A microfluidic platform for functional testing of cancer drugs on intact tumor slices.

Present approaches to assess cancer treatments are often inaccurate, costly, and/or cumbersome. Functional testing platforms that use live tumor cells are a promising tool both for drug development and for identifying the optimal therapy for a given patient, i.e. precision oncology. However, current methods that utilize patient-derived cells from dissociated tissue typically lack the microenvironment of the tumor tissue and/or cannot inform on a timescale rapid enough to guide decisions for patient-specific therapy. We have developed a microfluidic platform that allows for multiplexed drug testing of intact tumor slices cultured on a porous membrane. The device is digitally-manufactured in a biocompatible thermoplastic by laser-cutting and solvent bonding. Here we describe the fabrication process in detail, we characterize the fluidic performance of the device, and demonstrate on-device drug-response testing with tumor slices from xenografts and from a patient colorectal tumor.

Multiplexed drug testing of tumor slices using a microfluidic platform.

Current methods to assess the drug response of individual human cancers are often inaccurate, costly, or slow. Functional approaches that rapidly and directly assess the response of patient cancer tissue to drugs or small molecules offer a promising way to improve drug testing, and have the potential to identify the best therapy for individual patients. We developed a digitally manufactured microfluidic platform for multiplexed drug testing of intact cancer slice cultures, and demonstrate the use of this platform to evaluate drug responses in slice cultures from human glioma xenografts and patient tumor biopsies. This approach retains much of the tissue microenvironment and can provide results rapidly enough, within days of surgery, to guide the choice of effective initial therapies. Our results establish a useful preclinical platform for cancer drug testing and development with the potential to improve cancer personalized medicine.

Expression of neurogenic basic helix-loop-helix genes in primitive neuroectodermal tumors.

The basic helix-loop-helix (bHLH) class of transcription factors plays a pivotal role in tissue-specific determination and differentiation. Moreover, dysregulated expression or loss of function of these factors contributes to leukemogenesis and solid tumor development. Neurogenesis is regulated by genes of the NEUROD/atonal and ACHAETE SCUTE families. We analyzed expression of human NEUROD1, NEUROD2, NEUROD3, and ACHAETE SCUTE 1 (HASH1) in cerebellar and cerebral primitive neuroectodermal tumors (PNETs), gliomas, and cell lines derived from a variety of neuroectodermal tumors by Northern analysis and in situ hybridization. NEUROD1 was expressed in each of the 12 medulloblastoma specimens, whereas NEUROD2 and NEUROD3/neurogenin were expressed in partly overlapping subsets of medulloblastomas. All of the tumors that presented with distant metastases expressed NEUROD3. The only other NEUROD3-positive tumor progressed early in treatment. Human ACHAETE SCUTE homologue (HASH1) was not expressed in medulloblastomas (infratentorial PNETs) but was expressed in three of five supratentorial PNETs. Neuroectodermal tumor cell lines derived from other sites (e.g., neuroblastoma and retinoblastoma) expressed NeuroD and ACHAETE SCUTE family members. No NEUROD message was detected in glial tumors or cell lines. Neurogenic bHLH transcription factor expression patterns suggest that specific family members may contribute to or reflect biological differences that arise during malignant transformation.

Operation and permanent low activity 125I brachytheraphy for recurrent high-grade astrocytomas.

Twenty-two adult patients with recurrent high grade astrocytomas [18 glioblastoma multiforme (GBM) and 4 anaplastic astrocytoma (AA) at time of implant] underwent therapy at the University of Washington from October 1991 through March 1995, with repeat craniotomy, maximal debulking of tumor, and placement of permanent low activity 125I seeds. Median age was 41 years and median Karnofsky performance status was 90. Median survival for the entire group was 65 weeks from the time of implant. For the subgroup of GBM patients, median survival was 64 weeks from the time of implant. One-year survival from the date of implant was 57% for the entire group and 59% for those with GBM. The site of first failure after implant was local (within 2 cm of the resection cavity) in 70%, distant (noncontiguous, beyond 2 cm) in 18% and concurrently local and distant in 12%. There was one case of symptomatic radiation injury that resolved with steroid therapy, and no patient required repeat craniotomy for parenchymal necrosis. For patients with recurrent GBM, treatment with resection and permanent low activity 125I brachytherapy yielded improved survival compared to an internal historical control group treated with resection and chemotherapy (p = 0.023). Craniotomy with maximal tumor debulking and placement of low activity 125I seeds yields encouraging results with minimal morbidity in patients with recurrent high-grade astrocytomas.

