Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytc leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n = 278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.

http://www.rticc.org/2006-2012/docs/programas/documento-121381205176b457a69c8cff2223a24563374476707973e5f9.pdf

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program

Deconstructing a Disease: RAR, Its Fusion Partners, and Their Roles in the Pathogenesis of Acute Promyelocytic Leukemia

PURPOSE: To determine the safety and efficacy of arsenic trioxide (ATO) in patients with relapsed acute promyelocytic leukemia (APL). PATIENTS AND METHODS: Forty patients experiencing first (n = 21) or ≥ second (n = 19) relapse were treated with daily infusions of ATO to a maximum of 60 doses or until all leukemic cells in bone marrow were eliminated. Patients who achieved a complete remission (CR) were offered one consolidation course of ATO that began 3 to 4 weeks later. Patients who remained in CR were eligible to receive further cycles of ATO therapy on a maintenance study. RESULTS: Thirty-four patients (85%) achieved a CR. Thirty-one patients (91%) with CRs had posttreatment cytogenetic tests negative for t(15;17). Eighty-six percent of the patients who were assessable by reverse transcriptase polymerase chain reaction converted from positive to negative for the promyelocytic leukemia/retinoic acid receptor-alpha transcript by the completion of their consolidation therapy. Thirty-two patients receive...

United States Multicenter Study of Arsenic Trioxide in Relapsed Acute Promyelocytic Leukemia

Distinctive gene expression profiles of CD34 cells from patients with myelodysplastic syndrome characterized by specific chromosomal abnormalities

Recent studies suggest that some serous ovarian carcinomas (SOCs) arise from the fallopian tube (FT) epithelium rather than the ovarian surface epithelium. This hypothesis places emphasis on the FT secretory epithelial cell as a cell-of-origin. Herein, we report the development of a novel ex vivo primary human FT epithelium culture system that faithfully recapitulates the in vivo epithelium, as shown by morphological, ultrastructural and immunophenotypic analyses. Mass spectrometry-based proteomics reveal that these cultures secrete proteins previously identified as biomarkers for ovarian cancer. We also use this culture system to study the response of the FT epithelium to genotoxic stress and find that the secretory cells exhibit a distinct response to DNA damage when compared with neighboring ciliated cells. The secretory cells show a limited ability to resolve the damage over time, potentially leaving them more susceptible to accumulation of additional mutagenic injury. This divergent response is confirmed with in situ studies using tissue samples, further supporting the use of this ex vivo culture system to investigate FT epithelial pathobiology. We anticipate that this novel culture system will facilitate the study of SOC pathogenesis, and propose that similar culture systems could be developed for other organ site-specific epithelia.

Primary ex vivo cultures of human fallopian tube epithelium as a model for serous ovarian carcinogenesis

Acute promyelocytic leukemia (APL) is characterized by a block to
myeloid differentiation caused by expression of the fusion oncoprotein
promyelocytic leukemia-retinoic acid receptor α (PML-RARα). The
purpose of this study was to identify genes that are regulated in a
PML-RARα-dependent fashion by retinoic acid (RA), because such genes
may be integrally involved in APL pathogenesis and/or myeloid
differentiation. A cDNA microarray approach was used to identify genes
induced in response to RA in TF1 myeloid leukemia cells expressing
PML-RARα (TF1-PR cells). The B94 gene (TNFAIP2; Unigene
Hs.101382), originally identified as a tumor necrosis factorα
-inducible gene in endothelial cells, was one of several genes found
to be induced by RA specifically in TF1-PR cells, but not in TF1-neo
(control) cells. The induction of B94 was most pronounced in cells
expressing the PML-RARα short isoform and was negligible in cells
that expressed a mutant PML-RARα protein containing a deletion of the
PML coiled-coil domain. B94 induction by RA occurred within 1 h,
did not require new protein synthesis, and was inhibited by actinomycin
D, suggesting rapid transcriptional activation. B94 was also induced by
RA in NB4, UF1, and HL-60 cells, but not in other hematopoietic cell
lines tested, suggesting that its up-regulation by RA may be specific
to cells that express PML-RARα or are at the late myeloblast or
promyelocyte stage of myeloid development. A screen of bone marrow
cells from normal donors or patients with acute myelogenous leukemia
showed that B94 was highly expressed in normal marrow and in marrow
from patients with acute myelogenous leukemia French-American-British
subtypes M 0 -M 2 , but was repressed in marrow
cells from APL patients. Treatment of APL blasts in vitro 
with all- trans -RA resulted in up-regulation of B94 mRNA.
These results suggest that B94 plays a role in myeloid development and
support the hypothesis that B94 is a target gene of PML-RARα in APL.

https://cancerres.aacrjournals.org/content/canres/60/7/1824.full.pdf

Identification of B94 (TNFAIP2) as a potential retinoic acid target gene in acute promyelocytic leukemia.

Constitutive Expression of the Promyelocytic Leukemia–Associated Oncogene PML-RARα in TF1 Cells: Isoform-Specific and Retinoic Acid–Dependent Effects on Growth, bcl-2 Expression, and Apoptosis

Pre-Clinical Validation of a Novel, Highly Sensitive Assay to Detect PML-RARα mRNA Using Real-Time Reverse-Transcription Polymerase Chain Reaction

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Papers

Identification of B94 (TNFAIP2) as a potential retinoic acid target gene in acute promyelocytic leukemia.

Constitutive Expression of the Promyelocytic Leukemia–Associated Oncogene PML-RARα in TF1 Cells: Isoform-Specific and Retinoic Acid–Dependent Effects on Growth, bcl-2 Expression, and Apoptosis

Pre-Clinical Validation of a Novel, Highly Sensitive Assay to Detect PML-RARα mRNA Using Real-Time Reverse-Transcription Polymerase Chain Reaction