Hh Gene Research Articles

ELISL: early-late integrated synthetic lethality prediction in cancer.

Anti-cancer therapies based on synthetic lethality (SL) exploit tumour vulnerabilities for treatment with reduced side effects, by targeting a gene that is jointly essential with another whose function is lost. Computational prediction is key to expedite SL screening, yet existing methods are vulnerable to prevalent selection bias in SL data and reliant on cancer or tissue type-specific omics, which can be scarce. Notably, sequence similarity remains underexplored as a proxy for related gene function and joint essentiality. We propose ELISL, Early-Late Integrated SL prediction with forest ensembles, using context-free protein sequence embeddings and context-specific omics from cell lines and tissue. Across eight cancer types, ELISL showed superior robustness to selection bias and recovery of known SL genes, as well as promising cross-cancer predictions. Co-occurring mutations in a BRCA gene and ELISL-predicted pairs from the HH, FGF, WNT, or NEIL gene families were associated with longer patient survival times, revealing therapeutic potential. Data: 10.6084/m9.figshare.23607558 & Code: github.com/joanagoncalveslab/ELISL.

Evaluation of flaxseed lignan-enriched extract targeting autophagy, apoptosis, and hedgehog pathways against experimentally induced obesity.

This research investigated secoisolariciresinol diglucoside (SDG) flax extract effects on apoptosis, hedgehog (Hh), autophagy, and the anti-oxidation process in experimentally induced obesity. Forty rats were separated into two sets regarding either receiving a normal balanced diet or a high-fat diet (HFD) and then distributed into four groups: GI: The control group had a regular diet for 12 weeks. GII: animals received a high-fat meal and saline by gastric gavage. GIII: HFD obese rats treated with SDG extract orally (10 mg/kg/b.w.) and 1.18 mg SDG/kg in the diet for 4 weeks GIV: Normal balanced diet rats received SDG extract orally (10 mg/kg/b.w.) and 1.18 mg SDG/kg of chow for 12 weeks in addition to their regular balanced diet. The administration of SDG extract exhibited a significant drop in body weight, glucose, lipid profile, and leptin compared to the obese group. It also improved the antioxidant levels (lowering the levels of malondialdehyde while increasing the total antioxidant capacity) and anti-inflammatory status (decreasing interleukin-6 and tumor necrosis factor-alpha). SDG extract downregulates the expression of HH genes (protein patched homolog 1, Hh-interacting protein, glioma-associated oncogene homolog 1, and smoothened receptor) in conjunction with the modulation of autophagy genes and apoptotic proteins. SDG extract showed improved anti-inflammatory and antioxidant status and downregulated the expression of HH genes while modulating autophagy genes and apoptotic proteins among obese rats, suggesting that it may be used to avert and manage obesity and its correlated complications by modulating oxidation, inflammation, autophagy, and apoptosis. Advanced future research on the SDG autophagy pathway to address obesity and its complications is mandatory.

HDAC6 inhibition decreases leukemic stem cell expansion driven by Hedgehog hyperactivation by restoring primary ciliogenesis

Aberrant activation of the Hh pathway promotes cell proliferation and multi-drug resistance (MDR) in several cancers, including Acute Myeloid Leukemia (AML). Notably, only one Hh inhibitor, glasdegib, has been approved for AML treatment, and most patients eventually relapse, highlighting the urgent need to discover new therapeutic targets. Hh signal is transduced through the membrane of the primary cilium, a structure expressed by non-proliferating mammalian cells, whose stabilization depends on the activity of HDAC6. Here we describe a positive correlation between Hh, HDAC6, and MDR genes in a cohort of adult AML patients, human leukemic cell lines, and a zebrafish model of Hh overexpression. The hyper-activation of Hh or HDAC6 in zebrafish drove the increased proliferation of hematopoietic stem and progenitor cells (HSPCs). Interestingly, this phenotype was rescued by inhibition of HDAC6 but not of Hh. Also, in human leukemic cell lines, a reduction in vitality was obtained through HDAC6, but not Hh inhibition. Our data showed the presence of a cross-talk between Hh and HDAC6 mediated by stabilization of the primary cilium, which we detect for the first time in zebrafish HSPCs. Inhibition of HDAC6 activity alone or in combination therapy with the chemotherapeutic agent cytarabine, efficiently rescued the hematopoietic phenotype. Our results open the possibility to introduce HDAC6 as therapeutic target to reduce proliferation of leukemic blasts in AML patients.

