Distinct Molecular Abnormalities Research Articles

Understanding the Continuum between High-Risk Myelodysplastic Syndrome and Acute Myeloid Leukemia

Myelodysplastic syndrome (MDS) is a clonal hematopoietic neoplasm characterized by bone marrow dysplasia, failure of hematopoiesis and variable risk of progression to acute myeloid leukemia (AML). Recent large-scale studies have demonstrated that distinct molecular abnormalities detected at earlier stages of MDS alter disease biology and predict progression to AML. Consistently, various studies analyzing these diseases at the single-cell level have identified specific patterns of progression strongly associated with genomic alterations. These pre-clinical results have solidified the conclusion that high-risk MDS and AML arising from MDS or AML with MDS-related changes (AML-MRC) represent a continuum of the same disease. AML-MRC is distinguished from de novo AML by the presence of certain chromosomal abnormalities, such as deletion of 5q, 7/7q, 20q and complex karyotype and somatic mutations, which are also present in MDS and carry crucial prognostic implications. Recent changes in the classification and prognostication of MDS and AML by the International Consensus Classification (ICC) and the World Health Organization (WHO) reflect these advances. Finally, a better understanding of the biology of high-risk MDS and the mechanisms of disease progression have led to the introduction of novel therapeutic approaches, such as the addition of venetoclax to hypomethylating agents and, more recently, triplet therapies and agents targeting specific mutations, including FLT3 and IDH1/2. In this review, we analyze the pre-clinical data supporting that high-risk MDS and AML-MRC share the same genetic abnormalities and represent a continuum, describe the recent changes in the classification of these neoplasms and summarize the advances in the management of patients with these neoplasms.

Endometrial carcinoma: molecular subtypes, precursors and the role of pathology in early diagnosis.

Endometrial carcinoma (EC) is classified into a wide range of morphological variants; this list has expanded over the past decade with the inclusion of mesonephric-like and dedifferentiated carcinoma as EC variants in the fifth edition of the WHO Classification of Female Genital Tumours, and recognition that carcinosarcoma is a biphasic carcinoma rather than a sarcoma. Each EC variant has distinct molecular abnormalities, including TCGA-based molecular subtypes, allowing further subclassification and adding complexity. In contrast to this rapid progress in understanding EC, there are only two recognized EC precursor lesions: endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (EAH/EIN) and serous intraepithelial carcinoma, a situation that has not changed for many years. Diagnosis of EC precursors is a cornerstone of surgical pathology practice, with early diagnosis contributing to the relatively favorable prognosis of EC. In this review we relate the precursor lesions to each of the EC morphological variants and molecular subtypes, discuss how successful early diagnosis is for each variant/molecular subtype and how it might be improved, and identify knowledge gaps where there is insufficient understanding of EC histogenesis. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 2510: High-depth whole genome sequencing of clinically-annotated high-grade serous ovarian cancers