Multimodality management of recurrent adult malignant gliomas: results of a phase II multiagent chemotherapy study and analysis of cytoreductive surgery.

Fifty-one adult patients with recurrent malignant gliomas were treated in a Phase II trial of multidrug chemotherapy (6-thioguanine, dibromodulcitol, procarbazine, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 5-fluorouracil, and hydroxyurea). Thirty-one patients underwent radical tumor debulking, before the administration of chemotherapy. Fifty-seven percent of all patients had either an objective radiographic response or stabilization of disease after the institution of therapy. The overall median survival time (MST) was 40 weeks; it was 79 and 33 weeks for anaplastic astrocytoma and glioblastoma patients, respectively. The overall median time to tumor progression (MTP) was 19 weeks--32 weeks for anaplastic astrocytoma patients and 13 weeks for glioblastoma patients. Serious chemotoxicity occurred in 35% of patients without permanent morbidity or mortality. The factors that affected response (including disease stabilization), MTP, and MST were identified through a multivariate statistical analysis. A longer MTP was associated with higher Karnofsky scores, lower grade initial histology, lack of prior chemotherapy, greater degree of myelotoxicity, smaller postoperative tumor volumes, greater extent of surgical resection, and a local versus diffuse recurrence pattern. A longer MST was associated with higher Karnofsky scores, lower grade histology at the time of recurrence, greater degree of myelotoxicity, and lobar versus deep tumor location. Response (including disease stabilization) correlated with higher Karnofsky scores, lower grade histology (initial and current), prior lower grade histology, smaller preoperative tumor volume, longer intervals from the time of initial diagnosis, and absence of prior chemotherapy. These results suggest that, in addition to established prognostic factors such as Karnofsky scores, other factors including prior chemotherapy administration, patterns of tumor recurrence, and tumor location may be important variables to consider in future Phase II-III clinical trials. Of the treatment variables analyzed, greater surgical debulking and smaller postoperative tumor volumes were associated with prolonged MTP but not MST, and greater myelotoxicity had a positive association with all outcomes. The significance of this latter relationship and its relevance to chemotherapy dosing will require further study. Standardization in the design and reporting of clinical trials and the use of computer-assisted tumor volume calculations to assess the extent of surgical resection and the response to therapy are advocated.

Postoperative deficits and functional recovery following removal of tumors involving the dominant hemisphere supplementary motor area.

The supplementary motor area (SMA) is a region located within each cerebral hemisphere at the posterior mesial border of the frontal lobe adjacent to the falx. The functional significance of this area has been somewhat unclear, and information regarding its influence on motor output has largely been based on evoked responses to direct stimulation in primates and humans. In this series of patients with primary and metastatic tumors involving the dominant hemisphere SMA, a distinct pattern of postoperative deficits and recovery has emerged which emphasizes the role of this critical area in the initiation of motor activity, including speech. Based upon this analysis, ablation of this region after first identifying the primary motor cortex may be accomplished without risk of permanent loss of motor activity or speech function, despite the initial severe deficits.

Resolution of petrous internal carotid artery stenosis after transluminal angioplasty. Case report.

Percutaneous transluminal angioplasty is commonly used for treatment of peripheral vascular disease, but only recently has it been applied to craniocervical lesions. The successful use of percutaneous transluminal angioplasty for treatment of an isolated high-grade stenosis of the petrous internal carotid artery is described in a patient with progressive ischemic symptoms despite maximum medical management. At his 2-year follow-up examination, the patient remained asymptomatic with angiographic evidence of progressive resolution of the stenotic lesion and indirect evidence of improved hemispheric blood flow ipsilateral to the lesion. Percutaneous transluminal angioplasty may provide an effective means of treatment for selective intracranial artherosclerotic stenosis.

Measurement of tumor resection volumes from computerized images. Technical note.

