Thymic Enlargement Research Articles

Timing of the Loss of Pten Is Critical in Determining the Disease Phenotype in Mice- a Mouse Model for Pediatric Mixed MDS/MPN

Cancer is caused by accumulated genomic and epigenetic abnormalities during the development of an individual, particularly during the neonatal period, when developmental plasticity is actively occurring. Myeloid-specific deletion of pten in embryos or after 3 weeks of age causes acute monocytic or myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) following a transient myeloproliferative neoplasm (MPN) in adult mice, which can mimic the human diseases to varying degrees. However, it is not clear how the timing of genomic and epigenetic abnormalities contributes to the disease phenotype in a mouse that is of an age comparable to human children. We hypothesized that during the development/aging process, the timing of when the genomic abnormality or “hit” occurs, such as loss of Pten or Nf1, is a critical determinant of the disease phenotype. We tested this by investigating the effect of somatic deletion of Pten at an age of 8 days, one of the most vulnerable stages for malignancy development in mice with or without a germline mutant Nf1. Through crossbreeding, we generated mice with Ptenfl/flNf1Fcr/+Mx1-Cre+ on a C57BL6/129 genetic background, and conditionally deleted Pten in a myeloid-specific manner by intraperitoneal injection of Poly(I:C). Mice with a pten deletion and mutant Nf1 (ptenkoNf1mut, hereafter referred as double mutant) showed signs of sickness at the end of the 2nd week of life, and all died by age 3-5 weeks (equivalent to 1-3 years old in humans). The natural survival in double mutant mice (n=10) was significantly shorter than those with wild type pten and Nf1 (ptenwt; Nf1wt, hereafter referred as WT, n=6, median 0.9 vs >14 months, p<0.001). At autopsy, the double mutant mice had obvious hepatosplenomegaly compared to the WT littermates, but no lymphadenopathy nor thymus enlargement was detected. We then analyzed 15 littermate pairs with/without mutations at age 3-4 weeks. The spleen and liver sizes in the double mutants were significantly increased compared to that in WT (median weights: spleen-198 vs 69mg, p< 0.001; liver-836 vs 532mg, p=0.013). Peripheral blood (PB) analysis showed no difference in WBC in both groups, but significant decreases in double mutant mice in RBC and Hgb (median RBC-5.6 vs 7.6x106/ul, p=0.010; Hgb-10.1 vs 12.6g/ul, p=0.022), but with a normal MCV. HE and IHC stained tissue sections of formalin-fixed organs with antibodies to B220, CD3e, or F4/80 found increased cellularity in bone marrow (BM), and severe infiltration of mature myeloid cells in double mutant spleens, livers, and lungs. Flow cytometric analysis showed that double mutant mice had significantly increased cells marking for monocyte/macrophages(Mac-1+Gr-1int, or Mac-1+CD115+) and granulocytes (Mac-1+ Gr-1+)[p≤0.001] in BM and PB; both T-cells (CD3e+) and B-cells (CD19) were significantly reduced in PB and spleen (p<0.01); platelets (CD41+Ter119-) were reduced in PB. An erythroid differentiation block [indicative of myelodysplasia (MDS) or leukemia] was found in double mutant mice (significant increases in Ter119+CD71high cells). Pten deleted mice without mutant Nf1had a similar phenotype, but survived longer than double mutant mice (median 1.2 vs 0.9 months, p<0.001). Colony formation assays showed no cytokine hypersensitivity in BM or spleen cells in double mutants in response to GM-CSF or IL-3. Based on the Bethesda proposals for classification of non-lymphoid hematopoietic neoplasms in mice, we believe that we have developed a mouse model of myeloproliferative disease with features of a pediatric unclassifiable mixed MDS/MPN. It also mimics to some degree human Juvenile Myelomonocytic Leukemia (JMML), in terms of age of onset and organ infiltration with myelomonocytic cells, but fails to mimic JMML with respect to GM-CSF hypersensitivity.We demonstrate that deletion of pten at an early age in the mouse causes a fatal mixed MDS/MPN that is different from the previously reported mouse models, in which myeloid-specific deletions of Pten were induced in embryos or after 3 weeks of age, and these mice developed only a transient MPN, all eventually dying of AML or ALL at adult ages. To the best of our knowledge, this is the first model in young mice bearing a somatic mutation that resembles a human pediatric unclassifiable MDS/MPN and more closely represents the true genetic and epigenetic background, thus providing a novel tool to study these mechanisms underlying pediatric MDS/MPN. DisclosuresNo relevant conflicts of interest to declare.