Blocking chondrocyte hypertrophy in conditional Evc knockout mice does not modify cartilage damage in osteoarthritis.

Chondrocytes in osteoarthritic (OA) cartilage acquire a hypertrophic-like phenotype, where Hedgehog (Hh) signaling is pivotal. Hh overexpression causes OA-like cartilage lesions, whereas its downregulation prevents articular destruction in mouse models. Mutations in EVC and EVC2genes disrupt Hh signaling, and are responsible for the Ellis-van Creveld syndrome skeletal dysplasia. Since Ellis-van Creveld syndrome protein (Evc) deletion is expected to hamper Hh target gene expression we hypothesized that it would also prevent OA progression avoiding chondrocyte hypertrophy. Our aim was to study Evc as a new therapeutic target in OA, and whether Evc deletion restrains chondrocyte hypertrophy and prevents joint damage in an Evc tamoxifen induced knockout (EvccKO ) model of OA. For this purpose, OA was induced by surgical knee destabilization in wild-type (WT) and EvccKO adult mice, and healthy WT mice were used as controls (n=10knees/group). Hypertrophic markers and Hh genes were measured by qRT-PCR, and metalloproteinases (MMP) levels assessed by western blot. Human OA chondrocytes and cartilage samples were obtained from patients undergoing knee joint replacement surgery. Cyclopamine (CPA) was used for Hh pharmacological inhibition and IL-1 beta as an inflammatory insult. Our results showed that tamoxifen induced inactivation of Evc inhibited Hh overexpression and partially prevented chondrocyte hypertrophy during OA, although it did not ameliorate cartilage damage in DMM-EvccKO mice. Hh pathway inhibition did not modify the expression of proinflammatory mediators induced by IL-1 beta in human OA chondrocytes in culture. We found that hypertrophic-IHH-and inflammatory-COX-2-markers co-localized in OA cartilage samples. We concluded that tamoxifen induced inactivation of Evc partially prevented chondrocyte hypertrophy in DMM-EvccKO mice, but it did not ameliorate cartilage damage. Overall, our results suggest that chondrocyte hypertrophy per se is not a pathogenic event in the progression of OA.

Primary cilium-mediated MSC mechanotransduction is dependent on Gpr161 regulation of hedgehog signalling.

The benefits of physical loading to skeletal mass are well known. The primary cilium has emerged as an important organelle in bone mechanobiology/mechanotransduction, particularly in mesenchymal stem/stromal cells, yet the molecular mechanisms of cilium mechanotransduction are poorly understood. In this study, we demonstrate that Gpr161 is a mechanoresponsive GPCR, that localises to the cilium, and is involved in fluid shear-induced cAMP signalling and downstream osteogenesis. This Gpr161-mediated mechanotransduction is dependent on IFT88/cilium and may act through adenylyl cyclase 6 (AC6) to regulate cAMP and MSC osteogenesis. Moreover, we demonstrate that Hh signalling is positively associated with osteogenesis and that Hh gene expression is mechanically regulated and required for loading-induced osteogenic differentiation through a mechanism that involves IFT88, Gpr161, AC6, and cAMP. Therefore, we have delineated a molecular mechanism of MSC mechanotransduction which likely occurs at the cilium, leading to MSC osteogenesis, highlighting novel mechanotherapeutic targets to enhance osteogenesis.

