RepSox slows decay of CD34+ acute myeloid leukemia cells and decreases T cell immunoglobulin mucin-3 expression.

Despite initial response to therapy, most acute myeloid leukemia (AML) patients relapse. To eliminate relapse-causing leukemic stem/progenitor cells (LPCs), patient-specific immune therapies may be required. In vitro cellular engineering may require increasing the "stemness" or immunogenicity of tumor cells and activating or restoring cancer-impaired immune-effector and antigen-presenting cells. Leukapheresis samples provide the cells needed to engineer therapies: LPCs to be targeted, normal hematopoietic stem cells to be spared, and cancer-impaired immune cells to be repaired and activated. This study sought to advance development of LPC-targeted therapies by exploring nongenetic ways to slow the decay and to increase the immunogenicity of primary CD34(+) AML cells. CD34(+) AML cells generally displayed more colony-forming and aldehyde dehydrogenase activity than CD34(-) AML cells. Along with exposure to bone marrow stromal cells and low (1%-5%) oxygen, culture with RepSox (a reprogramming tool and inhibitor of transforming growth factor-ß receptor 1) consistently slowed decline of CD34(+) AML and myelodysplastic syndrome (MDS) cells. RepSox-treated AML cells displayed higher CD34, CXCL12, and MYC mRNA levels than dimethyl sulfoxide-treated controls. RepSox also accelerated loss of T cell immunoglobulin mucin-3 (Tim-3), an immune checkpoint receptor that impairs antitumor immunity, from the surface of AML and MDS cells. Our results suggest RepSox may reduce Tim-3 expression by inhibiting transforming growth factor-ß signaling and slow decay of CD34(+) AML cells by increasing CXCL12 and MYC, two factors that inhibit AML cell differentiation. By prolonging survival of CD34(+) AML cells and reducing Tim-3, RepSox may promote in vitro immune cell activation and advance development of LPC-targeted therapies.

c-MYC Amplification in Acute Myelogenous Leukemia Evolving from Double Minutes (dmins) to Homogeneously Staining Region (hsr).

A bone marrow biopsy of a 68-year-old woman revealed 59% blasts and immature monocytes, consistent with acute myeloid leukemia (AML) with monocytic features. Occasional hypolobated megakaryocytes and decreased iron stores were also present. A peripheral blood sample showed 7% blasts in addition to monocytosis, macrocytic anemia and thrombocytopenia. Molecular testing was negative for FLT3-ITD, NPM1 and CEBPA. Fluorescence in situ hybridization (FISH) probes were negative for t(8;21), t(15;17), inversion 16 and 11q23 rearrangements. The karyotype was 46,XX,del(20)(q11.2q13.1),~50dmin[3]/47,idem,+4[13]/47,idem,+22[2]/46,XX[2]. FISH confirmed that the double minutes were c-MYC positive with cryptic deletion of the c-MYC FISH signal on one of the chromosome 8s. Two months post-diagnosis, 57% of our patient's cells were still positive for c-MYC amplification; however, by four months only 8% of cells were positive for c-MYC amplification. After seven months, the patient's karyotype had the 20q deletion, X chromosome loss and a ring chromosome that consisted of a homogeneously staining region (hsr) containing c-MYC amplification. This case demonstrates that gene amplification in the form of double minutes can transform into a more stable hsr.

Persistent mosaicism for 12p duplication/triplication chromosome structural abnormality in peripheral blood.

We present a rare case of mosaicism for a structural abnormality of chromosome 12 in a patient with phenotypic features of Pallister-Killian syndrome. A six-month-old child with dysmorphic features, exotropia, hypotonia, and developmental delay was mosaic for both a normal karyotype and a cell line with 12p duplication/triplication in 25 percent of metaphase cells. Utilization of fluorescence in situ hybridization (FISH) identified three copies of probes from the end of the short arm of chromosome 12 (TEL(12p13) locus and the subtelomere (12p terminal)) on the structurally abnormal chromosome 12. Genome-wide SNP array analysis revealed that the regions of duplication and triplication were of maternal origin. The abnormal cell line in our patient was present at 25 percent at six months and 19 months of age in both metaphase and interphase cells from peripheral blood, where typically the isochromosome 12p is absent in the newborn. This may suggest that the gene(s) resulting in a growth disadvantage of abnormal cells in peripheral blood of patients with tetrasomy 12p may not have the same influence when present in only three copies.

Patterns of BCR/ABL Gene Rearrangements in Chronic Myeloid Leukemia with Complex t(9;22) Using Fluorescence In Situ Hybridization (FISH).

