Adelheid Wöhrer

Immunohistochemical testing of BRAF V600E status in 1,120 tumor tissue samples of patients with brain metastases

Brain metastases (BM) are frequent and carry a dismal prognosis. BRAF V600E mutations are found in a broad range of tumor types and specific inhibitors targeting BRAF V600E protein exist. We analyzed tumoral BRAF V600E-mutant protein expression using the novel mutation-specific antibody VE1 in a series of 1,120 tumor specimens (885 BM, 157 primary tumors, 78 extra-cranial metastases) of 874 BM patients. In 85 cases, we performed validation of immunohistochemical results by BRAF exon 15 gene sequencing. BRAF V600E protein was expressed in BM of 42/76 (55.3%) melanomas, 1/15 (6.7%) ovarian cancers, 4/72 (5.5%) colorectal cancers, 1/355 (0.3%) lung cancers, 2/6 thyroid cancers and 1/2 choriocarcinomas. BRAF V600E expression showed high intra-tumoral homogeneity and was similar in different tumor manifestations of individual patients. VE1 immunohistochemistry and BRAF exon 15 sequencing were congruent in 68/70 (97.1%) cases, but VE1 immunostaining identified small BRAF V600E expressing tumor cell aggregates in 10 cases with inconclusive genetic results. Melanoma patients with BRAF V600E mutant protein expressing tumors were significantly younger at diagnosis of the primary tumor and at operation of BM than patients with non-mutated tumors. In conclusion, expression of BRAF V600E mutant protein occurs in approximately 6% of BM and is consistent in different tumor manifestations of the same patient. Thus, BRAF V600E inhibiting therapies seem feasible in selected BM patients. Immunohistochemical visualization of V600E-mutant BRAF protein is a promising tool for patient stratification. An integrated approach combining both, VE1 immunohistochemistry and genetic analysis may increase the diagnostic accuracy of BRAF mutation analysis.

Programmed death ligand 1 expression and tumor-infiltrating lymphocytes in glioblastoma

BACKGROUND Immune checkpoint inhibitors targeting programmed cell death 1 (PD1) or its ligand (PD-L1) showed activity in several cancer types. METHODS We performed immunohistochemistry for CD3, CD8, CD20, HLA-DR, phosphatase and tensin homolog (PTEN), PD-1, and PD-L1 and pyrosequencing for assessment of the O6-methylguanine-methyltransferase (MGMT) promoter methylation status in 135 glioblastoma specimens (117 initial resection, 18 first local recurrence). PD-L1 gene expression was analyzed in 446 cases from The Cancer Genome Atlas. RESULTS Diffuse/fibrillary PD-L1 expression of variable extent, with or without interspersed epithelioid tumor cells with membranous PD-L1 expression, was observed in 103 of 117 (88.0%) newly diagnosed and 13 of 18 (72.2%) recurrent glioblastoma specimens. Sparse-to-moderate density of tumor-infiltrating lymphocytes (TILs) was found in 85 of 117 (72.6%) specimens (CD3+ 78/117, 66.7%; CD8+ 52/117, 44.4%; CD20+ 27/117, 23.1%; PD1+ 34/117, 29.1%). PD1+ TIL density correlated positively with CD3+ (P < .001), CD8+ (P < .001), CD20+ TIL density (P < .001), and PTEN expression (P = .035). Enrichment of specimens with low PD-L1 gene expression levels was observed in the proneural and G-CIMP glioblastoma subtypes and in specimens with high PD-L1 gene expression in the mesenchymal subtype (P = 5.966e-10). No significant differences in PD-L1 expression or TIL density between initial and recurrent glioblastoma specimens or correlation of PD-L1 expression or TIL density with patient age or outcome were evident. CONCLUSION TILs and PD-L1 expression are detectable in the majority of glioblastoma samples but are not related to outcome. Because the target is present, a clinical study with specific immune checkpoint inhibitors seems to be warranted in glioblastoma.