Highlights of This Issue

Most patients on androgen deprivation therapy eventually develop castration-resistant prostate cancer (CRPC). Here, the recently approved androgen antagonist enzalutamide was evaluated in combination with immunotherapy in a mouse transgenic prostate cancer model. This article described a novel immunogenic modulation property of enzalutamide, where treatment of enzalutamide improves tumor cells' sensitivity to T-cell mediated attack. In vivo, enzalutamide reduced genitourinary tissue weight, enlarged thymus, and increased levels of T-cell excision circles. The combination of enzalutamide and immunotherapy significantly improved overall survival of transgenic mice with advanced disease. These data support the combination of enzalutamide and immunotherapy as a promising treatment for CRPC.The role of human endogenous retroviruses (HERV) in cancer is uncertain. To explore the expression and immunogenicity of a recently described GAG protein encoded by HERV-K, Reis and colleagues analyzed cancer patient tissues and sera for reactivity to GAG-HERV-K. GAG-HERV-K was highly expressed in prostate cancer tissues and regulated both by promoter demethylation and androgen stimulation. A subset of prostate cancer patients had serum antibodies to GAG-HERV-K, which correlated with advanced disease stages and worse survival. This work characterizes one of the first human tumor antigens of retroviral origin, with potential as a biomarker for progression of prostate cancer.CD40, a member of the TNF receptor superfamily, is a major determinant of antitumor immunity. In this phase I study, Beatty and colleagues investigated the tolerability and clinical impact of an agonist CD40 monoclonal antibody (CP-870,893) when administered in combination with gemcitabine for the treatment of patients with chemotherapy-naive advanced pancreatic carcinoma. Treatment was well-tolerated and associated with preliminary evidence of efficacy. Using novel analyses of metabolic imaging, treatment response heterogeneity was observed between primary and metastatic lesions. Findings from this study highlight the biologic heterogeneity of pancreatic carcinoma and demonstrate a role for metabolic imaging in understanding treatment responses.Building on their recent findings that PARP1 is a potential therapeutic target in small cell lung cancer (SCLC), Cardnell and colleagues evaluated the activity of a novel PARP inhibitor and identified biomarkers of response. SCLC xenograft models exhibited exquisite sensitivity to the PARP inhibitor, BMN 673. Further, the authors identified a group of DNA repair proteins that are potential predictive biomarkers and discovered a role for cotargeting the PI3K pathway to enhance sensitivity to PARP inhibition. The biomarkers identified here show great promise as predictors of response and have potential to impact ongoing trials of BMN 673 and other PARP inhibitors in SCLC.

Hypokalemic Periodic Paralysis Induced by Thymic Hyperplasia and Relieved by Thymectomy

Hypokalemic periodic paralysis is a muscle channelopathy based on mutations or predisposing variants or secondary to potassium wasting. In contrast to myasthenia gravis, an association with thymic hyperplasia has not yet been reported, to our knowledge. We report a male patient in his mid-20s with progressive episodes of flaccid muscle weakness, associated low serum potassium levels, and a pathologic decrement in the long exercise test. Because the familial inheritance in the family was initially unknown, thorough diagnostic tests were performed including contrast-enhanced computed tomography scan, which displayed a mass in the anterior mediastinum. The test results for autoantibodies against myasthenia gravis (acetylcholine receptor, muscle-specific tyrosine kinase, and low-density lipoprotein receptor-related protein 4) and other end plate channelopathies were negative, and test results for hypokalemia-inducing hormones (thyroid, corticotropin, and cortisol) were negative. Surgery identified a thymus of 13 × 8 × 3 cm(3). Histologic analysis was consistent with thymic hyperplasia of the follicular subtype and immunohistologic analysis showed cytokeratin 5/6 in hyperplastic epithelial cells. A 2-year follow-up revealed the postoperative absence of weakness episodes. As in 30% of familial cases, molecular genetics testing failed to identify a mutation in periodic paralysis genes. Thymic hyperplasia can clinically manifest susceptibility to hypokalemic periodic paralysis. For patients with late onset or increasing weakness episodes, we recommend imaging to assess for thymic enlargement and thymectomy at thymic hyperplasia.