Chronic myeloid leukemia (CML) is characterized by the reciprocal translocation t(9;22)(q34;q11.2) which fuses the ABL1 oncogene on chromosome 9 with the BCR gene on chromosome 22. It is the BCR/ABL protein that drives the neoplasm and the ABL/BCR is not necessary for the disease. In the majority of CML cases, the BCR/ABL fusion gene is cytogenetically recognizable as a small derivative chromosome 22(der 22), which is known as the Philadelphia (Ph) chromosome. However, approximately 2-10% of patients with CML involve cryptic or complex variant translocations with deletions on the der(9) and/or der(22) occuring in roughly 10-15% of CML cases. Fluorescence in situ hybridization (FISH) analysis can help identify deletions and complex or cryptic rearrangements. Various BCR/ABL FISH probes are available, which include dual color single fusion, dual color extra signal (ES), dual color dual fusion and tri color dual fusion probes. To test the utility of these probes, six patients diagnosed with CML carrying different complex variant Ph translocations were studied by G-banding and FISH analysis using the BCR/ABL ES, BCR/ABL dual color dual fusion, and BCR/ABL tricolor probes. There are differences among the probes in their ability to detect variant rearrangements, with or without accompanying chromoso me 9 and/or 22 deletions, and low level disease.

Case report: cytogenetic and molecular analysis of proximal interstitial deletion of 4p, review of the literature and comparison with wolf-hirschhorn syndrome.

We report on a two-year-old female with a de novo proximal interstitial deletion of the short arm of chromosome 4 and a tetralogy of Fallot malformation. The patient had a karyotype of 46,XX,del(4)(p14p15.33) that was further characterized by array comparative genomic hybridization (aCGH). Phenotypic abnormalities for our patient are compared with those of previously reported patients with similar proximal 4p deletions as well as more distal deletions. The functions of genes that are deleted within this segment are reviewed.

Cryptic subtelomeric rearrangements and X chromosome mosaicism: a study of 565 apparently normal individuals with fluorescent in situ hybridization.

Five percent of patients with unexplained mental retardation have been attributed to cryptic unbalanced subtelomeric rearrangements. Half of these affected individuals have inherited the rearrangement from a parent who is a carrier for a balanced translocation. However, the frequency of carriers for cryptic balanced translocations is unknown. To determine this frequency, 565 phenotypically normal unrelated individuals were examined for balanced subtelomeric rearrangements using Fluorescent In Situ hybridization (FISH) probes for all subtelomere regions. While no balanced subtelomeric rearrangements were identified, three females in this study were determined to be mosaic for the X chromosome. Mosaicism for XXX cell lines were observed in the lymphocyte cultures of 3 in 379 women (0.8%), which is a higher frequency than the 1 in 1000 (0.1%) reported for sex chromosome aneuploidies. Our findings suggest that numerical abnormalities of the X chromosome are more common in females than previously reported. Based on a review of the literature, the incidence of cryptic translocation carriers is estimated to be approximately 1/8,000, more than ten-fold higher than the frequency of visible reciprocal translocations.

Human telomere length correlates to the size of the associated chromosome arm.

The majority of human telomere length studies have focused on the overall length of telomeres within a cell. In fact, very few studies have examined telomere length for individual chromosome arms. The objective of this study was to examine the relationship between chromosome arm size and the relative length of the associated telomere. Quantitative Fluorescence In Situ Hybridization (Q-FISH) was used to measure the relative telomere length of each chromosome arm in metaphases from cultured lymphocytes of 17 individuals. A statistically significant positive correlation (r = 0.6) was found between telomere length and the size of the associated chromosome arm, which was estimated based on megabase pair measurements from (http://www.ncbi.nlm.nih.gov/projects/mapview/).

Del(5q) is associated with clinical and histological parameters in small cell neuroendocrine lung carcinoma.

To elucidate the relationship between del(5q) and the clinical and histological features of small cell neuroendocrine lung carcinoma, 33 tissue samples from patients with this tumor were evaluated. By using fluorescence in situ hybridization, del(5q) was identified in almost 50% of cases (15/33, 45%). Clinically, patients with tumors showing del(5q) were older (mean age = 71 years) with a correspondingly greater pack-year smoking history (mean = 61) than patients with tumors (mean age = 59 years, mean pack-years = 44) without del(5q). Histologically, tumors with del(5q) had a greater frequency of spindle cell morphology (11/14 [79%] vs 6/16 [38%], P < .025) than those without del(5q). This is the first study to find an association between del(5q) and tumor histology in small cell neuroendocrine lung carcinoma.

BRCA1 contributes to cell cycle arrest and chemoresistance in response to the anticancer agent irofulven.