Abstract Background: Prior molecular characterization efforts for high-grade serous ovarian cancer (HGSOC) were restricted to those who had upfront surgical debulking with variable treatment paradigms. Thus, we sought to examine molecular and cellular differences between clinically defined groups (tumor tissues from patients who had complete gross resection (CGR) versus those who were triaged to neoadjuvant chemotherapy (NACT) and experienced either excellent or poor response. Methods: Tumor biopsies were collected from three patient groups managed under a systematic surgical algorithm: CGR after primary surgery (R0, n=10); poor tumor response to NACT (NACT-PR, n=10); excellent tumor response to NACT (NACT-ER, n=10). Primary and multiple metastatic tumor sites per each patient were obtained pre-treatment and subjected to comprehensive omics analyses including high-pass whole-genome (WGS) and targeted deep DNA sequencing. Results: An average of 71 nonsynonymous somatic mutations from each sample for 75 samples with high-purity tumors (≥75%) by WGS were identified from each sample for the entire cohort. Fourteen ovarian-cancer-associated genes were found mutated in our patient cohort and, as expected, the most frequently mutated gene was TP53 in both primary and metastatic sites in all three groups. TP53 nonsense mutations were exclusively identified in the NACT-ER (36.0%) and NACT-PR groups (15.4%), while in the R0 group most TP53 mutations were missense mutations (62.5%). Nonsense mutations in CSMD3 and PIK3CA were exclusively identified in both primary and metastatic sites in the NACT-PR group. The most frequent copy number variations (CNVs) in the R0 were copy number gain/loss of CSMD3 (67%) and copy number loss of NF1 (54%) and CDK12 (50%) in both primary and metastatic sites. Interestingly, copy number losses of NF1 were significantly lower in the NACT groups (18%, p=0.002), especially in the NACT-PR (8%, p=0.0004), when compared to the R0 group (54%). We also identified significant less observation of chromothripsis-like patterns, and a significantly higher level of strong-binding neoantigens in the R0 than in the NACT groups. Conclusions: Our findings using HGSOC samples obtained from patients treated on a prospective algorithm identified distinct molecular abnormalities, and could have prognostic and therapeutic implications for patients with HGSOC. Citation Format: Sanghoon Lee, Li Zhao, Shannon N. Westin, Nicholas W. Bateman, Amir A. Jazaeri, Nicole D. Fleming, Karen H. Lu, Robert L. Coleman, Gordon B. Mills, Jianhua Zhang, Thomas P. Conrads, George L. Maxwell, P. Andrew Futreal, Anil K. Sood. High-depth whole genome sequencing of clinically-annotated high-grade serous ovarian cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2510.

On the Shoulders of Giants: The Evolution of Renal Cell Carcinoma Treatment-Cytokines, Targeted Therapy, and Immunotherapy.

The treatment of advanced renal cell carcinoma (RCC) has evolved dramatically over the past 30 years, as has a better understanding of the biology of the disease, knowledge of multiple subtypes with distinct molecular abnormalities, and improved comprehension of the perturbed pathways that lead to the development and growth of RCC. This is no longer a monolithic disease, although the majority of tumors are of the clear cell subtype. However, progress is being made in other subtypes as well, as molecular profiles are better understood and as new agents show activity. Immunotherapies remain a major category of treatment, from cytokines to checkpoint inhibitors to ex vivo activated cellular therapy. Antiangiogenesis tyrosine kinase inhibitors are also an important part of the armamentarium. Because these approaches have evolved, we are now in the era of combination therapy using agents of differing mechanisms to try to achieve synergy to increase response rates and create durable responses leading to prolonged survival. Renal cell carcinoma as a tumor is unique in that there has always been a subset of patients who achieve complete responses that last for many years without subsequent treatment. Thus, the goal of further development is to enlarge this subset using new therapeutic approaches and to achieve further durable responses and treatment-free survival.

Molecular Analysis of Clinically Defined Subsets of High-Grade Serous Ovarian Cancer

SUMMARYThe diversity and heterogeneity within high-grade serous ovarian cancer (HGSC), which is the most lethal gynecologic malignancy, is not well understood. Here, we perform comprehensive multi-platform omics analyses, including integrated analysis, and immune monitoring on primary and metastatic sites from highly clinically annotated HGSC samples based on a laparoscopic triage algorithm from patients who underwent complete gross resection (R0) or received neoadjuvant chemotherapy (NACT) with excellent or poor response. We identify significant distinct molecular abnormalities and cellular changes and immune cell repertoire alterations between the groups, including a higher rate of NF1 copy number loss, and reduced chromothripsis-like patterns, higher levels of strong-binding neoantigens, and a higher number of infiltrated T cells in the R0 versus the NACT groups.

Altered miR-21, miRNA-148a Expression in Relation to KRAS Mutation Status as Indicator of Adenoma-Carcinoma Transitional Pattern in Colorectal Adenoma and Carcinoma Lesions.

