Richard P. Moser

Comparison of cell adhesion molecule expression between glioblastoma multiforme and autologous normal brain tissue

We investigated glioblastoma multiforme (GBM) for a pattern of consistent alterations in cell adhesion molecules (CAM) expression that might distinguish tumor from normal autologous brain tissue. We used frozen section immunohistochemistry with anti-CAM and computerized image analysis to quantify staining intensity which we expressed as relative intensity units (RIU). Our results showed that normal brain tissue generally did not express alpha 1 beta 1, intercellular CAM-1 (ICAM-1), and sialylated Lewisx, slightly expressed alpha 2, alpha 4, alpha 5, alpha 6 beta 1, alpha v beta 3, lymphocyte function-associated antigen-3 (LFA-3), Lewisx, sialylated LewisLewisx, had a good expression of alpha 3 beta 1 and CD44, and strongly expressed neural CAM (NCAM). GBM expressed alpha 2, alpha 3, alpha 5, alpha 6 beta 1, alpha v beta 3, ICAM-1, LFA-3, CD44, Lewisx, sialylated Lewisx, and sialylated LewisLewisx significantly higher (2-11-fold RIU) than normal brain tissue. ICAM-1 and LFA-3 were the most distinctive markers of GBM. The small blood vessel endothelial cells of the normal brain and the GBM showed a few differences. The tumor endothelium expression of alpha 2 beta 1, alpha 4 beta 1, and LFA-3 RIU appeared twice higher than in normal endothelium and alpha 6 beta 1 showed an average of 40% RIU decrease in comparison to normal. These results show that the expression of several CAM is consistently altered in GBM and its microvasculature when compared with autologous normal brain tissue.

Outcome and patterns of failure following limited-volume irradiation for malignant astrocytomas

Between January 1982 and June 1986, 60 consecutive patients with high-grade astrocytomas [39 glioblastoma multiforme (GBM), 21 anaplastic astrocytoma (AA)] were treated with radiation therapy after biopsy (13 patients) or resection (47 patients). Fifty-three patients were treated with limited-volume irradiation, and seven patients received whole-brain irradiation. The mean tumor dose was 65.4 Gy. In 35 patients, chemotherapy was given as part of their initial treatment. The 1- and 2-year survivals for GBM patients were 40 and 14%, respectively. Survival figures for AA patients were 76 and 52% at 1 and 2 years, respectively. The progression-free rate at 1 year was 13% in GBM and 29% in AA patients. Thirty-four of 48 patients who received limited-volume irradiation had evidence of progression on postirradiation CT scans. Six patients (3 GBM, 3 AA) had evidence of a new intracranial metastatic site on CT scan. In three patients the metastasis was within the previously irradiated volume, and in the other three patients, it was outside this volume. All six had evidence of progression of their primary tumor at the original location on CT scan prior to the discovery of the metastatic site. Twenty-one patients (15 GBM, 6 AA) had at least one postirradiation reoperation for a recurrent mass. Nineteen patients had recurrent tumors in the primary site, and two patients had necrosis but no tumor. Patients who received limited-volume irradiation for high-grade astrocytomas achieved the same survival results as patients treated previously with whole brain irradiation. New intracranial metastases did not influence the outcome, since these were always antedated by tumor progression at the primary site.

Anterior transcranial (craniofacial) resection of tumors of the paranasal sinuses: Surgical technique and results