Tumor suppressor gene BRCA1 is frequently mutated in familial breast and ovarian cancer. BRCA1 plays pivotal roles in maintaining genomic stability by interacting with numerous proteins in cell cycle control and DNA repair. Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863) is one of a new class of anticancer agents that are analogs of mushroom-derived illudin toxins. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor cell types. The exact nature of irofulven-induced DNA damage is not completely understood. We demonstrated previously that irofulven activates ATM and its targets, NBS1, SMC1, CHK2, and p53. In this study, we hypothesize that irofulven induces DNA double-strand breaks and that BRCA1 may affect chemosensitivity by controlling cell cycle checkpoints, DNA repair, and genomic stability in response to irofulven treatment. We observed that irofulven induces the formation of chromosome breaks and radials and the activation and foci formation of gamma-H2AX, BRCA1, and RAD51. We also provided evidence that irofulven induces the generation of DNA double-strand breaks. By using BRCA1-deficient or -proficient cells, we demonstrated that in response to irofulven, BRCA1 contributes to the control of S and G(2)/M cell cycle arrest and is critical for repairing DNA double-strand breaks and for RAD51-dependent homologous recombination. Furthermore, we found that BRCA1 deficiency results in increased chromosome damage and chemosensitivity after irofulven treatment.

Fanconi anemia D2 protein confers chemoresistance in response to the anticancer agent, irofulven.

The Fanconi anemia-BRCA pathway of genes are frequently mutated or epigenetically repressed in human cancer. The proteins of this pathway play pivotal roles in DNA damage signaling and repair. Irofulven is one of a new class of anticancer agents that are analogues of mushroom-derived illudin toxins. Preclinical studies and clinical trials have shown that irofulven is effective against several tumor cell types. The exact nature of irofulven-induced DNA damage is not completely understood. Previously, we have shown that irofulven activates ATM and its targets, NBS1, SMC1, CHK2, and p53. In this study, we hypothesize that irofulven induces DNA double-strand breaks and FANCD2 may play an important role in modulating cellular responses and chemosensitivity in response to irofulven treatment. By using cells that are proficient or deficient for FANCD2, ATR, or ATM, we showed that irofulven induces FANCD2 monoubiquitination and nuclear foci formation. ATR is important in mediating irofulven-induced FANCD2 monoubiquitination. Furthermore, we showed that FANCD2 plays a critical role in maintaining chromosome integrity and modulating chemosensitivity in response to irofulven-induced DNA damage. Therefore, this study suggests that it might be clinically significant to target irofulven therapy to cancers defective for proteins of the Fanconi anemia-BRCA pathway.

The spectrum of WRN mutations in Werner syndrome patients.

The International Registry of Werner syndrome (www.wernersyndrome.org) has been providing molecular diagnosis of the Werner syndrome (WS) for the past decade. The present communication summarizes, from among 99 WS subjects, the spectrum of 50 distinct mutations discovered by our group and by others since the WRN gene (also called RECQL2 or REQ3) was first cloned in 1996; 25 of these have not previously been published. All WRN mutations reported thus far have resulted in the elimination of the nuclear localization signal at the C-terminus of the protein, precluding functional interactions in the nucleus; thus, all could be classified as null mutations. We now report two new mutations in the N-terminus that result in instability of the WRN protein. Clinical data confirm that the most penetrant phenotype is bilateral ocular cataracts. Other cardinal signs were seen in more than 95% of the cases. The median age of death, previously reported to be in the range of 46-48 years, is 54 years. Lymphoblastoid cell lines (LCLs) have been cryopreserved from the majority of our index cases, including material from nuclear pedigrees. These, as well as inducible and complemented hTERT (catalytic subunit of human telomerase) immortalized skin fibroblast cell lines are available to qualified investigators.

Molecular characterization of an 11q interstitial deletion in a patient with the clinical features of Jacobsen syndrome.

The 11q terminal deletion disorder or Jacobsen syndrome is a contiguous gene disorder. It is characterized by psychomotor retardation, cardiac defects, blood dyscrasias (Paris-Trousseau syndrome) and craniofacial anomalies. We report on a female patient with an approximately 10 Mb interstitial deletion with many of the features of Jacobsen syndrome: A congenital heart defect, dysmorphic features, developmental delay, and Paris-Trousseau syndrome. The karyotype of the patient is 46,XX,del(11)(q24.1q24.3). The interstitial deletion was confirmed using FISH probes for distal 11q, and the breakpoints were characterized by microarray analysis. This is the first molecularly characterized interstitial deletion in a patient with the clinical features of Jacobsen syndrome. The deletion includes FLI-1, but not JAM-3, which will help to determine the critical genes involved in this syndrome.

The Pittsburgh Oral-Facial Cleft study: expanding the cleft phenotype. Background and justification.

