Yojiro Akagi

Donor-derived adult T-cell leukaemia

In July, 2008, a 55-year-old woman was referred to us with a 2-month history of progressive aphasia after a febrile episode. She had been diagnosed with chronic adult T-cell leukaemia in June, 1999, and had had a nonmyeloablative peripheral-blood stem-cell transplant with conditioning of pentostatin and 2 Gy total body irradiation in March, 2002. The stem cells had been harvested from her brother, an asymptomatic carrier of human T-cell lymphotropic virus type 1 (HTLV-1). After treatment, she had complete remission. Ciclosporin was discontinued 6 months after the transplant ation because of graft-versus-host disease. Our patient had had no bacterial or viral infections. She had no skin lesions, lymphadenopathy, or splenomegaly. Her leucocyte count was 8·74×109/L with 2·5% adult T-cell leukaemia-like cells. The leukaemia was therefore con sidered to be in remission. CD3-positive cells in the peripheral blood were confi rmed as donor derived by XY-fl uorescence insitu hybridisation (XY-FISH). Serum IgG was 13·7 g/L, IgA was 2·82 g/L, and IgM was 1·16 g/L. Our patient was immunocompetent; absolute number of circulating cells positive for CD4 was 1·74×109/L, CD8 0·475×109/L, CD19 0·522×109/L, and CD56 0·326×109/L. Brain MRI showed hyperintense, non-enhancing lesions in the left temporoparietal lobe and right frontal lobe (fi gure A), which suggested encephalitis, progressive multiple leukoencephalopathy, demyelinating disorder, or lymphoma. Serum concentrations of calcium, lactate dehydro genase, soluble interleukin-2 receptor, and β-2 microglobulin were not raised. CSF examination, culture, and PCR, abdominal ultrasonography, endoscopy, and 18F-fl urodeoxyglucose (18F-FDG) PET were normal. Craniotomy and biopsy of the left temporal lobe was done in July, 2008. Histopathology showed perivascular accumulation and cerebral infi ltration by mononuclear tumour cells with large, indented nuclei (fi gure B). Tumour cells were positive for CD3, CD4, CD8, and CD45RO, but negative for B-cell markers (CD20 and CD79a), suggesting cerebral invasion by adult T-cell leukaemia cells (webappendix). XY-FISH showed an XY genotype of the tumour cells (fi gure C), suggesting that they had originated from the donated leucocytes. After whole brain irradiation and localised irradiation of the left temporoparietal lobe, our patient’s aphasia improved remarkably, and repeat MRI showed shrinkage of the brain lesions. In October, 2010, our patient was fully recovered. For patients with adult T-cell leukaemia, asymptomatic carriers of HTLV-1 have been widely considered acceptable as donors in allogeneic stem-cell transplantation. The immune system is thought to have an important role in leukaemia occurrence in HTLV-1-seropositive carriers. The only reported case of donor-derived adult T-cell leukaemia was in 2006, in an immunosuppressed recipient of a peripheral-blood stem-cell transplant. No cases of donorderived adult T-cell leukaemia have been reported in immunocompetent patients. The prognosis tends to be better for these patients than for those with CNS involvement subsequent to systemic adult T-cell leukaemia. Only two cases of isolated CNS involvement have been reported so far. The function of CD4-positive cells was not fully analysed, so there is a possibility of some functional defi cit in immune reconstitution. Our patient’s case suggests that the consent practices in patients who receive HTLV-1-positive cells should be re-evaluated.

A comprehensive analysis identifies BRAF hotspot mutations associated with gliomas with peculiar epithelial morphology.