Large-scale pan-cancer analysis reveals broad prognostic association between TGF-\u03b2 ligands, not Hedgehog, and GLI1/2 expression in tumors

GLI1 expression is broadly accepted as a marker of Hedgehog pathway activation in tumors. Efficacy of Hedgehog inhibitors is essentially limited to tumors bearing activating mutations of the pathway. GLI2, a critical Hedgehog effector, is necessary for GLI1 expression and is a direct transcriptional target of TGF-β/SMAD signaling. We examined the expression correlations of GLI1/2 with TGFB and HH genes in 152 distinct transcriptome datasets totaling over 23,500 patients and representing 37 types of neoplasms. Their prognostic value was measured in over 15,000 clinically annotated tumor samples from 26 tumor types. In most tumor types, GLI1 and GLI2 follow a similar pattern of expression and are equally correlated with HH and TGFB genes. However, GLI1/2 broadly share prognostic value with TGFB genes and a mesenchymal/EMT signature, not with HH genes. Our results provide a likely explanation for the frequent failure of anti-Hedgehog therapies in tumors, as they suggest a key role for TGF-β, not Hedgehog, ligands, in tumors with elevated GLI1/2-expression.

Step-wise evolution of neural patterning by Hedgehog signalling in chordates

In vertebrate embryos, Hedgehog (Hh) is expressed in some anterior basal plate domains and by notochord and floorplate cells, and ventral neural cells are patterned by the activities of Hh-regulated transcription factors. Hh signalling is antagonized by signals from the dorsal neural tube and loss of Hh leads to loss of ventral patterning as dorsal pattern expands. These mechanisms are critical for producing the neurons that implement motor responses to sensory inputs but understanding how they evolved has been hindered by lack of insight from commonly studied invertebrates where nervous system morphology and genetic mechanisms are non-conserved with vertebrates. The invertebrate chordate amphioxus, which expresses Hh in its notochord and floorplate, provides a window into the prevertebrate condition. We examined amphioxus neural development by manipulating Hh and downstream genes involved in neural pattern and cell identity. We show that Hh signalling regulates the differentiation of some neurons in amphioxus, including a subset of motor neurons. This demonstrates some conservation of mechanism between vertebrates and amphioxus. However, other aspects of neural patterning differ between the lineages. We suggest the complexity of Hh-dependent neural patterning in vertebrates evolved in a step-wise manner. Alongside other previously described regulatory changes, initial recruitment of Hh along the length of the axis occurred in an ancestor to the chordates to regulate the differentiation of a subset of neurons. This was followed, in the vertebrate lineage, by additional changes to the downstream gene regulatory network of transcription factors, giving Hh a broader role in dorsal-ventral neural patterning.

The Correlation Between mRNA Hedgehog (Hh) Gene Expression with the Growth of Length of Tissue Regeneration Digit Tip Mice (Mus musculus) Post Amputation

Mice can regenerate tissue in the third phalanges of digit tip after amputation. The tissue regeneration process involved various cells, molecules, proteins, and genes that were thought to have a role in the process. Hedgehog (Hh) gene was thought to have a role in tissue regeneration, but research in adult tissue regeneration was limited. Hh gene had a role in mitosis and cell morphogenesis during the embryo growth. The expression of this gene continued in the adult cell population. We analyzed the Hh gene expression in tissue regeneration of the third phalange digit tip mice post-amputation until day 25. We used 30 males of mice (Mus musculus var Webster) as an animal model. We observed the histological analysis and Hh gene expression in each growth day. Spearman correlation test results in a strong and significant correlation between the growth length of digit tip mice and mRNA expression of the Hh gene (p <0.005; r= 0.874).
 Keywords: Hedgehog, tissue regeneration, digit tip, proliferation,

Functional role of hedgehog pathway in osteoarthritis.

