Chromosomal composition of a series of 22 human low-grade gliomas

G-banded chromosomal analysis was performed on direct and/or in vitro cultures of 22 low-grade gliomas, including nine grade I-II astrocytomas, nine oligodendrogliomas, one mixed tumor oligodendroglioma-astrocytoma, and three ependymomas. Normal diploid stem lines were present in most astrocytomas and oligodendrogliomas, whereas, all three ependymomas displayed polyploid modal numbers. However, secondary cell lines showed the presence of clonal recurrent numerical abnormalities, mainly polysomy 7, monosomy 10 and 22, and loss of the Y chromosome. Clonal structural rearrangements were present with a low incidence; they mainly involved chromosomes #1 and #7. These patterns of chromosome involvement seem to correlate with the scarce previous cytogenetic banding data available from low-grade gliomas. They are also similar to the chromosome alterations found in high-grade gliomas.     

Absence of expression of SMARCB1/INI1 in malignant rhabdoid tumors of the central nervous system, kidneys and soft tissue: an immunohistochemical study with implications for diagnosis.

Malignant rhabdoid tumors are high-grade neoplasms of the central nervous system (CNS), kidneys and soft tissue that usually occur in children. The histologic diagnosis of malignant rhabdoid tumor depends on identification of characteristic rhabdoid cells-large cells with eccentrically located nuclei and abundant, eosinophilic cytoplasm-and immunohistochemistry with antibodies to vimentin, keratin and epithelial membrane antigen. In most malignant rhabdoid tumors, the SMARCB1/INI1 gene, located in chromosome band 22q11.2, is inactivated by deletions and/or mutations, so genetic diagnosis is often possible. However, tissue may not be available for genetic analysis or studies not confirmatory. We assessed SMARCB1/INI1 expression in 17 rhabdoid tumors and 57 other tumors of the CNS, kidney or soft tissue using immunohistochemistry. In total, 12 brain, three renal and two soft tissue rhabdoid tumors were examined along with four glioblastomas, four pilocytic astrocytomas, four oligodendrogliomas, two ependymomas, two choroid plexus papillomas, five pituitary adenomas, four germinomas, four renal carcinomas with Xp11.2 translocations, two clear cell sarcomas, two Wilms' tumors, one renal medullary carcinoma, two desmoplastic small round cell tumors, two alveolar rhabdomyosarcomas, two embryonal rhabdomyosarcomas, one low-grade chondrosarcoma, two extraskeletal myxoid chondrosarcomas, one mesenchymal chondrosarcoma, four malignant peripheral nerve sheath tumors, five metastatic carcinomas and four epithelioid sarcomas, two primary and two metastatic. The neoplastic cells of all rhabdoid tumors, the four epithelioid sarcomas and the renal medullary carcinoma did not express SMARCB1/INI1 by immunohistochemistry; neoplastic cells of all other tumors expressed SMARCB1/INI1. Immunohistochemistry to assess expression of SMARCB1/INI1 may be useful in the diagnosis of rhabdoid tumors of the CNS, kidneys and soft tissue.     

Nogo-A: a useful marker for the diagnosis of oligodendroglioma and for identifying 1p19q codeletion