Sporadic colorectal cancer (CRC) is a fatal disease, mostly known as the silent killer, due to the fact that this disease is asymptomatic before diagnosis in advanced stage. Screening and the early detection of CRC and colorectal adenoma (CRA) by non-aggressive molecular biomarkers' signature is useful for improvement of survival rate in CRC patients. To achieve such a goal, a better understanding of distinct molecular abnormalities as candidate biomarkers in CRC development is crucial. In this study, seventy-five archived FFPE CRC samples, including colorectal adenocarcinoma, adenomatous polyps (adenoma), and adjacent non-neoplastic mucosa were collected for the investigation by Sanger sequencing at the DNA level and by real-time PCR at the RNA level. The results of the KRAS mutational analysis have shown that the majority of somatic mutations in the KRAS affect only one codon, mainly codon 12(p.G12D) with low frequency in adenomas (13.3%) versus CRCs (36%). The results of dysregulated epigenetic changes of miR-21 clearly showed upregulation of expression in colorectal adenocarcinoma, compared to non-neoplastic mucosa, in colorectal adenoma vs non-neoplastic mucosa: (p < 0.001) and in CRC versus adenoma (p < 0.001); while miR-148a expression were significantly downregulated in CRC, compared to non-neoplastic mucosa, in colorectal adenoma vs non-neoplastic mucosa, and in adenoma vs CRC (p < 0.001). Our findings support the important role of miR-21 in stages I-II of CRC, and the KRAS G12D mutant, and differential miR-148a expression, in advanced stages of CRC.

Early Detection and Molecular Characterization of Therapy-Related Leukemia in Children Reveals Patterns of Disease Transformation and Guides Future Surveillance Protocols

BackgroundTherapy-related myelodysplastic syndrome (MDS) and acute leukemias represent a major cause of non-relapse morbidity and mortality in childhood cancer survivors, and have been associated with exposure to cytotoxic therapies (e.g. radiation, alkylators, topoisomerase inhibitors). Neuroblastoma (NB) patients receive multimodality therapy with intensive chemotherapy, radiation, and immunotherapy, and have had high rates of treatment-related leukemias (Kushner, Cheung et al. 1998). Whilst specific therapeutic modalities have been associated with distinct cytogenetic and molecular abnormalities, our understanding of the relationships between timing of mutation acquisition, dynamics of clonal selection in relation to specific therapeutic modalities, and how these in unison result in overt leukemia, remains limited. Motivated to study these relationships and inform future screening guidelines, we characterized serial bone marrow (BM) samples obtained during surveillance for NB recurrence and therapy-related leukemias.MethodsWe studied a total of 219 serial samples from 55 NB patients treated at MSKCC over a 21-year period. These included 19 patients with MDS or leukemic transformation (median time following NB diagnosis 4.4 years), 15 with transient cytogenetic abnormalities (median time to abnormality 3.1 years), and 21 matched controls (median disease-free follow-up 8.1 years). On average, we analyzed 4 samples per transformation patient, representative of pre-treatment timepoints at NB diagnosis, during NB treatment, and throughout follow-up, with a lead time of 18 years - 1 month prior to transformation, and at time of leukemic transformation. Comprehensive genomic profiling with targeted gene sequencing (MSK-IMPACT Heme), RNA-seq, and Archer FusionPlex was performed to capture acquired gene mutations, chromosome-level copy number alterations (CNA), and fusion genes at the time of diagnosis. Backtracking studies were performed in longitudinal samples with complete molecular and clonal characterization.ResultsWe detected at least one disease-defining alteration in all cases with MDS or leukemic transformation at time of diagnosis, with a total of 61 putative oncogenic events across all patients (median 3 alterations per patient, range 1-12). As expected, the most frequent events were MLL fusions (n=6 patients), and mutations in TP53 (n=5 patients). The remaining cases harbored chromosomal aneuploidies or acquired gene mutations in NPM1, IDH1, PTPN11, NRAS, BCOR, CUX1, STAG2, WT1, amongst other genes, at a median variant allele frequency 24% (range 4-68%). Backtracking studies identified at least one of these mutations in 81% of patients at a sampling time point prior to diagnosis. In contrast, only two patients (2/15) from the cohort with transient cytogenetic abnormalities had acquired somatic mutations detected at a median VAF of 3%, with resolution of the molecular alterations in subsequent samples. One of the control cases also had an identified mutation, though this patient died of NB with limited hematologic follow-up.The median time of detection of a putative driver alteration was 6 months prior to leukemic transformation, with the earliest identified at 2.8 years prior to disease transformation. At least 3 cases of MLL fusions were detected 2.2, 14.5, and 20.9 months prior to diagnosis. Mutations in TP53 co-occurred with CNAs in all patients in our cohort, and has been shown to be predictive of chemoresistant disease. Mutations in TP53 were also identified in at least 2 pre-leukemic samples per patient in 4 of 5 cases, at a median VAF 5% (range 5-20%). In all of these cases, TP53 mutations preceded clinically detectable CNA. Genetic evolution led clonal dominance, which, intriguingly, often preceded disease presentation in the context of normal hematopoeisis. We also found evidence of clonal drifts, possibly as a consequence of treatment effects.ConclusionsOur preliminary data demonstrate that NB patients at risk of developing secondary leukemia can be identified by molecular profiling of BM aspirates obtained during routine disease surveillance for NB. These findings present an opportunity for the development of early detection studies for patients with pediatric malignancies undergoing intensive therapy and importantly inform studies into mechanisms of leukemic transformation and specific gene-treatment effects. DisclosuresCheung:Ymabs: Patents & Royalties.