Transfacial approaches, traditionally used for malignant tumors of the paranasal sinuses, provide limited exposure when several sinuses are involved and are unsuitable for tumors that erode through the floor of the anterior cranial fossa. A transcranial approach may aid in the removal of such lesions. To better understand the risks and benefits of this surgical approach, we reviewed all patients (n = 76) who underwent a transcranial approach as part of the excision of paranasal sinus lesions between 1984 and 1993 at our institution. The spectrum of disease included adenocarcinoma (13 patients), squamous cell carcinoma and olfactory neuroblastoma (11 patients each), adenoid cystic carcinoma and poorly differentiated forms of carcinoma (6 patients each), melanoma (5 patients), and miscellaneous others (24 patients). Most patients had ethmoid sinus involvement; tumors were also commonly found in the cribriform plate, sphenoid sinus, and nasal fossa. In each patient, a bifrontal craniotomy was performed with extradural dissection to the floor of the anterior fossa and osteotomies for resection of involved elements. In 47 patients (62%), disease in the orbit, the anterior nasal cavity, or the soft tissues of the face required transfacial as well as transcranial resections. Bony defect in the anterior fossa floor was repaired with a pedicled pericranial flap. Patients with major complications included six patients with epipericranial and/or epidural hematomas requiring evacuation, three with transient cerebrospinal fluid leaks, two who developed bifrontal cerebral infarcts, and one who died soon after surgery. No meningitis was seen. To date, 26 patients (34%) have died; of those living (mean follow-up, 34 mo), 42 (84%) remain in full remission. The transcranial approach can achieve removal of erosive, invasive tumors from this area with predictable morbidity and may be considered whenever sinus tumors breach the anterior cranial base or extend beyond the reach of conventional transfacial approaches.: Transfacial approaches, traditionally used for malignant tumors of the paranasal sinuses, provide limited exposure when several sinuses are involved and are unsuitable for tumors that erode through the floor of the anterior cranial fossa. A transcranial approach may aid in the removal of such lesions. To better understand the risks and benefits of this surgical approach, we reviewed all patients (n = 76) who underwent a transcranial approach as part of the excision of paranasal sinus lesions between 1984 and 1993 at our institution. The spectrum of disease included adenocarcinoma (13 patients), squamous cell carcinoma and olfactory neuroblastoma (11 patients each), adenoid cystic carcinoma and poorly differentiated forms of carcinoma (6 patients each), melanoma (5 patients), and miscellaneous others (24 patients). Most patients had ethmoid sinus involvement; tumors were also commonly found in the cribriform plate, sphenoid sinus, and nasal fossa. In each patient, a bifrontal craniotomy was performed with extradural dissection to the floor of the anterior fossa and osteotomies for resection of involved elements. In 47 patients (62%), disease in the orbit, the anterior nasal cavity, or the soft tissues of the face required transfacial as well as transcranial resections. Bony defect in the anterior fossa floor was repaired with a pedicled pericranial flap. Patients with major complications included six patients with epipericranial and/or epidural hematomas requiring evacuation, three with transient cerebrospinal fluid leaks, two who developed bifrontal cerebral infarcts, and one who died soon after surgery. No meningitis was seen. To date, 26 patients (34%) have died; of those living (mean follow-up, 34 mo), 42 (84%) remain in full remission. The transcranial approach can achieve removal of erosive, invasive tumors from this area with predictable morbidity and may be considered whenever sinus tumors breach the anterior cranial base or extend beyond the reach of conventional transfacial approaches.

Predominance of a type 2 intratumoural immune response in fresh tumour-infiltrating lymphocytes from human gliomas.

Increasing evidence suggests the existence of polarized human T cell responses described as Th1‐type (promoting cell‐mediated immunity) and Th2‐type (promoting humoral immunity), characterized by a dominant production of either interferon‐gamma (IFN‐γ) or IL‐4, respectively. Little is known about the intratumoural activation of infiltrating lymphocytes (TIL) in human gliomas. Therefore, we assessed fresh TIL at cellular and molecular levels to find out if they were activated and polarized into a type 1 or 2 immune response. Flow cytometry analysis of TIL revealed that the major subset was made of T lymphocytes. Double labelling with α‐CD3 and adhesion/activation markers revealed T cell subsets expressing CD49a, CD49b, CD54, and CD15, some of which were almost absent in autologous T peripheral blood lymphocytes (T‐PBL). Furthermore, the proportions of T‐TIL expressing CD56, CD65, or CD25 were several‐fold higher than in T‐PBL. Intratumoural functional activation of TIL was tested by semiquantitative assessment in relative units (RU) of lymphokine gene activation with mRNA reverse transcriptase‐polymerase chain reaction (RT‐PCR). All TIL populations except one significantly expressed IL‐4 1 to 2 logs of RU above healthy PBL baseline. Similarly, all patients expressed granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in a range comparable to IL‐4. However, most TIL populations did not express IFN‐γ, IL‐2, and tumour necrosis factor‐beta (TNF‐β) at higher levels than healthy normal PBL. The increased proportion of T cells expressing activation markers and the consistent detection of significant IL‐4 and GM‐CSF lymphokine gene activation in TIL populations suggested a predominant type 2 intratumoural immune response that does not promote cell‐mediated tumouricidal activity and may contribute to the inefficiency of the antiglioma immune response.

