Mirjam Renovanz

Intratumoral Heterogeneity, Its Contribution to Therapy Resistance and Methodological Caveats to Assessment

Cancer is one of the most urgent health issues of today. According to WHO, the number of cancer cases is expected to increase by 75% in the next two decades (1). Despite some remarkable achievements in the fields of cancer prevention and early detection, the goal of developing effective anti-cancer therapies still remains unmet. Tumor recurrence due to treatment resistance is the most common cause of death from cancer. Delineating cellular and molecular mechanisms underlying tumor recurrence is of prime importance for the ability to improve the efficacy of existing therapies and develop new strategies to cancer treatment.

MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response

BackgroundThe DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value.ResultsWe analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan–Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP.ConclusionsCompared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.

Navigated versus non-navigated intraoperative ultrasound: is there any impact on the extent of resection of high-grade gliomas? A retrospective clinical analysis.

INTRODUCTION The extent of tumor resection is a significant predictor of survival in high-grade gliomas. In recent years, several authors showed the benefit of intraoperative ultrasound partially matched with magnetic resonance imaging (MRI). The aim of this study was to find out if intraoperative neuronavigation in combination with intraoperative ultrasound has any impact on the complete resection of gliomas. A comparison between the ultrasound-controlled resection of brain tumors and operations controlled by navigated ultrasound was performed. MATERIALS AND METHODS A total of 92 patients (54 men and 39 women) with a mean age of 53.2 years underwent 93 operations over a period of 4 years (2007-2010). They harbored a tumor with suspicion of glioma; 32 of them had undergone previous surgery, and additional chemotherapy, and 29 of them had undergone irradiation. Overall, 49 operations were performed with navigated ultrasound (group A) and 44 with non-navigated ultrasound (group B). A standardized early postoperative MRI was performed . Complete or gross total resection (GTR) was defined by a resection of ≥ 95% of the tumor. Skin incision and craniotomy were planned after registration of the neuronavigation system. The ultrasound system was used systematically before and after opening the dura, and during and at the end of resection. RESULTS GTR could be achieved in 28 of 49 cases in group A and in 23 of 44 cases in group B. In group A, sensitivity and specificity of tumor remnants detected by ultrasound were higher than in group B. Concerning recurrent gliomas, the sensitivity of ultrasound visualizing tumor remnants was lower than in primary tumors. In case of preoperatively planned GTR, in both groups (navigated and non-navigated ultrasound) similar tumor remnant sizes were postoperatively detected by MRI. In nine cases the removal was incomplete because of eloquently located tumors. There was no significant difference between navigated and not-navigated ultrasound concerning GTR (p > 0.05). CONCLUSION Navigated ultrasound is an important technical tool that helps in intraoperative orientation. Further prospective investigation is needed to assess the impact on GTR.

CD133 expression is not synonymous to immunoreactivity for AC133 and fluctuates throughout the cell cycle in glioma stem-like cells

A transmembrane protein CD133 has been implicated as a marker of stem-like glioma cells and predictor for therapeutic response in malignant brain tumours. CD133 expression is commonly evaluated by using antibodies specific for the AC133 epitope located in one of the extracellular domains of membrane-bound CD133. There is conflicting evidence regarding the significance of the AC133 epitope as a marker for identifying stem-like glioma cells and predicting the degree of malignancy in glioma cells. The reasons for discrepant results between different studies addressing the role of CD133/AC133 in gliomas are unclear. A possible source for controversies about CD133/AC133 is the widespread assumption that expression patterns of the AC133 epitope reflect linearly those of the CD133 protein. Consequently, the readouts from AC133 assessments are often interpreted in terms of the CD133 protein. The purpose of this study is to determine whether and to what extent do the readouts obtained with anti-AC133 antibody correspond to the level of CD133 protein expressed in stem-like glioma cells. Our study reveals for the first time that CD133 expressed on the surface of glioma cells is poorly immunoreactive for AC133. Furthermore, we provide evidence that the level of CD133 occupancy on the surface of glioma cells fluctuates during the cell cycle. Our results offer a new explanation for numerous inconsistencies regarding the biological and clinical significance of CD133/AC133 in human gliomas and call for caution in interpreting the lack or presence of AC133 epitope in glioma cells.

