A time-and-motion approach to micro-costing of high-throughput genomic assays.

BACKGROUND: Genomic technologies are increasingly used to guide clinical decision-making in cancer control. Economic evidence about the cost-effectiveness of genomic technologies is limited, in part because of a lack of published comprehensive cost estimates. In the present micro-costing study, we used a time-and-motion approach to derive cost estimates for 3 genomic assays and processes-digital gene expression profiling (gep), fluorescence in situ hybridization (fish), and targeted capture sequencing, including bioinformatics analysis-in the context of lymphoma patient management. METHODS: The setting for the study was the Department of Lymphoid Cancer Research laboratory at the BC Cancer Agency in Vancouver, British Columbia. Mean per-case hands-on time and resource measurements were determined from a series of direct observations of each assay. Per-case cost estimates were calculated using a bottom-up costing approach, with labour, capital and equipment, supplies and reagents, and overhead costs included. RESULTS: The most labour-intensive assay was found to be fish at 258.2 minutes per case, followed by targeted capture sequencing (124.1 minutes per case) and digital gep (14.9 minutes per case). Based on a historical case throughput of 180 cases annually, the mean per-case cost (2014 Canadian dollars) was estimated to be $1,029.16 for targeted capture sequencing and bioinformatics analysis, $596.60 for fish, and $898.35 for digital gep with an 807-gene code set. CONCLUSIONS: With the growing emphasis on personalized approaches to cancer management, the need for economic evaluations of high-throughput genomic assays is increasing. Through economic modelling and budget-impact analyses, the cost estimates presented here can be used to inform priority-setting decisions about the implementation of such assays in clinical practice.

SNP analysis of minimally evolved t(14;18)(q32;q21)-positive follicular lymphomas reveals a common copy-neutral loss of heterozygosity pattern.

Follicular lymphoma (FL) cases with a t(14;18)(q32;q21) and minimal or no additional karyotypic alterations, such as copy number gains and losses and/or chromosomal rearrangements, may exhibit pathologic features and a clinical behavior similar to those with more complex karyotypes. This study sought to investigate whether the copy-neutral loss of heterozygosity (cnLOH) profiles of these minimally evolved t(14;18)(q32;q21)-positive follicular lymphoma (MEV-FL) cases are similar to or different from the majority of FL cases with more karyotypic alterations. Affymetrix SNP 6.0 array analysis was applied to the tumor genomes of 23 MEV-FL biopsy samples to assess for the presence of cnLOH. These cases carried either a single or no chromosomal abnormality in addition to t(14;18)(q32;q21) as determined by karyotyping. We found that, although these MEV-FL cases had simple karyotypes, they showed very similar cnLOH profiles as compared to cytogenetically complex cases. The most frequent regions affected by cnLOH were 1p (17%), 6p (17%), 12q (13%) and 16p (13%). Our study suggests that cnLOH alterations may serve as important contributors to the pathological and clinical manifestations of FL.

Therapy-related leukemia: clinical characteristics and analysis of new molecular risk factors in 96 adult patients.

Therapy-related leukemia or myelodysplasia (t-leuk/MDS) is a serious problem that is increasing in frequency. We studied the clinical characteristics of 96 patients (pts) with a mean age of 48 years, and analyzed the molecular parameters that could predispose to t-leuk/MDS. Hematological malignancies were the most common primary (53%), followed by breast and ovarian cancer (30% combined). The mean latency until the development of t-AML was 45.5 months. Median survival was 10 months. Cytogenetics was abnormal in 89% of pts. FLT3 internal tandem duplications were found in six of 41 (14.6%) pts, of whom four had an abnormal karyotype. Analysis of drug metabolism and disposition genes showed a protective effect of the CYP3A4 1*B genotype against the development of t-leuk/MDS, whereas the CC genotype of MDR1 C3435T and the NAD(P)H:quinone oxidoreductase1 codon 187 polymorphism were both noncontributory. Microsatellite instability (MSI) analysis using fluoresceinated PCR with ABI sequence analyzer demonstrated that 41% of pts had high levels of MSI in four or more of 10 microsatellite loci. Immunohistochemistry demonstrated reduced expression of MSH2 and MLH1 in 6/10 pts with MSI as compared to 0/5 of pts without MSI. In conclusion, genetic predisposition as well as epigenetic events contribute to the etiology of t-AML/MDS.

Clinical implications of fluorescence in situ hybridization analysis in 13 chronic myeloid leukemia cases: Ph-negative and variant Ph-positive.