Brain tumors harbor various BRAF alterations, the vast majority of which are the BRAF kinase‐activating V600E mutation. BRAF mutations are most frequently detected in certain subtypes of low‐grade glioma, such as pilocytic astrocytoma (PA), pleomorphic xanthoastrocytoma (PXA), ganglioglioma (GG) and dysembryoplastic neuroepithelial tumor (DNT). However, it is unclear whether gliomas harboring BRAF mutations can be invariably regarded as these glioma subtypes or their derivatives. To address this question, we analyzed 274 gliomas in our institutional case series. We performed high‐resolution melting analyses and subsequent direct Sanger sequencing on DNA isolated from snap‐frozen tumor tissues. As expected, BRAF mutations were detected in the aforementioned low‐grade gliomas: in 4/27 PAs, 2/3 PXAs, 4/8 GGs, and 1/6 DNTs. In addition to these gliomas, 1/2 astroblastomas (ABs) and 2/122 glioblastomas (GBs) harbored BRAF mutations. Pathological investigation of the two GBs revealed that one was a GB displaying epithelial features that presumably arose from a precedent GG, whereas the other GB, which harbored a rare G596 A mutation, showed marked epithelial features, including astroblastic rosettes. Our results indicate that in addition to being present in established BRAF‐associated gliomas, BRAF mutations might be associated with epithelial features in high‐grade gliomas, including sheet‐like arrangement of polygonal tumor cells with a plump cytoplasm and astroblastic rosettes, and thus could potentially serve as a genetic marker for these features.

Precise Detection of IDH1/2 and BRAF Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data.

High resolution melting (HRM) is a simple and rapid method for screening mutations. It offers various advantages for clinical diagnostic applications. Conventional HRM analysis often yields equivocal results, especially for surgically obtained tissues. We attempted to improve HRM analyses for more effective applications to clinical diagnostics. HRM analyses were performed for IDH1R132 and IDH2R172 mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. BRAFV600E mutations were analyzed in 52 additional brain tumor samples. The melting profiles were used for differential calculus analyses. Negative second derivative plots revealed additional peaks derived from heteroduplexes in PCR products that contained mutations; this enabled unequivocal visual discrimination of the mutations. We further developed a numerical expression, the HRM-mutation index (MI), to quantify the heteroduplex-derived peak of the mutational curves. Using this expression, all IDH1 mutation statuses matched those ascertained by sequencing, with the exception of three samples. These discordant results were all derived from the misinterpretation of sequencing data. The effectiveness of our approach was further validated by analyses of IDH2R172 and BRAFV600E mutations. The present analytical method enabled an unequivocal and objective HRM analysis and is suitable for reliable mutation scanning in surgically obtained glioma tissues. This approach could facilitate molecular diagnostics in clinical environments.

Posterior transjugular and transcervical approach for glomus tumours within the head and neck.

Abstract Glomus tumours within the head and neck are highly vascular in nature and are surrounded by vital neurovascular structures. The aim of this study is to review the step-by-step surgical techniques for a posterior transjugular approach and transcervical approach and to clarify the advantages of these approaches in the treatment of glomus tumours within the head and neck. The advantage of these approaches is that a wide operative field from the jugular bulb to the cervical portion can be obtained. In addition, the bloodless operative field that is achieved by the preoperative embolisation appeared to contribute to reducing the risk of cranial nerve injury.

Deferred radiotherapy and upfront procarbazine–ACNU–vincristine administration for 1p19q codeleted oligodendroglial tumors are associated with favorable outcome without compromising patient performance, regardless of WHO grade

Recently updated phase III trials revealed the favorable effect of add-on procarbazine-lomustine-vincristine chemotherapy (CT) to radiotherapy (RT) in treating anaplastic oligodendrogliomas with 1p19q codeletion (codel). However, the underlying rationality of deferring RT and upfront CT administration for these tumors is yet to be elucidated. Here, we retrospectively analyzed the long-term outcome of our case series with oligodendroglial tumors treated with deferred RT and upfront procarbazine+nimustine+vincristine (PAV) in the introduction administration. We enrolled 36 patients with newly diagnosed oligodendroglial tumors (17, grade II and 19, grade III) treated during 1999–2012 and followed up for a median period of 69.0 months. Their clinical and genetic prognostic factors were analyzed, and progression-free survival, overall survival (OS), and deterioration-free survival (DFS) were evaluated. Regardless of the WHO grade, the 25 patients with 1p19q codel tumors never received RT initially, and of these 25, 23 received PAV treatment upfront. The 75% OS of patients with 1p19q codel tumor was 135.3 months (did not reach the median OS), indicating a favorable outcome. Multivariate analysis revealed that IDH mutation and 1p19q, not WHO grade, are independent prognostic factors; furthermore, IDH and 1p19q status stratified the cohort into 3 groups with significantly different OS. The DFS explained the prolonged survival without declining performance in patients with both grade II and III 1p19q codel tumors. Deferred RT and upfront PAV treatment for 1p19q codel oligodendrogliomas were associated with favorable outcomes without compromising performance status, regardless of WHO grade.

