MEN1 mutations mediate clinical resistance to menin inhibition.

Chromatin-binding proteins are critical regulators of cell state in haematopoiesis1,2. Acute leukaemias driven by rearrangement of the mixed lineage leukaemia 1 gene (KMT2Ar) or mutation of the nucleophosmin gene (NPM1) require the chromatin adapter protein menin, encoded by the MEN1 gene, to sustain aberrant leukaemogenic gene expression programs3-5. In a phase 1 first-in-human clinical trial, the menin inhibitor revumenib, which is designed to disrupt the menin-MLL1 interaction, induced clinical responses in patients with leukaemia with KMT2Ar or mutated NPM1 (ref. 6). Here we identified somatic mutations in MEN1 at the revumenib-menin interface in patients with acquired resistance to menin inhibition. Consistent with the genetic data in patients, inhibitor-menin interface mutations represent a conserved mechanism of therapeutic resistance in xenograft models and in an unbiased base-editor screen. These mutants attenuate drug-target binding by generating structural perturbations that impact small-molecule binding but not the interaction with the natural ligand MLL1, and prevent inhibitor-induced eviction of menin and MLL1 from chromatin. To our knowledge, this study is the first to demonstrate that a chromatin-targeting therapeutic drug exerts sufficient selection pressure in patients to drive the evolution of escape mutants that lead to sustained chromatin occupancy, suggesting a common mechanism of therapeutic resistance.

Mutant NPM1 Directly Regulates Oncogenic Transcription in Acute Myeloid Leukemia.

The dysregulation of developmental and stem cell-associated genes is a common phenomenon during cancer development. Around half of patients with acute myeloid leukemia (AML) express high levels of HOXA cluster genes and MEIS1. Most of these AML cases harbor an NPM1 mutation (NPM1c), which encodes for an oncoprotein mislocalized from the nucleolus to the cytoplasm. How NPM1c expression in hematopoietic cells leads to its characteristic gene-expression pattern remains unclear. Here, we show that NPM1c directly binds to specific chromatin targets, which are co-occupied by the histone methyltransferase KMT2A (MLL1). Targeted degradation of NPM1c leads to a rapid decrease in gene expression and loss of RNA polymerase II, as well as activating histone modifications at its targets. We demonstrate that NPM1c directly regulates oncogenic gene expression in collaboration with the MLL1 complex and define the mechanism by which MLL1-Menin small-molecule inhibitors produce clinical responses in patients with NPM1-mutated AML. SIGNIFICANCE: We uncovered an important functional role of mutant NPM1 as a crucial direct driver of oncogenic gene expression in AML. NPM1c can bind to chromatin and cooperate with the MLL complex, providing the first functional insight into the mechanism of Menin-MLL inhibition in NPM1c leukemias. See related article by Wang et al., p. 724. This article is highlighted in the In This Issue feature, p. 517.

IL-13/IL-4 signaling contributes to fibrotic progression of the myeloproliferative neoplasms.

Myelofibrosis (MF) is a disease associated with high unmet medical needs because allogeneic stem cell transplantation is not an option for most patients, and JAK inhibitors are generally effective for only 2 to 3 years and do not delay disease progression. MF is characterized by dysplastic megakaryocytic hyperplasia and progression to fulminant disease, which is associated with progressively increasing marrow fibrosis. Despite evidence that the inflammatory milieu in MF contributes to disease progression, the specific factors that promote megakaryocyte growth are poorly understood. Here, we analyzed changes in the cytokine profiles of MF mouse models before and after the development of fibrosis, coupled with the analysis of bone marrow populations using single-cell RNA sequencing. We found high interleukin 13 (IL-13) levels in the bone marrow of MF mice. IL-13 promoted the growth of mutant megakaryocytes and induced surface expression of transforming growth factor ß and collagen biosynthesis. Similarly, analysis of samples from patients with MF revealed elevated levels of IL-13 in the plasma and increased IL-13 receptor expression in marrow megakaryocytes. In vivo, IL-13 overexpression promoted disease progression, whereas reducing IL-13/IL-4 signaling reduced several features of the disease, including fibrosis. Finally, we observed an increase in the number of marrow T cells and mast cells, which are known sources of IL-13. Together, our data demonstrate that IL-13 is involved in disease progression in MF and that inhibition of the IL-13/IL-4 signaling pathway might serve as a novel therapeutic target to treat MF.