The differential diagnosis between oligodendrogliomas and other gliomas remains a critical issue. The aim of this study is to verify the diagnostic value of Olig-2, Nogo-A, and synaptophysin and their role in identifying 1p19q codeletion. A total of 168 cases of brain tumors were studied: 24 oligodendrogliomas, 23 anaplastic oligodendrogliomas, 2 oligoastrocytomas, 2 anaplastic oligoastrocytomas, 30 glioblastoma multiforme, 2 diffuse astrocytomas, 4 anaplastic astrocytomas, 10 pilocytic astrocytomas, 9 ependymomas, 12 anaplastic ependymomas, 10 central neurocytomas, 10 meningiomas, 10 choroid plexus papillomas, 10 dysembryoplastic neuroepithelial tumors, and 10 metastases. All cases were immunostained with Olig-2, Nogo-A, and synaptophysin. In 79 cases, the status of 1p/19q had already been assessed by fluorescence in situ hybridization. Thus, in selected cases, fluorescence in situ hybridization was repeated in areas with numerous Nogo-A-positive neoplastic cells. Nogo-A was positive in 18 (75%) of 24 oligodendrogliomas, 8 (80%) of 10 dysembryoplastic neuroepithelial tumors, 6 (20%) of 30 glioblastoma multiforme, and 2 (20%) of 10 pilocytic astrocytomas. Olig-2 stained 22 (91.6%) of 24 oligodendrogliomas and all dysembryoplastic neuroepithelial tumors but also 24 (80%) of 30 glioblastoma multiforme and 8 (80%) of 10 pilocytic astrocytomas. Finally, synaptophysin stained 13 (54.1%) of 24 oligodendrogliomas, 3 (10%) of 30 glioblastoma multiforme, 1 (10%) of 10 pilocytic astrocytomas, and all neurocytomas. Among the 79 tested cases, original fluorescence in situ hybridization showed 1p/19q codeletion in 12 (52.2%) of 23 oligodendrogliomas, 8 (38%) of 21 anaplastic oligodendrogliomas, and 1 (4%) of 25 glioblastoma multiforme. However, after carrying out the Nogo-A-driven fluorescence in situ hybridization, 1p/19q codeletion was observed in 8 additional cases. Nogo-A is more useful and specific than Olig-2 in differentiating oligodendrogliomas from other gliomas. Furthermore, using a Nogo-A-driven fluorescence in situ hybridization analysis, it is possible to identify a larger number of 1p19q codeletions in gliomas.     

Genomic aberrations in diffuse low-grade gliomas

The current classification of diffuse low-grade gliomas is based mainly on histopathological criteria, which cannot accurately predict the highly variable clinical course observed in patients with such tumors. In an attempt to increase pathogenetic understanding of these tumors, we investigated 38 WHO Grade II astrocytomas, oligodendrogliomas, and oligoastrocytomas using a combination of G-band chromosome analysis and high-resolution comparative genomic hybridization (HR-CGH). Abnormal karyotypes were found in 41% of tumors. Karyotypes of astrocytomas and oligodendrogliomas were near-diploid whereas oligoastrocytomas also displayed near-tetraploid clones. The most common aberrations were losses of chromosomes X, Y, 3, 4, 6, and 11 and gains of chromosomes 8 and 12. The only recurrent structural rearrangement was del(6)(q21). HR-CGH analysis verified karyotyping findings but also revealed frequent losses at 1p, 17q, and 19q and gains of 7q, 10p, 11q, and 20p. Among the tumors were two gemistocytic astrocytomas, a subgroup of diffuse astrocytomas with a particular predisposition for progression but not studied cytogenetically before; one showed a near-diploid, complex karyotype with structural aberrations of chromosomes 1, 3, and 11 whereas both displayed simple aberrations including loss of 11p by HR-CGH. Our findings suggest that within diffuse low-grade gliomas are genetically distinct entities that do not fit the currently used classification. In addition, tumors with complex chromosomal aberrations had a higher tendency for aggressive tumor behavior (shorter progression-free survival) than tumors displaying simple aberrations only (P = 0.07). This could help identify genetic subsets of patients with low-grade glioma who might benefit from early antineoplastic therapy.     

Comparative genomic hybridization detects specific cytogenetic abnormalities in pediatric ependymomas and choroid plexus papillomas

Pathogenesis and genetic abnormalities of ependymomas are not well known and differential diagnosis with choroid plexus tumors may be difficult when these tumors are located in the ventricles. We analyzed 16 samples of primary pediatric ependymomas and seven choroid plexus tumors for significant gains or losses of genomic DNA, using comparative genomic hybridization (CGH). Four ependymoma samples were obtained after surgery for relapse, including one patient whose tumor was analyzed at diagnosis and at first and second relapses. Three out of 16 ependymomas and none of the choroid plexus tumors appeared normal by CGH. In the remaining ependymomas, the number of regions with genomic imbalance was limited. The most frequent copy number abnormality in ependymomas was 22q loss. In one patient from whom multiple samples could be analyzed during tumor progression, no abnormality was present at diagnosis; gain of chromosome 9 and loss of 6q were observed at first relapse and, at second relapse, additional genomic imbalances were loss of 3p, 10q, and chromosome 15. In choroid plexus tumors, recurrent abnormalities were gains of chromosome 7 and region 12q. The recurrent chromosomal abnormalities were clearly different between ependymomas and choroid plexus papillomas (CPP). Recurrent loss of 22q suggests that this region harbors tumor suppressor genes important in the pathogenesis of ependymomas; however, other pathogenic pathways may exist involving 6q and chromosome 10 losses or gain of 1q and chromosome 9. CPP can be distinguished from ependymoma on the basis of CGH abnormalities.     