Invasion of the cavernous sinus space in pituitary adenomas: endoscopic verification and its correlation with an MRI-based classification

OBJECT An important prognostic factor for the surgical outcome and recurrence of a pituitary adenoma is its invasiveness into parasellar tissue, particularly into the space of the cavernous sinus (CS). The aims of this study were to reevaluate the existing parasellar classifications using an endoscopic technique and to evaluate the clinical and radiological outcomes associated with each grade. METHODS The authors investigated 137 pituitary macroadenomas classified radiologically at least on one side as Grade 1 or higher (parasellar extension) and correlated the surgical findings using an endoscopic technique, with special reference to the invasiveness of the tumor into the CS. In each case, postoperative MRI was performed to evaluate the gross-total resection (GTR) rate and the rate of endocrinological remission (ER) in functioning adenomas. RESULTS The authors found a 16% rate of CS invasion during surgery for these macroadenomas. Adenomas radiologically classified as Grade 1 were found to be invasive in 1.5%, and the GTR/ER rate was 83%/88%. For Grade 2 adenomas, the rate of invasion was 9.9%, and the GTR/ER rate was 71%/60%. For Grade 3 adenomas, the rate of invasion was 37.9%, and the GTR/ER rate was 75%/33%. When the superior compartment of the CS (Grade 3A) was involved, the authors found a rate of invasion that was lower (p < 0.001) than that when the inferior compartment was involved (Grade 3B). The rate of invasion in Grade 3A adenomas was 26.5% with a GTR/ER rate of 85%/67%, whereas for Grade 3B adenomas, the rate of surgically observed invasion was 70.6% with a GTR/ER rate of 64%/0%. All of the Grade 4 adenomas were invasive, and the GTR/ER rate was 0%. A comparison of microscopic and endoscopic techniques revealed no difference in adenomas with Grade 1 or 4 parasellar extension. In Grade 2 adenomas, however, the CS was found by the endoscopic technique to be invaded in 9.9% and by microscopic evaluation to be invaded in 88% (p < 0.001); in Grade 3 adenomas, the difference was 37.9% versus 86%, respectively (p = 0.002). Grade 4 adenomas had a statistically significant lower rate of GTR than those of all the other grades. In case of ER only, Grade 1 adenomas had a statistically significant higher rate of remission than did Grade 3B and Grade 4 adenomas. CONCLUSIONS The proposed classification proved that with increasing grades, the likelihood of surgically observed invasion rises and the chance of GTR and ER decreases. The direct endoscopic view confirmed the low rate of invasion of Grade 1 adenomas but showed significantly lower rates of invasion in Grade 2 and 3 adenomas than those previously found using the microscopic technique. In cases in which the intracavernous internal carotid artery was encased (Grade 4), all the adenomas were invasive and the GTR/ER rate was 0%/0%. The authors suggest the addition of Grades 3A and 3B to distinguish the strikingly different outcomes of adenomas invading the superior CS compartments and those invading the inferior CS compartments.

Value and limitations of immunohistochemistry and gene sequencing for detection of the IDH1-R132H mutation in diffuse glioma biopsy specimens.

To assess the value of anti-isocitrate dehydrogenase 1 (IDH1) immunohistochemistry for evaluating diffuse gliomas, we analyzed anti-IDH1-R132H immunohistochemistry using monoclonal antibodies DIA-H09 and IMab-1 and IDH1 gene sequencing in formalin-fixed and paraffin-embedded biopsy samples of 95 diffuse gliomas. We found concordant immunostaining results using the 2 antibodies in 94 (98.9%) of the 95 cases, but DIA-H09 generally showed a higher signal-to-background ratio than IMab-1 did. Fifty-five percent of cases showed anti-IDH1-R132H immunostaining of virtually all tumor cells and 15% of only a fraction of tumor cells. All cases with complete or partial immunostaining of the tumor tissue carried the IDH1-R132H mutation. In all cases with negative immunostaining results (approximately 30%), genetic analysis showed IDH1 wild-type or non-R132H-IDH1 mutations. In a single tiny biopsy, both anti-IDH1-R132H antibodies showed immunoreactivity, but genetic testing was inconclusive. Our data confirm anti-IDH1-R132H immunostaining as a reliable method for evaluation of IDH1 gene mutation status. They also suggest the following: (i) in some cases, nonspecific background staining or regional heterogeneity of IDH1-R132H protein expression may necessitate confirmatory genetic analysis; (ii) for individual cases, anti-IDH1-R132H immunostaining may not reliably identify infiltrating tumor cells admixed with preexisting or reactive glial cells; and (iii) in tiny biopsies, immunohistochemistry may be more sensitive for detection of IDH1-R132H mutation than genetic analysis.