Resident Self-Tissue of Proinflammatory Cytokines Rather Than Their Systemic Levels Correlates with Development of Myelofibrosis in Gata1low Mice.

Serum levels of inflammatory cytokines are currently investigated as prognosis markers in myelofibrosis, the most severe Philadelphia-negative myeloproliferative neoplasm. We tested this hypothesis in the Gata1low model of myelofibrosis. Gata1low mice, and age-matched wild-type littermates, were analyzed before and after disease onset. We assessed cytokine serum levels by Luminex-bead-assay and ELISA, frequency and cytokine content of stromal cells by flow cytometry, and immunohistochemistry and bone marrow (BM) localization of GFP-tagged hematopoietic stem cells (HSC) by confocal microscopy. Differences in serum levels of 32 inflammatory-cytokines between prefibrotic and fibrotic Gata1low mice and their wild-type littermates were modest. However, BM from fibrotic Gata1low mice contained higher levels of lipocalin-2, CXCL1, and TGF-ß1 than wild-type BM. Although frequencies of endothelial cells, mesenchymal cells, osteoblasts, and megakaryocytes were higher than normal in Gata1low BM, the cells which expressed these cytokines the most were malignant megakaryocytes. This increased bioavailability of proinflammatory cytokines was associated with altered HSC localization: Gata1low HSC were localized in the femur diaphysis in areas surrounded by microvessels, neo-bones, and megakaryocytes, while wild-type HSC were localized in the femur epiphysis around adipocytes. In conclusion, bioavailability of inflammatory cytokines in BM, rather than blood levels, possibly by reshaping the HSC niche, correlates with myelofibrosis in Gata1low mice.

Epigenetic reprogramming of host and viral genes by Human Cytomegalovirus infection in Kasumi-3 myeloid progenitor cells at early times post-infection.

HCMV establishes latency in myeloid cells. Using the Kasumi-3 latency model, we previously showed that lytic gene expression is activated prior to establishment of latency in these cells. The early events in infection may have a critical role in shaping establishment of latency. Here, we have used an integrative multi-omics approach to investigate dynamic changes in host and HCMV gene expression and epigenomes at early times post infection. Our results show dynamic changes in viral gene expression and viral chromatin. Analyses of Pol II, H3K27Ac and H3K27me3 occupancy of the viral genome showed that 1) Pol II occupancy was highest at the MIEP at 4 hours post infection. However, it was observed throughout the genome; 2) At 24 hours, H3K27Ac was localized to the major immediate early promoter/enhancer and to a possible second enhancer in the origin of replication OriLyt; 3) viral chromatin was broadly accessible at 24 hpi. In addition, although HCMV infection activated expression of some host genes, we observed an overall loss of de novo transcription. This was associated with loss of promoter-proximal Pol II and H3K27Ac, but not with changes in chromatin accessibility or a switch in modification of H3K27.Importance.HCMV is an important human pathogen in immunocompromised hosts and developing fetuses. Current anti-viral therapies are limited by toxicity and emergence of resistant strains. Our studies highlight emerging concepts that challenge current paradigms of regulation of HCMV gene expression in myeloid cells. In addition, our studies show that HCMV has a profound effect on de novo transcription and the cellular epigenome. These results may have implications for mechanisms of viral pathogenesis.

LKB1/STK11 Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms.

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. SIGNIFICANCE: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression.This article is highlighted in the In This Issue feature, p. 1307.