The impact of nandrolone decanoate and growth hormone on biosynthesis of steroids in rats

Growth hormone (GH) and anabolic androgenic steroids (AAS) are commonly used in sports communities. Several studies have suggested an association between GH and AAS. We have investigated the impact of GH in rats treated with nandrolone decanoate (ND). Male Wistar rats received ND (15mg/kg) every third day during three weeks and were subsequently treated with recombinant human GH (1.0IU/kg) for ten consecutive days. Plasma samples were collected and peripheral organs (i.e. heart, liver, testis and thymus) were dissected and weighed. Concentration of thirteen endogenous steroids was measured in the rat plasma samples using high specificity LC–MS/MS methods. Seven steroids were detected and quantified, and concentrations of estrone, testosterone, and androstenedione were significantly different among the groups, while concentrations of pregnenolone, DHEA, 17-hydroxyprogesterone and corticosterone were not altered. Administration of rhGH alone altered the plasma steroid distribution, and the results demonstrated significantly increased concentrations of plasma estrone as well as decreased concentrations of testosterone and androstenedione in the ND-treated rats. Administration of rhGH to ND-pretreated rats did not reverse the alteration of the steroid distribution induced by ND. Administration of ND decreased the weight of the thymus, and addition of rhGH did not reverse this reduction. However, rhGH administration induced an enlargement of thymus. Taken together, the plasma steroid profile differed in the four groups, i.e. control, AAS, rhGH and the combination of AAS and rhGH treatment.

Severe thymic hyperplasia in a newborn calf associated with impaired T-cell differentiation

A 1-day-old female Holstein-Friesian calf was presented for severe dyspnea. Physical examination revealed respiratory distress, moderate edema of the ventral neck, and swollen jugular veins. The calf died and was submitted for necropsy. A severely enlarged thymus (40 cm × 20 cm × 10 cm) weighing 1.37 kg was detected on gross examination. Histomorphology was normal but no tingible body macrophages were observed in the medullary areas. Immunohistochemistry was characterized by the lack of thymic cluster of differentiation 3 and major histocompatibility complex class II expression compared to age-matched controls. The findings were consistent with severe thymic hyperplasia, a rare congenital condition that is also described in children. Immunohistochemical findings were suggestive of impaired T-cell development and selection associated with lack of apoptosis of thymic cells (lack of tingible body macrophages). Thymic hyperplasia in juvenile animals should be considered among the differential diagnoses of mediastinal masses as a rare cause of respiratory distress in newborn calves.

Radiological features of thymic langerhans cell histiocytosis

TI was reported in 18/1,264 (1.4%) LCH patients. All nine patients with TI at initial LCH presentation were below 2 years of age and had multisystem LCH (9/242, 4%). Images (sonography, CT, MRI) for central review were available in 15 cases. Characteristic findings of TI were thymus enlargement (67%), few to many cysts (80%), and few to many calcifications (100%). Sonographic and MRI findings were in excellent agreement. We recommend adding sonography of the thymus to the standard for initial clinical evaluation of LCH patients below the age of 2 years.

Thymus and mediastinal node involvement in childhood langerhans cell histiocytosis: Long‐term follow‐up from the French national cohort

BackgroundMediastinal involvement (MI) in Langerhans cell histiocytosis (LCH) has been rarely reported. Here, we describe the clinical, radiological, and biological presentation, and the outcome of childhood LCH with MI.MethodFrom the French LCH register, which includes 1,423 patients aged less than 18 years, we retrieved the medical charts of patients with mediastinal enlargement detected on chest X-rays.ResultsThirty-seven patients were retrieved, including 18 males; median age of diagnosis was 0.7 years, and median follow-up time was 6.2 years. The prevalence of MI varied with the age at diagnosis, ranging from 7% below 1 year old to less than 1% at >5 years. Thirteen cases (35%) were diagnosed because of MI-related symptoms, including respiratory distress (N = 4), superior venous cava syndrome (N = 2), and/or cough and polypnea (N = 10). CT scans performed in 32 cases at diagnosis showed tracheal compression (N = 5), cava thrombosis (N = 2), and/or calcification (N = 16). All patients presented multi-system disease at LCH diagnosis, and 35/37 were initially treated with vinblastine and corticosteroids. Death occurred in five cases, due to MI (N = 1) or hematological refractory involvement (N = 4). The overall 5-year survival was 87.1%, and immunodeficiency was not detected as a sequel.ConclusionsMI in LCH mainly occurs in young children, and diagnosis was based on CT showing thymus enlargement and calcifications. Pediatr Blood Cancer 2013;60:1759–1765. © 2013 Wiley Periodicals, Inc.

