Fibroepithelial Stromal Polyp of the Vulvovaginal Region as Part of the RB1 Family of Tumors: Friend or Foe?

Fibroepithelial stromal polyps (FSPs) are benign mesenchymal lesions occurring in the vulvovaginal region. Following the identification of loss of Retinoblastoma 1 (RB1) on immunohistochemical staining in routine practice, we stained a series of FSPs and performed additional fluorescence in situ hybridization (FISH) and copy number variation (CNV) sequencing to detect losses/deletions in the Retinoblastoma transcriptional corepressor 1 (RB1) gene. Fifteen FSP cases were stained for RB1, and subsequently, 9 cases were examined by FISH to detect a loss of RB1 (13q). Next, CNV sequencing was performed to assess genomic alterations. The mean age of the patients was 50 years. Loss of RB1 expression on immunohistochemistry was seen in 13 cases, and heterogeneous RB1 staining in the remaining 2 cases. FISH showed deletion of RB1 in all of the cases. CNV sequencing failed in almost all cases due to a low tumor content. Based on our findings, we hypothesize that FSPs are part of a spectrum of genetically related lesions, namely the 13q/RB1 family of tumors (which includes pleomorphic fibromas and spindle cell/pleomorphic lipomas). Due to the clinical, morphologic, and molecular overlap, we suggest that FSPs are pleomorphic fibromas occurring in the specialized stroma of the genital region.

Targeting hyperactive platelet-derived growth factor receptor-ß signaling in T-cell acute lymphoblastic leukemia and lymphoma.

T cell acute lymphoblastic leukemia (T-ALL) and T cell lymphoblastic lymphoma (T-LBL) are rare aggressive hematological malignancies. Current treatment consists of intensive chemotherapy, leading to 80% overall survival but are associated with severe toxic side effects. Furthermore, 10-20% of patients still die from relapsed or refractory disease providing a strong rationale for more specific, targeted therapeutic strategies with less toxicities. Here, we report a novel MYH9::PDGFRB fusion in a T-LBL patient and demonstrate that this fusion product is constitutively active and sufficient to drive oncogenic transformation in vitro and in vivo. Expanding our analysis more broadly across T-ALL, we found a T-ALL cell line and multiple patient derived xenograft models with PDGFRB hyperactivation in the absence of a fusion, with high PDGFRB expression in TLX3 and HOXA T-ALL molecular subtypes. To target this PDGFRB hyperactivation, we evaluated the therapeutic effects of a selective PDGFRB inhibitor, CP-673451, both in vitro and in vivo and demonstrated sensitivity if the receptor is hyperactivated. Altogether, our work reveals that hyperactivation of PDGFRB is an oncogenic driver in T-ALL/T-LBL and that screening T-ALL/TLBL patients for phosphorylated PDGFRB levels can serve as a biomarker for PDGFRB inhibition as a novel targeted therapeutic strategy in their treatment regimen.

Targeting cytokine- and therapy-induced PIM1 activation in preclinical models of T-cell acute lymphoblastic leukemia and lymphoma.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell acute lymphoblastic lymphoma (T-LBL) are aggressive hematological malignancies that are currently treated with high-dose chemotherapy. Over the last several years, the search toward novel and less-toxic therapeutic strategies for T-ALL/T-LBL patients has largely focused on the identification of cell-intrinsic properties of the tumor cell. However, non-cell-autonomous activation of specific oncogenic pathways might also offer opportunities that could be exploited at the therapeutic level. In line with this, we here show that endogenous interleukin 7 (IL7) can increase the expression of the oncogenic kinase proviral integration site for Moloney-murine leukemia 1 (PIM1) in CD127+ T-ALL/T-LBL, thereby rendering these tumor cells sensitive to in vivo PIM inhibition. In addition, using different CD127+ T-ALL/T-LBL xenograft models, we also reveal that residual tumor cells, which remain present after short-term in vivo chemotherapy, display consistent upregulation of PIM1 as compared with bulk nontreated tumor cells. Notably, this effect was transient as increased PIM1 levels were not observed in reestablished disease after abrogation of the initial chemotherapy. Furthermore, we uncover that this phenomenon is, at least in part, mediated by the ability of glucocorticoids to cause transcriptional upregulation of IL7RA in T-ALL/T-LBL patient-derived xenograft (PDX) cells, ultimately resulting in non-cell-autonomous PIM1 upregulation by endogenous IL7. Finally, we confirm in vivo that chemotherapy in combination with a pan-PIM inhibitor can improve leukemia survival in a PDX model of CD127+ T-ALL. Altogether, our work reveals that IL7 and glucocorticoids coordinately drive aberrant activation of PIM1 and suggests that IL7-responsive CD127+ T-ALL and T-LBL patients could benefit from PIM inhibition during induction chemotherapy.