FBXO11 is a candidate tumor suppressor in the leukemic transformation of myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous myeloid malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of transformation to secondary acute myeloid leukemia (sAML). A number of genetic abnormalities have been identified in MDS and sAML, but sensitive sequencing methods can detect these mutations in nearly all healthy individuals by 60 years of age. To discover novel cellular pathways that accelerate MDS and sAML, we performed a CRISPR/Cas9 screen in the human MDS-L cell line. We report here that loss of the F-Box protein FBXO11, a component of the SCF ubiquitin ligase complex, confers cytokine independent growth to MDS-L cells, suggesting a tumor suppressor role for FBXO11 in myeloid malignancies. Putative FBXO11 substrates are enriched for proteins with functions in RNA metabolism and, of note, spliceosome mutations that are commonly found in MDS/sAML are rare in patients with low FBXO11 expression. We also reveal that loss of FBXO11 leads to significant changes in transcriptional pathways influencing leukocyte proliferation, differentiation, and apoptosis. Last, we find that FBXO11 expression is reduced in patients with secondary AML. We conclude that loss of FBXO11 is a mechanism for disease transformation of MDS into AML, and may represent a future therapeutic target.

Activation of JAK/STAT Signaling in Megakaryocytes Sustains Myeloproliferation In Vivo.

PURPOSE: The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear. EXPERIMENTAL DESIGN: We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage-committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN. RESULTS: JAK2V617F -mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis. CONCLUSIONS: Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.

Aurora Kinase A Inhibition Provides Clinical Benefit, Normalizes Megakaryocytes, and Reduces Bone Marrow Fibrosis in Patients with Myelofibrosis: A Phase I Trial.

PURPOSE: Myelofibrosis is characterized by bone marrow fibrosis, atypical megakaryocytes, splenomegaly, constitutional symptoms, thrombotic and hemorrhagic complications, and a risk of evolution to acute leukemia. The JAK kinase inhibitor ruxolitinib provides therapeutic benefit, but the effects are limited. The purpose of this study was to determine whether targeting AURKA, which has been shown to increase maturation of atypical megakaryocytes, has potential benefit for patients with myelofibrosis. PATIENTS AND METHODS: Twenty-four patients with myelofibrosis were enrolled in a phase I study at three centers. The objective of the study was to evaluate the safety and preliminary efficacy of alisertib. Correlative studies involved assessment of the effect of alisertib on the megakaryocyte lineage, allele burden, and fibrosis. RESULTS: In addition to being well tolerated, alisertib reduced splenomegaly and symptom burden in 29% and 32% of patients, respectively, despite not consistently reducing the degree of inflammatory cytokines. Moreover, alisertib normalized megakaryocytes and reduced fibrosis in 5 of 7 patients for whom sequential marrows were available. Alisertib also decreased the mutant allele burden in a subset of patients. CONCLUSIONS: Given the limitations of ruxolitinib, novel therapies are needed for myelofibrosis. In this study, alisertib provided clinical benefit and exhibited the expected on-target effect on the megakaryocyte lineage, resulting in normalization of these cells and reduced fibrosis in the majority of patients for which sequential marrows were available. Thus, AURKA inhibition should be further developed as a therapeutic option in myelofibrosis.See related commentary by Piszczatowski and Steidl, p. 4868.

GATA-1: A potential novel biomarker for the differentiation of essential thrombocythemia and myelofibrosis.