Newborn Screening for Primary Immunodeficiency Diseases: The Past, the Present and the Future

Primary immunodeficiency diseases (PID) are a heterogeneous group of disorders caused by inborn errors of immunity, with affected children presenting with severe, recurrent or unusual infections. Over 300 distinct genetic molecular abnormalities resulting in PID have been identified, and this number continues to rise. Newborn screening for PID has been established in many countries, with the majority of centers using a PCR-based T cell receptor excision circle (TREC) assay to screen for severe combined immunodeficiency (SCID) and other forms of T cell lymphopenia. Multiplexed screening including quantitation of kappa-recombining exclusion circles (KREC) has also been described, offering advantages over TREC screening alone. Screening technologies are also expanding to include protein-based assays to identify complement deficiencies and granulocyte disorders. Given the rapid advances in genomic medicine, a potential future direction is the application of next-generation sequencing (NGS) technologies to screen infants for a panel of genetic mutations, which would enable identification of a wide range of diseases. However, several ethical and economic issues must be considered before moving towards this screening strategy.

What is the role of TDP-43 in C9orf72-related amyotrophic lateral sclerosis and frontemporal dementia?

This scientific commentary refers to ‘Timing and significance of pathological features in C9orf72 expansion-associated frontotemporal dementia’, by Vatsavayai et al. (doi:10.1093/brain/aww250) . The C9orf72 hexanucleotide repeat expansion mutation is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), accounting for up to 12% of all patients in an average ALS clinic population. At post-mortem, patients with the C9orf72 hexanucleotide repeat expansion have the typical pathology seen in sporadic ALS and FTD, in which transactive response DNA-binding protein of 43 kDa (TDP-43; encoded by TARDBP ) is translocated from its normal nuclear location to form cytoplasmic aggregates. However, they also show additional pathological features in the form of RNA foci, containing the transcribed hexanucleotide repeats, and aggregates, which stain with antibodies against dipeptide repeat proteins, the product of non-ATG translation. The order in which these distinct molecular abnormalities develop, and whether each of these pathological features is a necessary or sufficient condition for the development of clinical disease, are currently key questions in ALS/FTD research. In this issue of Brain , Vatsavayai et al. describe two strikingly different pathological cases of C9orf72 -positive FTD with excellent ante-mortem characterization that provide novel and provocative contributions to this debate (Vatsavayai et al. , 2016). Transcription of the C9orf72 hexanucleotide repeat expansion can be detected as sense and antisense RNA foci in the nucleus and cytoplasm. Despite being located in the non-coding region of the C9orf72 gene, the repeat sequence is translated through a non-canonical process called ‘repeat associated non-ATG’ (RAN) translation, into five different species of dipeptide …

Expression analysis of BECN1 in acute myeloid leukemia: association with distinct cytogenetic and molecular abnormalities.