Targeted therapy in juvenile myelomonocytic leukemia: Where are we now?

Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive clonal neoplasm of early childhood, classified as an overlap myeloproliferative/myelodysplastic neoplasm by the World Health Organization. In 90% of the patients with JMML, typical initiating mutations in the canonical Ras pathway genes NF1, PTPN11, NRAS, KRAS, and CBL can be identified. Hematopoietic stem cell transplantation (HSCT) currently is the established standard of care in most patients, although long-term survival is still only 50-60%. Given the limited therapeutic options and the important morbidity and mortality associated with HSCT, new therapeutic approaches are urgently needed. Hyperactivation of the Ras pathway as disease mechanism in JMML lends itself to the use of targeted therapy. Targeted therapy could play an important role in the future treatment of patients with JMML. This review presents a comprehensive overview of targeted therapies already developed and evaluated in vitro and in vivo in patients with JMML.

Correction to: The pitfalls and promise of liquid biopsies for diagnosing and treating solid tumors in children: as review.

In the original version of this article, a reader pointed out that there was a mistake in the phrasing in a paragraph. This could potentially be harmful to children. The authors agree to change the wording. "vitreous fluid" will be changed to "aqueous humor".

The pitfalls and promise of liquid biopsies for diagnosing and treating solid tumors in children: a review.

Cell-free DNA profiling using patient blood is emerging as a non-invasive complementary technique for cancer genomic characterization. Since these liquid biopsies will soon be integrated into clinical trial protocols for pediatric cancer treatment, clinicians should be informed about potential applications and advantages but also weaknesses and potential pitfalls. Small retrospective studies comparing genetic alterations detected in liquid biopsies with tumor biopsies for pediatric solid tumor types are encouraging. Molecular detection of tumor markers in cell-free DNA could be used for earlier therapy response monitoring and residual disease detection as well as enabling detection of pathognomonic and therapeutically relevant genomic alterations.Conclusion: Existing analyses of liquid biopsies from children with solid tumors increasingly suggest a potential relevance for molecular diagnostics, prognostic assessment, and therapeutic decision-making. Gaps remain in the types of tumors studied and value of detection methods applied. Here we review the current stand of liquid biopsy studies for pediatric solid tumors with a dedicated focus on cell-free DNA analysis. There is legitimate hope that integrating fully validated liquid biopsy-based innovations into the standard of care will advance patient monitoring and personalized treatment of children battling solid cancers.What is Known:• Liquid biopsies are finding their way into routine oncological screening, diagnosis, and disease monitoring in adult cancer types fast.• The most widely adopted source for liquid biopsies is blood although other easily accessible body fluids, such as saliva, pleural effusions, urine, or cerebrospinal fluid (CSF) can also serve as sources for liquid biopsiesWhat is New:• Retrospective proof-of-concept studies in small cohorts illustrate that liquid biopsies in pediatric solid tumors yield tremendous potential to be used in diagnostics, for therapy response monitoring and in residual disease detection.• Liquid biopsy diagnostics could tackle some long-standing issues in the pediatric oncology field; they can enable accurate genetic diagnostics in previously unbiopsied tumor types like renal tumors or brain stem tumors leading to better treatment strategies.

Pinpointing a potential role for CLEC12B in cancer predisposition through familial exome sequencing.

Predisposition to cancer is only partly understood, and thus, the contribution of still undiscovered cancer predisposing variants necessitates further research. In search of such variants, we performed exome sequencing on the germline DNA of a family with two children affected by ganglioneuroma and neuroblastoma. Applying stringent selection criteria, we identified a potential deleterious, missense mutation in CLEC12B, coding for a lectin C-type receptor that is predicted to regulate immune function. Although further screening in a larger population and functional characterization is needed, we propose CLEC12B as a candidate cancer predisposition gene.

