastases (BM) between 1990 and 2013. BMI at diagnosis of BM and graded prognostic assessment (GPA) were calculated. Definitions were underweight (BMI <18.50), weight within normal range (BMI 18.50-24.99) and overweight (BMI ≥ 25.00). A total of 624 patients (men 401/624 [64.3%]; women 223/624 [35.7%]; median age of 61 [range 33-88]) were analysed. Histology was non-small cell lung cancer in 417/622 (66.8%), small cell lung cancer (SCLC) in 205/624 (32.9%) and not otherwise specified in 2/624 (0.3%) patients. About 313/624 (50.2%) had normal BMI, 272/624 (43.5%) were overweight and 39/624 (6.3%) were underweight. Underweight patients had shorter median overall survival (3 months) compared to patients with normal BMI (7 months) and overweight (8 months; p < .001; log rank test). At multivariate analysis, higher GPA class (HR 1.430; 95% cumulative incidence, CI 1.279-1.598; p < .001; Cox regression model), SCLC histology (HR 1.310; 95% CI 1.101-1.558) and presence of underweight (HR 1.845; 95% CI 1.317-2.585; p = .014; Cox regression model) were independent prognostic factors. Underweight at diagnosis of BM in lung cancer is associated with an unfavourable prognosis.

Brain tumours at 7T MRI compared to 3T--contrast effect after half and full standard contrast agent dose: initial results.

OBJECTIVES: To compare the contrast agent effect of a full dose and half the dose of gadobenate dimeglumine in brain tumours at 7 Tesla (7 T) MR versus 3 Tesla (3T). METHODS: Ten patients with primary brain tumours or metastases were examined. Signal intensities were assessed in the lesion and normal brain. Tumour-to-brain contrast and lesion enhancement were calculated. Additionally, two independent readers subjectively graded the image quality and artefacts. RESULTS: The enhanced mean tumour-to-brain contrast and lesion enhancement were significantly higher at 7 T than at 3T for both half the dose (91.8 ± 45.8 vs. 43.9 ± 25.3 [p = 0.010], 128.1 ± 53.7 vs. 75.5 ± 32.4 [p = 0.004]) and the full dose (129.2 ± 50.9 vs. 66.6 ± 33.1 [p = 0.002], 165.4 ± 54.2 vs. 102.6 ± 45.4 [p = 0.004]). Differences between dosages at each field strength were also significant. Lesion enhancement was higher with half the dose at 7 T than with the full dose at 3T (p = .037), while the tumour-to-brain contrast was not significantly different. Subjectively, contrast enhancement, visibility, and lesion delineation were better at 7 T and with the full dose. All parameters were rated as good, at the least. CONCLUSION: Half the routine contrast agent dose at 7 T provided higher lesion enhancement than the full dose at 3T which indicates the possibility of dose reduction at 7 T. KEY POINTS: • The contrast effect of gadobenate dimeglumine was assessed at 7 T and 3T. • In brain tumours, contrast effect was higher at 7 T than at 3T. • Tumour-to-brain contrast at 7 T half dose and 3T full dose were comparable. • 7 T half dose lesion enhancement was higher than 3T full dose enhancement. • Our results indicate the possibility of contrast agent dose reduction at 7 T.

Prognostic significance of Ki67 proliferation index, HIF1 alpha index and microvascular density in patients with non-small cell lung cancer brain metastases.

