Histopathological-Molecular Genetic Correlations in Referral Pathologist-Diagnosed Low-Grade “Oligodendroglioma” (original) (raw)

Journal Article

Molecular Neuro-Oncology Laboratory (HS, LBJ, ANC, DNL), Department of Pathology and Neurosurgical Service, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; Department of Oncology (MCZ, JGC), University of Western Ontario and London Regional Cancer Centre, London, Ontario, Canada; Department of Biostatistics (RAB), Harvard School of Public Health, Boston, Massachusetts.

Correspondence to: David N. Louis, MD, Molecular Neuro-Oncology Laboratory, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129.

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Magdalena C. Zlatescu, MD

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J. Gregory Cairncross, MD

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Revision received:

17 September 2001

Accepted:

20 September 2001

Published:

01 January 2002

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Hikaru Sasaki, Magdalena C. Zlatescu, Rebecca A. Betensky, Loki B. Johnk, Andrea N. Cutone, J. Gregory Cairncross, David N. Louis, Histopathological-Molecular Genetic Correlations in Referral Pathologist-Diagnosed Low-Grade “Oligodendroglioma”, Journal of Neuropathology & Experimental Neurology, Volume 61, Issue 1, January 2002, Pages 58–63, https://doi.org/10.1093/jnen/61.1.58
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Abstract

Allelic loss of chromosome 1p predicts increased chemosensitivity and better survival in oligodendroglial tumors. Clinical testing for 1p loss in oligodendroglial tumors at our hospital has allowed us to postulate that certain histological appearances are associated with 1p allelic status. Forty-four cases received for genetic testing were diagnosed by referring pathologists as pure low-grade oligodendroglioma. Central neuropathological review divided the series equally into 22 cases with classical oligodendroglioma histology and 22 with more astrocytic features. Molecular genetic analyses demonstrated 1p loss in 19 of 22 classic oligodendrogliomas (86%) and maintenance of both 1p alleles in 16 of 22 gliomas with astrocytic features (73%). No glial fibrillary acidic protein-positive cell type (gliofibrillary oligodendrocyte, minigemistocyte, cellular processes) was associated with 1p allelic status. Fourteen of the 44 cases were treated with chemotherapy at tumor progression: 3 “astrocytic” gliomas with 1p loss responded to PCV chemotherapy and 2 classic oligodendrogliomas that maintained both 1p alleles included a responder and a non-responder. These results suggest that histological appearance correctly predicts genotype in approximately 80% of low-grade gliomas, but that tumor genotype more closely predicts chemosensitivity. As a result, such objective molecular genetic analyses should be incorporated into patient management and into clinical trials of low-grade diffuse gliomas.

Introduction

For a number of reasons, oligodendrogliomas have been of great interest to the neuro-oncology community over the past decade. Among the malignant gliomas, oligodendrogliomas remain the only subtype that commonly responds to chemotherapy (1). Oligodendrogliomas also have, in general, more favorable prognoses than diffuse astrocytic tumors (2). Thus, neuro-oncologists and patients have become alert to the importance of recognizing oligodendroglial tumors, since the diagnosis affects both therapeutic decisions and estimation of prognosis. At the same time, the emphasis on the importance of oligodendroglioma diagnosis has been a substantial challenge to pathologists, since the criteria used to recognize these lesions are subjective and no reliable immunohistochemical markers exist. As a result, pathologists, rather than risk missing this clinically important diagnosis, have generally loosened their criteria for oligodendroglioma over the past few years and make the diagnosis more frequently than in the past. Importantly, it is possible that such loosened criteria may not yield groups of “oligodendrogliomas” that respond as frequently to chemotherapy and have as favorable prognoses as more traditionally defined oligodendrogliomas.

Oligodendrogliomas are genetically characterized by frequent allelic losses of chromosome 1p and 19q. Allelic loss of chromosome 1p in anaplastic oligodendroglioma is a predictor of far greater likelihood of chemotherapeutic response to procarbazine, lomustine (CCNU), and vincristine (PCV regimen). In addition, combined losses of 1p and 19q are powerful predictors of longer survival in anaplastic oligodendroglioma (1). Patients with low-grade oligodendrogliomas that have allelic loss of chromosome 1p and 19q also show a trend toward better survival (3). Thus, clinical testing for allelic loss of 1p and 19q provides relevant clinical data and, as a result, has begun to be incorporated in the clinical armamentarium.