Thirteen chronic myeloid leukemia (CML) patients, 10 with variant Philadelphia (Ph) translocations and 3 Ph negative cases, were analyzed by fluorescence in situ hybridization (FISH) with the use of BCR and ABL cosmid probes and a chromosome 22 painting probe. In the variant Ph translocations, the BCR-ABL fusion gene was located on the Ph chromosome; in 1 CML Ph-negative patient, the BCR-ABL fusion gene was located on the Ph chromosome; and, in 2 patients, it was located on chromosome 9. The chromosome 22 painting probe was detected on the third-party chromosome of the variant translocation, and in none of the variant translocations was there any detectable signal on chromosome 9. In CML patients with clonal evolution of a simple Ph, a signal of the chromosome 22 painting probe was detected on the der(9) of the Ph translocation. It was concluded that the variant Ph translocations evolved simultaneously in a three-way rearrangement. The clinical parameters of the 13 patients were similar to those of a large group of CML patients with a simple Ph translocation. It is suggested that, to determine the prognosis of CML patients with a complex karyotype, FISH analysis with a chromosome 22 painting probe be performed.

A novel translocation (1;2)(p34;p21-22) in acute myelomonoblastic leukemia.

A novel and as yet unrecorded translocation, (1;2)(p34;p21-22), detected in a patient with acute myeloid leukemia (AML) is reported. The leukemia--in this case, AML-M4--showed a rapidly progressive fatal course despite an early transient response to aggressive chemotherapy. In this patient, the leukemic cells showed a novel balanced translocation, (1;2)(p34;p21-22), in most of the metaphases at the time of diagnosis and during subsequent relapse. Interferon-inducible double-stranded RNA-dependent protein kinase (ds RNA-PK) is located in the chromosome region, 2p21-22, that was involved in the translocation in this case. The possible role of ds RNA-PK in leukemogenesis is briefly mentioned.

Adoptive cell-mediated immunotherapy with interleukin-2 (IL-2) for relapsing lymphoblastic crisis following mismatched unrelated bone marrow transplantation in a chronic myelogenous leukemia patient.

A 21-year-old woman with chronic myelogenous leukemia (CML) relapsed into lymphoblastic crisis with new chromosomal translocations, 4 months following mismatched unrelated allogeneic bone marrow transplantation (BMT). Adoptive cell-mediated immunotherapy with mismatched unrelated donor lymphocytes followed by 3 days of in vivo interleukin-2 (IL-2) resulted in complete remission including disappearance of the Philadelphia chromosome as determined by cytogenetic analysis and the bcr/abl translocation detected by PCR. Lymphoblastic crisis following mismatched, unrelated BMT is relatively rare. Moreover lymphoblastic malignancies usually respond less favorably to cell-mediated immunotherapy. This case is the first reported CML lymphoblastic crisis following mismatched unrelated BMT that responded to cell-mediated immunotherapy and IL-2. Some possible mechanisms and new therapeutic directions are discussed.

De novo acute myeloid leukemia with near-pentaploidy: diploid karyotype and lymphoblastic phenotype at relapse.

Hyperploidy is a rare finding in leukemias, with isolated cases of tetraploidy reported in acute myeloblastic and acute lymphblastic leukemias. We report the first case of acute myeloid leukemia with near-pentaploidy (5 n+/-) which was present in 100% of metaphases at diagnosis. By light microscopy, the leukemic blasts were exceptionally large and coarsely granulated. Following one cycle of induction chemotherapy, complete morphologic and cytogenetic remission was documented. Four weeks later relapse occured, at which time the karyotype was diploid and the morphological and immunophenotypic characteristics were those of a lymphoid leukemia. However, the presence of three aberrant chromosomes (5q+, 6q+ and 20q+) confirmed that this was clonal evolution of the original myeloid leukemia. To the best of our knowledge, this case represents the first report of near-pentaloidy in de novo, pretreatment human leukemia.

Microsatellite instability and p53 mutations in therapy-related leukemia suggest mutator phenotype.

During the last decade the frequency of therapy-related acute leukemia (t-leuk) and myelodysplastic syndrome (t-MDS) has been increasingly observed. Over the past 15 years, we treated 56 patients with t-leuk who had received prior chemotherapy (39%), radiotherapy (11%), or both (45%). The drugs received included alkylating agents and topoisomerase II inhibitors. The primary tumors included hematological malignancies (49%) and solid tumors such as breast or ovarian cancer. The median age at diagnosis of the primary tumor was relatively young (43 years +/- 18). Twelve patients had more than one primary tumor and 31 patients had a family history of malignancy. Karyotypic abnormalities were found in 91% of the patients. Prognosis was uniformly poor, with an overall median survival of 10 months. Twelve of the 18 patients examined (67%) had a multidrug resistance phenotype. P53 genes of the leukemic cells, as well as the original tumors, were analyzed in 21 patients using polymerase chain reaction (PCR) with single-stranded conformation polymorphism analysis followed by sequencing. P53 mutations were identified in 38% of these patients, a relatively high prevalence compared with other forms of MDS or de novo acute myeloid leukemia. Mutations were nongermline and restricted to the leukemic cells. We identified different p53 mutations in the various primary tumors of individual patients. The presence of a mutator phenotype was assessed by PCR analysis of microsatellites in eight loci (one trinucleotide repeat sequence, four dinucleotide, and three mononuclear repeat sequences). Microsatellite instability in two to seven loci were found in 15 of 16 (94%) of the patients. This instability is compatible with a mutator phenotype, which predisposes the patients to the development of malignancies including t-leuk.

dup(10q) lacking alpha-satellite DNA in bone marrow cells of a patient with acute myeloid leukemia.