LIN28B overexpression defines a novel fetal-like subgroup of juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive stem cell disease of early childhood. RAS activation constitutes the core component of oncogenic signaling. In addition, leukemic blasts in one-fourth of JMML patients present with monosomy 7, and more than half of patients show elevated age-adjusted fetal hemoglobin (HbF) levels. Hematopoietic stem cell transplantation is the current standard of care and results in an event-free survival rate of 50% to 60%, indicating that novel molecular-driven therapeutic options are urgently needed. Using gene expression profiling in a series of 82 patient samples, we aimed at understanding the molecular biology behind JMML and identified a previously unrecognized molecular subgroup characterized by high LIN28B expression. LIN28B overexpression was significantly correlated with higher HbF levels, whereas patients with monosomy 7 seldom showed enhanced LIN28B expression. This finding gives a biological explanation of why patients with monosomy 7 are rarely diagnosed with high age-adjusted HbF levels. In addition, this new fetal-like JMML subgroup presented with reduced levels of most members of the let-7 microRNA family and showed characteristic overexpression of genes involved in fetal hematopoiesis and stem cell self-renewal. Lastly, high LIN28B expression was associated with poor clinical outcome in our JMML patient series but was not independent from other prognostic factors such as age and age-adjusted HbF levels. In conclusion, we identified elevated LIN28B expression as a hallmark of a novel fetal-like subgroup in JMML.

Tandem repeats of Allium fistulosum associated with major chromosomal landmarks.

Tandem repeats are often associated with important chromosomal landmarks, such as centromeres, telomeres, subtelomeric, and other heterochromatic regions, and can be good candidates for molecular cytogenetic markers. Tandem repeats present in many plant species demonstrate dramatic differences in unit length, proportion in the genome, and chromosomal organization. Members of genus Allium with their large genomes represent a challenging task for current genetics. Using the next generation sequencing data, molecular, and cytogenetic methods, we discovered two tandemly organized repeats in the Allium fistulosum genome (2n = 2C = 16), HAT58 and CAT36. Together, these repeats comprise 0.25% of the bunching onion genome with 160,000 copies/1 C of HAT58 and 93,000 copies/1 C of CAT36. Fluorescent in situ hybridization (FISH) and C-banding showed that HAT58 and CAT36 associated with the interstitial and pericentromeric heterochromatin of the A. fistulosum chromosomes 5, 6, 7, and 8. FISH with HAT58 and CAT36 performed on A. cepa (2n = 2C = 16) and A. wakegi (2n = 2C = 16), a natural allodiploid hybrid between A. fistulosum and A. cepa, revealed that these repeats are species specific and produced specific hybridization patterns only on A. fistulosum chromosomes. Thus, the markers can be used in interspecific breeding programs for monitoring of alien genetic material. We applied Non-denaturing FISH that allowed detection of the repeat bearing chromosomes within 3 h. A polymorphism of the HAT58 chromosome location was observed. This finding suggests that the rapid evolution of the HAT58 repeat is still ongoing.

The H3K27me3 demethylase UTX is a gender-specific tumor suppressor in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that is mainly diagnosed in children and shows a skewed gender distribution toward males. In this study, we report somatic loss-of-function mutations in the X-linked histone H3K27me3 demethylase ubiquitously transcribed X (UTX) chromosome, in human T-ALL. Interestingly, UTX mutations were exclusively present in male T-ALL patients and allelic expression analysis revealed that UTX escapes X-inactivation in female T-ALL lymphoblasts and normal T cells. Notably, we demonstrate in vitro and in vivo that the H3K27me3 demethylase UTX functions as a bona fide tumor suppressor in T-ALL. Moreover, T-ALL driven by UTX inactivation exhibits collateral sensitivity to pharmacologic H3K27me3 inhibition. All together, our results show how a gender-specific and therapeutically relevant defect in balancing H3K27 methylation contributes to T-cell leukemogenesis.

Heterogeneous cytogenetic subgroups and outcomes in childhood acute megakaryoblastic leukemia: a retrospective international study.