Essentials The BCR-ABL negative myeloproliferative neoplasms are subjected to unknown phenotypic modifiers. GATA-1 is upregulated in ET patients, regardless of treatment regimen or mutational status. Myelofibrosis (MF) megakaryocytes displayed decreased GATA-1 staining. GATA-1 may have utility as a diagnostic marker in ET and in its differential diagnosis from MF. ABSTRACT: Background The BCR-ABL-negative myeloproliferative neoplasms, i.e., polycythemia vera, essential thrombocythemia (ET), and myelofibrosis (MF), are characterized by mutations in JAK2, CALR, or MPL. However, an as yet unknown factor drives the precise disease phenotype. The hematopoietic transcription factor GATA-1 and its downstream targets NFE2 and FLI1 are responsible for determining erythroid and megakaryocyte lineages during hematopoietic stem cell differentiation. Previous studies have demonstrated a low level of GATA-1 expression in megakaryocytes from patients with MF. Objectives and methods The expression of GATA-1, NFE2 and FLI1 was studied for changes in the peripheral blood (PB) of ET patients. Peripheral blood samples were obtained from 36 ET patients, 14 MF patients, and seven healthy control donors. Total RNA from PB mononuclear cells (PBMCs) was extracted, and quantitative polymerase chain reaction was used to determine relative changes in gene expression. Protein levels of GATA-1 were also determined in bone marrow sections from ET and MF patients. Results GATA-1 mRNA was upregulated in ET patients, regardless of treatment regimen or mutational status. FLI1 expression was significantly downregulated, whereas NFE2 expression was unaffected by changes in GATA-1 mRNA levels. Megakaryocytes from ET patients showed increased protein levels of GATA-1 as compared with those from MF patients. Conclusions Our results confirmed, in PB, our previous data demonstrating elevated levels of GATA-1 mRNA in total bone marrow of ET patients. GATA-1 mRNA levels are independent of cytoreductive therapies, and may have utility as a diagnostic marker in ET and in its differential diagnosis from MF.

Constitutively Photomorphogenic 1 Reduces the Sensitivity of Chronic Lymphocytic Leukemia Cells to Fludarabine Through Promotion of Ubiquitin-Mediated P53 Degradation.

BACKGROUND/AIMS: Chronic Lymphocytic leukemia (CLL) is characterized by accumulation of cells in the G0/G1 phase of the cell cycle and resistance to apoptosis due to gene mutation or abnormal gene expression. In our previous study, constitutively photomorphogenic 1 (COP1) was shown to be upregulated in Binet C-phase CLL patients. Based on the negative regulation of COP1 in the repair of DNA damage, we further studied the function of COP1 in CLL cell apoptosis induced by fludarabine in vitro and in vivo. METHODS: We analyzed the sensitivity of primary CLL cells to the fludarabine by CCK-8, and detected the expression of p53 in cells after drug treatment by western blot. Next, we constructed COP1 overexrpessing CLL cell line HG3, and analyzed the effect of COP1 overexpression on the HG3 cell's apoptosis, and HG3 transplant mice survival with drug treatment. RESULTS: Here, we found that primary CLL cells with high expression of COP1 showed low sensitivity to the drug and presented delayed enrichment of p53 protein than cells with low COP1 expressed. COP1 overexpression reduced HG3 cell sensitivity to the fludarabine treatment and inhibited cell apoptosis, and also retarded itself via autoubiquitination. The further study showed that COP1 promoted ubiquitin-dependent p53 degradation, which further disrupts the formation of the p53-Brn-3a complex and activation of Bcl-2 transcription. Moreover, mice engrafted with cells overexpressing COP1 showed a shortened survival, increased tumor cells burden in spleen and bone marrow (BM), and reduced tumor cell apoptosis even when fludarabine combined cyclophosphamide (F+C) therapy was administered. CONCLUSION: This study demonstrates that COP1 contributes to drug resistance of CLL cells to the fludarabine treatment in vitro and in vivo.

Dual Targeting of Aurora Kinases with AMG 900 Exhibits Potent Preclinical Activity Against Acute Myeloid Leukemia with Distinct Post-Mitotic Outcomes.