Massachusetts General Hospital (MGH) began offering clinical testing for 1p loss in oligodendroglial tumors about two and a half years ago and has received approximately 1 case per week from hospitals around the United States and Canada. While the majority of cases have been anaplastic oligodendrogliomas, tumors diagnosed as lower-grade oligodendrogliomas have also been received for genetic evaluation. Such clinical testing has allowed our supervising neuropathologists to hypothesize that certain histological appearances of diffuse low-grade gliomas could be associated with 1p allelic status. Although all of these tumors arrived from outside hospitals with a diagnosis of “oligodendroglioma,” their appearances varied considerably, most likely reflecting the loosened criteria mentioned above. The presence of this population of referral pathologist-diagnosed oligodendrogliomas allowed us to inquire how oligodendrogliomas were being diagnosed in the referring community, relative to our practice and relative to objective parameters such as genetic status and therapeutic response. Using this population, we therefore sought to determine how closely standard hematoxylin and eosin (H&E) staining as well as glial fibrillary acidic protein (GFAP) immunohistochemistry reflected 1p status, and if histological appearance or 1p status correlated better with therapeutic parameters.

Materials and Methods

Tissues and Clinical Parameters

Forty-four low-grade gliomas were referred for clinical 1p testing. The cases originated from 16 different institutions across the United States and Canada. All 44 tumors were diagnosed as pure low-grade oligodendroglioma (WHO grade II) by the referring pathologists (4). Central histological review by one of us (DNL) confirmed that none of the tumors were classical biphasic oligoastrocytomas; in addition, while some had worrisome histological features such as mitotic activity, none met criteria for anaplastic oligodendroglioma, WHO grade III (4). Of the 44 tumors, 42 had no prior adjuvant therapy (radiation or chemotherapy), whereas information about prior treatment was not available for 2 cases. PCV chemotherapy was given to 14 of the 44 tumors at tumor progression, with a median interval of 47 months from surgery to chemotherapy (range: 2 months to 11 yr and 3 months). Eleven of the 14 PCV-treated tumors showed neuroradiological responses, defined by a decrease in tumor size of 50% or greater (5).

Assessment of Histopathological Features

Each case was assessed, using H&E stains and GFAP immunohistochemistry, by a single neuropathologist (DNL) blinded to the molecular genetic findings. Over the past 2 yr, it had been our impression that oligodendrogliomas with classical features more frequently demonstrated loss of 1p. Special attention was therefore paid to classical oligodendroglioma features, such as uniform and rounded nuclei, often with small nucleoli, surrounded by perinuclear halos, and in an even tissue distribution (Fig. 1). Cases with such features were designated as classic oligodendroglioma. Such tumors sometimes had other histological findings such as calcifications and “chickenwire” vasculature, but these were not considered necessary for designation as classic oligodendroglioma. Cases were also assessed for histopathological features commonly found in astrocytic tumors, such as irregular distribution of mildly pleomorphic, more hyperchromatic nuclei, often with tapering eosinophilic cell processes (Fig. 1). Such tumors were considered to have a more astrocytic histological appearance and, in our hospital practice, would be diagnosed as diffuse astrocytoma (4). These cases did not contain significant numbers of reactive-appearing astrocytes.

Fig. 1.

Low-grade “oligodendrogliomas” referred for genetic testing. Case 1 (1A, H&E; 1B, GFAP): classic oligodendroglioma with 1p loss showing rounded nuclei with perinuclear halos and microcalcification (1A). Reactive GFAP-positive astrocytes among predominantly GFAP-negative tumor cells (1B). Case 2 (2A, H&E; 2B, GFAP): classic oligodendroglioma with 1p loss showing even distribution of tumor cells with rounded, monotonous nuclei (2A). Minigemistocytes with GFAP-positive cytoplasm (2B). Case 3 (3A, H&E; 3B, GFAP): glioma with astrocytic features, without 1p loss. Irregularly distributed tumor cells with mild nuclear pleomorphism and occasional cellular processes (3A). Tumor cells with tapering cytoplasmic processes are highlighted with GFAP staining (3B, arrows). Case 4 (H&E): glioma with astrocytic features that has 1p loss, showing nuclear pleomorphism, irregular clustering of cells and some visible cellular processes. Case 5 (H&E): glioma with astrocytic features that has 1p loss. Cytoplasmic cellular processes are readily detectable in many tumor cells. This tumor showed 90% response to PCV chemotherapy at the time of progression. Original magnification: ×400 with enlargement in 4, 5.

