Generation and External Validation of a Histologic Transformation Risk Model for Patients with Follicular Lymphoma.

Follicular lymphoma (FL) is the most frequent indolent lymphoma. Some patients (10%-15%) experience histologic transformation (HT) to a more aggressive lymphoma, usually diffuse large B-cell lymphoma (DLBCL). This study aimed to validate and improve a genetic risk model to predict HT at diagnosis.We collected mutational data from diagnosis biopsies of 64 FL patients. We combined them with the data from a previously published cohort (total n = 104; 62 from nontransformed and 42 from patients who did transform to DLBCL). This combined cohort was used to develop a nomogram to estimate the risk of HT. Prognostic mutated genes and clinical variables were assessed using Cox regression analysis to generate a risk model. The model was internally validated by bootstrapping and externally validated in an independent cohort. Its performance was evaluated using a concordance index and a calibration curve. The clinicogenetic nomogram included the mutational status of 3 genes (HIST1HE1, KMT2D, and TNFSR14) and high-risk Follicular Lymphoma International Prognostic Index and predicted HT with a concordance index of 0.746. Patients were classified as being at low or high risk of transformation. The probability HT function at 24 months was 0.90 in the low-risk group vs 0.51 in the high-risk group and, at 60 months, 0.71 vs 0.15, respectively. In the external validation cohort, the probability HT function in the low-risk group was 0.86 vs 0.54 in the high-risk group at 24 months, and 0.71 vs 0.32 at 60 months. The concordance index in the external cohort was 0.552. In conclusion, we propose a clinicogenetic risk model to predict FL HT to DLBLC, combining genetic alterations in HIST1H1E, KMT2D, and TNFRSF14 genes and clinical features (Follicular Lymphoma International Prognostic Index) at diagnosis. This model could improve the management of FL patients and allow treatment strategies that would prevent or delay transformation.

Frizzled 1 and Wnt1 as new potential therapeutic targets in the traumatically injured spinal cord.

Despite the experimental evidence pointing to a significant role of the Wnt family of proteins in physiological and pathological rodent spinal cord functioning, its potential relevance in the healthy and traumatically injured human spinal cord as well as its therapeutic potential in spinal cord injury (SCI) are still poorly understood. To get further insight into these interesting issues, we first demonstrated by quantitative Real-Time PCR and simple immunohistochemistry that detectable mRNA expression of most Wnt components, as well as protein expression of all known Wnt receptors, can be found in the healthy human spinal cord, supporting its potential involvement in human spinal cord physiology. Moreover, evaluation of Frizzled (Fz) 1 expression by double immunohistochemistry showed that its spatio-temporal and cellular expression pattern in the traumatically injured human spinal cord is equivalent to that observed in a clinically relevant model of rat SCI and suggests its potential involvement in SCI progression/outcome. Accordingly, we found that long-term lentiviral-mediated overexpression of the Fz1 ligand Wnt1 after rat SCI improves motor functional recovery, increases myelin preservation and neuronal survival, and reduces early astroglial reactivity and NG2+ cell accumulation, highlighting the therapeutic potential of Wnt1 in this neuropathological situation.

Craniopharyngiomas: A clinicopathological and molecular study of 52 cases - Experience in the Complejo Hospitalario de Toledo and Hospital Universitario 12 de Octubre (Madrid).

Craniopharyngiomas (CPs) are histologically benign tumors that are associated with high levels of morbidity. Two clinicopathological variants - adamantinomatous (ACP) and papillary (PCP) - have been described. They differ in their molecular features, whereby activating mutations in BRAF (V600E) and CTNNB1 genes characterize PCP and ACP, respectively. Recently, both variants have been shown to express elevated PD-L1 protein expression, but ACP also exhibited tumor cell-intrinsic PD-1 expression. In this study we analyze these molecular alterations in 52 cases with a long follow-up and examine their associations with immunohistochemical and clinical characteristics. ACPs comprise 73.1% of cases, while 21.2% are PCPs. Aberrant nuclear immunoreactivity for ß-catenin was observed in all ACPs. BRAF p.V600E mutations were observed in 90.9% of PCPs. Only one ACP case featured both alterations. Both types of CP exhibited strong nuclear staining for p63 with diffuse and basal distribution. ACP and PCP consistently expressed PD-L1, most in a substantial percentage of tumor cells, with a distinctive spatial distribution of expression in each subtype; only ACP demonstrated PD-1 expression. There was no evidence of differences in clinical prognosis between ACPs and PCPs. The identification of hallmark molecular signatures in the two CP variants is useful for sub-categorization in routine histopathology reporting. It is also pertinent to personalized therapy and for the development of improved non-invasive therapeutic strategies in this disease.