Comprehensive clinical studies of patients with acute megakaryoblastic leukemia (AMKL) are lacking. We performed an international retrospective study on 490 patients (age ≤18 years) with non-Down syndrome de novo AMKL diagnosed from 1989 to 2009. Patients with AMKL (median age 1.53 years) comprised 7.8% of pediatric AML. Five-year event-free (EFS) and overall survival (OS) were 43.7% ± 2.7% and 49.0% ± 2.7%, respectively. Patients diagnosed in 2000 to 2009 were treated with higher cytarabine doses and had better EFS (P = .037) and OS (P = .003) than those diagnosed in 1989 to 1999. Transplantation in first remission did not improve survival. Cytogenetic data were available for 372 (75.9%) patients: hypodiploid (n = 18, 4.8%), normal karyotype (n = 49, 13.2%), pseudodiploid (n = 119, 32.0%), 47 to 50 chromosomes (n = 142, 38.2%), and >50 chromosomes (n = 44, 11.8%). Chromosome gain occurred in 195 of 372 (52.4%) patients: +21 (n = 106, 28.5%), +19 (n = 93, 25.0%), +8 (n = 77, 20.7%). Losses occurred in 65 patients (17.5%): -7 (n = 13, 3.5%). Common structural chromosomal aberrations were t(1;22)(p13;q13) (n = 51, 13.7%) and 11q23 rearrangements (n = 38, 10.2%); t(9;11)(p22;q23) occurred in 21 patients. On the basis of frequency and prognosis, AMKL can be classified to 3 risk groups: good risk-7p abnormalities; poor risk-normal karyotypes, -7, 9p abnormalities including t(9;11)(p22;q23)/MLL-MLLT3, -13/13q-, and -15; and intermediate risk-others including t(1;22)(p13;q13)/OTT-MAL (RBM15-MKL1) and 11q23/MLL except t(9;11). Risk-based innovative therapy is needed to improve patient outcomes.

In vitro human embryonic stem cell hematopoiesis mimics MYB-independent yolk sac hematopoiesis.

Although hematopoietic precursor activity can be generated in vitro from human embryonic stem cells, there is no solid evidence for the appearance of multipotent, self-renewing and transplantable hematopoietic stem cells. This could be due to short half-life of hematopoietic stem cells in culture or, alternatively, human embryonic stem cell-initiated hematopoiesis may be hematopoietic stem cell-independent, similar to yolk sac hematopoiesis, generating multipotent progenitors with limited expansion capacity. Since a MYB was reported to be an excellent marker for hematopoietic stem cell-dependent hematopoiesis, we generated a MYB-eGFP reporter human embryonic stem cell line to study formation of hematopoietic progenitor cells in vitro. We found CD34(+) hemogenic endothelial cells rounding up and developing into CD43(+) hematopoietic cells without expression of MYB-eGFP. MYB-eGFP(+) cells appeared relatively late in embryoid body cultures as CD34(+)CD43(+)CD45(-/lo) cells. These MYB-eGFP(+) cells were CD33 positive, proliferated in IL-3 containing media and hematopoietic differentiation was restricted to the granulocytic lineage. In agreement with data obtained on murine Myb(-/-) embryonic stem cells, bright eGFP expression was observed in a subpopulation of cells, during directed myeloid differentiation, which again belonged to the granulocytic lineage. In contrast, CD14(+) macrophage cells were consistently eGFP(-) and were derived from eGFP-precursors only. In summary, no evidence was obtained for in vitro generation of MYB(+) hematopoietic stem cells during embryoid body cultures. The observed MYB expression appeared late in culture and was confined to the granulocytic lineage.

CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol.

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23-3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia. This trial was registered at www.ClinicalTrials.gov as #NCT00003728.

Treatment of human embryos with the TGFß inhibitor SB431542 increases epiblast proliferation and permits successful human embryonic stem cell derivation.

STUDY QUESTION: Is there an effect of the TGFß inhibitor SB431542 (SB) on the epiblast compartment of human blastocysts, and does it affect subsequent human embryonic stem cell (hESC) derivation? SUMMARY ANSWER: SB increases the mean number of NANOG-positive cells in the inner cell mass (ICM), and allows for subsequent hESC derivation. WHAT IS KNOWN ALREADY: It is known that inhibition of TGFß by SB has a positive effect on mouse ESC self-renewal, while active TGFß signalling is needed for self-renewal of primed ESC. STUDY DESIGN, SIZE, DURATION: From December 2011 until March 2012, 263 donated spare embryos were used from patients who had undergone IVF/ICSI in our centre. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donated human embryos were cultured in the presence of SB or Activin A, and immunocytochemistry was performed on Day 6 blastocysts for NANOG and GATA6. Moreover, blastocysts were used for the derivation of hESC, with or without exposure to SB. MAIN RESULTS AND THE ROLE OF CHANCE: Immunocytochemistry revealed a significantly higher number of NANOG-positive ICM cells in the SB group compared with the control (12.0 ± 5.9 versus 6.1 ± 4.7), while no difference was observed in the Activin A group compared with other groups (6.7 ± 3.7). The number of GATA6-positive ICM cells did not differ between the SB, Activin A and control group (8.8 ± 4.3, 8.0 ± 4.6 and 7.2 ± 4.0, respectively). Blocking TGFß signalling did not prevent subsequent hESC line derivation. LIMITATIONS, REASONS FOR CAUTION: The number of human blastocysts available for this study was too low to reveal if the observed increase in NANOG-positive epiblast cells after exposure to SB affected the efficiency of hESC derivation (12.5% compared with 16.7%). WIDER IMPLICATIONS OF THE FINDINGS: This work can contribute to the derivation of naive hESC lines in the future. STUDY FUNDING/COMPETING INTEREST(S): M.V.d.J. is holder of a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT, grant number SB093128), Belgium. G.D. and this research are supported by the Research Foundation Flanders (FWO), grant number FWO-3G062910) and a Concerted Research Actions funding from BOF (Bijzonder Onderzoeksfonds University Ghent, grant number BOF GOA 01G01112). S.M.C.d. S.L. is supported by the Netherlands Organization of Scientific Research (NWO) (ASPASIA 015.007.037) and the Interuniversity Attraction Poles (PAI) (no. P7/07). P.D.S. is holder of a fundamental clinical research mandate by the FWO. We would like to thank Ferring Company (Aalst, Belgium) for financial support of this study. The authors do not have any competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