Aurora kinase A and B have essential and non-overlapping roles in mitosis, with elevated expression in a subset of human cancers, including acute myeloid leukemia (AML). In this study, pan-aurora kinase inhibitor (AKI) AMG 900 distinguishes itself as an anti-leukemic agent that is more uniformly potent against a panel of AML cell lines than are isoform-selective AKIs and classic AML drugs. AMG 900 inhibited AML cell growth by inducing polyploidization and/or apoptosis. AMG 900 and aurora-B-selective inhibitor AZD1152-hQPA showed comparable cellular effects on AML lines that do not harbor a FLT3-ITD mutation. AMG 900 was active against P-glycoprotein-expressing AML cells resistant to AZD1152-hQPA and was effective at inducing expression of megakaryocyte-lineage markers (CD41, CD42) on human CHRF-288-11 cells and mouse Jak2 V617F cells. In MOLM-13 cells, inhibition of p-histone H3 by AMG 900 was associated with polyploidy, extra centrosomes, accumulation of p53 protein, apoptosis, and cleavage of Bcl-2 protein. Co-administration of cytarabine (Ara-C) with AMG 900 potentiated cell killing in a subset of AML lines, with evidence of attenuated polyploidization. AMG 900 inhibited the proliferation of primary human bone marrow cells in culture, with a better proliferation recovery profile relative to classic antimitotic drug docetaxel. In vivo, AMG 900 significantly reduced tumor burden in a systemic MOLM-13 xenograft model where we demonstrate the utility of 3'-deoxy-3'-18F-fluorothymidine [18F]FLT positron emission tomographic (PET)-CT imaging to measure the antiproliferative effects of AMG 900 in skeletal tissues in mice.

Angiogenic activity in vivo of the particulate matter (PM10).

BACKGROUND: Particulate matter (PM) is the most efficient vehicle for the inhalation and absorption of toxic substances into the body. METHOD: The present study was aimed at testing the hypothesis that PM10 samples collected on quartz filters exert an angiogenic activity in vivo in the chick embryo chorioallantoic membrane (CAM) assay. RESULTS: When the low, medium, and high PM10 concentrations filters were tested in the CAM assay, an increasing number of microvessels was detectable after 4 days of applications of the filters. Moreover, at histological level, numerous microvessels and a dense inflammatory infiltrate were recognizable in the CAM mesenchyme. CONCLUSION: Our data show a clear dose-response relationship between the dose variable (PM10 and Bap) and the outcome variable. So far, the PM10 target value is determined on the basis of regulatory agreements and is not health-based. In addition, the mere gravimetric measure of PM10 cannot be considered a fully reliable surrogate of the overall toxicity of the mixture.

Downregulation of GATA1 drives impaired hematopoiesis in primary myelofibrosis.

Primary myelofibrosis (PMF) is a clonal hematologic malignancy characterized by BM fibrosis, extramedullary hematopoiesis, circulating CD34+ cells, splenomegaly, and a propensity to evolve to acute myeloid leukemia. Moreover, the spleen and BM of patients harbor atypical, clustered megakaryocytes, which contribute to the disease by secreting profibrotic cytokines. Here, we have revealed that megakaryocytes in PMF show impaired maturation that is associated with reduced GATA1 protein. In investigating the cause of GATA1 downregulation, our gene-expression study revealed the presence of the RPS14-deficient gene signature, which is associated with defective ribosomal protein function and linked to the erythroid lineage in 5q deletion myelodysplastic syndrome. Surprisingly, reduced GATA1 expression and impaired differentiation were limited to megakaryocytes, consistent with a proproliferative effect of a GATA1 deficiency on this lineage. Importantly, expression of GATA1 effectively rescued maturation of PMF megakaryocytes. Together, these results suggest that ribosomal deficiency contributes to impaired megakaryopoiesis in myeloproliferative neoplasms.

Non-random spatial relationships between mast cells and microvessels in human endometrial carcinoma.