Inactivation of the RB family prevents thymus involution and promotes thymic function by direct control of Foxn1 expression

Thymic involution during aging is a major cause of decreased production of T cells and reduced immunity. Here we show that inactivation of Rb family genes in young mice prevents thymic involution and results in an enlarged thymus competent for increased production of naive T cells. This phenotype originates from the expansion of functional thymic epithelial cells (TECs). In RB family mutant TECs, increased activity of E2F transcription factors drives increased expression of Foxn1, a central regulator of the thymic epithelium. Increased Foxn1 expression is required for the thymic expansion observed in Rb family mutant mice. Thus, the RB family promotes thymic involution and controls T cell production via a bone marrow-independent mechanism, identifying a novel pathway to target to increase thymic function in patients.

Von Hippel-Lindau disease associated with myasthenia gravis not related to thymoma

BACKGROUND Von Hippel-Lindau disease (VHL) is a rare autosomal dominant inherited disorder characterized by an increased risk of tumours in a number of locations (eyes, brain, adrenal gland, pancreas, liver, kidneys, or other areas of the body). It is caused by germline mutation in the VHL gene. The VHL gene is a tumour suppressor gene that has been identified on the short arm of chromosome 3. CASE REPORT We report a case of a 60 year-old female with the clinical diagnosis of VHL type 1 (cerebellar haemangioblastoma, pancreatic cysts with subsequent steatorrhoea, and bilateral renal carcinoma) who developed weakness and fatigability of skeletal muscles, left lid ptosis, snarling expression and nasal timbre speech. Acetylcholine receptor antibodies were negative in serum, while the electrodiagnostic test demonstrated an alteration of neuromuscolar junction which was consistent with the diagnosis of myasthenia gravis. Contrast-enhanced TC scan of the anterior mediastinum was performed, which excluded thymus enlargement. VHL gene evaluation in this patient identified a new mutation (c279delC9) and polymorphism c291C&gt;G. At present the patient still suffers from ataxia and dysmetria due to cerebellar involvement in VHL, while fatigue and lid ptosis improved after the treatment with oral pyridostigmine 60 mg tid. DISCUSSION AND CONCLUSIONS To our knowledge this is the first report of a case of VHL associated with myasthenia gravis without thymoma. A case of VHL associated with a form of myasthenia gravis related to thymoma has been recently reported. In our case the absence of acetylcholine receptor antibodies may suggest a genetic origin also for the myasthenia gravis.

Surgery of Myasthenia Gravis Associated or Not With Thymoma: A Retrospective Study of 43 Cases

Thymectomy is a surgical treatment of myasthenia gravis. Our goal is to report our experience in the surgical treatment of myasthenia gravis with or without thymoma and a review of the literature. This is a retrospective study over a period of 10 years (2001-2010) on 43 patients: 28 women and 15 men with a mean age of 39.3 years (range 16-68 years). The myasthenia gravis was confirmed by clinical, electromyographic data and the presence of antibodies to acetylcholine receptors. Computed tomography objectified thymic mass in 14 cases (32.5%) enlarged thymus without visible mass in eight cases (18.6%). All patients received anticholinesterase, cortico steroids in 25 cases and in three cases plasmapheresis was required. The surgical approach was total sternotomy (n=32 cases), cervicotomy (n=2), cervical and manubriotomy (n=1), a manubriotomy (n=3) and a thoracotomy in five cases (lateralised thymoma). All patients underwent a total thymectomy associated or not with resection of the tumour. Intensive Care Unit was necessary for at least 24h up to six days. The postoperative course was marked by a myasthaenic crisis (n=2) and respiratory failure (n=3) with a favourable outcome. The prognosis was marked by a complete remission in 14 cases, partial remission in 11 patients, stabilisation (n=16 cases) and increasing crisis in two patients. Thymectomy certainly allows clinical improvement and reduced crisis of myasthenia gravis. Long term monitoring will confirm the benefit of non-oncological thymectomy alone or in combination with standard treatments for patients with generalised myasthenia gravis without thymoma.