Neurotoxicity of intraventricularly administered alpha-interferon for leptomeningeal disease

Nine patients with leptomeningeal disease are reported who were treated with intraventricular alpha‐interferon (α‐IFN). In seven of these patients, a progressive vegetative state developed during treatment. The patients became unresponsive to verbal commands but opened their eyes with auditory or tactile stimulation. It took an average of 3 weeks for these patients to become verbally responsive after treatment was discontinued. Electroencephalographic findings showed evidence of irritative involvement of the deep midline nuclei in 80% of patients. Periventricular white matter changes developed during treatment in three of six patients who underwent computed tomographic scans. All patients with this severe neurotoxicity received whole‐brain irradiation before treatment. Possible mechanisms for the development of this neurotoxic syndrome are discussed. The neurotoxicity of α‐IFN and brain irradiation may be additive, suggesting a cautious approach when using this combination for treatment.

Growth inhibitory effect of recombinant α and β interferon on human glioma cells

SummaryGrowth inhibitory activity of recombinant α and β interferon on two human glioma cell lines, EFC-2 and KE cells, was determined by two different growth assays. Recombinant β interferon showed β slight growth inhibitory effect on EFC-2 cells at day 3, and maximum inhibition was seen on day 6 with an ID50 of 50 U/ml. Recombinant α interferon showed no significant growth inhibition at any concentration. KE cells were resistant to both recombinant α and β interferon. The growth inhibitory activity of recombinant β interferon on EFC-2 cells was not blocked by recombinant α interferon, although recombinant α and β interferons shared same receptors on EFC-2 cells. Addition of DFMO (α-difluoromethylornithine) to interferon in the media showed additive effect rather than synergistic effect in growth inhibition of glioma cells. Out of 7 glioma cell lines tested, 4 showed heterogeneous sensitivity to recombinant β interferon, and all were resistant to recombinant α interferon. These results suggest β differential sensitivity of EFC-2 cells to recombinant β interferon, as well as a heterogeneous sensitivity to recombinant β interferon among different glioma cell lines.

A pilot study of recombinant interferon beta (IFN-βser) in patients with recurrent glioma

Recombinant interferon beta (IFN-βser) has been administered by intravenous bolus injection three times weekly at a dose of 90 × 106 IU to 14, patients with recurrent malignant glioma in an ongoing study. The treatment period has ranged from 1 to 40 weeks. The most common adverse experiences were fever, chills, malaise, and headache. Fever, chills and headache were worse with the first two doses and were usually relieved with acetaminophen. All patients tolerated subsequent treatments without any difficulties. No neurologic or hematologic toxicities were observed. Of ten evaluable patients, five had progressive disease in 4 to 8 weeks; three had stable disease for 12 to 21 weeks; one has had a minor response for 13 weeks; and one has had a complete resolution of tumor for 150 + weeks. IFN-βser appears to have activity in human glioma and is well tolerated at this dosage and schedule.

Characterization of interleukin-2-initiated versus OKT3-initiated human tumor-infiltrating lymphocytes from glioblastoma multiforme: growth characteristics, cytolytic activity, and cell phenotype.