The hedgehog signalling pathway is one of the key regulators of metazoan development, and it plays an important role in the regulation of a variety of developmental and physiological processes. But it is aberrantly activated in many human diseases, including osteoarthritis (OA). In this study, we have reviewed the association of hedgehog signalling pathway in the development and progression of OA and evaluated the efforts to target this pathway for the prevention of OA. Usually in OA, activation of hedgehog induces up-regulation of the expression of hypertrophic markers, including type X collagen, increases production of nitric oxide and prostaglandin E2, several matrix-degrading enzymes including matrix metalloproteinase and a disintegrin and metalloproteinase with thrombospondin motifs in human knee joint cartilage leading to cartilage degeneration, and thus contributes in OA. Targeting hedgehog signalling might be a viable strategy to prevent or treat OA. Chemical inhibitors of hedgehog signalling is promising, but they cause severe side effects. Knockdown of HH gene is not an option for OA treatment in humans because it is not possible to delete HH in larger animals. Efficient knockdown of HH achieved by local delivery of small interfering RNA in future studies utilizing large animal OA models might be a more efficient approach for the prevention of OA. However, it remains a major problem to develop one single scaffold due to the different physiological functions of cartilage and subchondral bones possess. More studies are necessary to identify selective inhibitors for efficiently targeting the hedgehog pathway in clinical conditions.

The search for the Booroola (FecB) mutation

Structural patterning and functional programming of uterine tissues are mechanistically coupled. These processes ensure anteroposterior differentiation of uterine tissues from adjacent segments of the developing female reproductive tract (FRT) and radial patterning that establishes uterine-specific histoarchitecture and functionality. Uterine organogenesis begins prenatally and is completed postnatally. Genes required for FRT development include Pax2, Lim 1 and Emx2, genes in the abdominal-B Hoxa cluster, and members of both Wnt and Hedgehog (Hh) gene families. Disruption of morphoregulatory gene expression patterns can prevent FRT development entirely or compromise uterine organogenesis specifically. Oestrogen receptor-α (ER) -dependent events associated with development of the neonatal porcine uterus can be altered by administration of oestrogen (E) or relaxin (RLX). Expression of the RLX receptor is detectable in porcine endometrium at birth, before onset of ER expression and uterine gland genesis. Uterotrophic effects of both E and RLX can be inhibited with the ER antagonist ICI 182,780, indicating that RLX may act via crosstalk with the ER system in neonatal tissues. Exposure of neonatal gilts to E alters temporospatial patterns of Hh, Wnt and Hoxa expression in the uterine wall. Oestrogen given for two weeks from birth produced hypoplastic adult porcine uteri that were less responsive to periattachment conceptus signals as reflected by reduced growth response and luminal fluid protein accumulation, altered endometrial gene expression, and reduced capacity for conceptus support. Data reinforce the concept that factors affecting signalling events in uterine tissues that produce changes in morphoregulatory gene expression patterns during critical organisational periods can alter the developmental trajectory of the uterus with lasting consequences. Thus, uterine tissues can be programmed epigenetically for success or failure during perinatal life.

From the Editor’s desk…: June 2019

From the Editor’s desk…: June 2019

Tick-tock hedgehog-mutual crosstalk with liver circadian clock promotes liver steatosis

The mammalian circadian clock controls various aspects of liver metabolism and integrates nutritional signals. Recently, we described Hedgehog (Hh) signaling as a novel regulator of liver lipid metabolism. Herein, we investigated crosstalk between hepatic Hh signaling and circadian rhythm. Diurnal rhythms of Hh signaling were investigated in liver and hepatocytes from mice with ablation of Smoothened (SAC-KO) and crossbreeds with PER2::LUC reporter mice. By using genome-wide screening, qPCR, immunostaining, ELISA and RNAi experiments in vitro we identified relevant transcriptional regulatory steps. Shotgun lipidomics and metabolic cages were used for analysis of metabolic alterations and behavior. Hh signaling showed diurnal oscillations in liver and hepatocytes in vitro. Correspondingly, the level of Indian Hh, oscillated in serum. Depletion of the clock gene Bmal1 in hepatocytes resulted in significant alterations in the expression of Hh genes. Conversely, SAC-KO mice showed altered expression of clock genes, confirmed by RNAi against Gli1 and Gli3. Genome-wide screening revealed that SAC-KO hepatocytes showed time-dependent alterations in various genes, particularly those associated with lipid metabolism. The clock/hedgehog module further plays a role in rhythmicity of steatosis, and in the response of the liver to a high-fat diet or to differently timed starvation. For the first time, Hh signaling in hepatocytes was found to be time-of-day dependent and to feed back on the circadian clock. Our findings suggest an integrative role of Hh signaling, mediated mainly by GLI factors, in maintaining homeostasis of hepatic lipid metabolism by balancing the circadian clock. The results of our investigation show for the first time that the Hh signaling in hepatocytes is time-of-day dependent, leading to differences not only in transcript levels but also in the amount of Hh ligands in peripheral blood. Conversely, Hh signaling is able to feed back to the circadian clock.