Clinicopathological Conference: A 9-y-old Girl with Transient Chest Pain

Dr Bakhshi: A 9-y-girl from Amritsar presented with transient chest pain of 1-d duration in August 2008. Her chest pain was relieved without any medications and was subsequently asymptomatic. There was no history of cough, dyspnea, orthopnea, anorexia, rash, fever, weight loss or night sweats. There was no significant past or family history. The girl was immunized to date and there was no history of contact with a case of tuberculosis. She was a well-nourished child, with normal vitals, no adenopathy, pallor, organomegaly, a normal chest and physical examination. A chest X-ray done at Amritsar was suggestive of mediastinal mass (Fig. 1a) for which the patient underwent a CT scan showing widening of the superior mediastinum which was interpreted as enlarged thymus with homogenous attenuation. In addition there were opacities in the anterior segment of the left upper lobe. The patient was referred to Sir Ganga Ram Hospital (SGRH). The patient was well looking and essentially normal on physical examination. In the initial and subsequent investigations, the complete blood count (CBC) was normal (Table 1). The erythrocyte sedimentation rate (ESR) was consistently high. Lactate Dehydrogenase (LDH) and uric acid were normal and Mantoux test was negative. She was started on some oral antibiotics for 10 d. She was also advised a biopsy by the doctors at the hospital, but the family refused. The child continued to follow up in Amritsar for a period of 1 y from August 2008 till July 2009. For almost a year the patient remained absolutely well. She was on some homeopathic medications, of which we have no details. Serial chest X-rays done during this period after a 3 mo showed a mediastinal bulge and some shadowing in the left hilar region. The lung fields showed increase in the bronchovascular markings in the left hemithorax. The diaphragms and the bones were normal. Eight months later the mediastinal bulge had increased, the hilum was prominent, the diaphragm was flattened and there was a haze in the left hemithorax with loss of lung volume and the costophrenic angle on the left side was not well seen. Three months later, in Sep 2009 (Fig. 1b), the mediastinal bulge had increased; the diaphragm was showing tenting, and the left hemithorax had became more opaque with some shadows. Thus it was a mediastinal mass with slowly growing pathology. There was gradual involvement of the lung in the form of a subpulmonic effusion or pleural effusion. Possibility of left phrenic nerve palsy due to mediastinal mass could be entertained but would not explain the lung pathology. Serial CT scans from Aug till Sep 2009 clearly showed a homogenous mediastinal mass (Fig. 2a). The color pointed towards a lymph nodal mass in general. It did not have any necrosis and there was some rim enhancement. The lung window showed no abnormality. Three months later, in Dec 2008, the mass had increased in size; there was a lung nodule (Fig. 2b). In July 2009, 1 y after the initial visit at V. Dinand : S. P. Yadav :A. Sachdeva (*) Pediatric Hematology Oncology and Bone Marrow Transplant Unit, Department of Pediatrics, Institute of Child Health, Sir Ganga Ram Hospital, New Delhi 110 060, India e-mail: anupamace@yahoo.co.in

Thyroid Cancer after Exposure to External Radiation: A Pooled Analysis of Seven Studies

The thyroid gland of children is especially vulnerable to the carcinogenic action of ionizing radiation. To provide insights into various modifying influences on risk, seven major studies with organ doses to individual subjects were evaluated. Five cohort studies (atomic bomb survivors, children treated for tinea capitis, two studies of children irradiated for enlarged tonsils, and infants irradiated for an enlarged thymus gland) and two case-control studies (patients with cervical cancer and childhood cancer) were studied. The combined studies include almost 120,000 people (approximately 58,000 exposed to a wide range of doses and 61,000 nonexposed subjects), nearly 700 thyroid cancers and 3,000,000 person years of follow-up. For persons exposed to radiation before age 15 years, linearity best described the dose response, even down to 0.10 Gy. At the highest doses (> 10 Gy), associated with cancer therapy, there appeared to be a decrease or leveling of risk. For childhood exposures, the pooled excess relative risk per Gy (ERR/Gy) was 7.7 (95% CI = 2.1, 28.7) and the excess absolute risk per 10(4) PY Gy (EAR/10(4) PY Gy) was 4.4 (95% CI = 1.9, 10.1). The attributable risk percent (AR%) at 1 Gy was 88%. However, these summary estimates were affected strongly by age at exposure even within this limited age range. The ERR was greater (P = 0.07) for females than males, but the findings from the individual studies were not consistent. The EAR was higher among women, reflecting their higher rate of naturally occurring thyroid cancer. The distribution of ERR over time followed neither a simple multiplicative nor an additive pattern in relation to background occurrence. Only two cases were seen within 5 years of exposure. The ERR began to decline about 30 years after exposure but was still elevated at 40 years. Risk also decreased significantly with increasing age at exposure, with little risk apparent after age 20 years. Based on limited data, there was a suggestion that spreading dose over time (from a few days to > 1 year) may lower risk, possibly due to the opportunity for cellular repair mechanisms to operate. The thyroid gland in children has one of the highest risk coefficients of any organ and is the only tissue with convincing evidence for risk about 1.10 Gy.