In acute myeloid leukemia (AML), it has been shown that AML-derived cells often remain sensitive to autophagy-inducing stimuli, leading to the idea that harnessing the autophagy can be pertinent to AML cytotoxic therapy. Despite this promising notion, to date, there is no comprehensive study addressing autophagy-related genes expression status in AML. As a critical mediator, BECN1 influences the onset and advance of autophagy and several studies have pointed to the BECN1 recurrent allelic deletion and expression variation in a broad range of tumors. To explore this caveat, we chose this alteration-prone gene to investigate in our study. We have analyzed the expression status of BECN1 in a series of 128 de novo AML patients using real-time quantitative polymerase chain reaction (qRT-PCR). In our favorable subgroup, BECN1 expression did not alter (P = 0.301), but in intermediate and unfavorable patients, we have had BECN1 low expression compared to the normal controls (P = 0.008 and P < 0.001, respectively). We found evidence for the association of reduced expression of BECN1 with FLT3-ITD mutation (19 of 27 patients), monosomal karyotype (all of 11 patients), higher age, and WBC count. Overall, remarkable association of reduced expression of BECN1 with FLT3-ITD mutation and monosomal karyotype and their functional relationship is interesting which should be addressed and verified in future studies.

Molecular Characteristics of Large Parathyroid Adenomas

The clinical entity of large parathyroid adenomas (LPTAs) has not been well defined. It is speculated that LPTAs would have biochemical, histological, and molecular characteristics different from small adenomas. Our study aimed to find out occurrence of atypia and carcinomas in large parathyroid lesions and the presence of distinct molecular abnormalities in LPTAs. We divided the parathyroid lesions into large (>7 g, i.e., LPTAs) and small (<7 g) adenomas. We performed parafibromin, APC (adenomatous polyposis coli), galectin 3, and PGP9.5 (protein gene product 9.5) analysis by immunohistochemistry in adenomas without atypia, atypical adenomas, and carcinomas. Mean serum calcium, alkaline phosphatase, and intact PTH were significantly higher in large parathyroid tumor group. The presence of both atypical adenoma and carcinoma was higher in large parathyroid tumor group. There was higher percentage of atypia in patients with LPTAs >10 g (33%), and 68% of tumors showed at least one marker suggestive of malignancy in this group. Detailed analysis of immunohistochemical features of LPTA >10 g revealed that six patients showed complete loss of parafibromin immunoreactivity (out of these four showed atypia), while seven showed partial loss. In histopathologically proven malignancy (n = 9), six patients showed complete loss of parafibromin staining, 5 (55%) APC negativity, and 45% showed both galectin 3 and PGP9.5 positivity. Three out of these showed all IHC markers s/o malignancy, and all of them had evidence of metastases or recurrence. 32% of atypical adenoma and 13% of atypical adenoma showed complete loss of parafibromin staining, however none developed metastases or recurrence in follow-up (median follow-up 40 months). Loss of parafibromin staining (complete or partial) was higher in LPTA group (56%) than that in small adenoma (39%); however, it was not statistically significant. APC, galectin 3, and PGP9.5 markers suggestive were higher in LPTA group but were not significant. LPTAs may show some morphological and immunohistochemical features suggestive of malignancy and can be considered a separate entity. However, the immunohistochemical markers are unable to clearly segregate those LPTAs that may show premalignant potential. Further, we would like to recommend that LPTAs showing complete parafibromin loss together with atypia should be kept under close follow-up.