Epigenetic downregulation of TET3 reduces genome-wide 5hmC levels and promotes glioblastoma tumorigenesis.

Loss of 5-hydroxymethylcytosine (5hmC) has been associated with mutations of the ten-eleven translocation (TET) enzymes in several types of cancer. However, tumors with wild-type TET genes can also display low 5hmC levels, suggesting that other mechanisms involved in gene regulation might be implicated in the decline of this epigenetic mark. Here we show that DNA hypermethylation and loss of DNA hydroxymethylation, as well as a marked reduction of activating histone marks in the TET3 gene, impair TET3 expression and lead to a genome-wide reduction in 5hmC levels in glioma samples and cancer cell lines. Epigenetic drugs increased expression of TET3 in glioblastoma cells and ectopic overexpression of TET3 impaired in vitro cell growth and markedly reduced tumor formation in immunodeficient mice models. TET3 overexpression partially restored the genome-wide patterns of 5hmC characteristic of control brain samples in glioblastoma cell lines, while elevated TET3 mRNA levels were correlated with better prognosis in glioma samples. Our results suggest that epigenetic repression of TET3 might promote glioblastoma tumorigenesis through the genome-wide alteration of 5hmC.

Loss of 5hmC identifies a new type of aberrant DNA hypermethylation in glioma.

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.

Spatio-temporal and Cellular Expression Patterns of PTK7 in the Healthy and Traumatically Injured Rat and Human Spinal Cord.

Despite the emerging role of protein tyrosine kinase 7 (PTK7) as a Wnt co-receptor and the relevant functions of the Wnt family of proteins in spinal cord injury (SCI), the potential involvement of PTK7 in SCI is currently unknown. As a first essential step to shed light on this issue, we evaluated the spatio-temporal and cellular expression patterns of PTK7 in healthy and traumatically injured rat and human spinal cords. In the uninjured rats, PTK7 expression was observed in the ependymal epithelium, endothelial cells, meningeal fibronectin-expressing cells, and specific axonal tracts, but not in microglia, astrocytes, neurons, oligodendrocytes, or NG2+ cells. After rat SCI, the mRNA expression of PTK7 was significantly increased, while its spatio-temporal and cellular protein expression patterns also suffered evident changes in the injured region. Briefly, the expression of PTK7 in the affected areas was observed in axons, reactive astrocytes, NG2+ and fibronectin-expressing cells, and in a subpopulation of reactive microglia/macrophages and blood vessels. Finally, in both healthy and traumatically injured human spinal cords, PTK7 expression pattern was similar to that observed in the rat, although some specific differences were found. In conclusion, we demonstrate for the first time that PTK7 is constitutively expressed in the healthy adult rat and human spinal cord and that its expression pattern clearly varied after rat and human SCI which, to our knowledge, constitutes the first experimental evidence pointing to the potential involvement of this co-receptor in physiological and pathological spinal cord functioning.

Simultaneous occurrence of cutaneous mastocytosis and juvenile xanthogranuloma in a child: Random or true association?

Juvenile xanthogranuloma (JXG) and cutaneous mastocytosis (CM) are two distinct conditions that have rarely been reported in association. We report a child with CM and disseminated JXG, who showed a significant decrease in serum tryptase levels and regression of JXG lesions over time. Due to the paucity of reports, a true association between these two conditions has not been validated, although a potential induction of histiocytic lesions by mast cell degranulation has been proposed.

