Richard Meyermann

NOA-04 Randomized Phase III Trial of Sequential Radiochemotherapy of Anaplastic Glioma With Procarbazine, Lomustine, and Vincristine or Temozolomide

PURPOSE The standard of care for anaplastic gliomas is surgery followed by radiotherapy. The NOA-04 phase III trial compared efficacy and safety of radiotherapy followed by chemotherapy at progression with the reverse sequence in patients with newly diagnosed anaplastic gliomas. PATIENTS AND METHODS Patients (N = 318) were randomly assigned 2:1:1 (A:B1:B2) to receive conventional radiotherapy (arm A); procarbazine, lomustine (CCNU), and vincristine (PCV; arm B1); or temozolomide (arm B2) at diagnosis. At occurrence of unacceptable toxicity or disease progression, patients in arm A were treated with PCV or temozolomide (1:1 random assignment), whereas patients in arms B1 or B2 received radiotherapy. The primary end point was time to treatment failure (TTF), defined as progression after radiotherapy and one chemotherapy in either sequence. RESULTS Patient characteristics in the intention-to-treat population (n = 274) were balanced between arms. All histologic diagnoses were centrally confirmed. Median TTF (hazard ratio [HR] = 1.2; 95% CI, 0.8 to 1.8), progression-free survival (PFS; HR = 1.0; 95% CI, 0.7 to 1.3, and overall survival (HR = 1.2; 95% CI, 0.8 to 1.9) were similar for arms A and B1/B2. Extent of resection was an important prognosticator. Anaplastic oligodendrogliomas and oligoastrocytomas share the same, better prognosis than anaplastic astrocytomas. Hypermethylation of the O(6)-methylguanine DNA-methyltransferase (MGMT) promoter (HR = 0.59; 95% CI, 0.36 to 1.0), mutations of the isocitrate dehydrogenase (IDH1) gene (HR = 0.48; 95% CI, 0.29 to 0.77), and oligodendroglial histology (HR = 0.33; 95% CI, 0.2 to 0.55) reduced the risk of progression. Hypermethylation of the MGMT promoter was associated with prolonged PFS in the chemotherapy and radiotherapy arm. CONCLUSION Initial radiotherapy or chemotherapy achieved comparable results in patients with anaplastic gliomas. IDH1 mutations are a novel positive prognostic factor in anaplastic gliomas, with a favorable impact stronger than that of 1p/19q codeletion or MGMT promoter methylation.

A Functional Role of HLA-G Expression in Human Gliomas: An Alternative Strategy of Immune Escape

HLA-G is a nonclassical MHC molecule with highly limited tissue distribution that has been attributed chiefly immune regulatory functions. Glioblastoma is paradigmatic for the capability of human cancers to paralyze the immune system. To delineate the potential role of HLA-G in glioblastoma immunobiology, expression patterns and functional relevance of this MHC class Ib molecule were investigated in glioma cells and brain tissues. HLA-G mRNA expression was detected in six of 12 glioma cell lines in the absence of IFN-γ and in 10 of 12 cell lines in the presence of IFN-γ. HLA-G protein was detected in four of 12 cell lines in the absence of IFN-γ and in eight of 12 cell lines in the presence of IFN-γ. Immunohistochemical analysis of human brain tumors revealed expression of HLA-G in four of five tissue samples. Functional studies on the role of HLA-G in glioma cells were conducted with alloreactive PBMCs, NK cells, and T cell subpopulations. Expression of membrane-bound HLA-G1 and soluble HLA-G5 inhibited alloreactive and Ag-specific immune responses. Gene transfer of HLA-G1 or HLA-G5 into HLA-G-negative glioma cells (U87MG) rendered cells highly resistant to direct alloreactive lysis, inhibited the alloproliferative response, and prevented efficient priming of cytotoxic T cells. The inhibitory effects of HLA-G were directed against CD8 and CD4 T cells, but appeared to be NK cell independent. Interestingly, few HLA-G-positive cells within a population of HLA-G-negative tumor cells exerted significant immune inhibitory effects. We conclude that the aberrant expression of HLA-G may contribute to immune escape in human glioblastoma.

Efficacy and Tolerability of Temozolomide in an Alternating Weekly Regimen in Patients With Recurrent Glioma

PURPOSE Evaluation of toxicity and efficacy of an alternating weekly regimen of temozolomide administered 1 week on and 1 week off in patients with recurrent glioma. PATIENTS AND METHODS Ninety adult patients with recurrent gliomas accrued in one center received chemotherapy with temozolomide at 150 mg/m(2)/d (days 1 through 7 and 15 through 21 every 4 weeks) with individual dose adjustments according to hematologic toxicity. RESULTS A total of 906 treatment weeks were delivered. Grade 4 hematotoxicity according to the Common Terminology Criteria for Adverse Events (CTCAE; version 3.0) was observed in 24 treatment weeks (2.6%). CTCAE grade 4 lymphopenia eventually developed in 11 patients (12%). There were neither cumulative lymphopenias nor opportunistic infections. The progression-free survival (PFS) rate at 6 months for glioblastoma patients was 43.8%. The median PFS in these patients was 24 weeks (95% CI, 17 to 26 weeks), the median survival time from diagnosis of progression was 38 weeks (95% CI, 30 to 46 weeks), and the 1-year survival rate from progression was 23%. O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter methylation in the tumor tissue was not associated with longer PFS (log-rank P = .37). CONCLUSION These data imply that the alternating weekly schedule is feasible, safe, and effective and clearly warrants investigation in randomized studies. Compared with more protracted low-dose temozolomide schedules, the 1-week-on/1-week-off schedule may be less toxic. We provide preliminary evidence that this dose-dense schedule is also active in patients with tumors lacking MGMT gene promoter methylation.