Myasthenia Graves with Malignant Thymoma and Spinal Metastasis (P04.097)

Objective: Case report of rare spinal metastasis of malignant thymoma. Background Malignant Thymoma is a rare mediastinal tumor. About 15% have extrathoracic metastasis in liver, kidney and bone. Spinal metastasis are exceptional. We present 2 cases of malignant thymoma with spinal metastasis and poor response to chemotherapy and radiotherapy. Design/Methods: CASE REPORT: Case 1: 55years old Emirati presented in 1995 with difficulty in chewing and shortness of breath and was diagnosed as sero-positive myasthenia with thymoma. She underwent surgery followed by chemotherapy and radiotherapy. 2002 she presented with paraspinal pain with multiple metastasis in liver, para aortic lymph nodes. She had extensive surgery followed by chemotherapy and radiotherapy. 2009, she had new liver metastasis and she was started on Sutent. June 2011 she presented with progressive weakness of both legs, back pain and was found to have spinal metastasis from T5-T8. She is on chemotherapy at present. Case 2: 22 years old lebenese lady presented with persistant cough in 1998 and MRI thoracic- revealed enlarged thymus extending to the posterior chest wall. Surgery revealed Cortical Thymoma-lymphocyte type. This recurred in 2000 and she underwent chemotherapy and radiotherapy. Aug 2005 she developed, numbness, weakness in both lower extremities and bladder invovement. CT Chest showed multinodular paraspinal mass going into the intraspinal compartment at D6/7 for which she underwent surgery ,chemotherapy and radiotherapy. April 2007 developed huge cervical lymphadenopathy and biopsy revealed diffuse large cell lymphoma. This was followed by chemotherapy but patient succumbed in 2008. Results: Both our cases relapsed inspite of all available therapy given. Conclusions: Spinal metastasis with malignant thymoma is rare but any back pain or symptoms of spinal cord involvement should alert the physician of spinal metastasis. No definitive treatment is present. Disclosure: Dr. Thakre has nothing to disclose. Dr. Inshasi has nothing to disclose.

Consultation with the Specialist: Thyroid Nodules

Based on strong research evidence, thyroid nodules in children and teenagers are more likely to be malignant than in adults. Based on strong research evidence, a history of ionizing radiation to the head or neck is an independent risk factor for the development of thyroid malignancies. There is strong research evidence, including a recent meta-analysis, supporting the use of fine-needle aspiration biopsy in the evaluation of all pediatric and adolescent patients presenting with a thyroid nodule. The surgical management and postoperative care of pediatric and adolescent patients who have well-differentiated thyroid carcinomas remains controversial, because the rarity of the disease limits the ability to conduct randomized, prospective research studies. Numerous studies have demonstrated that, despite presenting with more advanced disease, pediatric and adolescent patients with thyroid carcinoma have a higher survival rate than adults. The American Thyroid Association has issued strong evidence-based recommendations for the management of medullary thyroid carcinoma, including RET mutation testing and early prophylactic total thyroidectomy in children with high-risk mutations.

Thymic Enlargement in Two Cases of Graves' Disease

Thymic Enlargement in Two Cases of Graves' Disease

Evidence for Cooperation of Receptor Tyrosine Kinases and Activating NOTCH Mutations to Hyperactivate mTOR in T-Cell Leukemia: A Rationale Basis for Targeted Therapy