Mast cells (MCs) accumulate in the stroma surrounding tumors, where they secrete angiogenic cytokines and proteases, and an increased number of MCs have been demonstrated in angiogenesis associated with solid and hematological tumors. The aim of this study is to contribute to the knowledge of distribution of MCs in tumors, investigating the pattern of distribution of tryptase-positive MCs around the blood vessels in human endometrial carcinoma samples by introducing a quantitative approach to characterize their spatial distribution. The results have shown that in human endometrial cancer bioptic specimens the spatial distribution of MCs shows significant deviation from randomness as compared with control group in which, instead, the spatial distribution of MCs is consistent with a random distribution. These findings confirm that MCs enhance tumor angiogenesis and their preferential localization along blood vessels and sites of new vessel formation sustaining the suggestion for an association between MCs and angiogenesis. However, this spatial association between vessels and MCs might simply reflect migrating MCs from the blood stream at vessel growing sites.

Isolation and characterization of neural stem cells from dystrophic mdx mouse.

The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and ß-dystroglycan (αDG, ßDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier control occurring in the adult dystrophic brain.

Interval sentinel lymph nodes in melanoma: a digital pathology analysis of Ki67 expression and microvascular density.

The presence of interval sentinel lymph nodes in melanoma is documented in several studies, but controversies still exist about the management of these lymph nodes. In this study, an immunohistochemical evaluation of tumor cell proliferation and neo-angiogenesis has been performed with the aim of establishing a correlation between these two parameters between positive and negative interval sentinel lymph nodes. This retrospective study reviewed data of 23 patients diagnosed with melanoma. Bioptic specimens of interval sentinel lymph node were retrieved, and immunohistochemical reactions on tissue sections were performed using Ki67 as a marker of proliferation and CD31 as a blood vessel marker for the study of angiogenesis. The entire stained tissue sections for each case were digitized using Aperio Scanscope Cs whole-slide scanning platform and stored as high-resolution images. Image analysis was carried out on three selected fields of equal area using IHC Nuclear and Microvessel analysis algorithms to determine positive Ki67 nuclei and vessel number. Patients were divided into positive and negative interval sentinel lymph node groups, and the positive interval sentinel lymph node group was further divided into interval positive with micrometastasis and interval positive with macrometastasis subgroups. The analysis revealed a significant difference between positive and negative interval sentinel lymph nodes in the percentage of Ki67-positive nuclei and mean vessel number suggestive of an increased cellular proliferation and angiogenesis in positive interval sentinel lymph nodes. Further analysis in the interval positive lymph node group showed a significant difference between micro- and macrometastasis subgroups in the percentage of Ki67-positive nuclei and mean vessel number. Percentage of Ki67-positive nuclei was increased in the macrometastasis subgroup, while mean vessel number was increased in the micrometastasis subgroup. The results of this study suggest that the correlation between tumor cell proliferation and neo-angiogenesis in interval sentinel lymph nodes in melanoma could be used as a good predictive marker to distinguish interval positive sentinel lymph nodes with micrometastasis from interval positive lymph nodes with macrometastasis subgroups.

First evidence of in vivo pro-angiogenic activity of cerebrospinal fluid samples from multiple sclerosis patients.

Increased vascular density and endothelial cell proliferation have been demonstrated in multiple sclerosis (MS) white matter, as well as an elevated vascular endothelial growth factor expression was detected in reactive astrocytes of both active and inactive chronic demyelinated lesions and in sera of MS patients during clinical relapses. In this study, we have investigated the angiogenic activity of cerebrospinal fluid (CSF) samples from MS patients with different stages of disease by means of the chick embryo chorioallantoic membrane (CAM), a well-known assay to study angiogenesis in vivo. Results have shown that CSF samples from MS patients induced a significant (p < 0.05) angiogenic response in CAM in comparison with CSF from neurological controls. The vessel density was higher (p < 0.0001) in secondary (23.60 ± 1.14) and primary (23.50 ± 1.87) progressive patients in comparison with relapsing MS (17.25 ± 1.75) and clinically isolated syndrome suggestive of MS (13.00 ± 1.79), and a significant correlation (r = 0.611, p = 0.005) was found between the angiogenic response and disability level. The results of this preliminary report demonstrate for the first time an angiogenic activity in vivo of CSF samples from MS patients and confirm the importance of angiogenesis as a key event in MS pathogenesis and progression.