Comprehensive screening shows that mutations in the known syndromic genes are rare in infants presenting with hyperinsulinaemic hypoglycaemia.

SummaryObjectiveHyperinsulinaemic hypoglycaemia (HH) can occur in isolation or more rarely feature as part of a syndrome. Screening for mutations in the “syndromic” HH genes is guided by phenotype with genetic testing used to confirm the clinical diagnosis. As HH can be the presenting feature of a syndrome, it is possible that mutations will be missed as these genes are not routinely screened in all newly diagnosed individuals. We investigated the frequency of pathogenic variants in syndromic genes in infants with HH who had not been clinically diagnosed with a syndromic disorder at referral for genetic testing.DesignWe used genome sequencing data to assess the prevalence of mutations in syndromic HH genes in an international cohort of patients with HH of unknown genetic cause.PatientsWe undertook genome sequencing in 82 infants with HH without a clinical diagnosis of a known syndrome at referral for genetic testing.MeasurementsWithin this cohort, we searched for the genetic aetiologies causing 20 different syndromes where HH had been reported as a feature.ResultsWe identified a pathogenic KMT2D variant in a patient with HH diagnosed at birth, confirming a genetic diagnosis of Kabuki syndrome. Clinical data received following the identification of the mutation highlighted additional features consistent with the genetic diagnosis. Pathogenic variants were not identified in the remainder of the cohort.ConclusionsPathogenic variants in the syndromic HH genes are rare; thus, routine testing of these genes by molecular genetics laboratories is unlikely to be justified in patients without syndromic phenotypes.

Identification and validation of a Hedgehog pathway-based 3-gene prognostic signature for gastric cancers.

Developing prognostic factors for patients with gastric cancer (GC) is crucial for the accurate identification of subgroups with distinct clinical outcomes and the development of effective treatment strategies. The aim of this study was to determine novel gene expression signatures from the hedgehog (Hh) signaling pathway as predictors of risk with biological significance. The Cancer Genome Atlas (TCGA) GC (STAD) cohort was used as the training dataset to select for significant prognostic Hh genes. Three Hh genes, indian hedgehog (IHH), patched 1 (PTCH1) and smoothened frizzled class receptor (SMO), were identified to be significant prognostic factors. On this basis, a 3-Hh-gene set was constructed and the high-risk patients of the training cohort were distinguished against low-risk cases [hazard ratio (HR)=1.73, 95% confidence interval (CI)=1.26-2.39, P=0.00069]. Then the gene signature was externally validated in a combined dataset from Gene Expression Omnibus (n=631), and experimentally confirmed in an independent cohort of 126 clinical GC samples by immunohistochemistry (IHC). Validation in the combined GEO dataset yielded consistent results (HR=1.45, 95% CI=1.17-1.81, P=0.00068), and remained significant for stages I-III, HER2-positive and surgery alone subgroups. Subsequently, we further demonstrated that this mRNA-based gene set could be successfully transferred to an IHC-based signature in our local cohort (HR=2.04, 95% CI=1.09-3.82, P=0.02). In addition, this signature served as an independent prognostic indicator for overall survival in the multivariate Cox analysis (HR=2.133, 95% CI=1.110-4.099, P=0.02). In conclusion, we successfully generated a stable III-Hh-gene model with the ability to separate patients into prognostic subgroups, which may have notable biological importance and be easily utilized clinically.

Clinical and mutational spectrum of highly differentiated, paired box 3:forkhead box protein o1 fusion-negative rhabdomyosarcoma: A report from the Children's Oncology Group.