DNA methylation profiling identifies two splenic marginal zone lymphoma subgroups with different clinical and genetic features.

Splenic marginal zone lymphoma is a rare lymphoma. Loss of 7q31 and somatic mutations affecting the NOTCH2 and KLF2 genes are the commonest genomic aberrations. Epigenetic changes can be pharmacologically reverted; therefore, identification of groups of patients with specific epigenomic alterations might have therapeutic relevance. Here we integrated genome-wide DNA-promoter methylation profiling with gene expression profiling, and clinical and biological variables. An unsupervised clustering analysis of a test series of 98 samples identified 2 clusters with different degrees of promoter methylation. The cluster comprising samples with higher-promoter methylation (High-M) had a poorer overall survival compared with the lower (Low-M) cluster. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set of 36 patients. In the whole series, the High-M phenotype was associated with IGHV1-02 usage, mutations of NOTCH2 gene, 7q31-32 loss, and histologic transformation. In the High-M set, a number of tumor-suppressor genes were methylated and repressed. PRC2 subunit genes and several prosurvival lymphoma genes were unmethylated and overexpressed. A model based on the methylation of 3 genes (CACNB2, HTRA1, KLF4) identified a poorer-outcome patient subset. Exposure of splenic marginal zone lymphoma cell lines to a demethylating agent caused partial reversion of the High-M phenotype and inhibition of proliferation.

Clinical, immunophenotypic, and molecular characteristics of well-differentiated systemic mastocytosis.

BACKGROUND: Well-differentiated systemic mastocytosis (WDSM) is a rare variant of systemic mastocytosis (SM) characterized by bone marrow (BM) infiltration by mature-appearing mast cells (MCs) often lacking exon 17 KIT mutations. Because of its rarity, the clinical and biological features of WDSM remain poorly defined. OBJECTIVE: We sought to determine the clinical, biological, and molecular features of a cohort of 33 patients with mastocytosis in the skin in association with BM infiltration by well-differentiated MCs and to establish potential diagnostic criteria for WDSM. METHODS: Thirty-three patients with mastocytosis in the skin plus BM aggregates of round, fully granulated MCs lacking strong CD25 and CD2 expression in association with clonal MC features were studied. RESULTS: Our cohort of patients showed female predominance (female/male ratio, 4:1) and childhood onset of the disease (91%) with frequent familial aggregation (39%). Skin involvement was heterogeneous, including maculopapular (82%), nodular (6%), and diffuse cutaneous (12%) mastocytosis. KIT mutations were detected in only 10 (30%) of 33 patients, including the KIT D816V (n = 5), K509I (n = 3), N819Y (n = 1), and I817V (n = 1) mutations. BM MCs displayed a unique immunophenotypic pattern consisting of increased light scatter features, overexpression of cytoplasmic carboxypeptidase, and aberrant expression of CD30, together with absent (79%) or low (21%) positivity for CD25, CD2, or both. Despite only 9 (27%) of 33 patients fulfilling the World Health Organization criteria for SM, our findings allowed us to establish the systemic nature of the disease, which fit with the definition of WDSM. CONCLUSIONS: WDSM represents a rare clinically and molecularly heterogeneous variant of SM that requires unique diagnostic criteria to avoid a misdiagnosis of cutaneous mastocytosis per current World Health Organization criteria.

Diagnosis and classification of mastocytosis in non-specialized versus reference centres: a Spanish Network on Mastocytosis (REMA) study on 122 patients.