GFAP immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections with monoclonal anti-human GFAP antibody (DAKO USA, Carpinteria, CA; 1:1,000 dilution) and visualized by the avidin-biotin complex technique with 3, 3′-diaminobenzidine tetrahydrochloride/H2O2 solution. The presence or absence of the following GFAP-positive features was carefully assessed: gliofibrillary oligodendrocytes (GFO), minigemistocytes, and tapering GFAP-positive cytoplasmic processes (reminiscent of those seen in small reactive astrocytes). By definition, GFO and minigemistocytes did not have such GFAP-positive processes. Each GFAP-positive feature was scored as −, not obvious; +, present in <10% of tumor cells; or ++, present in >10% of tumor cells.

Loss of Heterozygosity Studies

Tumor DNA was extracted from formalin-fixed, paraffin-embedded sections of histologically verified cellular tumor (6), and constitutional DNA was extracted from blood leukocytes according to standard protocols. Allelic losses of chromosomes 1p and 19q were assessed in loss of heterozygosity assays using microsatellite markers on 1p36 (D1S2734, D1S199, D1S508) and 19q13.3 (D19S219, D19S112, D19S412), as previously described (6).

Statistical Analysis

The Fisher exact test was used to test for associations among genotype, histology, and GFAP immunohistochemistry.

Results

Histopathological-Molecular Genetic Correlation

Central neuropathological review of H&E stains divided the series into 2 even groups: 22 tumors with classical oligodendroglioma histopathology (classic oligodendroglioma) and 22 tumors with more astrocytic histopathological features. Those with classical oligodendroglioma appearances were predicted to have allelic loss of chromosome 1p, whereas those with more astrocytic features were predicted to have maintenance of both 1p alleles. Molecular genetic analyses demonstrated 1p loss in 19 of 22 tumors (86%) with classical oligodendroglioma histopathology, and maintenance of both 1p alleles in 16 of 22 lesions (73%) with more astrocytic features (Fig. 1; Table 1). Histopathological grouping based on H&E stains and 1p status were significantly associated with one another (p < 0.001). Nonetheless, 9 tumors had discrepant histopathology and 1p status; these tumors were all adult-onset, hemispheric gliomas except for 1 classic oligodendroglioma lacking 1p loss that arose in a 17-yr-old girl. Twenty-four of the 25 tumors with 1p loss also had allelic loss of chromosome 19q, and the sole tumor with 1p loss that maintained both 19q alleles had classical oligodendroglioma histopathology in the sampled tissue (Table 1). One of the 19 tumors without 1p loss had 19q loss; this was one of the 22 tumors with more astrocytic features (Table 1). GFAP immunohistochemistry was performed on 36 tumors for which extra tissue sections were available. Twenty-four of the 36 tumors had GFO, 14 had minigemistocytes, and 26 had GFAP-positive cellular processes (Fig. 1). There was no significant association between 1p allelic status and the presence of any of these GFAP-positive cell types (Table 2), although the presence of GFAP-positive cytoplasmic processes was, not surprisingly, associated with astrocytic histopathology (Table 3, p = 0.03).