Identification in Human Brain Tumors of DNA Sequences Specific for SV40 Large T Antigen

Simian virus 40 (SV40) sequences have recently been identified in a variety of human neoplasms, including mesothelioma, osteosarcoma, and brain tumors, but significant discrepancies exist regarding the frequency at which this occurs. The SV40 genome is 70% homologous to JC and BK, two related polyomaviruses that are highly prevalent in humans and which may cause in immune-compromised patients progressive multifocal leukoencephalopathy (PML) and cystitis, respectively. We have established a specific and sensitive method to identify SV40 sequence in DNA extracted from histological sections, using PCR followed by Southern hybridization to probes specific to the large T region. We found SV40 largeT antigen sequences in all brain tumor types investigated. High frequencies were found in low-grade astrocytomas, anaplastic astro-cytomas and secondary glioblastomas derived thereof (13/22, 59%) while somewhat lower frequencies were found in gemistocytic astrocytomas (9/28, 32%) and oligodendrogliomas (3/12, 25%). Primary glioblastomas, giant cell glioblastomas, and gliosarcomas, which clinically develop de novo, contained SV40 sequences in 11–25% of cases. Presence of viral DNA was also observed in pediatric brain tumors, including ependymomas (9/16, 56%), choroid plexus papillomas (6/16, 38%), and medulloblastomas (5/17, 29%). In 8 tumor biopsies with SV40 sequences, the adjacent normal brain tissue was also analyzed but was devoid of viral DNA in all but one case. BK and JC virus sequences were rarely detected, the overall frequencies being 3% and 2%, respectively. It remains to be shown whether the presence of SV40 contributes significantly to malignant transformation or whether certain human neoplasms provide a microenvironment that favors viral replication in humans with latent SV40 infection.     

100例中枢神经系统肿瘤的胶质纤维酸性蛋白免疫组化观察

对100例中枢神经系统肿瘤进行GFAP免疫组化观察。结果表明,GFAP主要分布于各种类型的星形细胞瘤,多形性胶质母细胞瘤,混合性胶质瘤及部分室管膜瘤。GFAP染色强度与瘤细胞分化程度有关。本文还探讨了室管膜瘤与少突胶质瘤存在GFAP问题。研究表明GFAP可作为诊断神经胶质瘤的一种有用标记物。     

SV40大T抗原在人脑肿瘤中与p53形成特异性复合物

目的 探讨SV40早期区域基因编码产物大T抗原（Tag) 表达及与抑癌蛋白p53的相互作用在人脑肿瘤发生发展中的意义。方法 采用免疫共沉淀及Western印迹法检测43例人脑肿瘤组织及5例正常人脑组织中Tag的表达，并对18例Tag阳性瘤组织检测Tag-p53复合物的存在。结果 Tag在5例室管膜瘤及2例脉络丛乳头状瘤中全部表达，垂体腺瘤（5／6）、星形胶质细胞瘤（7／10）、脑膜瘤（4／6）、多形性胶质母细胞瘤（3／5）及髓母细胞瘤（2／5）均有Tag的表达，3例少枝胶质细胞瘤、1例松果体瘤及5例正常人脑组织无Tag表达；检测18例Tag阳性瘤组织均发现Tag与p53形成特异性复合物。结论 在人脑肿瘤组织中Tag广泛表达，Tag可与p53形成特异性复合物，Tag-p53特异性复合物的形成导致p53失活，可能是SV40致人脑肿瘤发生的一个重要机理。     

Significance of immunohistochemistry in neuro-oncology. V. Keratin as a marker for epithelial differentiation of primary and secondary intracranial and intraspinal tumors