Local distribution of microglia in the normal adult human central nervous system differs by up to one order of magnitude.

Abstract. Although microglia are considered to be a sensitive sensor for pathological processes in the central nervous system, there are only a few studies about the distribution and density of microglia in the normal human brain. Therefore, a study of local density of microglial cells was conducted by investigating 20 normal human brains with no clinical neurological symptoms or diseases and no neuropathological alterations. Microglial cells were visualized by immunolabeling of proteins which are known to be expressed either constitutively or facultatively, such as CD68, major histocompatibility complex class II (MHC-II), leukocyte common antigen (LCA), leukocyte chemotactic factor (LCF), macrophage inhibitory factor-related protein (MRP) 8, MRP14, CD4 and allograft-inflammatory factor-1 (AIF-1). CD68, MHC-II and AIF-1 showed the highest densities with significant regional differences ranging from 0.5% to 16.6% of all cells in the brain parenchyma with significantly more microglia in white than in gray matter. LCF and LCA showed a similar pattern of distribution as the proteins described above, but with lower percentages of microglial cells. CD4 was not found in the brain parenchyma. We conclude that CD68, MHC-II and AIF-1 define the main microglial cell population, whereas LCF and LCA are expressed by a subpopulation of microglial cells. The brains showed no or a negligible vascular expression of MRP8 and MRP14. Information about the local microglia density in the normal human brain can serve as a reference for the evaluation of pathological microglial responses.

Long-term expression of heme oxygenase-1 (HO-1, HSP-32) following focal cerebral infarctions and traumatic brain injury in humans.

Abstract Extracellular heme derived from hemoglobin following hemorrhage or released from dying cells induces the expression of heme oxygenase-1 (HO-1, HSP-32) which metabolizes heme to the gaseous mediator carbon monoxide (CO), iron (Fe) and biliverdin. Biliverdin and its product bilirubin are powerful antioxidants. Thus, expression of HO-1 is considered to be a protective mechanism against oxidative stress and has been described in microglia, astrocytes and neurons following distinct experimental models of pathological alterations to the brain such as subarachnoidal hemorrhage, ischemia and traumatic brain injury (TBI) and in human neurodegenerative diseases. We have now analyzed the expression of HO-1 in human brains following TBI (n = 28; survival times: few minutes up to 6 months) and focal cerebral infarctions (FCI; n = 17; survival time: < 1 day up to months) by ¶immunohistochemistry. Follwing TBI, accumulation of ¶HO-1+ microglia/macrophages at the hemorrhagic lesion was detected as early as 6 h post trauma and was still pronounced after 6 months. In contrast, after FCI HO-1+ microglia/macrophages accumulated within focal hemorrhages only and were absent in non-hemorrhagic regions. Further, HO-1 was weakly expressed in astrocytes in the perifocal penumbra. In contrast to experimental data derived from rat focal ischemia, these results indicate a prolonged HO-1 expression in humans after brain injury.

Phase II Trial of Lomustine Plus Temozolomide Chemotherapy in Addition to Radiotherapy in Newly Diagnosed Glioblastoma: UKT-03

PURPOSE To evaluate toxicity and efficacy of the combination of lomustine, temozolomide (TMZ) and involved-field radiotherapy in patients with newly diagnosed glioblastoma (GBM). PATIENTS AND METHODS Thirty-one adult patients (median Karnofsky performance score 90; median age, 51 years) accrued in two centers received involved-field radiotherapy (60 Gy in 2-Gy fractions) and chemotherapy with lomustine 100 mg/m2 (day 1) and TMZ 100 mg/m2/d (days 2 to 6) with individual dose adjustments according to hematologic toxicity. RESULTS A median of five courses (range, one to six courses) were delivered. WHO grade 4 hematotoxicity was observed in five patients (16%) and one of these patients died as a result of septicemia. Nonhematologic toxicity included one patient with WHO grade 4 drug-induced hepatitis (leading to discontinuation of lomustine and TMZ) and one patient with WHO grade 2 lung fibrosis (leading to discontinuation of lomustine). The progression-free survival (PFS) rate at 6 months was 61.3%. The median PFS was 9 months (95% CI, 5.3 to 11.7 months), the median overall survival time (MST) was 22.6 months (95% CI, 12.5 to not assessable), the 2-year survival rate was 44.7%. O6-methylguanine-DNA methyltransferase (MGMT) gene-promoter methylation in the tumor tissue was associated with longer PFS (P = .014, log-rank test) and MST (P = .037). CONCLUSION The combination of lomustine, TMZ, and radiotherapy had acceptable toxicity and yielded promising survival data in patients with newly diagnosed GBM. MGMT gene-promoter methylation was a strong predictor of survival.

