Gerda Ricken

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.

Involvement of the endosomal-lysosomal system correlates with regional pathology in creutzfeldt-jakob disease

The endosomal-lysosomal system (ELS) has been suggested to play a role in the pathogenesis of prion diseases. The purpose of this study was to examine how experimental observations can be translated to human neuropathology and whether alterations of the ELS relate to neuropathologic changes. Combined with stereologic techniques, we examined components of the ELS in human sporadic Creutzfeldt-Jakob disease brains. We immunostained for the early endosomal marker Rab5 and lysosomal enzymes cathepsin D and B. We determined neuron-specific changes in their expression and correlated these with the severity of neuropathologic changes. In regions with mild pathology and scant abnormal prion protein (PrPSc) deposition, neurons showed an increased volume of Rab5-immunopositive early endosomes. In contrast, neurons in regions with prominent pathology had an increased volume of cathepsin D- or B-immunoreactive lysosomes. The intraneuronal distribution of cathepsin D and B diverges between Purkinje cells and frontal cortical neurons in sporadic Creutzfeldt-Jakob disease brains. We demonstrated focal intra- and perineuronal colocalization of cathepsin D and PrPSc. Our results indicate that effects in the ELS correlate with regional pathology. Overloading of this system might impair the function of lysosomal enzymes and thus may mimic some features of lysosomal storage disorders.

Density of tumor-infiltrating lymphocytes correlates with extent of brain edema and overall survival time in patients with brain metastases

The immune microenvironment of the brain differs from that of other organs and the role of tumor-infiltrating lymphocytes (TILs) in brain metastases (BM), one of the most common and devastating complication of cancer, is unclear. We investigated TIL subsets and their prognostic impact in 116 BM specimens using immunohistochemistry for CD3, CD8, CD45RO, FOXP3, PD1 and PD-L1. The Immunoscore was calculated as published previously. Overall, we found TIL infiltration in 115/116 (99.1%) BM specimens. PD-L1 expression was evident in 19/67 (28.4%) BM specimens and showed no correlation with TIL density (p > 0.05). TIL density was not associated with corticosteroid administration (p > 0.05). A significant difference in infiltration density according to TIL subtype was present (p < 0.001; Chi Square); high infiltration was most frequently observed for CD3+ TILs (95/116; 81.9%) and least frequently for PD1+ TILs (18/116; 15.5%; p < 0.001). Highest TIL density was observed in melanoma, followed by renal cell cancer and lung cancer BM (p < 0.001). The density of CD8+ TILs correlated positively with the extent of peritumoral edema seen on pre-operative magnetic resonance imaging (p = 0.031). The density of CD3+ (15 vs. 6 mo; p = 0.015), CD8+ (15 vs. 11 mo; p = 0.030) and CD45RO+ TILs (18 vs. 8 mo; p = 0.006) showed a positive correlation with favorable median OS times. Immunoscore showed significant correlation with survival prognosis (27 vs. 10 mo; p < 0.001). The prognostic impact of Immunoscore was independent from established prognostic parameters at multivariable analysis (HR 0.612, p < 0.001). In conclusion, our data indicate that dense TILs infiltrates are common in BM and correlate with the amount of peritumoral brain edema and survival prognosis, thus identifying the immune system as potential biomarker for cancer patients with CNS affection. Further studies are needed to substantiate our findings.

PD1 (CD279) and PD-L1 (CD274, B7H1) expression in primary central nervous system lymphomas (PCNSL).

BACKGROUND Primary central nervous system lymphoma (PCNSL) is a malignant brain tumor with limited treatment options and shows prominent infiltration by tumor infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). Programmed death 1 (PD1; CD279) and its ligand PD-L1 (B7H1, CD274) promote escape of tumor cells from immune surveillance in several tumor types, but no data are available on PCNSL. Agents inhibiting PD1 and PD-L1 are showing compelling antitumor activity in current clinical trials in solid and hematological cancers. METHODS We investigated PD1 (clone NAT ab52587) and PD-L1 (clone 5H1) expression in large neurosurgical resection specimens of 20 immunocompetent historical PCNSL patients using immunohistochemistry. RESULTS We found expression of PD1 and/or PD-L1 on tumor cells, TILs, or TAMs in a total of 18/20 (90%) of PCNSL cases. In 12/20 (60%) cases, intratumoral PD1-positive TILs were present (low density: 9/20, 45%; moderate density: 2/20, 10%; high density: 1/20, 5%) with additional peritumoral accumulation of PD1-positive lymphocytes in all of 12 cases with evaluable adjacent brain tissue on the tissue section. PD-L1 expressing intratumoral TAMs were found in 4/20 (20%) tumors. In 2/20 (10%) and 4/20 (20%) specimens, we observed striking PD-L1 or PD1 expression on PCNSL tumor cells, respectively. Median number of CD8-positive TILs was 517/mm2 (range 75 - 2,470) and did not correlate with PD1 or PD-L1 expression (p > 0.05, Kruskal- Wallis test). CONCLUSIONS PD1 and PDL1 are immunohistochemically detectable in PCNSL and may be involved in creating an immunosuppressive microenvironment. Specific immune checkpoint inhibitors may be considered for experimental therapy approaches in this disease.