TABLE 1

Histopathological-Molecular Genetic Correlations (n = 44)1,2

TABLE 1

Histopathological-Molecular Genetic Correlations (n = 44)1,2

TABLE 2

GFAP Immunohistochemistry vs 1p Status (n = 36)1,2

TABLE 2

GFAP Immunohistochemistry vs 1p Status (n = 36)1,2

TABLE 3

GFAP Immunohistochemistry vs “Trained” H&E Histological Classification (n = 36)1

TABLE 3

GFAP Immunohistochemistry vs “Trained” H&E Histological Classification (n = 36)1

Chemotherapeutic Response

Chemotherapeutic response was evaluated in 13 of the 14 tumors treated with PCV chemotherapy at conversion to more aggressive lesions. Eleven tumors responded and 2 did not respond. Ten of the 11 tumors that responded to chemotherapy had allelic loss of chromosome 1p, and both of the 2 tumors that lacked response to chemotherapy maintained both alleles of 1p. Five of the 13 tumors with known response status had histopathological-molecular genetic discrepancies and thus provided a small group to sample the possible predictive power of histopathological appearance versus 1p status. Strikingly, 3 tumors with more astrocytic histopathology but with 1p loss responded to PCV chemotherapy. One of the 2 tumors with classic oligodendroglioma pathology that maintained both 1p alleles did not respond to chemotherapy, but the other tumor responded.

Discussion

The present study, comparing molecular genetic and histopathological features in a referral pathologist-based series of non-anaplastic diffuse gliomas, suggests that the diagnosis of “oligodendroglioma” is being made more frequently in the community of pathologists currently referring these tumors to MGH for molecular diagnosis than in our laboratory at central review. In this series, only half of 44 tumors diagnosed as “oligodendroglioma” in the United States and Canada were considered classic oligodendrogliomas on central neuropathological review. Importantly, these diagnoses were compared with objective molecular genetic parameters: allelic losses of chromosomes 1p and 19q. Central review diagnosis of oligodendroglioma correlated closely with allelic status of 1p and 19q, with 86% of such tumors harboring 1p loss. In contrast, only 57% of referral pathologist-diagnosed “oligodendrogliomas” had 1p loss. The present figure of 86% of our central neuropathologist-diagnosed oligodendrogliomas with 1p loss also contrasts with a figure of 73% 1p loss in a predominantly low-grade collection of oligodendrogliomas diagnosed by consensus of 3 experienced neuropathologists (7). The comparison figures demonstrate that pathologists can be educated to recognize histopathological patterns that reflect specific tumorigenic events.

In our experience, the overwhelming majority (86%) of diffuse, non-anaplastic gliomas with classical oligodendroglioma histology have allelic loss of chromosome 1p. These tumors have relatively evenly distributed, uniform and rounded nuclei and frequent perinuclear halos. Such tumors are not controversial diagnoses and resemble the typical depictions of oligodendroglioma in standard textbooks. In common parlance, these cases are “first year pathology resident” diagnoses. Other histological features, such as delicate branching vasculature and calcifications, may be present but are not essential to the diagnosis. On the other hand, the diffuse gliomas with more astrocytic histological appearances, which would have been diagnosed by us as “diffuse astrocytoma,” have irregularly distributed, mildly pleomorphic and irregular, hyperchromatic nuclei, often with tapering eosinophilic cytoplasmic processes that can be highlighted with GFAP immunohistochemistry. Notably, these cases did not simply represent the infiltrating edge of an otherwise classical oligodendroglioma, and copious reactive astrocytes were not present. Presumably, the presence of some rounded nuclei and some perinuclear halos in such cases suggested a diagnosis of “oligodendroglioma” to the referring pathologists. More than 70% of such cases maintained both 1p alleles. Thus, although it is perhaps premature to suggest that such cases are not oligodendrogliomas, these tumors clearly differ from classic oligodendrogliomas at a genetic level. Nonetheless, the classic oligodendrogliomas lacking 1p loss did not appear histologically different from those with 1p loss, nor did the tumors with more astrocytic appearance that had 1p loss appear histologically different from those lacking 1p loss.

In the present study, none of the various GFAP-positive cell types was significantly associated with 1p allelic status, consistent with the previous report that presence of minigemistocytes and/or GFO does not correlate with patient survival (8). The presence of GFAP-positive cell processes correlated with the central review impression of astrocytic features, but in nearly all cases the processes were clearly visible on H&E stains. Therefore, GFAP patterns are not likely to predict 1p allelic status in low-grade diffuse gliomas directly, although GFAP staining can confirm astrocytic histological features such as cell processes, which in turn correlate with maintenance of both 1p alleles.