BACKGROUND: Survival upon diagnosis of brain metastases (BM) in patients with non-small cell lung cancer (NSCLC) is highly variable and established prognostic scores do not include tissue-based parameters. METHODS: Patients who underwent neurosurgical resection as first-line therapy for newly diagnosed NSCLC BM were included. Microvascular density (MVD), Ki67 tumor cell proliferation index and hypoxia-inducible factor 1 alpha (HIF-1 alpha) index were determined by immunohistochemistry. RESULTS: NSCLC BM specimens from 230 patients (151 male, 79 female; median age 56 years; 199 nonsquamous histology) and 53/230 (23.0%) matched primary tumor samples were available. Adjuvant whole-brain radiation therapy (WBRT) was given to 153/230 (66.5%) patients after neurosurgical resection. MVD and HIF-1 alpha indices were significantly higher in BM than in matched primary tumors. In patients treated with adjuvant WBRT, low BM HIF-1 alpha expression was associated with favorable overall survival (OS), while among patients not treated with adjuvant WBRT, BM HIF-1 alpha expression did not correlate with OS. Low diagnosis-specific graded prognostic assessment score (DS-GPA), low Ki67 index, high MVD, low HIF-1 alpha index and administration of adjuvant WBRT were independently associated with favorable OS. Incorporation of tissue-based parameters into the commonly used DS-GPA allowed refined discrimination of prognostic subgroups. CONCLUSION: Ki67 index, MVD and HIF-1 alpha index have promising prognostic value in BM and should be validated in further studies.

Arterial spin-labeling assessment of normalized vascular intratumoral signal intensity as a predictor of histologic grade of astrocytic neoplasms.

BACKGROUND AND PURPOSE: Pulsed arterial spin-labeling is a noninvasive MR imaging perfusion method performed with the use of water in the arterial blood as an endogenous contrast agent. The purpose of this study was to determine the inversion time with the largest difference in normalized intratumoral signal intensity between high-grade and low-grade astrocytomas. MATERIALS AND METHODS: Thirty-three patients with gliomas, histologically classified as low-grade (n = 7) or high-grade astrocytomas (n = 26) according to the World Health Organization brain tumor classification, were included. A 3T MR scanner was used to perform pulsed arterial spin-labeling measurements at 8 different inversion times (370 ms, 614 ms, 864 ms, 1114 ms, 1364 ms, 1614 ms, 1864 ms, and 2114 ms). Normalized intratumoral signal intensity was calculated, which was defined by the signal intensity ratio of the tumor and the contralateral normal brain tissue for all fixed inversion times. A 3-way mixed ANOVA was used to reveal potential differences in the normalized vascular intratumoral signal intensity between high-grade and low-grade astrocytomas. RESULTS: The difference in normalized vascular intratumoral signal intensity between high-grade and low-grade astrocytomas obtained the most statistically significant results at 370 ms (P = .003, other P values ranged from .012-.955). CONCLUSIONS: The inversion time by which to differentiate high-grade and low-grade astrocytomas by use of normalized vascular intratumoral signal intensity was 370 ms in our study. The normalized vascular intratumoral signal intensity values at this inversion time mainly reflect the labeled intra-arterial blood bolus and therefore could be referred to as normalized vascular intratumoral signal intensity. Our data indicate that the use of normalized vascular intratumoral signal intensity values allows differentiation between low-grade and high-grade astrocytomas and thus may serve as a new, noninvasive marker for astrocytoma grading.

Relative survival of patients with non-malignant central nervous system tumours: a descriptive study by the Austrian Brain Tumour Registry.

BACKGROUND: Unlike malignant primary central nervous system (CNS) tumours outcome data on non-malignant CNS tumours are scarce. For patients diagnosed from 1996 to 2002 5-year relative survival of only 85.0% has been reported. We investigated this rate in a contemporary patient cohort to update information on survival. METHODS: We followed a cohort of 3983 cases within the Austrian Brain Tumour Registry. All patients were newly diagnosed from 2005 to 2010 with a histologically confirmed non-malignant CNS tumour. Vital status, cause of death, and population life tables were obtained by 31 December 2011 to calculate relative survival. RESULTS: Overall 5-year relative survival was 96.1% (95% CI 95.1-97.1%), being significantly lower in tumours of borderline (90.2%, 87.2-92.7%) than benign behaviour (97.4%, 96.3-98.3%). Benign tumour survival ranged from 86.8 for neurofibroma to 99.7% for Schwannoma; for borderline tumours survival rates varied from 83.2 for haemangiopericytoma to 98.4% for myxopapillary ependymoma. Cause of death was directly attributed to the CNS tumour in 39.6%, followed by other cancer (20.4%) and cardiovascular disease (15.8%). CONCLUSION: The overall excess mortality in patients with non-malignant CNS tumours is 5.5%, indicating a significant improvement in survival over the last decade. Still, the remaining adverse impact on survival underpins the importance of systematic registration of these tumours.