Patterns of cyclooxygenase-1 and -2 expression in human gliomas in vivo.

Abstract Cyclooxygenases (COX, prostaglandin endoperoxide synthases, PGG/H synthases) are potent mediators of inflammation. While COX-1 is constitutively expressed in a wide range of tissues, COX-2 is cytokine inducible. Although COX-1 expression is observed in normal tissue, enhanced COX-2 expression has been attributed a key role in the development of edema, impeding blood flow and immunomodulation observed in pathologically altered tissues. Here, we have analyzed the expression of COX-1 and COX-2 in 50 gliomas and 10 control brains with no neuropathological alterations by immunohistochemistry; 22 glioblastoma multiforme, 9 anaplastic astrocytomas, 5 protoplasmic astrocytomas, 1 gemistocytic astrocytoma and 13 fibrillary astrocytomas were included in the study. Compared with control brains, accumulation of COX-1 was detected in 20–50% of all cells in both low- and high-grade gliomas. Double-labeling experiments revealed COX-1 expression in subsets of macrophages/ microglial cells within the tumor parenchyma and in areas of infiltrative tumor growth. Of the COX-1-positive cells, 90% expressed MHC class II antigens. No COX-1 immunoreactivity was observed in tumor cells. COX-2-positive cells accumulated in tumor cells and in single macrophages/microglial cells in the immediate vicinity of necroses. Further studies are required to determine whether COX-2 is involved in the development of necrosis or, more likely, whether COX-2 is a part of the tumor tissue response to necrosis.

Application of mutant IDH1 antibody to differentiate diffuse glioma from nonneoplastic central nervous system lesions and therapy-induced changes.

Differentiation of gliomas and reactive gliosis may be challenging both at primary tumor occurrence and at posttherapy biopsy. The most frequent IDH1 mutation found in the majority of WHO grade II and III gliomas can be visualized with an antibody specifically detecting mutant IDH1R132H protein. In this study, mIDH1R132H immunoreactivity in 120 reactive gliosis specimens of various etiologies is compared with Wilms Tumor 1 (WT1) and p53 expression, both markers applied for the differentiation of reactive gliosis and glioma. Although WT1 and p53 positive glial cells were found in 17% and 63% of cases respectively, all samples were negative for mIDH1R132H. Furthermore, we investigated 19 posttherapy gliomas (6 WHO II, 13 WHO III) with extensive reactive changes and detected mIDH1R132H positive cells in 13 specimens. In 5 of these cases, tumor cells were missed by conventional staining, showing the improved sensitivity of mIDH1R132H. Thus, mIDH1R132H is a tumor-specific marker that is superior to other established markers to differentiate reactive from neoplastic cells in grade II and III gliomas and allows identifying tumor cells in posttherapy specimens with extensive reactive changes. As IDH mutations are not characteristic of grade IV primary glioblastomas, this antibody cannot differentiate primary glioblastoma from reactive gliosis. Thus, caution has to be taken and a combined panel with other markers is needed.

The allograft inflammatory factor-1 family of proteins

The allograft inflammatory factor‐1 (AIF‐1) is a 17 kDa interferon‐γ‐inducible Ca2+‐binding EF‐hand protein that is encoded within the HLA class III genomic region. Three proteins are probably identical with AIF‐1 termed Iba1 (ionized Ca2+‐binding adapter), MRF‐1 (microglia response factor) and daintain. Considerable but not complete sequence identity with AIF‐1 has been described for IRT‐1 (interferon‐responsive transcript), BART‐1 (balloon angioplasty‐responsive transcript), and other, yet unassigned alternatively spliced variants. In this review, genomic and functional characteristics of AIF‐1‐related proteins are summarized and a common nomenclature is proposed.

Dynamics of microglial activation after human traumatic brain injury are revealed by delayed expression of macrophage-related proteins MRP8 and MRP14

Abstract Human traumatic brain injury (TBI) is ideally suited for investigation of the kinetics of human microglial cell activation as the onset of lesion formation is precisely defined. The present study provides evidence of a distinct delay in macrophage/microglia response following TBI. Eighteen brains of patients who had survived TBI for 1 h to 6 months were analysed by immunohistology. Samples of contusional and non-contusional areas were studied using antibodies directed against antigens of microglia/ macrophages [major histocompatibility complex class II, CD4, interleukin (IL)-16, macrophage-related protein (MRP) 8 and MRP14]. IL-16, a natural ligand to CD4, was expressed constitutively by numerous microglial cells in all cases throughout the brain. CD4 could be detected regularly on perivascular cells. MRP8 and MRP14, which are only expressed on activated macrophages and microglial cells, could be detected only within brains with a survival time of more than 72 h post TBI. In addition, proliferation of microglia detected by MIB-1 was not present until 72 h. This delayed expression of the activation markers MRP8 and MRP14 and the proliferation marker MIB-1 is comparable to experimental closed head injuries but strictly different from acute activation found in ischemic brains.