Abstract 1381▪▪This icon denotes a clinically relevant abstractT-cell acute lymphoblastic leukemia (T-ALL) remains a therapeutic challenge. T-ALLs are characterized by recurring chromosomal rearrangements causing aberrant expression of transcription factors (Myb; TAL/SCL; HOX) dividing patients into different subgroups. Activating mutations in NOTCH, the master regulator of T-cell development, are found in more than 60% of T-ALLs independently of subtype. Most T-ALLs display a hyperactivation of the PI3K-AKT-mTOR pathway, a potential target for therapeutic intervention. The master regulator of PI3K-AKT signalling is PTEN, which is frequently inactivated in cancer. Recent data suggests that complete PTEN loss due to mutation is rare in primary human T-ALL, whereas PTEN-inhibiting posttranslational modifications are more common (Barata et al., J. Clin. Invest. 2008, 118). As these modifications decrease, but do not abolish the phosphatase activity of PTEN, we hypothesized that further input from tyrosine kinases, particularly receptor tyrosine kinases (RTK), may be needed to sustain PI3K-AKT-mTOR activation.In order to investigate how RTK-signaling may contribute to the pathogenesis of T-ALL we used an established murine bone marrow transplantation model (Li et al. Blood 2009, 113). To mimic tyrosine-kinase signaling we expressed δTrkA, a constitutively active TRKA receptor tyrosine kinase (TRK =tropomyosin-related kinase) from gammaretroviral or lentiviral vectors in c-kit+ Sca-1+ Lin− (KSL) cells. Intravenous injection of δTrkA-transduced hematopoietic cells in C57BL6 mice (n=10) induced transplantable T-ALL with a latency of about 120 days. The resulting T-ALLs could be propagated in culture as clonal cell lines. Signaling studies showed that δTRKA activates predominantly ERK upon expression in murine hematopoietic cell lines. However, the obtained δTRKA+ T-ALL lines (n=7) showed a profound shift in the use of downstream signaling cascades, displaying a very high activation of AKT-mTOR and absent ERK phosphorylation, resembling human T-ALL. High AKT activation was uniformly detected regardless of PTEN protein expression in all but one T-ALL (#003). To understand the rewired signaling network we looked for a potential contribution of insertional mutagenesis and chromosomal aberrations. Array-CGH showed homozygous deletions on chr14c2 involving the T-cell receptor alpha and delta genes in 3/3 cell lines and heterozygous deletions in Ikzf1 in 2/3 cell lines. Viral integration sites showed no common insertion pattern and no insertion in genes implicated in RTK-signaling. The expression of genes in proximity to viral integrations (±500 kb) appeared unaltered as determined by cDNA-microarray analysis of the T-ALL cell line #483 against wild type CD4+CD8+ thymocytes. Microarray analysis revealed enrichment of Notch1 target genes in the T-ALL cell line #483. Sequencing of Notch1 revealed both, PEST domain mutations and the recently described (Aster et al, Blood 2010, 116) RAG mediated 5’-deletions in cis, in all but one investigated T-ALL. Northern and Western Blots confirmed the expression of truncated Notch1 transcripts and protein, respectively. The one cell line (#003) which retained the original δTrkA signaling pattern had no Notch mutation and could only be cultured on OP9-Delta-like-1 stroma cells, highlighting the importance of Notch signaling. As this cell line was established from a mouse displaying an enlarged thymus, but no full manifestation of T-ALL, our data suggests that acquisition of Notch mutations is a late, but necessary step required for overt leukemia, whereas the initiating events may arise in kinase signaling pathways of prethymic progenitors. All T-ALL cell lines were sensitive to mTOR or Notch inhibition with Rapamycin or Compound E, respectively. Finally, we used phosphoprotein-arrays to monitor the phosphorylation of 42 RTK in childhood T-ALL samples with different activating NOTCH mutations (n=5) and detected several activated RTK (e.g. MSPR, FGFR, ErbB4, VEGFR) in the patient samples.Taken together, our findings suggest a cooperation of RTK and activating NOTCH mutations in mTOR activation seen in T-ALL and encourage further investigation of 1) aberrant RTK-signaling in T-ALL 2) the role of RTK activation in creating a preleukemic cell clone, 3) evaluation of combined therapy targeting RTKs and NOTCH, and 4) the role of activated NOTCH on mTORC2-AKT activation independently of PTEN. Disclosures:Baum:Patent office: Patents & Royalties.

MCL1 increases primitive thymocyte viability in female mice and promotes thymic expansion into adulthood

Increasing the pool of cells at early T-cell developmental stages enhances thymopoiesis and is especially beneficial when T-cell production is compromised by radiation or aging. Within the immature double-negative (DN; CD4(-)CD8(-)) thymocyte subpopulation, the DN1 subset contains the most primitive cells including the rare early T-cell progenitors (ETPs). In the present study, a human MCL1 transgene, under the control of its endogenous promoter, resulted in enlargement of an undistorted thymus in C57/BL6 mice. Enlargement occurred in females but not males, being seen at 1 month of age and maintained during progression into adulthood as the thymus underwent involution. The small DN1 subset was expanded disproportionally (ETPs increasing from ∼0.016 to 0.03% of thymocytes), while more mature thymocytes were increased proportionally (1.5-fold) along with the stroma. DN1 cells from transgenic females exhibited increased viability with maintained proliferation, and their survival in primary culture was extended. Exposure of transgenic females to γ-irradiation also revealed an expanded pool of radioresistant DN1 cells exhibiting increased viability. While the viability of DN1 cells from transgenic males was equivalent to that of their non-transgenic counterparts directly after harvest, it was enhanced in culture-suggesting that the effect of the transgene was suppressed in the in vivo environment of the male. Viability was increased in ETPs from transgenic females, but unchanged in more mature thymocytes, indicating that primitive cells were affected selectively. The MCL1 transgene thus increases the viability and pool size of primitive ETP/DN1 cells, promoting thymopoiesis and radioresistance in peripubescent females and into adulthood.