The authors describe a method for quantitation of the area and volume of the resection cavity in patients who have undergone surgery for brain tumors. Using a slide scanner and Image 1.27, a public domain program for the Apple Macintosh II computer, computerized tomography scans and magnetic resonance images can be digitized and analyzed for a particular region of interest, such as the area and volume of tumor on preoperative and postresection scans. Phantom scans were used to analyze the accuracy of the program and the program users. User error was estimated at 2%, program error was 4.5%. This methodology is proposed as a means of retrospectively calculating the extent of tumor resection.

Surgical management and seizure outcome in patients with tuberous sclerosis.

The authors report the results obtained in 11 patients with tuberous sclerosis (TS) who underwent cortical resection surgery for medically intractable epilepsy. Patients' ages at time of surgery ranged from 3 to 46 years (mean 19.6 years). Preoperative epileptiform electroencephalographic abnormalities were focal spike wave discharges in six patients (55%), multifocal in four patients (36%), and generalized in one patient (9%). In the multifocal and generalized groups, all patients (45%) were evaluated by means of subdural grid and strip electrode recordings, whereas electrophysiological localization in the remaining patients was derived from ictal and interictal scalp recordings. The seizure foci were found to be extratemporal in six patients (55%) and temporal in five patients (45%). Surgical intervention consisted of craniotomy and seizure foci resection guided by electrocorticographic monitoring and functional mapping in five awake (45%) and six asleep (55%) patients. Neuropathological examination of the resected seizure foci revealed cortical tubers in eight patients and diffuse gliosis in three patients. Follow up ranged from 8 to 127 months (mean 35 months). Six patients (55%) were seizure free, half of whom were not receiving antiepileptic drugs (AEDs); three patients (27%) had a greater than 70% reduction in seizure frequency, although they required AEDs; one patient (9%) had a 50% temporary reduction in seizure frequency during the initial 6-month postoperative period; and one patient (9%) was lost to follow-up study. From this small but adequately followed patient population with TS, the authors conclude that cortical resection of seizure foci tailored to electrocorticographic findings and functional mapping is encouraging for this difficult to manage patient population with medically intractable epilepsy.

Tentorial meningiomas.

Tentorial meningiomas are encountered relatively infrequently, but it is nonetheless important to be aware of their variable clinical presentations and the need for individualized preoperative assessment and surgical management. The challenges presented by these lesions are due in large part to the complexity and variety of neurovascular structures associated with the tentorium. As is true for all meningiomas, the goal of operation is complete resection with limited morbidity and mortality. Preoperative assessment with CT or MR imaging or both and angiography with embolization, when possible, is advocated for all patients. The choice of surgical approach is dictated by the location of the lesion, dural origin, and involvement of major neurovascular structures. With extended surgical approaches, combining infratentorial and supratentorial exposure, one can attempt complete tumor resection even in patients with extensive skull base involvement. However, if complete resection presents an unacceptable risk of neurologic morbidity, one must consider adjuvant therapy. Radiotherapy (conventional external beam or stereotactic) has been shown to be of some benefit in stabilizing residual disease or delaying recurrence; adjuvant chemotherapy with antiprogesterone agents is still undergoing clinical evaluation.

Two cases of esophageal carcinoma metastatic to the pineal region with a review of the literature.

BACKGROUND: Metastasis to the pineal region is rare, and there are no previously reported cases of esophageal carcinoma metastatic to the pineal region. CASE DESCRIPTION: We now present two cases of solitary esophageal carcinoma metastatic to the pineal region. In each case there was no evidence of disease progression at the primary site at the time of presentation, and neurologic symptoms were the first indication of recurrent disease. Both patients underwent infratentorial/supracerebellar resection of the pineal lesions and were subsequently referred for radiation therapy. CONCLUSIONS: These two cases emphasize that metastatic disease must be a likely differential consideration in a patient with a CNS lesion and a history of previous malignancy, even if the lesion is in an unusual location.

A mathematical modelling tool for predicting survival of individual patients following resection of glioblastoma: a proof of principle.