Epidermolysis bullosa and Kindler syndrome - where is the border in oral manifestaions

Epidermolysis bullosa (EB) represents a rare chronic autoimmune subepidermal vesiculobullous disorder that affects the skin and mucosa and is characterized by blistering and mechanical fragility of the skin. The most recent classification recognizes four major EB groups: intraepidermal EB (Simplex), junctional EB, dermolytic EB (Dystrophic), and mixed EB (Kindler syndrome). Kindler syndrome is a rare genodermatosis with clinical features of trauma-induced blistering, poikiloderma, skin atrophy, mucosal inflammation and varying degrees of photosensitivity. Although Kindler syndrome is classified as a subtype of epidermolysis bullosa, it has distinct clinicopathological and molecular abnormalities. The clinical features of the syndrome have been annotated by different authors but definite criteria to confirm the diagnosis have not yet been generally accepted.

Distinct Molecular Abnormalities Underlie Unique Clinical Features of Essential Thrombocythemia in Children

▪PurposeEssential thrombocythemia (ET) is rare in children. The clinical course and pathogenesis of childhood ET is far less clear. We aimed to analyze the clinical and molecular characteristics of children diagnosed with ET.Patients and MethodsSixty-three children diagnosed with ET (age < 15 years) were enrolled in the study. JAK2 V617F mutation was detected using quantitative real-time polymerase chain reaction. MPL and CALR mutations were assessed using Sanger sequencing. Fifty-five genes associated with myeloid malignancies were analyzed using targeted next-generation sequencing in 25 children.ResultsThe median follow-up time for 63 patients was 48 months (range, 12 to 302 months). Three children (4.8%) had thrombosis, and 2 (3.2%) progressed to myelofibrosis. One child developed both abdominal vein thrombosis and myelofibrotic transformation. JAK2 V617F mutation was found in 14 children (14/63, 22.2%). CALR mutations (1/49, 2.0%) and MPL mutations (0/49) were rarely detected. By targeted sequencing, somatic mutations were found in 13 genes and in 56.0% (14/25) of the patients (Figure 1A and 1B). After JAK2 V617F, the most frequently observed mutations were found in ASXL1 (4/25, 16.0%). Besides, mutations in U2AF1, FLT3, NRAS, MLL, GNAS, RUNX1, WT1, and ZRSR2 were first reported in childhood ET. These newly identified mutations, which are rarely seen in adult ET, are linked to more malignant diseases such as acute leukemia and myelodysplastic syndrome. The key pathway involved was the JAK2 signaling pathway, followed by epigenetic regulators. In 14 children with somatic mutations, 42.9% (6/14) had two or more mutations (Figure 1C). Co-occurrence of mutations in the JAK2 signaling pathway and epigenetic regulators was found in 4 children (4/14, 28.6%). Clonality assessment revealed that some children only had founding clones, but some had both founding clone and subclones (Figure 2). Analysis of allele frequencies showed that the JAK2 V617F mutation and CALR mutations were not the initial abnormalities in some children.ConclusionChildhood ET has a distinct molecular profile from that of adult patients. CALR and MPL are rarely mutated in childhood ET; instead, some previously undocumented mutations are detected. Genetic composition in childhood ET may be more complex than that in adults. The JAK2 V617F mutation and CALR mutations were not the initial abnormalities in some children. Whole exome sequencing would help to reveal the initial molecular events and to offer molecular targets for therapeutic intervention.KeywordsChildhood; Essential thrombocythemia; Molecular markers [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Renal cell carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Renal cell carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

High-risk acute myelogenous leukemia: treatment today … and tomorrow

High-risk acute myelogenous leukemia (AML) constitutes a distinct subset of disease based on clinical and biological characteristics and comprises a significant percentage of all cases of adult AML. Biologic features such as distinct clonal cytogenetic and molecular abnormalities identify a subgroup of AML patients characterized by poor response to induction chemotherapy and poor long-term survival after treatment with consolidation chemotherapy. Clinical variables that predict for poor response include AML relapsed after less than 1 year of remission and AML characterized by resistance to conventional agents. We review here our understanding of the defining biologic subtypes of AML and discuss how adequate initial evaluation can be used to inform the choice of treatment. By defining high-risk biologic and clinical variables, a strong case can be made for treating patients with investigational agents, with treatment directed at distinct cytogenetic or molecular abnormalities. Allogeneic transplantation is the only form of therapy available outside of the setting of a clinical trial that may offer a chance for long-term survival for patients with high-risk AML.