Tumour-infiltrating lymphocytes and expression of programmed death ligand 1 (PD-L1) in melanoma brain metastases

In this study we aimed to characterize immune infiltrates and expression of programmed death 1 (PD‐1) and programmed death ligand 1 (PD‐L1) in a series of melanoma BM to provide a basis for experimental therapy using immune checkpoint inhibitors.

Neuropathological criteria of anti-IgLON5-related tauopathy

We recently reported a novel neurological syndrome characterized by a unique NREM and REM parasomnia with sleep apnea and stridor, accompanied by bulbar dysfunction and specific association with antibodies against the neuronal cell-adhesion protein IgLON5. All patients had the HLA-DRB1*1001 and HLA-DQB1*0501 alleles. Neuropathological findings in two patients revealed a novel tauopathy restricted to neurons and predominantly involving the hypothalamus and tegmentum of the brainstem. The aim of the current study is to describe the neuropathological features of the anti-IgLON5 syndrome and to provide diagnostic levels of certainty based on the presence of associated clinical and immunological data. The brains of six patients were examined and the features required for the neuropathological diagnosis were established by consensus. Additional clinical and immunological criteria were used to define “definite”, “probable” and “possible” diagnostic categories. The brains of all patients showed remarkably similar features consistent with a neurodegenerative disease with neuronal loss and gliosis and absence of inflammatory infiltrates. The most relevant finding was the neuronal accumulation of hyperphosphorylated tau composed of both three-repeat (3R) and four-repeat (4R) tau isoforms, preferentially involving the hypothalamus, and more severely the tegmental nuclei of the brainstem with a cranio-caudal gradient of severity until the upper cervical cord. A “definite” diagnosis of anti-IgLON5-related tauopathy is established when these neuropathological features are present along with the detection of serum or CSF IgLON5 antibodies. When the antibody status is unknown, a “probable” diagnosis requires neuropathological findings along with a compatible clinical history or confirmation of possession of HLA-DRB1*1001 and HLA-DQB1*0501 alleles. A “possible” diagnosis should be considered in cases with compatible neuropathology but without information about a relevant clinical presentation and immunological status. These criteria should help to identify undiagnosed cases among archival tissue, and will assist future clinicopathological studies of this novel disorder.

1324PTUMOR-INFILTRATING LYMPHOCYTES (TILS) AND PD-L1 EXPRESSION IN NON- SMALL CELL LUNG CANCER BRAIN METASTASES (BM) AND MATCHED PRIMARY TUMORS (PT)

ABSTRACT Aim: To investigate TIL infiltration and the PD1/PD-L1 axis in NSCLC BM. Methods: We performed immunohistochemistry for CD3, CD8, CD45RO, PD1 and PDL1 in 73 NSCLC BM specimens and 25/73 (34%) matched PT specimens of 73 patients (46/73 63%) male; 27/73 (37%) female; 15/73 (21%) squamous histology; 58/73 (79%) non squamous histology). Results: BM and PT both showed accumulation of TILs in the tumor stroma, perivascular area and at the tumor border to the surrounding parenchyma. Strong, membranous PDL1 expression was observed in 25/46 (52%; 12/46 (26%) expression in >5% of tumor cells) BM and in 8/25 (32%; 3/25 (12%) expression in >5% of tumor cells) matched PT. Accentuation of PDL1+ tumor cells at the tumor border and in regions with high TIL density was evident in BM and PT. Significant correlation of infiltration density between BM and matched PT was observed for CD3+ TILs (p = 0.002) and CD8+ TILs (p = 0.003). Dense infiltration with CD3 + , CD8+ CD45R0+ and PD1+ TILs was more frequently observed in PT than in BM, while PD-L1 expression was more frequently observed in BM than in PT. No correlation of survival from diagnosis of BM and TIL infiltration or PDL1 expression was observed (p > 0.05). Conclusions: (1) NSCLC BM contain considerable TILS infiltrates and show high PD-L1 expression. (2) The composition of the immune response differ between tumor sites with BM showing less dense TIL infiltration but more PD-L1 expression than PT. Our data may be relevant for clinical trials with immune checkpoint inhibitors in NSCLC BM. Disclosure: All authors have declared no conflicts of interest.