The diagnosis of 'rare diseases', such as mastocytosis, remains a challenge. Despite this, the precise benefits of referral of mastocytosis patients to highly specialized reference centres are poorly defined and whether patients should be managed at non-specialized versus reference centres remains a matter of debate. To evaluate the quality and efficiency of diagnostic procedures performed at the reference centres for mastocytosis in Spain (REMA) versus other non-reference centres, we retrospectively analysed a series of 122 patients, for the overall degree of agreement obtained for the World Health Organization (WHO) diagnostic and classification criteria betwen the referring and REMA centres. Our results showed that not all WHO diagnostic criteria were frequently investigated at the referring centres. Among the five WHO diagnostic criteria, the highest degree of agreement was obtained for serum tryptase levels [median 90% (95% confidence interval 84-96%)]; in turn, the overall agreement was significantly lower for the major histopathological criterion [80% (72-89%)], and the other three minor criteria: cytomorphology [68% (56-80%)] immunophenotyping of BM mast cells [75% (62-87%)] and detection of the KIT mutation [34% (8-60%)]. Referral of patients with diagnostic suspicion of mastocytosis to a multidisciplinary reference centre improves diagnostic efficiency and quality.

Antineutrophil cytoplasmic antibody negative pauci-immune extracapillary glomerulonephritis.

AIM: Pauci-immune extracapillary glomerulonephritis (PEGN) is one of the most common causes of rapidly progressive glomerulonephritis and is usually associated with circulating anti-neutrophil cytoplasmic antibodies (ANCAs). However, a significant number of individuals with PEGN test negative for ANCA and this study aimed to analyze the characteristics of this subgroup of patients. METHODS: Patients from two centres who were diagnosed with PEGN between 1997 and 2014 were studied retrospectively. Clinicopathological characteristics and renal outcome were compared between patients presenting with pauci-immune necrotizing extracapillary glomerulonephritis associated or not with the presence of circulating ANCA. RESULTS: Among the 114 patients with PEGN, 29 (25.4%) were ANCA negative. Compared with the 85 ANCA-positive patients, ANCA-negative patients were younger at the onset (54.8 ± 17.2 vs. 62 ± 14.0 years; P < 0.05). The median level of urinary protein excretion was significantly higher among ANCA-negative patients (3.1 vs. 1 g/24 h; P < 0.001), whereas no differences were found in renal function and need for dialysis between ANCA-negative and positive groups. Extrarenal involvement was present independently of ANCA status. Histological analysis showed that ANCA-negative patients were more likely to have mesangial proliferation (P < 0.05). Renal and global survival were similar between ANCA-negative and positive patients, and treatment response and relapse rates were comparable in both groups. CONCLUSIONS: ANCA-negative pauci-immune extracapillary glomerulonephritis is not a rare condition and is part of a systemic vasculitis disease. Although ANCA-negative patients have renal and histological characteristics that differ from ANCA-positive patients, renal survival and treatment response in PEGN are independent of ANCA status.

Impact on prognosis of the regional distribution of MGMT methylation with respect to the CpG island methylator phenotype and age in glioma patients.

Clinical and molecular prognostic factors in gliomas include age, IDH mutation, the glioma CpG island methylator phenotype (G-CIMP+) and promoter methylation of the O(6)-methylguanine DNA-methyltransferase (MGMT) gene. Among these markers, a predictive value was reported in glioblastomas (GBM) for MGMT promoter methylation, in particular in elderly GBM patients. In this study, methylation data from 46 glioma samples with the Illumina 450K platform were obtained and extended using external data to include a total of 247 glioma samples. Methylation analysis of the whole MGMT gene with this platform revealed two strongly survival-associated CpG regions within the promoter and the gene body, which were confirmed in a reported dataset of high grade-gliomas. Methylation at the promoter (CpG 25, cg12981137 and the prognostic model MGMT-STP27) and at the gene body CpG 165 (cg07933035), were significantly associated with better overall survival, and strongly correlated with G-CIMP+ status. In this series, the prognostic value of MGMT methylation at the promoter was not observed in G-CIMP- cases, although around 50 % of them were MGMT-methylated. These results were also obtained in an homogeneously-treated series of chemoradiated G-CIMP- GBMs analyzed by MSP and qMSP, and confirmed in a reported pyrosequencing-analyzed series of gliomas. Interestingly, in contrast to the MGMT promoter, gene body methylation was of prognostic value in G-CIMP-patients older than 65 years. Our study highlights the relevance of the prognostic value of the different regions of methylation throughout the MGMT gene that could be affected by specific G-CIMP profiles and age groups.