Ki67 index in intracranial ependymoma: a promising histopathological candidate biomarker

Aims:  The Ki67 tumour cell proliferation index is an independent prognostic factor in ependymoma patients. Essential prerequisites for validation of the Ki67 index as a histopathological biomarker are the reproducibility of this factor and its prognostic influence by different observers (proof of objective clinical and analytical performance). To this end, the aim was to analyse systematically inter‐ and intraobserver agreement and reproducibility of the prognostic impact of the Ki67 index in intracranial ependymoma.

ALK gene translocations and amplifications in brain metastases of non-small cell lung cancer.

BACKGROUND Increased incidence of brain metastases (BM) in non-small cell lung cancer (NSCLC) with ALK translocations was postulated, however, ALK gene aberrations in NSCLC-BM have not been investigated so far. METHODS We investigated ALK and EML4 gene aberrations (amplifications, translocations, inversions) by fluorescent in situ hybridization (FISH) (n=175) and ALK and EML4 protein expression by immunohistochemistry (n=221) in NSCLC BM and corresponding primary tumors. RESULTS ALK translocations were found in 4/151 (2.6%; 3 of them involving EML4) of BM of adenocarcinomas (AC), 1/9 (11.1%) of adenosquamous carcinomas (ASC), 0/5 of squamous cell carcinomas (SCC) and 0/10 of large cell carcinomas (LCC). Rearrangement of ALK without involvement of EML4 was seen in 1 AC-BM and rearrangement of EML4 without involvement of ALK in 3 AC-BM, 1 ASC-BM and 1 LCC. ALK amplifications without gene rearrangements were found in BM of 16/151 (10.6%) AC, 2/5 (40%) SCC, 0/9 ASC and one LCC. ALK translocation status was constant between BM and primary tumors in 16 evaluable cases including two cases with ALK-EML4 translocations Among these 16 cases ALK amplification was seen in two BM and none of the primary tumors. All cases with translocations but not with amplifications of ALK showed protein expression. We found no association of ALK gene status with patient age, gender or overall survival time. CONCLUSIONS ALK translocations and amplifications are found in approximately 3% and 11% of NSCLC-BM, respectively. While ALK translocations appear to be constant between primary tumors and BM, amplifications seem to be more prevalent in BM. ALK translocation, but not ALK amplification is associated with ALK protein overexpression. Further studies are needed to determine whether NSCLC-BM patients with ALK gene aberrations may benefit from specific inhibitor therapy.

BRAF-Mutated Pleomorphic Xanthoastrocytoma is Associated with Temporal Location, Reticulin Fiber Deposition and CD34 Expression

BRAF V600E mutation and homozygous deletion of CDKN2A (p16) are frequent molecular alterations in pleomorphic xanthoastrocytomas (PXAs). We investigated 49 PXAs for clinical, histological and immunohistochemical characteristics related to BRAF mutation status. BRAF mutation was detected by immunohistochemical assay and DNA sequencing in 38/49 (78%) tumors. All but one PXA located in the temporal lobe harbored a BRAF V600E mutation (23/24; 96%) compared with 10/19 nontemporal PXAs (53%; P = 0.0009). Histological and immunohistochemical analysis demonstrated increased reticulin deposition (76% vs. 27%; P = 0.003) and a more frequent expression of CD34 in BRAF‐mutant PXAs (76% vs. 27%; P = 0.003).

Plasma MicroRNA-21 concentration may be a useful biomarker in glioblastoma patients.