SummaryOutgrowth of tumor-infiltrating lymphocytes (TIL) from the human primary brain tumor glioblastoma multiforme was achieved by OKT3 initiation (10 ng/ml), followed by sustained expansion by interleukin-2 (IL-2; 200 U/ml). Tumor-infiltrating lymphocyte (TIL) initiation by this process was performed in parallel with the standard “IL-2-only” method. Of ten tumors, seven yielded TIL in response to OKT3/IL-2, whereas only three of these seven grew after initiation with IL-2 alone. On the basis of cell doubling times, at least 60 doublings, resulting in (hypothetically) up to 1023 TIL from as few as 2 × 105 cells in tumor suspensions, could be achieved using OKT3/IL-2. OKT3-initiated TIL proliferated in culture for as long as 288 days, although senescence of some cultures occurred at as early as 73 days. Significant heterogeneity of lymphocytes infiltrating the fresh tumors and heterogeneity of resultant TIL phenotype and function were apparent, yet several common trends were noted. In all cases after OKT3 initiation, significant net growth was not apparent until approximately 14 days. In contrast, in the three samples that grew in response to IL-2 alone, log-phase growth was always observed earlier. During the early phase of the cultures, all TIL expressed some killing activity toward a broad spectrum of tumors, including the autologous tumor. No consistent preference of TIL for lysis of autologous tumor was observed. Glioblastoma multiforme TIL cultures contained a mixture of CD8+ and CD4+ cells, with few CD16+ or NKH-1+. Of the six TIL examined in detail for population phenotype in relationship to time in culture, four eventually became exclusively CD4+. Further analysis of these CD4+ TIL indicated that all were of the helper-inducer class, 4B4+ and 2H4−. Concurrent with the decline in CD8+ cells, a decline in the cytolytic activity of these TIL cultures occurred. Furthermore, in two TIL that remained CD8+, a decline in the cytolytic activity also occurred. Therefore, loss of killing activity was not merely a reflection of the major cell phenotype changes. These results indicate that the OKT3/IL-2 process provides an alternative to IL-2 alone for TIL initiation and growth, as well as providing a novel system for further analysis of tumorderived lymphocyte and accessory cell functional potential.

Detection of p53 alterations in human astrocytomas using frozen tissue sections for the polymerase chain reaction

SummaryThe polymorphism of amino acid residue 72 on the humanp53 tumor-suppressor gene is a useful marker for detecting intragenic loss of heterozygosity (LOH). We examined the LOH of thep53 gene in human malignant astrocytomas by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using DNA extracted from frozen tissue sections under histologic examination. Eleven of 16 informative cases (69%) of the malignant astrocytomas were found to have LOH in thep53 gene. Sequential frozen sections were analyzed by immunohistochemistry using anti-p53 antibody PAb1801 to detect overexpression of the p53 protein, which is presumably altered if it is detectable. Ten of the 11 cases that had LOH of thep53 gene overexpressed the p53 protein. Moreover, 4 of the 11 patients with LOH of thep53 gene developed a second neoplasm in addition to an astrocytoma, possibly indicating genetic instability in these patients. These data suggest that alterations of thep53 gene may play an important role in the genesis of malignant astrocytoma. The combination of the PCR-RFLP method and immunohistochemical analysis using frozen tissue sections is a practical diagnostic tool for examination of human malignancies, including astrocytomas.

Effect of anti-CD3/anti-CD28/interleukin-2 stimulation of mononuclear cells on transforming growth factor β inhibition of lymphokine-activated killer cell generation

SummaryAfter simultaneous stimulation with anti-CD3 monoclonal antibody (mAb) at 10 ng/ml, anti-CD28 mAb at 125 ng/ml, and interleukin-2 (IL-2) at 20 U/ml, peripheral blood mononuclear cells (PBMC) were partially resistant to immunosuppression by transforming growth factor-β (TGFβ2). The doses of TGFβ2 that inhibit cytotoxicity of IL-2 stimulated cells by 60%–70% were much less effective when the same cells were stimulated with anti-CD3/anti-CD28/IL-2. This favorable stimulation generated a cell population characterized by high lytic activity, excellent expansion, and a greater resistance to immunosuppressive action of TGFβ2. The secretion of secondary cytokines important for LAK generation is considered a crucial event, at least partially responsible for the antagonization of TGFβ immunosuppression.