Derivation of Breast Cancer Cell Lines Under Physiological (5%) Oxygen Concentrations

Background: Most human breast cancer cell lines currently in use were developed and are cultured under ambient (21%) oxygen conditions. While this is convenient in practical terms, higher ambient oxygen could increase oxygen radical production, potentially modulating signaling pathways. We have derived and grown a series of four human breast cancer cell lines under 5% oxygen, and have compared their properties to those of established breast cancer lines growing under ambient oxygen.Methods: Cell lines were characterized in terms of appearance, cellular DNA content, mutation spectrum, hormone receptor status, pathway utilization and drug sensitivity.Results: Three of the four lines (NZBR1, NZBR2, and NZBR4) were triple negative (ER-, PR-, HER2-), with NZBR1 also over-expressing EGFR. NZBR3 was HER2+ and ER+ and also over-expressed EGFR. Cell lines grown in 5% oxygen showed increased expression of the hypoxia-inducible factor 1 (HIF-1) target gene carbonic anhydrase 9 (CA9) and decreased levels of ROS. As determined by protein phosphorylation, NZBR1 showed low AKT pathway utilization while NZBR2 and NZBR4 showed low p70S6K and rpS6 pathway utilization. The lines were characterized for sensitivity to 7-hydroxytamoxifen, doxorubicin, paclitaxel, the PI3K inhibitor BEZ235 and the HER inhibitors lapatinib, afatinib, dacomitinib, and ARRY-380. In some cases they were compared to established breast cancer lines. Of particular note was the high sensitivity of NZBR3 to HER inhibitors. The spectrum of mutations in the NZBR lines was generally similar to that found in commonly used breast cancer cell lines but TP53 mutations were absent and mutations in EVI2B, LRP1B, and PMS2, which have not been reported in other breast cancer lines, were detected. The results suggest that the properties of cell lines developed under low oxygen conditions (5% O2) are similar to those of commonly used breast cancer cell lines. Although reduced ROS production and increased HIF-1 activity under 5% oxygen can potentially influence experimental outcomes, no difference in sensitivity to estrogen or doxorubicin was observed between cell lines cultured in 5 vs. 21% oxygen

Fluorescence in situ hybridization studies: Break apart or not?

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AML-M2 with der(18)t(1;18)(q2?;p11.3) in addition to t(8;21) and del(9q)

We report a case of Acute Myeloid Leukemia with clinical features suggestive of AML-M3 and 46,XX,t(8;21),del(9q),der(18)t(1;18) karyotype leading to the final diagnosis AML-M2 in light of t(8;21). The Deletion (9q) is a frequent secondary anomaly to the t(8;21)(q22;q22) in AML-M2. In addition to these two AML-M2 related rearrangements we also observed der(18)t(1;18)(q2?;p11.3) which may be an unusual rearrangement. This rearrangement resulted into partial trisomy of chromosome #1(q2?) without the loss of any part of chromosome 18, morphologically. Rearrangements of long arm of chromosome 1 that result in complete or partial trisomy for 1q mostly involved the region q25-q32, which may confer a proliferation advantage

An ETV6-ABL1 fusion in a patient with chronic myeloproliferative neoplasm: Initial response to Imatinib followed by rapid transformation into ALL

We report the case of a 26 year-old patient presenting with a persistent leukocytosis and CML-like marrow but no evidence of a BCR/ABL1 fusion. Molecular cytogenetics revealed that a portion of the ETV6 locus was inserted into the ABL1 locus. An ETV6/ABL1 fusion transcript could subsequently be confirmed. The patient was started on imatinib and went into complete cytomorphological remission. QRT-PCR measurements showed a 4 log reduction of the ETV6/ABL1 fusion. 15 months later, the disease transformed into ALL and the patient expired. Thus, an ETV6/ABL1 fusion positive MPN has the potential to transform very rapidly into ALL