The present findings raise the questions of how one defines an oligodendroglioma, and whether a particular genetic signature can define a neuropathological entity. In the fields of hematological and soft tissue malignancies, various translocations and other genetic events indeed define diagnostic entities (9, 10). In neuro-oncology, there is certainly increasing evidence that tumor genotyping can stratify gliomas into clinically relevant groups (1, 3, 11, 12). This evidence is largely derived from studies of oligodendrogliomas, particularly anaplastic oligodendrogliomas. In these tumors, as noted earlier, there are strong associations between allelic status and both therapeutic response and survival. Allelic status of chromosomes 1p and 19q has become an important determinant in oligodendroglioma patient management, not only to encourage chemotherapy in some patients, but to minimize the toxicity of ineffective therapies in other patients (11). Such tailored approaches to patient treatment promise to improve the likelihood of response of individual patients to therapies and reduce unnecessary toxicities, thus maximizing patient quality of life.

The association of 1p and 19q loss with well-differentiated oligodendroglioma histology has been suggested from single institution studies (13), but it has not been determined whether histological appearance or genetic status is more relevant to clinical course. The present series enabled us to identify discrepant cases in which pathological appearance and actual allelic status did not match, that is, classical oligodendrogliomas maintaining both 1p alleles and more astrocytic tumors with 1p loss. Since prognostic studies of low-grade diffuse gliomas require long follow-up times, the present series of relatively recent cases cannot adequately evaluate differences in prognosis; the literature, however, suggests that 1p loss does confer better prognosis in series of consensus-diagnosed low-grade oligodendrogliomas (3). Importantly, we were able to assess therapeutic response in those patients who received chemotherapy at recurrence or progression. Strikingly, of the 5 tumors discrepant for genotype-phenotype that were treated with PCV therapy at tumor progression, all 3 tumors with 1p loss that were centrally diagnosed as having astrocytic features indeed responded to chemotherapy. The 2 tumors with classic oligodendroglioma histology that maintained both 1p alleles included 1 responder and 1 non-responder. Thus, the outcomes of 4 of 5 discordant cases were predicted by their genotype rather than their phenotype. Although these are small numbers of cases and although these chromosomal changes do not currently define oligodendroglioma, the data raise the testable hypothesis that 1p allelic status is a more clinically relevant predictor than histopathological diagnosis in diffuse low-grade gliomas (14).

A caveat to some of the molecular-histological comparisons relates to possible sampling error, a common problem in neuropathological diagnosis of brain tumor biopsies. Two of the 3 tumors in this series with classic oligodendroglioma histology that maintained both 1p alleles were represented by only small biopsy specimens, and it is possible that other regions of the tumor appeared astrocytic. Nonetheless, such possibilities again indicate a role for molecular genetic testing, especially since studies on glial neoplasms to date have shown greater homogeneity in genotype than in phenotype. For instance, microdissection of oligodendroglioma from astrocytoma components in oligoastrocytomas has revealed allelic losses of 1p and 19q in the astrocytic regions if present in the oligodendroglial areas (15). And in gliosarcomas, the glioblastomatous and sarcomatous regions typically have the same genetic alterations (16, 17). Such data argue strongly that molecular genetic testing can add objectivity sorely needed to overcome diagnostic problems such as phenotypic heterogeneity and tissue sampling biases.

Finally, it is important to emphasize that even neuropathologists “trained” to recognize particular histopathological appearances do not achieve 100% accuracy in predicting tumor genotype in low-grade diffuse gliomas. Rather, the correct prediction rate was only 80% for these non-anaplastic diffuse gliomas (35 of 44 cases; 76% of 1p-deleted and 84% of 1p-intact tumors). Moreover, for those cases with discrepant findings, tumor genotype seems more promising than histological appearance in predicting possible response to chemotherapy. While such results clearly need to be confirmed in larger and prospective series, the present findings encourage the use of molecular analyses in clinical trials that evaluate novel therapies, not only in oligodendroglioma studies but also in investigations of low-grade diffuse gliomas in general.

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Author notes

Supported by NIH CA57683 and MRC-MOP-37849.

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Histopathological-Molecular Genetic Correlations in Referral Pathologist-Diagnosed Low-Grade “Oligodendroglioma” (original) (raw)

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Abstract

Introduction