Intermediate filament keratin is regarded as a good marker for epithelial and mesothelial tumors. In the intracranial and intraspinal spaces keratin has been demonstrated only in the endocrine cells of the adenohypophysis, squamous epithelial islands in the pars tuberalis of the hypophysis and in the choroid plexus epithelium. Since gliomas and meningiomas do not express keratin, this marker provides an additional help for differentiating between primary and secondary CNS tumors. Indirect immunofluorescence using an anti-keratin serum was used in a retrospective search for keratin in 80 tumors of the cranium and intraspinal space. Of the primary CNS tumors keratin positivity occurred in craniopharyngiomas, epidermoid tumors, pituitary adenomas, chordomas, a plexus papilloma as well as in the majority of germ cell tumors. Only 3 renal cell carcinoma metastases of 21 metastatic epithelial cell tumors (7 bronchial carcinomas, 6 breast cancers, 6 renal carcinomas, 1 rectum carcinoma, 1 cervix carcinoma) were keratin-negative. Similar findings were made in two melanoma metastases which we examined, whereas in a seminoma metastasis a few keratin expressing cells were found. Primary CNS tumors such as myxopapillary ependymomas, medulloepitheliomas, malignant meningiomas and paragangliomas which are often difficult to distinguish from these metastases proved to be keratin negative.     

Analysis of the NF2 gene in oligodendrogliomas and ependymomas

Allelic losses of chromosome 22 are commonly found in ependymomas and oligodendrogliomas, suggesting that at least one tumor suppressor gene on chromosome 22 must be inactivated during the multistep process of tumorigenesis in these glial tumors. The neurofibromatosis 2 gene (NF2) located at 22q12, is a candidate tumor suppressor gene potentially involved in the pathogenesis of gliomas. Because there have been only a few studies of the NF2 gene in glial tumors other than astrocytoma, we screened the entire 17 NF2 exons for mutations in a series of 47 nonastrocytic tumors, including 40 oligodendrogliomas and 7 ependymomas. Only one mutation was detected, a 59-base pair insertion in exon 3 from a spinal anaplastic ependymoma. These results concur with previous findings proposing preferential inactivation of the NF2 gene in a subgroup of ependymomas, and suggest that the NF2 gene is not the target of chromosome 22 aberrations in oligodendrogliomas.     

Ezrin expression in stromal cells of capillary hemangioblastoma. An immunohistochemical survey of brain tumors.

Ezrin is a cytoskeleton-associated protein that appears to link actin filaments to the plasma membrane. Immunocytochemical studies suggest that ezrin is expressed in epithelial cells but not in mesenchymal cells. In addition, ezrin is expressed by certain epithelial tumors, such as renal cell adenocarcinomas. Ezrin serves as a tyrosine kinase substrate, and is phosphorylated in epidermal growth factor-stimulated cells. Ezrin may thus mediate regulatory signals in different cell functions. We studied the distribution of ezrin in 104 cases of primary tumors of the central nervous system (CNS) by immunocytochemistry. Special interest was focused on capillary hemangioblastoma, owing to its resemblance to renal cell adenocarcinoma, and on malignant gliomas, owing to their frequent epidermal growth factor receptor amplification. The stromal cells of hemangioblastomas were found to be strongly positive for ezrin. No expression was detected in gliomas and, except for hemangioblastomas, ezrin expression was restricted to those few CNS tumors that show epithelial differentiation, ie, choroid plexus papillomas, craniopharyngiomas, ependymomas, and cysts. The diffuse cytoplasmic expression of ezrin in the stromal cells of capillary hemangioblastoma may indicate that stromal cells overexpress ezrin or express ezrin with deficient binding properties.     

Significance of immunohistochemistry for neuro-oncology. VI. Occurrence, localization and distribution of glial fibrillary acid protein (GFAP) in 820 tumors

In a retrospective study 820 tumors were immunohistochemically examined with anti-GFAP. All 224 astrocytomas and 105 of 112 glioblastomas were, at least focally, positive. 72% of ependymomas and 64% of oligodendrogliomas contained tumor cells which expressed GFAP. In such entities the reaction is dependent on the histologic subtype. Only 26 of 114 medulloblastomas (22.8%) demonstrated scattered GFAP positive cells. GFAP was also demonstrated in the CNS in gangliogliomas, monstrocellular sarcomas, 3 of 6 PNET, one non-classifiable tumor in a child, 1 plexus papilloma, in scattered stromal cells in 15 of 26 hemangioblastomas as well as in the mature glial component of intracranial germ cell tumors. Outside of the CNS there was evidence of GFAP in 3 cases with nasal glial heterotopy and in the myxoidal part of a pleomorphic salivary gland adenoma. Neoplasms which proved negative to GFAP in our series included purely neural differentiated tumors meningioma, neurolemmomas, chordomas, paragangliomas, sarcomas, lymphomas, melanomas and carcinoma metastases. Separating GFAP-positive reactive astrocytes from the actual tumor cells has proved to be a problem in the routine use of GFAP in differential diagnosis. Absence of an immunohistochemical response does not exclude a tumor of glial origin. Tissue samples which are too small, particularly in the case of anaplastic astrocytomas and glioblastomas can give false negative results.     