Loss of sex chromosome in acute myeloid leukemia

Loss of sex chromosomes has been reported in normal and malignant marrows and its frequency increases with age in both situations. It is not clear whether the sex chromosome loss is a critical mutational event for neoplastic transformation or a genetic change related to ageing. The present study was undertaken to analyze incidence of loss of sex chromosomes in leukemia patients. Karyotypic analysis in bone marrow cells was carried out in total 270 AML patients registered at G.C.& R.I. during January 2000 to October 2003. Out of 270, 22 patients had loss of sex chromosome in addition to other disease specific chromosomal abnormalities. Out of 22 patients, 50% (11 of 22) were of the pediatric age (up to 14 years), and only 10% (3 of 22) patients were above the age of 50 years, maximum age being 65 years. On follow-up, only in patients with pathological remission normal 46XX/XY karyotypes were seen. Whereas in patients with persistent leukemic activity, clones with loss of sex chromosome were observed. The results indicate that sex chromosome loss in these cases may be equivalent of a clonal cytogenetic process rather than related to ageing process

Mutations in SEC24D, Encoding a Component of the COPII Machinery, Cause a Syndromic Form of Osteogenesis Imperfecta

As a result of a whole-exome sequencing study, we report three mutant alleles in SEC24D, a gene encoding a component of the COPII complex involved in protein export from the ER: the truncating mutation c.613C>T (p.Gln205*) and the missense mutations c.3044C>T (p.Ser1015Phe, located in a cargo-binding pocket) and c.2933A>C (p.Gln978Pro, located in the gelsolin-like domain). Three individuals from two families affected by a similar skeletal phenotype were each compound heterozygous for two of these mutant alleles, with c.3044C>T being embedded in a 14 Mb founder haplotype shared by all three. The affected individuals were a 7-year-old boy with a phenotype most closely resembling Cole-Carpenter syndrome and two fetuses initially suspected to have a severe type of osteogenesis imperfecta. All three displayed a severely disturbed ossification of the skull and multiple fractures with prenatal onset. The 7-year-old boy had short stature and craniofacial malformations including macrocephaly, midface hypoplasia, micrognathia, frontal bossing, and down-slanting palpebral fissures. Electron and immunofluorescence microscopy of skin fibroblasts of this individual revealed that ER export of procollagen was inefficient and that ER tubules were dilated, faithfully reproducing the cellular phenotype of individuals with cranio-lentico-sutural dysplasia (CLSD). CLSD is caused by SEC23A mutations and displays a largely overlapping craniofacial phenotype, but it is not characterized by generalized bone fragility and presented with cataracts in the original family described. The cellular and morphological phenotypes we report are in concordance with the phenotypes described for the Sec24d-deficient fish mutants vbi (medaka) and bulldog (zebrafish)

Contribution of mutant HSC clones to immature and mature cells in MDS and CMML, and variations with AZA therapy.

Myelodysplastic Neoplasms (MDS) and Chronic Myelomonocytic Leukemia (CMML) are clonal disorders driven by progressively acquired somatic mutations in hematopoietic stem cells (HSCs). Hypomethylating agents (HMA) can modify the clinical course of MDS and CMML. Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated HSCs. However, in patients with established disease it is unclear whether; (a) HSCs with multiple mutations progress through differentiation with comparable frequency to their less mutated counterparts, or (b) improvements in peripheral blood counts following HMA therapy is driven by residual wild-type HSCs or by clones with particular combinations of mutations. To address these questions, we characterised the somatic mutations of individual stem, progenitor (common myeloid progenitor, granulocyte monocyte progenitor, megakaryocyte erythroid progenitor), and matched circulating (monocyte, neutrophil, naïve B) hematopoietic cells in MDS and CMML via high-throughput single-cell genotyping, followed by bulk analysis in immature and mature cells before and after AZA treatment. The mutational burden was similar throughout differentiation, with even the most mutated stem and progenitor clones maintained their capacity to differentiate to mature cell types in vivo. Increased contributions from productive mutant progenitors appear to underlie improved hematopoiesis in MDS following HMA therapy