Deletion of genes encoding PU.1 and Spi-B in B cells impairs differentiation and induces pre-B cell acute lymphoblastic leukemia

AbstractThe E26 transformation-specific (Ets) transcription factor PU.1 is required to generate lymphoid progenitor cells from hematopoietic stem cells, but it is not required to generate B cells from committed B-cell lineage progenitors. We hypothesized that PU.1 function in B-cell differentiation is complemented by the related Ets transcription factor Spi-B. To test this hypothesis, mice were generated lacking both PU.1 and Spi-B in the B-cell lineage. Unlike mice lacking PU.1 or Spi-B, mice deficient in both PU.1 and Spi-B in the B-cell lineage had reduced frequencies of B cells as well as impaired B-cell differentiation. Strikingly, all PU.1 and Spi-B–deficient mice developed pre-B cell acute lymphoblastic leukemia before 30 weeks of age. Pre-B cells accumulated in the thymus resulting in massive thymic enlargement and dyspnea. These findings demonstrate that PU.1 and Spi-B are essential transcriptional regulators of B-cell differentiation as well as novel tumor suppressors in the B-cell lineage.

The autoimmune disease–associated PTPN22 variant promotes calpain-mediated Lyp/Pep degradation associated with lymphocyte and dendritic cell hyperresponsiveness

A variant of the PTPN22-encoded Lyp phosphatase (Lyp620W) confers risk for autoimmune disease, but the mechanisms underlying this association remain unclear. We show here that mice expressing the Lyp variant homolog Pep619W manifest thymic and splenic enlargement accompanied by increases in T-cell number, activation and positive selection and in dendritic- and B-cell activation. Although Ptpn22 (Pep) transcript levels were comparable in Pep619W and wild-type Pep619R mice, Pep protein levels were dramatically reduced in the mutant mice, with Pep619W protein being more rapidly degraded and showing greater association with and in vitro cleavage by calpain 1 than Pep619R. Similarly, levels of the Lyp620W variant were decreased in human T and B cells, and its calpain binding and cleavage were increased relative to wild-type Lyp620R. Thus, calpain-mediated degradation with consequently reduced Lyp/Pep expression and lymphocyte and dendritic cell hyperresponsiveness represents a mechanism whereby Lyp620W may increase risk for autoimmune disease.

Myasthenia Gravis–Associated Neuromyelitis Optica–Like Disease

Although overt involvement of the central nervous system (CNS) in myasthenia gravis (MG) is considered rare, hyperreflexia is a common and yet unexplained finding. Aquaporin 4 (AQP4), the target autoantigen in neuromyelitis optica, is expressed both in the CNS and in the neuromuscular junction. To evaluate the prevalence of even mild CNS involvement in patients with MG and to identify features indicative of neuromyelitis optica-like disease. Cohort study. Outpatient clinic. A cohort of 164 patients with MG. In 24 patients with MG, signs of CNS involvement were detected; 15 of these patients had at least 1 additional paraclinical indication of neuromyelitis optica-like disease (presence of antibodies against AQP4, pathological visual evoked potentials, or white matter lesions detected on brain and/or spinal magnetic resonance imaging scans) and fulfilled the inclusion and exclusion criteria for our study. Of the 15 patients who had at least 1 additional paraclinical indication of neuromyelitis optica-like disease, 14 had abnormal visual evoked potentials, and in 6 of 9 patients in whom magnetic resonance imaging was performed, there was evidence of lesions in the white matter of the brain and/or spinal cord. Anti-AQP4 antibodies were detected in 7 patients (out of the 14 tested). Thymic enlargement (hyperplasia or thymoma) was more frequent in patients with MG who had signs of CNS involvement than in patients with MG who did not. The incidence of CNS involvement in MG is higher than previously reported and is expressed predominantly as a pyramidal syndrome accompanied by optical tract involvement (frequently subclinical). These features bear some resemblance to neuromyelitis optica spectrum disease, supported also by the presence of anti-AQP4 antibodies in 7 of the 14 patients tested. This association may represent a new nosological entity or may indicate that an autoimmune process targeting AQP4 is an integral part of the immunopathogenetic mechanisms in MG.