Validation of In utero Tractography of Human Fetal Commissural and Internal Capsule Fibers with Histological Structure Tensor Analysis.

Diffusion tensor imaging (DTI) and tractography offer the unique possibility to visualize the developing white matter macroanatomy of the human fetal brain in vivo and in utero and are currently under investigation for their potential use in the diagnosis of developmental pathologies of the human central nervous system. However, in order to establish in utero DTI as a clinical imaging tool, an independent comparison between macroscopic imaging and microscopic histology data in the same subject is needed. The present study aimed to cross-validate normal as well as abnormal in utero tractography results of commissural and internal capsule fibers in human fetal brains using postmortem histological structure tensor (ST) analysis. In utero tractography findings from two structurally unremarkable and five abnormal fetal brains were compared to the results of postmortem ST analysis applied to digitalized whole hemisphere sections of the same subjects. An approach to perform ST-based deterministic tractography in histological sections was implemented to overcome limitations in correlating in utero tractography to postmortem histology data. ST analysis and histology-based tractography of fetal brain sections enabled the direct assessment of the anisotropic organization and main fiber orientation of fetal telencephalic layers on a micro- and macroscopic scale, and validated in utero tractography results of corpus callosum and internal capsule fiber tracts. Cross-validation of abnormal in utero tractography results could be achieved in four subjects with agenesis of the corpus callosum (ACC) and in two cases with malformations of internal capsule fibers. In addition, potential limitations of current DTI-based in utero tractography could be demonstrated in several brain regions. Combining the three-dimensional nature of DTI-based in utero tractography with the microscopic resolution provided by histological ST analysis may ultimately facilitate a more complete morphologic characterization of axon guidance disorders at prenatal stages of human brain development.

Visualization of neuritic plaques in Alzheimer’s disease by polarization-sensitive optical coherence microscopy

One major hallmark of Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA) is the deposition of extracellular senile plaques and vessel wall deposits composed of amyloid-beta (Aβ). In AD, degeneration of neurons is preceded by the formation of Aβ plaques, which show different morphological forms. Most of them are birefringent owing to the parallel arrangement of amyloid fibrils. Here, we present polarization sensitive optical coherence microscopy (PS-OCM) for imaging mature neuritic Aβ plaques based on their birefringent properties. Formalin-fixed, post-mortem brain samples of advanced stage AD patients were investigated. In several cortical brain regions, neuritic Aβ plaques were successfully visualized in tomographic and three-dimensional (3D) images. Cortical grey matter appeared polarization preserving, whereas neuritic plaques caused increased phase retardation. Consistent with the results from PS-OCM imaging, the 3D structure of senile Aβ plaques was computationally modelled for different illumination settings and plaque sizes. Furthermore, the birefringent properties of cortical and meningeal vessel walls in CAA were investigated in selected samples. Significantly increased birefringence was found in smaller vessels. Overall, these results provide evidence that PS-OCM is able to assess amyloidosis based on intrinsic birefringent properties.

Impaired oligodendroglial turnover is associated with myelin pathology in Focal Cortical Dysplasia and Tuberous Sclerosis Complex

Conventional antiepileptic drugs suppress the excessive firing of neurons during seizures. In drug‐resistant patients, treatment failure indicates an alternative important epileptogenic trigger. Two epilepsy‐associated pathologies show myelin deficiencies in seizure‐related brain regions: Focal Cortical Dysplasia IIB (FCD) and cortical tubers in Tuberous Sclerosis Complex (TSC). Studies uncovering white matter‐pathology mechanisms are therefore urgently needed to gain more insight into epileptogenesis, the propensity to maintain seizures, and their associated comorbidities such as cognitive defects. We analyzed epilepsy surgery specimens of FCD IIB (n = 22), TSC (n = 8), and other malformations of cortical development MCD (n = 12), and compared them to autopsy and biopsy cases (n = 15). The entire lesional pathology was assessed using digital immunohistochemistry, immunofluorescence and western blotting for oligodendroglial lineage, myelin and mTOR markers, and findings were correlated to clinical parameters. White matter pathology with depleted myelin and oligodendroglia were found in 50% of FCD IIB and 62% of TSC cases. Other MCDs had either a normal content or even showed reactive oligodendrolial hyperplasia. Furthermore, myelin deficiency was associated with increased mTOR expression and the lower amount of oligodendroglia was linked with their precursor cells (PDGFRa). The relative duration of epilepsy (normalized to age) also correlated positively to mTOR activation and negatively to myelination. Decreased content of oligodendroglia and missing precursor cells indicated insufficient oligodendroglial development, probably mediated by mTOR, which may ultimately lead to severe myelin loss. In terms of disease management, an early and targeted treatment could restore normal myelin development and, therefore, alter seizure threshold and improve cognitive outcome.