Pediatric paired box 3:forkhead box protein O1 fusion-negative (PF-) rhabdomyosarcoma (RMS) represents a diverse spectrum of tumors with marked differences in histology, myogenic differentiation, and clinical behavior. This study sought to evaluate the clinical and mutational spectrum of 24 pediatric PF- human RMS tumors with high levels of myogenic differentiation. Tumors were sequenced with OncoPanel v.2, a panel consisting of the coding regions of 504 genes previously linked to human cancer. Most of the tumors (19 of 24) arose at head/neck or genitourinary sites, and the overall survival rate was 100% with a median follow-up time of 4.6 years (range, 1.4-8.6 years). RAS pathway gene mutations were the most common mutations in PF-, highly differentiated RMS tumors. In addition, Hedgehog (Hh) and mechanistic target of rapamycin (mTOR) gene mutations with evidence for functional relevance (high-impact) were identified in subsets of tumors. The presence of Hh and mTOR pathway gene mutations was mutually exclusive and was associated with high-impact RAS pathway gene mutations in 3 of 4 Hh-mutated tumors and in 1 of 6 mTOR-mutated tumors. Interestingly, Hh and mTOR gene mutations were previously associated with rhabdomyomas, which are also known to preferentially arise at head/neck and genitourinary sites. Findings from this study further support the idea that PF-, highly differentiated RMS tumors and rhabdomyomas may represent a continuous spectrum of tumors. Cancer 2018;124:1973-81. © 2018 American Cancer Society.

The Noncell Autonomous Requirement of Proboscipedia for Growth and Differentiation of the Distal Maxillary Palp during Metamorphosis of Drosophila melanogaster.

The Drosophila maxillary palpus that develops during metamorphosis is composed of two elements: the proximal maxillary socket and distal maxillary palp. The HOX protein, Proboscipedia (PB), was required for development of the proximal maxillary socket and distal maxillary palp. For growth and differentiation of the distal maxillary palp, PB was required in the cells of, or close to, the maxillary socket, as well as the cells of the distal maxillary palp. Therefore, PB is required in cells outside the distal maxillary palp for the expression, by some mechanism, of a growth factor or factors that promote the growth of the distal maxillary palp. Both wingless (wg) and hedgehog (hh) genes were expressed in cells outside the distal maxillary palp in the lancinia and maxillary socket, respectively. Both wg and hh were required for distal maxillary palp growth, and hh was required noncell autonomously for distal maxillary palp growth. However, expression of wg-GAL4 and hh-GAL4 during maxillary palp differentiation did not require PB, ruling out a direct role for PB in the regulation of transcription of these growth factors.

Hedgehog participates in the establishment of left-right asymmetry during amphioxus development by controlling Cerberus expression.

Correct patterning of left-right (LR) asymmetry is essential during the embryonic development of bilaterians. Hedgehog (Hh) signaling is known to play a role in LR asymmetry development of mouse, chicken and sea urchin embryos by regulating Nodal expression. In this study, we report a novel regulatory mechanism for Hh in LR asymmetry development of amphioxus embryos. Our results revealed that Hh-/- embryos abolish Cerberus (Cer) transcription, with bilaterally symmetric expression of Nodal, Lefty and Pitx In consequence, Hh-/- mutants duplicated left-side structures and lost right-side characters, displaying an abnormal bilaterally symmetric body plan. These LR defects in morphology and gene expression could be rescued by Hh mRNA injection. Our results indicate that Hh participates in amphioxus LR patterning by controlling Cer gene expression. Curiously, however, upregulation of Hh signaling failed to alter the Cer expression pattern or LR morphology in amphioxus embryos, indicating that Hh might not provide an asymmetric cue for Cer expression. In addition, Hh is required for mouth opening in amphioxus, hinting at a homologous relationship between amphioxus and vertebrate mouth development.

A Rare Combination of Genetic Mutations in an Elderly Female: A Diagnostic Dilemma!