MicroRNA-128-3p is a novel oncomiR targeting PHF6 in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia arises from the leukemic transformation of developing thymocytes and results from cooperative genetic lesions. Inactivation of the PHF6 gene is frequently observed in T-cell acute lymphoblastic leukemia, suggesting an important tumor suppressive role for PHF6 in the pathobiology of this leukemia. Although the precise function of PHF6 is still unknown, this gene is most likely involved in chromatin regulation, a strongly emerging theme in T-cell acute lymphoblastic leukemia. In this context, our previous description of a cooperative microRNA regulatory network controlling several well-known T-cell acute lymphoblastic leukemia tumor suppressor genes, including PHF6, is of great importance. Given the high frequency of PHF6 lesions in T-cell acute lymphoblastic leukemia and the integration of PHF6 in this microRNA regulatory network, we aimed to identify novel oncogenic microRNAs in T-cell acute lymphoblastic leukemia which suppress PHF6. To this end, we performed an unbiased PHF6 3'UTR-microRNA library screen and combined the results with microRNA profiling data of samples from patients with T-cell acute lymphoblastic leukemia and normal thymocyte subsets. We selected miR-128-3p as a candidate PHF6-targeting, oncogenic microRNA and demonstrated regulation of PHF6 expression upon modulation of this microRNA in T-cell acute lymphoblastic leukemia cell lines. In vivo evidence of an oncogenic role of this microRNA in T-cell acute lymphoblastic leukemia was obtained through accelerated leukemia onset in a NOTCH1-induced T-cell acute lymphoblastic leukemia mouse model upon miR-128-3p over-expression. We conclude that miR-128-3p is a strong novel candidate oncogenic microRNA in T-cell acute lymphoblastic leukemia which targets the PHF6 tumor suppressor gene.

The Notch driven long non-coding RNA repertoire in T-cell acute lymphoblastic leukemia.

Genetic studies in T-cell acute lymphoblastic leukemia have uncovered a remarkable complexity of oncogenic and loss-of-function mutations. Amongst this plethora of genetic changes, NOTCH1 activating mutations stand out as the most frequently occurring genetic defect, identified in more than 50% of T-cell acute lymphoblastic leukemias, supporting a role as an essential driver for this gene in T-cell acute lymphoblastic leukemia oncogenesis. In this study, we aimed to establish a comprehensive compendium of the long non-coding RNA transcriptome under control of Notch signaling. For this purpose, we measured the transcriptional response of all protein coding genes and long non-coding RNAs upon pharmacological Notch inhibition in the human T-cell acute lymphoblastic leukemia cell line CUTLL1 using RNA-sequencing. Similar Notch dependent profiles were established for normal human CD34(+) thymic T-cell progenitors exposed to Notch signaling activity in vivo. In addition, we generated long non-coding RNA expression profiles (array data) from ex vivo isolated Notch active CD34(+) and Notch inactive CD4(+)CD8(+) thymocytes and from a primary cohort of 15 T-cell acute lymphoblastic leukemia patients with known NOTCH1 mutation status. Integration of these expression datasets with publicly available Notch1 ChIP-sequencing data resulted in the identification of long non-coding RNAs directly regulated by Notch activity in normal and malignant T cells. Given the central role of Notch in T-cell acute lymphoblastic leukemia oncogenesis, these data pave the way for the development of novel therapeutic strategies that target hyperactive Notch signaling in human T-cell acute lymphoblastic leukemia.