Role of Arsenic Trioxide in Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia that is characterized by distinct clinical, morphological, cytogenetic, and molecular abnormalities. It is associated with a striking risk of early hemorrhagic death due to disseminated intravascular coagulation and hyperfibrinolysis. The prognosis of APL has improved dramatically following the introduction of all-trans retinoic acid (ATRA) and its combination with anthracycline-based chemotherapy during induction and consolidation. Patients with high-risk APL, defined by a white cell count >10 × 10(9)/L at diagnosis, also appear to benefit from the addition of intermediate- or high-dose cytarabine during consolidation. Arsenic trioxide (ATO) has proved to be even more effective than ATRA as a single agent, and is now routinely used for the treatment of the 20%-30% of patients who manifest disease relapse after initial treatment with ATRA and chemotherapy. ATO has a toxicity profile that differs considerably from that of both ATRA and cytotoxic chemotherapy, and accordingly presents its own specific challenges during treatment. Optimizing a strategy for the incorporation of ATO into initial therapy is currently the focus of several cooperative group trials, with an emphasis on minimizing or even eradicating the use of chemotherapy. ATRA plus ATO without chemotherapy appears to be adequate during induction and consolidation for patients with standard-risk APL, but triple therapy that includes limited anthracycline or gemtuzumab ozogamicin (GO) during induction is required for high-risk APL. Uncertainty still exists regarding the minimum amount of chemotherapy and number of consolidation cycles necessary, the optimal scheduling of ATO, and the potential utility of oral ATO administration. Although prolonged oral maintenance therapy is usually included in most current APL treatment protocols, its value remains controversial, and the superior anti-leukemic efficacy of ATO-based therapy may facilitate its elimination in the future.

Pharmacological treatment of gastrointestinal stromal tumors: update from recent clinical trials

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the GI tract and until recently were uniformly found to have poor prognoses with no successful treatment options aside from surgical resection. Cytotoxic chemotherapeutics evaluated in this disease revealed minimal clinical responses. Given the clearer understanding of the distinct molecular abnormalities and biology of the tumor, treatment strategies to overcome this lack of clinical response have been achieved. As the field of oncology has shifted from the development of cytotoxic chemotherapeutics to the emergence of targeted agents, treatment options are available and are becoming effective at delaying progression of disease and decreasing mortality. GISTs can now be classified as a treatable malignancy. This review seeks to provide a comprehensive overview of GISTs, with a focus on recent clinical trials of pharmacological agents in their treatment.

CEBPα Is a Transcriptional Repressor of T-Cell Related Genes Explaining the Myeloid/T-Lymphoid Features of CEBPα-Silenced AML