Secretory meningioma with KLF4 K409Q mutation in collision with glioma.

AIMS: The simultaneous occurrence of two primary intracranial tumors is a rare event, especially if unrelated to radiotherapy or genetic disorders. We present two patients, both with two primary intracranial tumors simultaneously present at adjacent sites, in order to explore a possible mechanism of synchronous tumor formation. METHODS: We performed a molecular analysis of the K409Q mutation of the KLF4 gene, in addition to conventional immunohistochemistry. RESULTS: Preoperative gadolinium-enhanced magnetic resonance imaging revealed a necrotic mass with an irregular ring-like enhancement adjacent to a frontal meningioma in patient 1, and an infiltrative non-enhancing glial tumor with no evidence of another tumor in patient 2. Postoperative histological examination revealed the presence of two distinct tumors in both cases: secretory meningioma and glioblastoma in patient 1 and secretory meningioma and anaplastic astrocytoma in patient 2. Secretory meningiomas both showed the KLF4 K409Q mutation, while none of the glial tumors had it. CONCLUSIONS: To our knowledge, these are the first two cases reported of the simultaneous occurrence of secretory meningiomas with mutation of KLF4 in collision with a glioblastoma and an anaplastic astrocytoma, respectively. These collision tumors presumably have different molecular origins.

Nonaggressive systemic mastocytosis (SM) without skin lesions associated with insect-induced anaphylaxis shows unique features versus other indolent SM.

BACKGROUND: Indolent systemic mastocytosis (ISM) without skin lesions (ISMs(-)) shows a higher prevalence in males, lower serum baseline tryptase levels, and KIT mutation more frequently restricted to bone marrow (BM) mast cells (MCs) than ISM with skin lesions (ISMs(+)). Interestingly, in almost one-half of ISMs(-) patients, MC-mediator release episodes are triggered exclusively by insects. OBJECTIVE: We aimed to determine the clinical and laboratory features of ISMs(-) associated with insect-induced anaphylaxis (insectISMs(-)) versus other patients with ISM. METHODS: A total of 335 patients presenting with MC activation syndrome, including 143 insectISMs(-), 72 ISMs(-) triggered by other factors (otherISMs(-)), 56 ISMs(+), and 64 nonclonal MC activation syndrome, were studied. RESULTS: Compared with otherISMs(-) and ISMs(+) patients, insectISMs(-) cases showed marked male predominance (78% vs 53% and 46%; P < .001), a distinct pattern of MC-related symptoms, and significantly lower median serum baseline tryptase levels (22.4 vs 28.7 and 45.8 µg/L; P ≤ .009). Moreover, insectISMs(-) less frequently presented BM MC aggregates (46% vs 70% and 81%; P ≤ .001), and they systematically showed MC-restricted KIT mutation. CONCLUSIONS: ISMs(-) patients with anaphylaxis triggered exclusively by insects display clinical and laboratory features that are significantly different from other ISM cases, including other ISMs(-) and ISMs(+) patients, suggesting that they represent a unique subgroup of ISM with a particularly low BM MC burden in the absence of adverse prognostic factors.

Hepatitis C virus-related lymphoproliferative disorders encompass a broader clinical and morphological spectrum than previously recognized: a clinicopathological study.

We describe a retrospective series of B-cell lymphoproliferative disorders associated with hepatitis C virus infection. In addition to splenic marginal zone lymphoma, follicular lymphoma and diffuse large B-cell lymphoma, all of which showed some specific features, we found two poorly described groups of cases. The first featured disseminated marginal zone lymphoma without splenic marginal zone lymphoma features, defying the current marginal zone lymphoma classification; the other consisted of monoclonal B lymphocytes in the peripheral blood, bone marrow or other tissues, with no clinical or histological evidence of lymphoma, and exhibiting a pattern that requires proper identification in order to avoid the misdiagnosis of the lymphoma. Diagnosis of hepatitis C virus infection-associated lymphoproliferative disorders requires the integration of clinical, pathological and molecular findings to establish an adequate diagnosis and decide the appropriate therapy to be applied.