Factors associated with supportive care needs in glioma patients in the neuro-oncological outpatient setting

Objective of this study aimed at assessing glioma patients’ supportive care needs in a neurosurgical outpatient setting and identifying factors that are associated with needs for support. In three neuro-oncological outpatient departments, glioma patients were assessed for their psychosocial needs using the Supportive Care Needs Survey short-form (SCNS-SF34-G). Associations between clinical, sociodemographic, treatment related factors as well as distress (measured with the distress thermometer) and supportive care needs were explored using multivariable general linear models. One-hundred and seventy three of 244 eligible glioma patients participated, most of them with primary diagnoses of a high-grade glioma (81%). Highest need for support was observed in ‘psychological needs’ (median 17.5, range 5–45) followed by ‘physical and daily living needs’ (median 12.5, range 0–25) and ‘health system and information needs’ (median 11.3, range 0–36). Needs in the psychological area were associated with distress (R2 = 0.36) but not with age, sex, Karnofsky performance status (KPS), extend of resection, currently undergoing chemotherapy and whether guidance during assessment was offered. Regarding ‘health system and information needs’, we observed associations with distress, age, currently undergoing chemotherapy and guidance (R2 = 0.31). In the domain ‘physical and daily living needs’ we found associations with KPS, residual tumor, as well as with distress (R2 = 0.37). Glioma patients in neuro-oncological departments report unmet supportive care needs, especially in the psychological domain. Distress is the factor most consistently associated with unmet needs requiring support and could serve as indicator for clinical neuro-oncologists to initiate support.

Video-Assisted Navigation for Adjustment of Image-Guidance Accuracy to Slight Brain Shift

BACKGROUND: Information supplied by an image-guidance system can be superimposed on the operating microscope oculars or on a screen, generating augmented reality. Recently, the outline of a patients head and skull, injected in the oculars of a standard operating microscope, has been used to check the registration accuracy of image guidance. OBJECTIVE: To propose the use of the brain surface relief and superficial vessels for real-time intraoperative visualization and image-guidance accuracy and for intraoperative adjustment for brain shift. METHODS: A commercially available image-guidance system and a standard operating microscope were used. Segmentation of the brain surface and cortical blood vessel relief was performed manually on preoperative computed tomography and magnetic resonance images. The overlay of segmented digital and real operating-microscope images was used to monitor image-guidance accuracy. Adjustment for brain shift was performed by manually matching digital images on real structures. RESULTS: Experimental manipulation on a phantom proved that the brain surface relief could be used to restore accuracy if the primary registration shifted. Afterward, the technique was used to assist during surgery of 5 consecutive patients with 7 deep-seated brain tumors. The brain surface relief could be successfully used to monitor registration accuracy after craniotomy and during the whole procedure. If a certain degree of brain shift occurred after craniotomy, the accuracy could be restored in all cases, and corticotomies were correctly centered in all cases. CONCLUSION: The proposed method was easy to perform and augmented image-guidance accuracy when operating on small deep-seated lesions.

Evaluation of robot-guided minimally invasive implantation of 2067 pedicle screws

Objective Recent studies have investigated the role of spinal image guidance for pedicle screw placement. Many authors have observed an elevated placement accuracy and overall improvement of outcome measures. This study assessed a bi-institutional experience following introduction of the Renaissance miniature robot for spinal image guidance in Europe. Methods The medical records and radiographs of all patients who underwent robot-guided implantation of spinal instrumentation using the novel system (between October 2011 and March 2015 in Mainz and February 2014 and February 2016 in Regensburg) were reviewed to determine the efficacy and safety of the newly introduced robotic system. Screw position accuracy, complications, exposure durations to intraoperative radiation, and reoperation rate were assessed. Results Of the 413 surgeries that used robotic guidance, 406 were via a minimally invasive approach. In 7 cases the surgeon switched to conventional screw placement, using a midline approach, due to referencing problems. A total of 2067 screws were implanted using robotic guidance, and 1857 screws were evaluated by postoperative CT. Of the 1857 screws, 1799 (96.9%) were classified as having an acceptable or good position, whereas 38 screws (2%) showed deviations of 3-6 mm and 20 screws (1.1%) had deviations > 6 mm. Nine misplaced screws, implanted in 7 patients, required revision surgery, yielding a screw revision rate of 0.48% of the screws and 7 of 406 (1.7%) of the patients. The mean ± SD per-patient intraoperative fluoroscopy exposure was 114.4 (± 72.5) seconds for 5.1 screws on average and any further procedure required. Perioperative and direct postoperative complications included hemorrhage (2 patients, 0.49%) and wound infections necessitating surgical revision (20 patients, 4.9%). Conclusions The hexapod miniature robotic device proved to be a safe and robust instrument in all situations, including those in which patients were treated on an emergency basis. Placement accuracy was high; peri- and early postoperative complication rates were found to be lower than rates published in other series of percutaneous screw placement techniques. Intraoperative radiation exposure was found to be comparable to published values for other minimally invasive and conventional approaches.