Abstract 554Acute myeloid leukemia (AML) is a heterogeneous disease of different subtypes characterized by distinct cytogenetic or molecular abnormalities. We recently identified a previously unrecognized subtype with a unique epigenetic feature, i.e. silencing of the gene that encodes CCAAT-enhancer binding protein alpha (CEBPα) by DNA hypermethylation (denoted as the CEBPαsilenced group). The leukemic blast cells of these patients express myeloid as well as T-lymphoid markers. Moreover, gene expression and DNA methylation profiling put these leukemias in between AML and T-lymphoid leukemia (T-ALL). CEBPα is a transcription regulator that is essential for normal neutrophil development. We hypothesize that methylation and consequently silencing of the gene encoding CEBPα is abnormal and an important hit in the transformation of this unique leukemia subtype. The mechanism by which CEBPα silencing plays a role in transformation of cells with myeloid/T-lymphoid features is the aim of our study. We carried out gene expression profiling of the CEBPαsilenced group (n=10) and compared gene expression levels to normal CD34+ bone marrow cells (n=11) and the remaining AML group (n=506) using a three-way ANOVA and a post-hoc test (tukey-kramer method). We detected 686 differentially expressed genes with P < 0.05 after multiple testing. Of these 686 genes, 288 were up- and 401 down-regulated in the CEBPαsilenced group. We next asked the question which of those genes might be bona fide CEBPα targets and whether expression has been altered as the result of CEBPα silencing. We transduced estrogen-inducible C/EBPα in 32D cells and carried out ChIP-on-chip analysis using ER specific antibodies. The analysis yielded a collection of 529 CEBPα target genes that are significant enriched for C/EBPα binding (P < 0.05) using Hypergeometric Analysis of Tiling-arrays (HAT). We hypothesized that the direct target genes of CEBPα, derived from the 32D model system, were also present among deregulated genes in CEBPαsilenced human AMLs. We therefore overlaid the detected direct binding targets of CEBPα in 32D cells, with the differentially expressed genes in the CEBPαsilenced group and identified 49 overlapping genes (P=1×10−7) as putative direct targets. Among these 49 genes, 25 were down-regulated and 24 were upregulated in the primary CEBPαsilenced AML group. Both groups of genes were highly enriched for the CEBPα consensus binding sequence, i.e. 16/25 and 20/24 promoter regions respectively. The 25 downregulated genes, among which ACSL1, MYCT1 or Slc7a11, represent targets that are most likely normally activated by CEBPα, but are not transcribed in CEBPαsilenced human AMLs due to the absence of CEBPα. Among the genes that were upregulated in CEBPαsilenced leukemias are BCL2, CCR9, CEBPG or CD47. These putative target genes are repressed in CEBPα expressing cells and activated when CEBPα is silenced. This observation suggests that the transcription factor CEBPα may also acts as a repressor of gene transcription. We therefore studied the expression of two of those genes, i.e. CEBPG and CCR9 in Lin-Sca+Kit+ (LSK) bone marrow hematopoietic stem cells from wild type versus conditional Mx-Cre/CEBPα knock-out mice. Similar to what we observed in human CEBPαsilenced leukemias, we found that these two genes were switched off in LSK cells from CEBPα knock-out animals. Ingenuity pathway analysis of the 24 upregulated genes detected high enrichment (P < 0.001) of pathways involved with T-cell development. Our data clearly suggest that CEBPα may act as repressor of T-cell related genes through direct promoter interaction. We therefore propose that silencing of CEBPα by promoter hypermethylation is one of the transforming events that driving towards mixed myeloid/T-lymphoid leukemia. Disclosures:No relevant conflicts of interest to declare.

Overexpression of GATA2 predicts an adverse prognosis for patients with acute myeloid leukemia and it is associated with distinct molecular abnormalities

Overexpression of GATA2 predicts an adverse prognosis for patients with acute myeloid leukemia and it is associated with distinct molecular abnormalities

Kindler syndrome: a focal adhesion genodermatosis

Kindler syndrome (OMIM 173650) is an autosomal recessive genodermatosis characterized by trauma-induced blistering, poikiloderma, skin atrophy, mucosal inflammation and varying degrees of photosensitivity. Although Kindler syndrome is classified as a subtype of epidermolysis bullosa, it has distinct clinicopathological and molecular abnormalities. The molecular pathology of Kindler syndrome involves loss-of-function mutations in a newly recognized actin cytoskeleton-associated protein, now known as fermitin family homologue 1, encoded by the gene FERMT1. This protein mediates anchorage between the actin cytoskeleton and the extracellular matrix via focal adhesions, and thus the structural pathology differs from other forms of epidermolysis bullosa in which there is a disruption of the keratin intermediate filament-hemidesmosome network and the extracellular matrix. In the skin, fermitin family homologue 1 is mainly expressed in basal keratinocytes and binds to the cytoplasmic tails of beta1 and beta3 integrins as well as to fermitin family homologue 2 and filamin-binding LIM protein 1. It also plays a crucial role in keratinocyte migration, proliferation and adhesion. In this report, we review the clinical, cellular and molecular pathology of Kindler syndrome and discuss the role of fermitin family homologue 1 in keratinocyte biology.