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author:("gockel, C D")
1.  Risk-based classification of leukemia by cytogenetic and multiplex molecular methods: results from a multicenter validation study 
Blood Cancer Journal  2012;2(7):e78-.
Modern management of leukemia and selection of optimal treatment approaches entails the analysis of multiple recurrent cytogenetic abnormalities with independent diagnostic or prognostic value. We report the first multicenter validation of a multiplex molecular assay for 12 relevant fusion transcripts relative to cytogenetic methods. Performance was evaluated using a set of 280 adult and pediatric acute or chronic leukemias representative of the variety of presentations and pre-analytical parameters encountered in the clinical setting. The positive, negative and overall agreements were >98.5% with high concordance at each of the four sites. Positive detection of cases with low blast count or at relapse was consistent with a method sensitivity of 1%. There was 98.7% qualitative agreement with independent reference molecular tests. Apparent false negatives corresponded to rare alternative splicing isoforms not included in the panel. We further demonstrate that clinical sensitivity can be increased by adding those rare variants and other relevant transcripts or submicroscopic abnormalities. We conclude that multiplex RT-PCR followed by liquid bead array detection is a rapid and flexible method attuned to the clinical laboratory workflow, complementing standard cytogenetic methods and generating additional information valuable for the accurate diagnosis, prognosis and subsequent molecular monitoring of leukemia.
doi:10.1038/bcj.2012.24
PMCID: PMC3408638  PMID: 22852047
leukemia; diagnosis; prognosis; molecular classification; RT-PCR; multiplex
2.  Immunologic recovery following autologous stem-cell transplantation with pre- and posttransplantation rituximab for low-grade or mantle cell lymphoma 
Annals of Oncology  2009;21(6):1203-1210.
Background: Rituximab may improve transplant outcomes but may delay immunologic recovery.
Patients and methods: Seventy-seven patients with low-grade or mantle cell lymphoma received autologous stem-cell transplantation (ASCT) on a phase II study. Rituximab 375 mg/m2 was administered 3 days before mobilization-dose cyclophosphamide, then weekly for four doses after count recovery from ASCT. Immune reconstitution was assessed.
Results: Sixty percent of transplants occurred in first remission. Actuarial event-free survival (EFS) and overall survival (OS) were 60% and 73%, respectively, at 5 years, with 7.2-year median follow-up for OS in surviving patients. Median EFS was 8.3 years. Older age and transformed lymphomas were independently associated with inferior EFS, whereas day 60 lymphocyte counts did not predict EFS or late infections. Early and late transplant-related mortality was 1% and 8%, with secondary leukemia in two patients. B-cell counts recovered by 1–2 years; however, the median IgG level remained low at 2 years. Late-onset idiopathic neutropenia, generally inconsequential, was noted in 43%.
Conclusion: ASCT with rituximab can produce durable remissions on follow-up out to 10 years. Major infections do not appear to be significantly increased or to be predicted by immune monitoring.
doi:10.1093/annonc/mdp484
PMCID: PMC2875548  PMID: 19880437
immune reconstitution; lymphoma; rituximab; transplantation
3.  High frequencies of leukemia stem cells in poor-outcome childhood precursor-B acute lymphoblastic leukemias 
Leukemia  2010;24(11):1859-1866.
In order to develop a xenograft model to determine the efficacy of new therapies against primary human precursor-B acute lymphoblastic leukemia (ALL) stem cells (LSCs), we used the highly immunodeficient non-obese diabetic (NOD).Cg-PrkdcscidIL2rgtmlWjl/SzJ (NOD-severe combined immune deficient (scid) IL2rg−/−) mouse strain. Intravenous transplantation of 2 of 2 ALL cell lines and 9 of 14 primary ALL cases generated leukemia-like proliferations in recipient mice by 1–7 months after transplant. Leukemias were retransplantable, and the immunophenotypes, gene rearrangements and expression profiles were identical or similar to those of the original primary samples. NOD-scid mice transplanted with the same primary samples developed similar leukemias with only a slightly longer latency than did NOD-scid-IL2Rg−/− mice. In this highly sensitive NOD-scid-IL2Rg−/−-based assay, 1–100 unsorted primary human ALL cells from five of five tested patients, four of whom eventually experienced leukemia relapse, generated leukemias in recipient mice. This very high frequency of LSCs suggests that a hierarchical LSC model is not valuable for poor-outcome ALL.
doi:10.1038/leu.2010.184
PMCID: PMC3035974  PMID: 20739953
childhood acute lymphoblastic leukemia; leukemia stem cells; xenograft
4.  Detection of mutant K-ras DNA in plasma or serum of patients with colorectal cancer. 
British Journal of Cancer  1997;76(10):1293-1299.
Increased understanding of the molecular basis of colorectal cancer and recognition that extracellular DNA circulates in the plasma and serum of cancer patients enables new approaches to detection and monitoring. We used a polymerase chain reaction (PCR) assay to demonstrate mutant K-ras DNA in the plasma or serum of patients with colorectal cancer. Plasma or serum was fractionated from the blood of 31 patients with metastatic or unresected colorectal cancer and from 28 normal volunteers. DNA was extracted using either a sodium chloride or a gelatin precipitation method and then amplified in a two-stage PCR assay using selective restriction enzyme digestion to enrich for mutant K-ras DNA. Mutant K-ras DNA was detected in the plasma or serum of 12 (39%) patients, all confirmed by sequencing, but was not detected in any of the normal volunteers. K-ras mutations were detected in plasma or serum regardless of sex, primary tumour location, principal site of metastasis or proximity of chemotherapy and surgery to blood sampling. Tumour specimens available for 19 of the patients were additionally assayed for ras mutations and compared with blood specimens. Our results indicate mutant K-ras DNA is readily detectable by PCR in the plasma or serum of patients with advanced colorectal cancer. Thus, plasma- or serum-based nucleic acid amplification assays may provide a valuable method of monitoring and potentially detecting colorectal cancer.
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PMCID: PMC2228153  PMID: 9374374

Results 1-5 (5)