The Pittsburgh Oral-Facial Cleft study was begun in 1993 with the primary goal of identifying genes involved in nonsyndromic orofacial clefts in a variety of populations worldwide. Based on the results from a number of pilot studies and preliminary genetic analyses, a new research focus was added to the Pittsburgh Oral-Facial Cleft study in 1999: to elucidate the role that associated phenotypic features play in the familial transmission patterns of orofacial clefts in order to expand the definition of the nonsyndromic cleft phenotype. The purpose of this paper is to provide a comprehensive review of phenotypic features associated with nonsyndromic orofacial clefts. These features include fluctuating and directional asymmetry, non-right-handedness, dermatoglyphic patterns, craniofacial morphology, orbicularis oris muscle defects, dental anomalies, structural brain and vertebral anomalies, minor physical anomalies, and velopharyngeal incompetence.

Cell death as a possible mechanism for tissue limited mosaicism in Pallister-Killian syndrome.

Pallister-Killian syndrome is a chromosomal mosaic syndrome with a normal and an isochromosome 12p cell line, the latter rarely seen in peripheral blood. The isochromosome 12p cell line decreases with serial passages of fibroblasts in vitro and with age of patient in vivo. To evaluate cell death as a possible mechanism for loss of the abnormal cell line, amniocytes from a fetus with Pallister-Killian syndrome were identified as normal or aneuploid using a chromosome 12 alpha-satellite DNA probe by fluorescent in situ hybridization (FISH) and then subsequently stained with Annexin V, which stains the cytoplasm of cells that are dying. Although not conclusive, our preliminary results suggest that the abnormal cell line is going through apoptosis or necrosis at a higher rate than normal cells. Cell death may be a possible mechanism for decrease of the aneuploid cell line in patients with Pallister-Killian syndrome.

Prenatal detection of deletion 6q13q15 in a complex karyotype.

OBJECTIVES: Prenatal diagnosis of a pregnancy with elevated maternal serum alpha-fetoprotein identified a karyotype with a complex chromosomal rearrangement, a Robertsonian translocation and a 6q deletion involving bands q13q15. Sonography identified mild IUGR, polyhydramnios and micrognathia. The infant presented with multiple congenital anomalies, primarily limited to the head and neck, including hypertelorism, broad nose, micrognathia, cleft palate, microglossia and low-set ears with microtia. METHODS: Amniocytes of the fetus and blood of the patient and her parents were analyzed by cytogenetics and fluorescence in situ hybridization. RESULTS: The karyotype on the fetus was 45,XX,t(3;21;20)(p12;q11.2;p11.2), del(6)(q13q15),der(13;14) (q10;q10)mat. CONCLUSION: The 13;14 Robertsonian translocation was inherited from the mother and the three-way translocation appeared to be balanced. The patient had facial dysmorphology similar to that which has been described in 6 previously reported cases with the same deletion involving 6q13q15. There was no recognizable abnormality of limbs or digits, and the autopsy did not identify defects involving the internal organs.

Extra copy of 20q deletion, acute myelomonocytic leukemia (M4) and Niemann-Pick disease.

A 59-year-old hypertensive white male was diagnosed with acute myelogenous leukemia (AML), M4. A bone marrow aspirate showed a karyotype of 46,XY,del(20)(q11.2q13.3)[12]/ 47,XY,del(20)(q11.2q13.3)x2[8]. The majority of cases with 20q deletion are associated with myeloid disorders; however, an extra copy of the 20q deletion has rarely been reported. The patient expired seven days after admission to the hospital. At autopsy hepatosplenomegaly was present. Many foamy macrophages with bubbling cytoplasm in the spleen, liver, bone marrow and lymph nodes were suggestive of Niemann-Pick disease, type E. AML has not previously been reported with Niemann-Pick disease.

Use of the glycophorin A somatic mutation assay for rapid, unambiguous identification of Fanconi anemia homozygotes regardless of GPA genotype.

A 7-year-old girl was hospitalized with pancytopenia requiring blood transfusion. She and an older brother with suspicious symptoms were referred for laboratory testing to confirm a clinical diagnosis of Fanconi anemia (FA). Blood samples from these two children and one parent were examined with the GPA somatic mutation assay. The patient's total GPA somatic mutation frequency of 1.4 x 10(-4) was determined despite the confounding effects of her recent transfusion, and was greater than 10-fold higher than that of a population of pediatric controls, consistent with the known FA phenotype. Her brother was not informative for the standard GPA assay, which requires heterozygosity for the MN blood group, but was analyzed with a modified assay that measured only allele loss mutation. His mutation frequency, 6.8 x 10(-4) was also supportive of a diagnosis of FA. Both analyses also showed evidence of ongoing mutation through terminal erythroblast differentiation, a characteristic of patients with DNA repair syndromes which further confirmed the diagnoses. These conclusions were confirmed with traditional DEB-induced chromosome breakage studies. The quantitative and qualitative aspects of the GPA assay relevant for applying this test for FA diagnosis, and perhaps for carrier detection, are discussed.