Introduction: JAK2 is located on chromosome 9p24 and includes 25 exons encoding a protein of about 1132 amino acids. JAK2 is one of the four Janus family non-receptor protein tyrosine kinases. JAK2V617F is by far the most prevalent mutation in BCR-ABL1-negative Myeloproliferative neoplasms (occurs in ∼95% of patients with polycythemia vera, in ∼55% with essential thrombocythemia and in ∼65% with primary myelofibrosis) 1, 2. More than 80% of hemochromatosis patients are homozygous for a C282Y mutation in HFE gene, and a smaller proportion are compound heterozygous for both the C282Y mutation and an H63D mutation3. Here we present the first case of an elderly female with concomitant diagnosis of Polycythemia Vera (PV) and hemochromatosis. To our knowledge, there is no literature about the co-existence or associations of these diseases. Case Reports: 75 year old female, former smoker with PMH of hemochromatosis and COPD with recent exacerbation, presented to the oncology clinic after hospital discharge for continuing care of her hemochromatosis requiring phlebotomy. She reports to have had multiple phlebotomies in the past fifteen years. Patient denied any history of liver disease, diabetes, arthralgia, skin pigmentation or sleep problems. Vital signs and examination were within normal limits. Her initial work up reported significantly elevated hemoglobin of 17.4gm/dl, hematocrit of 56.1%, RBC count of 6.98M/UL with MCV 80.4 fl, MCH 24.9 pg and platelet count of 673 K/UL. Peripheral smear showed normal red cell morphology and few giant platelets. Subsequently, further lab testing revealed ferritin of 25.7ng/ml. Her elevated hematocrit was further evaluated and erythropoietin was surprisingly <1mIU/ml. Genetic testing for HFE gene mutation screen was positive for homozygous C282Y mutation. Due to high suspicion for Polycythemia Vera, JAK2 mutation was also tested, which to our surprise, came back positive for JAK2 V617F point mutation. Patient is diagnosed with Polycythemia Vera and Hereditary Hemochromatosis and is recommended to start Aspirin, continue phlebotomy to maintain Hematocrit below 45% and take hydroxyurea for thrombocytosis. Discussion: It is interesting to note the co-existence of two un-related diseases. Franchini M et al analyzed 52 patients with PV for 12 HH gene mutations and found no significant association between the two conditions4. Hannuksela J et al studied C282Y and H63D mutations in 232 patients with hematological malignancies and reported no significant association5. Beaton and Adams in their review article about the myths and realities of hemochromatosis reports an elevated hemoglobin, in hemochromatosis's patient as a myth, based on their review of 634 C282Y homozygous patients at London health Science center, with mean hemoglobin of 145±13 g/L6. Our case re-iterates the importance of clinical suspicion of polycythemia Vera in a hemochromatosis patient with elevated hematocrit and undetectable erythropoietin. The coincidence is, phlebotomy is the treatment for both conditions as long as patient is fairly asymptomatic.

Abstract 3189: The alternate activation of hedgehog pathway, either in CD138+ or in CD138-CD19+ multiple myeloma primary cells, impacts on disease outcome