The prediction of the outcome of individual patients with glioblastoma would be of great significance for monitoring responses to therapy. We hypothesise that, although a large number of genetic-metabolic abnormalities occur upstream, there are two 'final common pathways' dominating glioblastoma growth - net rates of proliferation (rho) and dispersal (D). These rates can be estimated from features of pretreatment MR images and can be applied in a mathematical model to predict tumour growth, impact of extent of tumour resection and patient survival. Only the pre-operative gadolinium-enhanced T1-weighted (T1-Gd) and T2-weighted (T2) volume data from 70 patients with previously untreated glioblastoma were used to derive a ratio D/rho for each patient. We developed a 'virtual control' for each patient with the same size tumour at the time of diagnosis, the same ratio of net invasion to proliferation (D/rho) and the same extent of resection. The median durations of survival and the shapes of the survival curves of actual and 'virtual' patients subjected to biopsy or subtotal resection (STR) superimpose exactly. For those actually receiving gross total resection (GTR), as shown by post-operative CT, the actual survival curve lies between the 'virtual' results predicted for 100 and 125% resection of the T1-Gd volume. The concordance between predicted (virtual) and actual survivals suggests that the mathematical model is realistic enough to allow precise definition of the effectiveness of individualised treatments and their site(s) of action on proliferation (rho) and/or dispersal (D) of the tumour cells without knowledge of any other clinical or pathological information.

Low grade gliomas: functional mapping resection strategies, extent of resection, and outcome.

The impact of surgery on outcome of adult patients with low-grade gliomas is controversial. Without prospective randomized treatment trials, one is primarily dependent on retrospective studies to address this issue. This paper reviews the recent clinical series of low-grade gliomas in which the association between extent of resection (EOR) and outcome could be analyzed. Functional stimulation mapping methods will be described to point out their critical role in maximizing the extent of resection while minimizing the risk associated with radical resection of low-grade gliomas. Studies from the CT-era analyzed with multivariate statistical methods were emphasized. The analysis of these studies points out that, for astrocytomas, there is no clear consensus that a greater EOR improves survival, but in most series under review, greater EOR significantly extended the survival of patients with oligodendroglioma. Unfortunately, there is little data which specifically analyzes and stratifies the outcome for other end-points such as time to progression, malignant degeneration, mortality and morbidity, and duration of high quality survival by EOR.

The cause of death in patients with glioblastoma is multifactorial: clinical factors and autopsy findings in 117 cases of supratentorial glioblastoma in adults.

To delineate the causes of death (COD) in adults with supratentorial glioblastoma multiforme (GM) we reviewed 117 consecutive cases examined at autopsy over a nineteen year period at the University of Washington. Twenty cases (17%) had expired unexpectedly without ante mortem diagnosis, 5 patients (4%) had been diagnosed as having lower grade astrocytomas prior to death. Other than the 20 patients without ante mortem diagnosis, all patients had a surgical procedure for treatment and/or diagnosis (biopsy 10%, craniotomy 90%). Postsurgical therapy varied, but there was no significant difference in median length of survival among the different treatment groups. Factors considered as potential COD were: herniation (axial, transtentorial, subfalcine, tonsillar), surgical complications (death within thirty days of surgery secondary to cerebral hemorrhage and/or edema), severe systemic illness, brainstem invasion by tumor, and neutron-induced cerebral injury (cerebral and brainstem gliosis were evident in these cases). A potential COD could be identified in 93% of patients. Patients with no ante mortem diagnosis were likely to have herniated (p = 0.01), whereas patients who underwent neutron irradiation were unlikely to have herniated (p = 0.001). No other variables were statistically significant predictors of herniation, including multifocal tumors (20 patients), and brainstem invasion by tumor (18 patients). No patients died as a result of treatment except those who underwent neutron radiotherapy and those who died postoperatively. Although significant mass effect, as evidenced by herniation, was apparent in 61% of patients, most of these patients had an additional identifiable COD. We conclude that the COD in patients with GM varies and is multifactorial.

Permanent low-activity (125)I seed placement for the treatment of pediatric brain tumors: preliminary experience.