Background: MicroRNAs are proposed as plasma biomarkers in cancer. Glioblastoma is the most frequent primary brain tumor of adults. MicroRNA-21 was shown to induce glioblastoma growth. Aims: To evaluate the circulating microRNA-21 in glioblastoma patients. Methods: MicroRNA-21 copies in glioblastoma plasma (n = 10, before surgery and during/after concomitant chemoradiotherapy), control plasma (n = 10), and tissue samples were analyzed using RT-qPCR. Results: MicroRNA-21 in glioblastoma was significantly higher than controls (p = .02) and decreased significantly in 9 patients (p = .05). One patient with increasing microRNA-21 developed a histopathologically proven recurrence after the second sample collection. Discussion: Circulating microRNA-21 might be a useful biomarker in glioblastoma.

Exploratory investigation of eight circulating plasma markers in brain tumor patients

Several blood biomarkers have been established for the early diagnosis, screening and follow-up of non central nervous system cancers. However, there is lack of knowledge on biochemical blood alterations in brain tumor patients. In this study, we prospectively collected blood plasma samples of 105 adult brain tumor patients with diffuse low-grade glioma (World Health Organization (WHO) II, n = 7), anaplastic glioma (WHO III, n = 10), glioblastoma multiforme (WHO IV, glioblastoma multiforme (GBM)) (n = 34), meningioma (WHO I, n = 8), atypical meningioma (WHO II, n = 5), and intracerebral metastasis (ICM; n = 41). In each case, we measured plasma concentrations of neuropeptide Y, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, placental growth factor (PlGF), S100B, secretagogin, interleukin 8, and glial fibrillary acidic protein (GFAP) using enzyme-linked immunosorbent assay. Plasma marker concentrations were correlated to patient parameters including neuropathological diagnosis and neuroradiological features. Most of the markers were detectable in all diagnostic categories in variable concentrations. GFAP plasma detectability was strongly associated with a diagnosis of GBM (p < 0.001). Plasma GFAP and plasma placental growth factor showed promising moderate potential in the differential diagnosis of unifocal GBM versus unifocal supratentorial ICM (area under the curve = 0.73, p < 0.05). To summarize, our data show that none of the investigated markers is suitable to substitute histological diagnosis. However, measurement of circulating GFAP and PlGF may support neuroradiological differential diagnosis of GBM versus ICM.

Value of 1H-magnetic resonance spectroscopy chemical shift imaging for detection of anaplastic foci in diffusely infiltrating gliomas with non-significant contrast-enhancement

Objective In diffusely infiltrating gliomas (DIG), positron emission tomography (PET) imaging is a powerful method for detection of anaplastic foci. Recently, 1H-magnetic resonance spectroscopy chemical shift imaging (CSI) using choline/creatine (Cho/Cr) or choline/N-acetylaspartate (Cho/NAA) ratios has emerged as a new non-invasive, widely available alternative. The authors therefore correlated CSI with 11C-methionine (MET)-PET data in a series of DIG with non-significant contrast-enhancement (CE). Methods Thirty-two patients with DIG were examined with single-slice CSI on a 3 T MRI and MET-PET. Maximum pathological intratumoural ratios of CSI (=CSImax) and maximum tumour-to-normal-brain PET ratios (=PETmax; T/N ratio) were determined. Coregistration of MRI with CSI and PET was performed, and the topographic overlap of CSImax and PETmax was analysed. Histological criteria of anaplasia as well as cell proliferation rate were assessed in tumour samples inside and outside CSImax. Results CSI showed a pathological ratio in all patients, whereas PET demonstrated a pathological T/N ratio in 21/32 patients. Topographical correlation of CSImax and PETmax revealed a ≥50% overlap in 18/21 and <50% overlap in 3/21 patients, respectively. Cho/Crmax and Cho/NAAmax showed a ≥50% overlap in 24/32 and a <50% overlap in 8/32 patients. Cell proliferation rate was significantly higher inside than outside the CSImax (13.6% vs 6.9%, p<0.001). Conclusion The results indicate that CSI is a promising method for detection of anaplastic foci within DIG with non-significant CE. Intraoperative use of CSI by multimodal neuronavigation may increase the reliability of detection of malignant areas in glioma surgery and therefore optimise allocation of patients to adjuvant treatments.