Epigenetic silencing of XAF1 in high-grade gliomas is associated with IDH1 status and improved clinical outcome

XAF1 (X-linked inhibitor of apoptosis (XIAP)-associated factor 1) is a tumor suppressor that counteracts the anti-apoptotic effects of XIAP and can sensitize cells to cell death triggering events. XAF1 knockdown abrogated the temozolomide (TMZ)-induced G2-arrest and prevented TMZ-induced apoptosis in the glioblastoma (GB) cell line LN229. Promoter methylation of XAF1 was found to be inversely correlated with mRNA expression in GB cells. We analyzed XAF1 methylation in a panel of 16 GB cell lines and 80 patients with first-diagnosed WHO grade III/IV high-grade gliomas using methylation-sensitive high-resolution melt (MS-HRM) analysis. In those patients, XAF1 promoter methylation was strongly associated with enhanced progression free and overall survival. Interestingly, XAF1 promoter methylation was strictly correlated with the occurrence of IDH1 mutations, indicating a causal link to the IDH1 mutant phenotype. XAF1 methylation was observed in 18 grade III tumors all of which showed heterozygous mutations in the IDH1 gene. 17 harbored a mutation leading to an arginine > histidine (R132H) and one carried a mutation causing an arginine > glycine (R132G) substitution. Furthermore, six out of six recurrent and IDH1 mutated grade III tumors also showed XAF1 promoter methylation. The data demonstrate that XAF1 promoter methylation determined by MS-HRM is a robust and precise indicator of IDH1 mutations in grade III gliomas. It is useful for complementing the immunohistochemistry-based detection of mutant IDH, uncovering rare 2-HG-producing IDH1 and potentially IDH2 mutations. The MS-HRM-based detection of XAF1 methylation could therefore be a reliable tool in assisting the sub-classification of high-grade gliomas.

Perspectives and Challenges in Molecular-Based Diagnostics and Personalized Treatment for Recurrent High-Grade Gliomas

Glioblastoma is the most common and most malignant type of intrinsic brain tumor in adults. The standard of care for glioblastoma consists of surgical debulking followed by combined radiochemotherapy. The clinical efficacy of standard therapies for newly diagnosed glioblastomas is rather modest with the highest survival rate at 5-years being less than 10%. Inevitable recurrence after cytotoxic therapies poses the major challenge in the clinical management of high grade gliomas. For recurrent glioblastomas, there is no standard therapy with lack of level one evidence for treatment efficacy. Recent evidence indicates that post-therapy recurrence in gliomas is a consequence of a plethora of molecular and cellular factors including intratumoural heterogeneity, functional hierarchy of distinct types of glioma cells, dynamic changes in the molecular landscapes and cellular composition of the tumour during therapy and the impact of particular treatment modalities. There is an emerging consensus that molecular distinctions within and between individual tumours is an important factor determining clinical outcomes. Consequently, integrated approaches based on the combination of molecular profiling with traditional methods such as immunohistochemical phenotyping, karyotyping and/or non-quantitative methylation-specific PCR have emerged as a promising venue towards increasing the predictive value of diagnostics for malignant brain tumors. The high level of inter-and intra-tumoural molecular diversity in gliomas underscores the need of integrating high throughput molecular profiling and pharmacogenomics into a diagnostic paradigm for gliomas and raises the possibility that molecular-instructed personalized treatments may provide clinical benefit to patients with glioblastoma, particularly in the setting of post-treatment recurrence. Here we discuss potential prospects and challenges of patient-tailored diagnostics and personalized treatment strategies for recurrent glioblastomas.

Evaluation of surgical decision making and resulting outcome in patients with highly eloquent glioblastoma: Results of a multicenter assessment

INTRODUCTION Treatment of glioblastoma(GB) patients amenable only for a subtotal resection(STR) is controversial. Since outcome of patients is affected by surgical management, our aim was to assess surgical decision making and resulting outcome in patients with highly eloquent GBs. PATIENTS AND METHODS We retrospectively assessed GB patients with intended sub-total resection (STR) or stereotactic biopsy (STX) of 3 neurooncological centers operated between 2008 and 2013. A volumetric assessment of overall extent of resection(oEoR), presence of complications, new permanent neurological deficits(nPNDs) was performed. A central reviewer reassessed all cases blinded and gave recommendation on surgical management and on a potential EoR(pEoR) based on imaging data. We compared outcome data using Mann-Whitney-U-test and Sign-Rank-Test. Survival was assessed based on Kaplan-Meier-estimates. RESULTS 97 patients were included. In 17 patients received STX, 70 patients a STR and 10 patients a near total resection (NTR, EoR>95%). Median OS was significantly different from STX patients only if NTR was reached (16 vs. 7 months, p=0.042). The central reviewer recommended a more aggressive strategy(NTR or STR resp.) in 41 patients and a less aggressive strategy in 13 patients. Overall, management recommendation was significantly different to clinical treatment (p<0.001). Mean pEoR was significantly higher than oEoR (85.7% vs. 71.3%, p=0.001). Regarding the different OR subgroups, no significant differences were found in the NTR group(12/13 ties, p=1) and in STX group (14/17 ties, p=0.125). In STR group, a significant difference was found (p=0.001). In 38/69 patients a NTR and in 13/77 patients a STX was recommended. CONCLUSION Surgery in GB patients with intended STR requires precise preoperative planning since potential EoR is mainly underestimated. Especially, patients with lesions amenable for a NTR should not be missed.