Distribution of beta2-microglobulin in cerebrospinal fluid and in cystic fluid of brain tumors. A preliminary study.

The concentrations of immuno-reactive beta2-microglobulin (beta2m) were measured in the cerebrospinal fluid (CSF) and the cystic fluid (CF) of Central Nervous System (CNS) tumours (gliomas, n = 5; craniopharyngiomas, n = 5) and in the culture medium of established cell lines derived from CNS tumors. These data are compared with plasma and CSF values of beta2m in normal subjects (n = 15) and in a group of peripheral solid tumors (metastatic breast carcinomas, n = 9). In the control group the absence of correlation between plasma and CSF values, suggests an independant production of beta2m in the two compartments considered. The capacity of CNS tumor cells to synthesize beta2m is demonstrated in vitro. In vitro beta-2m concentrations in the CF embryonic tumors (craniopharyngiomas) is significantly more elevated than in non malignant astrocytomas.     

Cytokeratin immunoreactivity in gliomas

Monoclonal antibodies (AE1/3, CAM 5.2 and PKK-1) and polyclonal antisera against the cytokeratin proteins were reacted with a range of astrocytic tumours, oligodendrogliomas and ependymomas. Seven of 12 cases (58%) of glioblastoma multiforme, five of eight (63%) anaplastic astrocytomas and two of five (40%) well-differentiated astrocytomas were immunoreactive with AE1/3 but not with the other anti-cytokeratin antibodies. In oligodendrogliomas, AE1/3 stained isolated astrocyte-like cells as well as scattered neoplastic oligodendrocytes in four of eight cases (50%) cases. Four ependymomas were negative for all cytokeratin markers examined. The immunostaining of astrocytomas and oligodendrogliomas with AE1/3 might represent co-expression of cytokeratin with glial fibrillary acidic protein by gliomas and calls for caution in the use of these antibodies in the differential diagnosis between gliomas and carcinomas.     

Molecular Markers of Gliomas

Gliomas are invasive recurrent brain tumors with high lethality. Gliomas have been morphologically divided into astrocytomas, oligodendrogliomas, and mixed oligoastrocytomas. Gliomas are graded according to the criteria of the World Health Organization criteria as grade I (pilocytic astrocytomas), grade II (low-grade gliomas), grade III (high-grade gliomas), and grade IV (glioblastoma). The mutations of IDH1/2 and TP53 genes and MGMT methylation have been described as prognostic markers of glioma. The aim of the present review is to analyze research and experimental results (Scopus, Web of Science, Pubmed) concerning somatic mutations, aberrant regulation of gene expression of signal pathways, diagnostic and prognostic markers of glioma progression and characterizing various morphological types and grades of glioma. In particular, the specificities of medulloblastomas and ependymomas have been considered in the present review.     

Immunostaining for α1-antichymotrypsin and α1-antitrypsin in gliomas

Antisera to α₁-antichymotrypsin, α₁-antitrypsin and lysozyme were reacted with 20 cases of glioblastoma multiforme, seven anaplastic astrocytomas, eight astrocytomas, six oligodendrogliomas, four ependymomas and the cerebral cortex from six normal autopsy brains. In addition, two pleomorphic xantho-astrocytomas and two heavily lipidized malignant gliomas were similarly examined. All astrocytic lesions were confirmed with anti-GFAP antisera. Thirty astrocytic tumours (77%), four oligodendrogliomas (67%) and three ependymomas (75%) reacted positively with anti-α₁-antichymotrypsin; 25 astrocytic tumours (64%), three oligodendrogliomas (50%) and three ependymomas (75%) showed positive staining for α₁-antitrypsin. The pattern of staining with either of these two markers did not correlate with tumour grading. None of the gliomas examined stained positively with anti-lysozyme. Non-neoplastic glial elements did not react with any of the three antisera. The results of this study suggest that staining for α₁-antichymotrypsin and α₁-antitrypsin is of little value in the differential diagnosis of neuroepithelial or mesenchymal lesions in the brain.     