A marker chromosome was identified in leukemic cells on an AML patient. The G-banding pattern resembled on i(10q), but its centromeric position was not clear; in some cells it had a telocentric shape, in others a metacentric or acentric shape. The origin of the marker chromosome was confirmed by FISH, using chromosome-10-specific painting. To determine the centromeric position, C-banding and alpha-satellite probes were applied in FISH, and none of them gave a positive signal. Despite the absence of the centromeric alpha-satellite sequences and the constricted feature of the centromere, the essential centromeric activity was retained in this chromosome, namely, the separation of sister chromatids in anaphase.

Predominant 45,X,--Y karyotype in donor cells after allogeneic BMT: cytogenetic and molecular analysis.

We describe two women; one (patient 1) with the diagnosis of acute myeloblastic leukemia (AML), the second (patient 2) with myelodysplastic syndrome (MDS). Both patients underwent allogeneic bone marrow transplantation (BMT), from their HLA-matched brothers. Cytogenetic analysis after the BMT revealed a chromosomal mosaicism in both patients, with the karyotype 46,XX/45,X with no sign of the Y chromosome. The origin of the clone with monosomy X was determined using cytogenetic analysis including heteromorphism and segregation of DNA polymorphic markers. The results led us to the conclusion that in both patients the origin of the 45,X clone was that of the donors. Patient 1 had MDS-like syndrome after the BMT and was stabilized in the chimeric state; to date she is doing well. Patient II also had MDS. However, in her case, it was her primary disease. The graft in patient II was rejected and she died 6 months after BMT.

Reversed BCR/ABL rearrangement detected by FISH in Philadelphia negative chronic myelocytic leukemia.

We investigated leukemic cells from a patient with chronic myelocytic leukemia (CML) and a normal 46,XX karyotype. Molecular studies revealed rearrangement of the M-bcr region and formation of BCR/ABL fusion mRNA with b3a2 configuration. Fluorescence in situ hybridization (FISH) using the abl probe showed signal on both chromosomes 9 band q34, while the bcr probe hybridized to one chromosome 22 and to one chromosome 9. In this case, as in three other cases recently described (Hagemeijer et al. and Nachava et al.), the bcr/abl rearrangement is shown to be on 9q34, instead of the usual location on 22q11.

Loss of Y chromosome. An age-related event or a cytogenetic marker of a malignant clone?

Cytogenetic analysis of bone marrow cells performed at the time of diagnosis of ALL in an 80-year-old male patient revealed two unrelated abnormal clones. One included the 9;22 translocation (Philadelphia chromosome) as the sole aberration and the second was missing the Y chromosome. The significance of this finding in the light of the role of -Y clone in malignancy is discussed.

Therapy-related myelodysplastic syndrome. Two cytogenetically unrelated abnormal clones in a patient with multiple myeloma.

A multiple myeloma patient presented for cytogenetic analysis at diagnosis of secondary MDS, which followed cytotoxic treatment including melphalan. Two abnormal unrelated clones were detected, one of them had 5q-, 7q- with clonal evolution of an additional aberration, t(12;13); in the second clone there was a translocation between the two homologues of chromosome 1 as the only aberration. We suggest that the clone with 5q- and 7q- represented the secondary MDS cells, whereas the abnormal clone with t(1;1) represented the plasmablasts of the multiple myeloma.

"Jumping translocation" in a 17-month-old child with mixed-lineage leukemia.

A 17-month-old child with acute biphenotypic (pre B-ALL/myelomonocytic) leukemia is reported. Extensive cytogenetic analysis performed at various stages of the disease revealed a clonal evolution at the time of initial diagnosis with two types of abnormal clones, one with trisomy 22 and two other related clones with trisomy 22 plus partial trisomy of the long arm of chromosome 1 associated with the telomeric segment of either chromosome 20q or 21p. At the time of relapse the only abnormal clone involved trisomy 22 and partial trisomy of 1q, but this time in association with the telomeric segment of 14p. The unique feature of these translocations is discussed and the possibility of the correlation between the different chromosomal abnormalities and the expression of biphenotypic markers is raised.