Prevalence and clinicopathological features of H3.3 G34-mutant high-grade gliomas: a retrospective study of 411 consecutive glioma cases in a single institution

A recurrent glycine-to-arginine/valine alteration at codon 34 (G34R/V) within H3F3A, a gene that encodes the replication-independent histone variant H3.3, reportedly occurs exclusively in pediatric glioblastomas. However, the clinicopathological and biological significances of this mutation have not been completely elucidated; especially, no such data exist for tumor samples from Japanese patients. We analyzed 411 consecutive glioma cases representing patients of all ages. Our results demonstrated that 14 patients (3.4%) harbored H3F3A mutations, of which four had G34R mutations and 10 had K27M mutations. G34R-mutant tumors were located in the parietal region in two patients and the basal ganglia in one patient. One patient showed multi-lobular extension similar to the pattern observed in gliomatosis cerebri. Regarding neuroradiological features, intratumoral calcification was evident in two cases and all cases showed no or scarce contrast enhancement on MRI. Histopathologically, the four G34R-mutant cases included three glioblastomas and one astroblastoma. We have also investigated alterations in histone methylation including H3K27me3, H3K9me3, and H3K4me3 in G34R-mutant samples by immunohistochemistry. These results indicate that G34R-mutant tumors are likely to show extensive infiltration and alterations in global histone trimethylation might also play an important role in G34R mutant tumors.

Detection of proneural/mesenchymal marker expression in glioblastoma: temporospatial dynamics and association with chromatin-modifying gene expression

Proneural and mesenchymal are two subtypes of glioblastoma identified by gene expression profiling. In this study, the primary aim was to detect markers to develop a clinically applicable method for distinguishing proneural and mesenchymal glioblastoma. The secondary aims were to investigate the temporospatial dynamics of these markers and to explore the association between these markers and the expression of chromatin-modifying genes. One hundred thirty-three glioma samples (grade II: 14 samples, grade III: 18, grade IV: 101) were analyzed. We quantified the expression of 6 signature genes associated with proneural and mesenchymal glioblastoma by quantitative reverse transcription-polymerase chain reaction. We assigned proneural (PN) and mesenchymal (MES) scores based on the average of the 6 markers and calculated a predominant metagene (P-M) score by subtracting the MES from the PN score. We used these scores to analyze correlations with malignant transformation, tumor recurrence, tumor heterogeneity, chromatin-modifying gene expression, and HDAC7 expression. The MES score positively correlated with tumor grade, whereas the PN score did not. The P-M score was able to distinguish the proneural and mesenchymal subtypes. It was decreased in cases of tumor recurrence and malignant transformation and showed variability within a tumor, suggesting intratumoral heterogeneity. The PN score correlated with the expression of multiple histone-modifying genes, whereas the MES score was associated only with HDAC7 expression. Thus, we demonstrated a simple and straightforward method of quantifying proneural/mesenchymal markers in glioblastoma. Of note, HDAC7 expression might be a novel therapeutic target in glioblastoma treatment.

Add-on bevacizumab can prevent early clinical deterioration and prolong survival in newly diagnosed partially resected glioblastoma patients with a poor performance status

Purpose The AVAglio trial established the beneficial effect of add-on bevacizumab (BEV) for the treatment of newly diagnosed glioblastomas (nd-GBMs) that led to the approval of BEV for the treatment of these patients in Japan. However, the rationality of using BEV as a first-line treatment for nd-GBMs remains controversial. The purpose of this study was to analyze the outcomes of a case series of nd-GBM patients. Patients and methods The outcomes of 69 nd-GBM patients treated after 2006 were retrospectively analyzed. Clinical and genetic analyses were performed, and estimates of progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan–Meier method. Since add-on BEV therapy was only used for partially resected GBMs (pr-GBMs) after its approval in 2013, the patients were subdivided into 3 treatment groups: Type I, partial removal with temozolomide (TMZ)/BEV and concurrent radiotherapy (CCRT); Type II, partial removal with TMZ and CCRT; and Type III, gross total removal with TMZ and CCRT. Results The PFS rate of Type I patients was significantly higher than that of Type II patients (P=0.014), but comparable to that of Type III patients. Differences in OS rates between Type I and Type II patients were less apparent (P=0.075), although the median OS of Type I patients was ~8 months higher than that of Type II patients (17.4 vs 9.8 months, respectively). The clinical deterioration rate during initial treatment was significantly (P=0.024) lower in Type I than in Type II patients (7.7% vs 47.4%, respectively). Differences in OS rates between Type I and Type II patients with a poor performance status (PS) were significant (P=0.017). Conclusion Our findings suggest that add-on BEV can prevent early clinical deterioration of pr-GBM patients and contribute to a prolonged survival, especially for those with a poor PS.