Brain-only metastatic breast cancer is a distinct clinical entity characterised by favourable median overall survival time and a high rate of long-term survivors.

BACKGROUND: The clinical course of breast cancer patients with brain metastases (BM) as only metastatic site (brain-only metastatic breast cancer (BO-MBC)) has been insufficiently explored. METHODS: All breast cancer patients with BM treated at our institution between 1990 and 2011 were identified. For each patient, full information on follow-up and administered therapies was mandatory for inclusion. Oestrogen receptor, progesterone receptor and Her2 status were determined according to standard protocols. Statistical analyses including computation of survival probabilities was performed. RESULTS: In total, 222 female patients (26% luminal; 47% Her2; 27% triple negative) with BM of MBC were included in this study. In all, 38/222 (17%) BM patients did not develop extracranial metastases (ECM) during their disease course and were classified as BO-MBC. Brain-only-MBC was not associated with breast cancer subtype or number of BM. The median overall survival of BO-MBC patients was 11 months (range 0-69) and was significantly longer than in patients with BM and ECM (6 months, range 0-104; P=0.007). In all, 7/38 (18%) BO-MBC patients had long-term survival of >3 years after diagnosis of BM and long-term survival was significantly more common in BO-MBC patients as compared with BM patients with ECM (P<0.001). CONCLUSIONS: Brain-only metastatic behaviour occurs in around 17% of breast cancer with BM and is not associated with breast cancer subtype. Exploitation of all multimodal treatment options is warranted in BO-MBC patients, as these patients have favourable prognosis and long-term survival is not uncommon.

Epithelial Growth Factor Receptor Inhibitors for treatment of recurrent or progressive high grade glioma: an exploratory study.

INTRODUCTION: Erlotinib and Gefitinib (EGFRi) are small molecules specifically inhibiting epidermal growth factor receptor (EGFR). We present here data of an exploratory study evaluating EGFRi monotherapy in patients with recurrent or progressive malignant glioma. PATIENTS: 21 patients with recurrent or progressive malignant glioma were included in this study. EGFRi treatment was started at a median of 1.8 years (range 0.54 to 10.95) after initial surgery. 20/21 patients had undergone radiotherapy and all patients had received at least one (range 1 to 5, median 2) line of systemic antineoplastic therapy. Patients received 100 or 150 mg Erlotinib or 250 mg Gefitinib orally per day. RESULTS: Median age at primary diagnosis was 47.9 years (range 31.9 to 76 years). 18 patients received a total of 92.8 months (median 3.03) of Erlotinib treatment and 3 patients received a total of 16.1 months (median 6.06) of Gefitinib treatment. The best responses were partial remission in one patient receiving Erlotinib and in two patients receiving Gefitinib, respectively. Median time to progression was 3.05 months. Six months after start of EGFRi treatment, 4/21 (19%) patients were progression-free and 6/21(29%) patients were alive. Expression of EGFRwt, EGFRvIII, PTEN, phospho-Akt or EGFRvIII/PTEN co-expression in tumor cells did not significantly associate with time to progression or survival time. In one patient EGFRi administration had to be discontinued due to toxicity (grade 3 rash). CONCLUSION: EGFRi monotherapy is associated with therapeutic efficacy in only a small fraction of patients with malignant gliomas. Biomarkers reliably predicting tumor response to EGFRi need to be identified.

Cardiac transplantation and simultaneous surgical repair of an aortic aneurysm.