Although external beam radiation therapy is effective in the treatment of many pediatric brain neoplasms its use in this patient population has been associated with the development of significant cognitive and endocrine dysfunction and is severely limited as an option in previously irradiated patients. Therefore, we have adopted a strategy for management of residual microscopic disease by implantation of low-activity (125)I seeds in the tumor bed at the time of surgery. Six patients aged 2-14 years with recurrent tumors including two supratentorial primitive neuroectodermal tumors (n = 2), one medulloblastoma, one malignant ependymoma (n = 1), glioblastoma (n = 1) and one pleomorphic xanthoastrocytoma were implanted at the time of reoperation. A total of 11-126 seeds were implanted resulting in total doses of 16-21.8 Gy (after theoretical infinite time) at a depth of 5 mm from the implanted resection bed. Five patients had prior external beam radiation while the other patient (2 years old at initial diagnosis) progressed after surgery and chemotherapy. Two patients had lasting local tumor control. One patient is alive at 390 weeks of follow-up and another who died of distant failure at 366 weeks had no recurrence on MRI at 333 weeks' follow-up. Only 2 patients had first local failures. These results suggest that the use of permanent low-activity (125)I seeds as an adjunct to surgery can provide good local tumor control and is a suitable treatment option for pediatric patients.

Gunshot wounds of the internal carotid artery at the skull base: management with vein bypass grafts and a review of the literature.

BACKGROUND: Penetrating trauma to the skull base and distal cervical internal carotid artery (ICA) can result in occlusion or pseudoaneurysm formation. The appropriate management strategy for these rare lesions is controversial and includes observation, anticoagulation, carotid ligation, balloon occlusion, or revascularization. METHODS: We present the management and outcomes of four consecutive patients, two with pseudoaneurysms and two with acute occlusions, after injury to the distal cervical/petrous ICA from gunshot wounds. Preoperative assessment determined intracranial collateral flow patterns and the patency of the distal portion of the petrous ICA. RESULTS: Two patients underwent cervical-to-petrous ICA vein bypass grafts without neurologic complications. Both grafts remain patent without evidence of emboli at 2 years and 3 months, respectively. Both of the conservatively managed patients died, one from a massive cerebral infarction and the other from intracerebral hemorrhage. CONCLUSIONS: These cases underscore the need for an aggressive approach to the assessment and management of patients with penetrating vascular skull-base injuries. Although the optimal treatment of remains controversial, when the goal is exclusion of the injured portion of the carotid artery and revascularization, the cervical to petrous ICA vein bypass graft is a valuable management option that can reduce the potential morbidity and mortality from acute ischemic or delayed embolic or hemorrhagic complications, provide immediate restoration of high flow, and allow good surgical access with minimal risk to intracranial structures.

Pleomorphic xanthoastrocytoma: DNA flow cytometry and outcome analysis of 12 patients.

BACKGROUND: Pleomorphic xanthoastrocytoma (PXA) is an astrocytic tumor occurring primarily in childhood and adolescence with some malignant histologic features but a relatively slow clinical course. However, some tumors progress more rapidly and can undergo malignant degeneration. The authors attempted to determine whether various histologic features or tumor cell proliferative indices might help identify lesions at risk for early progression and distinguish PXAs from malignant gliomas. METHODS: In a retrospective study of 12 patients with PXA, the tumor's histologic features and DNA flow cytometric parameters were compared with their clinical course. DNA flow cytometry values for the S- and G2-phase of the PXAs also were compared with control group samples of malignant and low grade astrocytomas. RESULTS: Of the 12 tumors at initial diagnosis, 5 were considered typical PXAs whereas 7 had some atypical features (4 with paucity of reticulin fibers, 1 with focal necrosis, and 2 with both atypical reticulin and focal necrosis). During the follow-up period (range, 3.75-11 years; mean, 6.8 years), 2 patients had recurrences; 1 atypical reticulin PXA progressed to glioblastoma after 6.5 years and the 1 tumor with focal necrosis recurred at 6 months and again at 2 years with typical histologic features. DNA flow cytometry parameters of the typical PXA group were similar to values for malignant astrocytoma and significantly higher than values for control specimens of low grade astrocytomas. There were no distinctive DNA flow cytometric features that could distinguish this last tumor from others with a more benign clinical course. CONCLUSIONS: Measurements of the S-phase and G2-phase obtained from DNA flow cytometry and atypical histologic features cannot reliably identify PXA patients at risk for early progression and overall are significantly higher than values obtained for low grade gliomas. Therefore, frequent (i.e., two to three times per year) postoperative clinical and radiologic examinations are necessary to judge the appropriateness of adjuvant therapy in patients with PXA. The paradox of slow growth but DNA flow cytometry consistent with aggressive malignant lesions may represent a cell-cycle arrest mechanism in these lesions that could be verified in subsequent studies.