High-resolution melting and immunohistochemical analysis efficiently detects mutually exclusive genetic alterations of adamantinomatous and papillary craniopharyngiomas

Craniopharyngioma consists of adamantinomatous and papillary subtypes. Recent genetic analysis has demonstrated that the two subtypes are different, not only in clinicopathological features, but also in molecular oncogenesis. Papillary craniopharyngioma (pCP) is characterized by a BRAF mutation, the V600E (Val 600 Glu) mutation. Adamantinomatous craniopharyngioma (aCP) can be distinguished by frequent β‐catenin gene (CTNNB1) mutations. Although these genetic alterations can be a diagnostic molecular marker, the precise frequency of these mutations in clinical specimens remains unknown. In this study, we first evaluated BRAF V600E and CTNNB1 mutations in four and 14 cases of pCP and aCP, respectively, using high‐resolution melting analysis followed by Sanger sequencing. The results showed that 100% (4/4) of pCP cases had BRAF V600E mutations, while 78% (11/14) of the aCP cases had CTNNB1 mutations, with these genetic alterations being subtype‐specific and mutually exclusive. Second, we evaluated BRAF V600E and CTNNB1 mutations by immunohistochemical analysis (IHC). All pCP cases showed positive cytoplasmic staining with the BRAF V600E‐mutant antibody (VE‐1), whereas 86% (12/14) of aCP cases showed positive cytoplasmic and nuclear staining for CTNNB1, suggesting a CTNNB1 mutation. Only one case of wild‐type CTNNB1 on the DNA analysis showed immunopositivity on IHC. We did not detect a coexistence of BRAF V600E and CTNNB1 mutations in any single tumor, which indicated that these genetic alterations were mutually exclusive. We also report our modified IHC protocol for VE‐1 staining, and present the possibility that BRAF V600E mutations can be used as a diagnostic marker of pCP in the differentiation of Rathke cleft cyst with squamous metaplasia.

Insular primary glioblastomas with IDH mutations: Clinical and biological specificities.

Isocitrate dehydrogenase (IDH) mutation is a good prognostic marker for glioblastoma (GBM). Although it is infrequent in primary tumors, it is found in most lower‐grade gliomas. Thus, it is unclear whether IDH mutation is a marker for a specific phenotype of apparently primary de novo GBMs (pGBMs), or a marker for secondary tumors (sGBMs). We addressed this issue by analyzing clinical, radiographic and molecular findings in our institutional case series. Our cases included 92 pGBMs, with five cases of IDH1 mutations at R132 and no IDH2 mutations. The median overall survival of these five patients was 29 months (range: 4 to >40 months), which is considered good prognoses. Clinical and radiographic characteristics were distinct from IDH‐wildtype (IDH‐wt) pGBMs. IDH‐mutant (IDH‐mut) tumors consistently involved insular lesions and were subdivided into: (i) the two cases of elderly patients with long clinical histories and features implying multistep tumor development; and (ii) the three cases of younger patients with diffusely swelling insular tumors, slight contrast enhancement and no necrosis. Genetic and expression analyses of IDH‐mut pGBMs were similar to those of sGBMs, suggesting that they are indeed distinct from their IDH‐wt counterparts. TERT promoter mutation, a genetic marker of oligodendroglial derivation, was detected in one long‐surviving case, but genetic alterations in the astrocyte‐sGBM pathway were generally prevalent in IDH‐mut pGBMs. Our results present a unique phenotype of IDH‐mut pGBMs arising from insular cortex region, the molecular backgrounds of which are similar to sGBMs.