Simultaneous cardiac transplantation and surgical repair of an aortic aneurysm has not been reported previously. At our institution, a 59-year-old patient with an aneurysm of the ascending aorta and aortic arch required orthotopic cardiac transplantation for end-stage cardiomyopathy. He underwent successful surgical replacement of his ascending aorta and transverse arch (in circulatory arrest and deep hypothermia) at the time of heart transplantation.

CD15 (LeuM1) immunoreactivity: prognostic factor for sporadic and hereditary medullary thyroid cancer? Study Group on Multiple Endocrine Neoplasia of Austria.

Patients treated for sporadic and hereditary medullary thyroid carcinoma (MTC) have varying rates of persistent disease, recurrence, and survival. The aim of this study was to correlate the immunoreactivity of the monoclonal antibody CD15 (LeuM1) to initial clinical findings and the outcome of treatment. The primary tumors of 75 patients with sporadic MTC, 7 with hereditary disease, and 3 members of MEN 2A families were studied. Of these subjects 74 (87%) showed no or little immunoreactivity (< 15% positive cells; score 0) in most tumors. The remaining 13% had surgery for tumors with more than 15% cells with positive staining (score I). There was no correlation between LeuM1 immunoreactivity and sex, age, and type of MTC. There was, however, a significant correlation with the pTNM classification and UICC staging. The prognosis for patients with score 0 was significantly better than score 1 patients. CD15 immunoreactivity appears to be a predictive factor in sporadic and hereditary MTC. Lymph node dissection seems to be more successful in patients with score 0 tumors than in those with score 1 tumors. The question of reoperation in patients with recurrence of disease (especially with biochemical recurrence or persistence) should be discussed on the basis of CD15 immunoreactivity.

[Regional lymph node micrometastases in malignant melanoma of the skin]. / Regionäre Lymphknotenmikrometastasen beim malignen Melanom der Haut.

Histological studies were conducted into 352 primary malignant melanomas of the skin together with 4,003 associated and surgically removed lymph nodes at the Institute of Pathology and Bacteriology of Kaiserin-Elisabeth-Spital, between 1982 and 1987. Particular attention was given to micrometastases of regional lymph nodes. 27 lymph node micrometastases were recorded from 25 patients. Spots of melanin were also found in lymphatic tissue, but no tumour cells were detectable. There is no clinical aid by which micrometastases of lymph nodes might be possibly diagnosed, and, on principle, they are greatly accidental findings even for the pathohistologist. However, undetected lymph node micrometastases may have a deteriorating impact upon the prognosis of malignant melanoma. Complete microscopic elucidation of surgically removed lymph nodes, there fore, is absolutely essential. Capillaries are present in abundance deep inside lymph nodes, and from these points of departure tumour cells may enter into the blood flow and cause haematogenic metastases. That is why the prognostic importance attributed to histologically verified micrometastases is just as great as that attached to macrometastases.

[The importance of atypical adenoma of the thyroid gland]. / Uber die Dignität des atypischen Adenoms der Schilddrüse.

Histological appraisals were made of 8,741 specimens of removed thyroid glands at the Pathologico-Bacteriological Institute of Kaiserin Elisabeth Spital, between 1978 and 1987. Included, according to classification, were 234 atypical adenomas, 47 encapsulated follicular carcinomas, and 158 invasive follicular carcinomas. Atypical adenoma was found to differ histologically from encapsulated follicular carcinoma by non-detectability of vascular invasions. The age distribution of the patients involved failed to provide any conclusive clue to the effect that atypical adenoma was a precursor of encapsulated follicular carcinoma. Females were more often affected than males with atypical adenoma. No recurrence, metastasation nor complaints are recordable at present from any of the surviving patients with atypical adenoma. Patients who died did not reveal any sign indicative of a thyroid disease as cause of death. The five-year survival rate has been 95 per cent for encapsulated follicular carcinoma. Diagnosis of atypical adenoma is not merely a qualitative problem but a quantitative problem as well. Atypical adenoma of the thyroid gland should be classified as a neoplasia in its own right and as absolutely benign.