NFκB expression is a feature of both activated B-cell-like and germinal center B-cell-like subtypes of diffuse large B-cell lymphoma.

The activation of nuclear factor kappa B (NFκB) transcription factor family is considered to have a key role in diffuse large B-cell lymphoma (DLBCL) pathogenesis and is associated with a specific molecular subtype, the activated B-cell-like (ABC) subtype. We evaluated the expression of NFκB by immunohistochemistry in a large series of DLBCL cases. The five different NFκB family members (NFκB1, NFκB2, RELA, RELB, and REL) showed a heterogeneous expression pattern with the vast majority of cases being positive for at least one factor. Two independent series of tumor samples were classified into germinal center B-cell-like (GCB) or ABC subtypes using different approaches, immunohistochemistry, or gene expression profiling, and the expression of NFκB family members was assessed. Notably, no significant differences regarding the expression of the different NFκB members were detected between the two subtypes, suggesting that NFκB signaling is a prominent feature not only in the ABC subtype, but also in the GCB tumors. Of the five transcription factors, only REL expression had a significant clinical impact on R-CHOP-treated diffuse large B-cell lymphoma, identifying a subgroup of patients with superior clinical outcome.

Nodal marginal zone lymphoma: gene expression and miRNA profiling identify diagnostic markers and potential therapeutic targets.

Nodal marginal zone lymphoma (NMZL) is a small B-cell neoplasm whose molecular pathogenesis is still essentially unknown and whose differentiation from other small B-cell lymphomas is hampered by the lack of specific markers. We have analyzed gene expression, miRNA profile, and copy number data from 15 NMZL cases. For comparison, 16 follicular lymphomas (FLs), 9 extranodal marginal zone lymphomas, and 8 reactive lymph nodes and B-cell subtypes were included. The results were validated by quantitative RT-PCR in an independent series, including 61 paraffin-embedded NMZLs. NMZL signature showed an enriched expression of gene sets identifying interleukins, integrins, CD40, PI3K, NF-κB, and TGF-ß, and included genes expressed by normal marginal zone cells and memory B cells. The most highly overexpressed genes were SYK, TACI, CD74, CD82, and CDC42EP5. Genes linked to G(2)/M and germinal center were down-regulated. Comparison of the gene expression profiles of NMZL and FL showed enriched expression of CHIT1, TGFB1, and TACI in NMZL, and BCL6, LMO2, and CD10 in FL. NMZL displayed increased expression of miR-221, miR-223, and let-7f, whereas FL strongly expressed miR-494. Our study identifies new candidate diagnostic molecules for NMZL and reveals survival pathways activated in NMZL.

Association of both BCL2 positive and negative follicular lymphoma to clasical Hodgkin lymphoma and/or gray zone lymphoma.

We present a series of 9 follicular lymphomas that progressed/transformed into classical Hodgkin lymphoma (CHL). Three cases of CHL showed a syncytial pattern (SCHL) making the differential diagnosis to Gray zone lymphoma (GZL) challenging. None of these three cases presented in the mediastinum. Based in all molecular data analyzed (BCL2/BCL6 FISH studies, IgH PCR and TNGS with a customized gene panel) we did find clonal relationship between the BCL2-positive FL cases and their CHL components in all cases. The three SCHL/GZL cases showed an activated phenotype according to Hans algorithm, presented the t(14; 18)(q32; q21), two out of three showed B cell markers and all expressed CD30 and p53. Interestingly, we identified three BCL2-negative FL cases with a further diagnosis of CHL expanding the spectrum of these association. In one of these three cases a different mutational profile was found in both the FL and the CHL components. All this data together suggests that CHL associated to BCL2-positive FL could be originated in a common progenitor cell (CPC) that give rise to both FL and CHL, acquiring this last component further genetic events in a linear fashion. On the other hand, no clonal relationship between CHL and BCL2-negative FL could be found, suggesting a fortuity association. Nevertheless, ample series of cases studied with more sensitive techniques are needed to confirm our hypothesis.