Intermediate filament proteins in choroid plexus and ependyma and their tumors.

The intermediate filament protein types of normal choroid plexus and ependymal tissue and their putative tumors were investigated. In normal human choroid plexus tissue, but not in ependyma, keratin could be demonstrated immunohistochemically. By immunoblotting, keratins 8, 18, and 19 were found, but glial fibrillary acidic protein (GFAP) was absent. In mouse and rat, choroid plexus epithelium and ependymal lining cells were keratin-positive. In addition, many ependymal cells were vimentin-positive. Keratin was immunohistochemically found in three of four choroid plexus papillomas, two of two choroid plexus carcinomas, and the lining cells of three neuroepithelial cysts. GFAP-positive cells were present in some choroid plexus tumors. In contrast, none of the eight ependymomas contained keratin, but all were strongly positive for GFAP. The results show that choroid plexus lining cells and choroid plexus tumors have true epithelial characteristics in their cytoskeleton, in contrast to ependymomas, which do not show keratin positivity but show glial filaments, as would be seen in astrocytic tumors.     

A marker for primary choroid plexus neoplasms.

Primary choroid plexus (CP) tumors are rare neoplasms that present in childhood or, less frequently, in adult life. The majority are benign and amenable to complete surgical excision, but occasionally more invasive variants are encountered. Although generally pathologically distinct, occasionally primary CP neoplasms may be difficult to distinguish from metastatic papillary carcinomas or papillary ependymomas. Conventional cytologic markers are not sufficiently specific to permit accurate diagnosis of primary CP tumors. The authors have reported that the CP is the unique site of synthesis within the brain of transthyretin (TTR, prealbumin), a transport protein for thyroxine and retinol. They therefore investigated the utility of TTR as a biochemical marker for CP tumors. They detected intense immunoreactivity for TTR at high dilutions of primary antiserum in the neoplastic epithelium of all of nine primary CP tumors (six papillomas and three carcinomas), but not in eight cellular or three papillary intracerebral ependymomas, meningiomas, oligodendrogliomas, astrocytomas, primary extracerebral papillary carcinomas (three thyroid, two breast) or five of six cerebral metastases from systemic papillary carcinomas. In one case of cerebral metastasis from papillary thyroid carcinoma, rare isolated immunoreactive cells were observed. Faint staining of the stromal-ependymal junction was seen in myxopapillary ependymomas of the filum terminale, which were otherwise nonreactive. By in situ hybridization, TTR mRNA was abundant in neoplastic CP epithelium, confirming local TTR synthesis. The authors conclude that TTR is synthesized by neoplastic CP epithelium and is an excellent marker for primary CP neoplasms.     

A cytogenetic study of 53 human gliomas

Cytogenetic studies were performed on human glioma samples obtained by stereotactic biopsy, stereotactic craniotomy, or routine craniotomy. Using in situ culture and robotic harvesting techniques, we obtained suitable metaphases in 50 (94%) of 53 tumors, including 28 diffuse astrocytomas, four juvenile pilocytic astrocytomas, two gliosarcomas, three other miscellaneous astrocytomas, eight oligodendrogliomas, four mixed oligodendroglioma-astrocytomas, and four ependymomas. Cytogenetic studies were performed only on primary cultures; the mean culture time was 9.6 days (range 1-31 days). One or more chromosomally abnormal clones were observed in 35 (66%) tumors. Eleven (21%) other specimens had random nonclonal chromosome abnormalities. In four (8%) specimens, no chromosome abnormalities were noted. The results of this study suggest that grade 3 and 4 tumors are more likely to contain an abnormal clone than tumors of grade 1 or 2 (p less than 0.01). The most common numeric chromosome abnormalities were -6, +7, -10, -13, -14, -15, -18, and -Y. The most common structural abnormalities involved 1p, 6q, 7q, 8p, 9p, 11p, 11q, 13q, and 19q. Four tumors had two or more independent clones and ten contained subclones demonstrating karyotype evolution. With in situ culture and robotic harvesting techniques, cytogenetic studies can be successful on nearly all human gliomas, including those derived from small stereotactic biopsies.