Abstract The iperactivation of Hedgehog (Hh) pathway, which controls the refuel of the tumor clone, might be critical to Multiple Myeloma (MM) recurrence. In order to dissect the role played by Hh pathway in different MM cells compartments, and to evaluate its clinical impact on patient outcomes, here we explore the transcriptomic and genomic profiles in both CD138+ plasma cells and CD138-19+ B progenitors cells obtained from newly diagnosed MM patients (pts), The study included a cohort of 126 pts, homogenously treated with bortezomib-based regimens and ASCT. DNA and RNA were obtained from both CD138+ plasma cell fraction and CD19+ B cells. Gene expression profiling (GEP) (HG U133 Plus 2.0) and genomic analysis (SNP 6.0) were performed on Affymetrix platform. Data were analysed by employing several software: dChip (GEP clustering), Ingenuity Pathway Analysis (GEP Enrichment) and Nexus (Copy number). By unsupervised hierarchical clustering, an Hh signature of 10 genes - SHH, IHH, DHH, SMO, PTCH1, PTCH2, SUFU, GLI1, GLI2 and GLI3 - was identified, and it was able to significantly cluster pts in two subgroups: cluster 1 (70 pts) and cluster 2 (56 pts. An overall significant activation of Hh pathway was shown in cluster 2, as compared to cluster 1. Of note, the Hh pathway was down regulated in CD19+ B cells obtained from pts included in cluster 2, while it was overexpressed in cluster 1 pts. Western blots on both cell fractions confirmed this opposite Hh genes behavior. A higher genomic instability (e.g. higher frequencies of both t(4;14) and del(17p)) was demonstrated in CD138+ cells from cluster 2 pts and, at least 5 known tumor suppressor genes, such as RB1, BRCA2, PDX1, FOXO1 and TP53 were affected. Conversely, cluster 1 pts were mainly characterized by hyperdiploid karyotypes. The more aggressive phenotype of cluster 2 pts was confirmed by an overall deregulation of cell adhesion processes (CD44, LIMS1, COL4A2, CTGF, COL1A1, FN1), increased proliferation (MYCBP, IL22, SDPR, SOX2, SOX6) and DNA repair mechanisms (SP1, SMARCD3, FOXA3). Hh pathway activation significantly influenced pts’ outcome, since those included in cluster 2 had a shorter PFS and OS compared to cluster 1. In fact, the 5-year PFS estimates were 31% vs 56% (p = 0.0062), whereas the OS probabilities were 66% and 83%, respectively (p = 0.0071). Of note, both hazard ratios for PFS and OS were doubled in pts included in cluster 2, as compared to pts included in cluster 1. Finally, multivariate analyses confirmed that being part of cluster 2 was an independent prognostic factor for both PFS and OS, along with del(17p) and ISS 3. Two alternate Hh-driven subtypes of MM might be identified at diagnosis, which correlated with pts outcomes. Stratification of pts according to their molecular background might help the fine-tuning of future clinical studies. Acknowledgements: FP7 NGS-PTL project, Progetto Regione-Università 2010/2012 L. Bolondi. Citation Format: Marina Martello, Daniel Remondini, Enrica Borsi, Mauro Procacci, Barbara Santacroce, Angela Flores Dico, Annalisa Pezzi, Elena Zamagni, Paola Tacchetti, Lucia Pantani, Giulia Marzocchi, Serena Rocchi, Katia Mancuso, Beatrice Anna Zannetti, Giovanni Martinelli, Michele Cavo, Carolina Terragna. The alternate activation of hedgehog pathway, either in CD138+ or in CD138-CD19+ multiple myeloma primary cells, impacts on disease outcome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3189.

Identification of novel mutations in hemochromatosis genes by targeted next generation sequencing in Italian patients with unexplained iron overload

Hereditary hemochromatosis, one of the commonest genetic disorder in Caucasians, is mainly associated to homozygosity for the C282Y mutation in the HFE gene, which is highly prevalent (allele frequency up to near 10% in Northern Europe) and easily detectable through a widely available "first level" molecular test. However, in certain geographical regions like the Mediterranean area, up to 30% of patients with a HH phenotype has a negative or non-diagnostic (i.e. simple heterozygosity) test, because of a known heterogeneity involving at least four other genes (HAMP, HJV, TFR2, and SLC40A1). Mutations in such genes are generally rare/private, making the diagnosis of atypical HH essentially a matter of exclusion in clinical practice (from here the term of "non-HFE" HH), unless cumbersome traditional sequencing is applied. We developed a Next Generation Sequencing (NGS)-based test targeting the five HH genes, and applied it to patients with clinically relevant iron overload (IO) and a non-diagnostic first level genetic test. We identified several mutations, some of which were novel (i.e. HFE W163X, HAMP R59X, and TFR2 D555N) and allowed molecular reclassification of "non-HFE" HH clinical diagnosis, particularly in some highly selected IO patients without concurring acquired risk factors. This NGS-based "second level" genetic test may represent a useful tool for molecular diagnosis of HH in patients in whom HH phenotype remains unexplained after the search of common HFE mutations.