Integration of ruxolitinib into dose-intensified therapy targeted against a novel JAK2 F694L mutation in B-precursor acute lymphoblastic leukemia.

A 17-year-old girl with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with persistent minimal residual disease (MRD) who underwent standard chemotherapy was found to have a BCR-ABL1-like gene expression pattern. Genome sequencing revealed a JAK2 mutation not previously described in BCP-ALL and a potential therapeutic target. Due to concern for an on-therapy relapse, the JAK2 inhibitor ruxolitinib was incorporated into a modified chemotherapy backbone to achieve complete remission prior to stem cell transplant. Treatment was well tolerated and she had undetectable MRD prior to a matched allogeneic stem cell transplant and remained in remission at day +100.

Significance of MYD88 L265P Mutation Status in the Subclassification of Low-Grade B-Cell Lymphoma/Leukemia.

CONTEXT: Lymphoplasmacytic lymphoma (LPL), marginal zone lymphoma (MZL), and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) are well-defined clinicopathologic entities. However, distinguishing LPL from MZL and from atypical cases of CLL can sometimes be difficult because of overlapping features. Recent studies have identified a recurrent L265P mutation in the MYD88 gene in most cases of LPL. Although this represents a promising diagnostic marker for LPL, the mutation is also reported in rare cases of MZL and CLL (as well as other types of B-cell lymphoma). Detection rates for this mutation have varied depending on the analytic methodology. OBJECTIVE: To assess the diagnostic utility of MYD88 L265P mutation in diagnosing low-grade B-cell lymphomas. DESIGN: We developed a novel pyrosequencing assay for the MYD88 L265P mutation and assessed its diagnostic utility in 317 cases of low-grade B-cell lymphoma (45 LPL [14%], 53 MZL [17%], and 219 CLL [69%]). We incorporated formal clinical and pathologic review of selected cases to ensure the most accurate diagnosis and subclassification. RESULTS: The MYD88 L265P mutation was identified in 43 cases of LPL (96%), including 3 nonimmunoglobulin-M LPL cases. In contrast, the mutation was present in only 2 cases of MZL (4%), and 5 cases of CLL (2%). Thus, pyrosequencing for the MYD88 L265P mutation demonstrates a high clinical sensitivity and specificity to distinguish LPL from MZL and CLL. CONCLUSIONS: This study confirms the strong association of the MYD88 L265P mutation with LPL, as well as the existence of rare cases of small B-cell lymphoma that complicate this association.

Pyrosequencing for classification of human FcγRIIIA allotypes: a comparison with PCR-based techniques.

BACKGROUND: Surface-specific antigens expressed by hematopoietic cells are attractive targets for antibody-mediated immunotherapy. Monoclonal antibodies (mAbs) involve various mechanisms to eliminate target cells, including antibody-dependent cellular cytotoxicity (ADCC)- and phagocytosis (ADCP)-mediated killing through natural killer (NK) and macrophage effector cells bearing FcγRIIIA (CD16). The clinical efficacy of ADCC is particularly impacted by a single nucleotide polymorphism (SNP) found in the gene encoding FcγRIIIA (FCGR3A), which generates a variable distribution of the 158 V/V, F/V or F/F CD16 allotypes (F = phenylalanine, V = valine) in the normal human population. Currently, most patients are not screened for CD16 allotypes, creating the potential to include in their treatment a mAb-based therapy that may have limited benefit. Therefore, it is important to identify CD16 allotypes when considering mAb therapies that require ADCC/ADCP. OBJECTIVE: The objective of this study was to develop a reliable PCR-based assay for classification of human FcγRIIIA allotypes. METHODS: We studied 42 normal human subjects for the incidence of FcγRIIIA-158 polymorphisms using comparative molecular approaches. RESULTS: The results of our study showed 100% accuracy in genotyping by pyrosequencing. In contrast, nested PCR-based allele-specific restriction assay and quantitative PCR techniques proved to be relatively less sensitive and less specific in distinguishing variant genotypes. CONCLUSION: Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in a given individual, we recommend pyrosequencing for CD16 allotype testing.

Microarray analysis of cutaneous squamous cell carcinomas reveals enhanced expression of epidermal differentiation complex genes.

Gene expression profiles were determined for 12 cutaneous squamous cell carcinomas (SCC) removed from sun-exposed sites on nonimmunosuppressed patients. Gene expression in each SCC was compared to that in sun-exposed skin from the same patient using the Affymetrix HGU133 2.0 PlusGeneChip. We identified 440 genes with increased expression in SCC and 738 with decreased expression; overall we identified a large number of small changes in gene expression rather than a few marked changes that distinguished SCC from sun-exposed skin. Analyzing this robust data set according to biofunctional pathways using DAVID, transcriptional control elements using oPOSSUM, and chromosomal location using GSEA suggested genetic and epigenetic mechanisms of gene expression regulation in SCC. Some altered patterns of gene expression in SCC were consistent with regulation of spatially separated genes by a number of developmentally important transcription factors (forkhead, HMG, and homeo factors) that negatively regulated gene expression and to a few factors that positively regulated expression (Creb-1, NFkappaB, RelA, and Sp-1). We also found that coordinately enhanced expression of epidermal differentiation complex genes on chromosome 1q21 was a hallmark of SCC. A novel finding in our study was enhanced expression of keratin 13 in SCC, a result validated by immunohistochemical staining of an SCC tumor tissue array.

Most morphologic features in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) do not reliably predict underlying FISH genetics or immunoglobulin heavy chain variable region somatic mutational status.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is common in the Western world. Genetic abnormalities detected by fluorescence in situ hybridization (FISH) and immunoglobulin heavy chain variable gene region (IGHV) mutational status are well-known independent prognostic indicators in CLL/SLL. Given the requirement for specialized testing to detect such aberrations, we investigated whether morphologic features may predict the presence of a more or less favorable genetic profile. Forty-one SLL cases were morphologically evaluated for expanded proliferation centers, increased large cells outside of proliferation centers, and nuclear contour irregularities (NCI) in small and large tumor cells. ZAP-70 immunohistochemistry and FISH (deletions of 13q14, p53 and ATM and trisomy 12) were successful in all cases. IGHV mutational status was determined in 26/41 cases. Significant NCI in both small and large cells correlated with the presence of an unfavorable FISH abnormality (ie, ATM or p53 deletions). However, despite good specificity (94%), the sensitivity (57%) of this finding is inadequate for routine use. No other significant associations with morphologic features were identified. Strong ZAP-70 positivity correlated with unmutated IGHV (P=0.001), rendering ZAP-70 IHC a useful surrogate for IGHV mutational status. ZAP-70 positivity predicted against finding a favorable FISH deletion 13q14 (P=0.023). Although we only studied 41 cases, we corroborated their validity using Kaplan-Meier overall survival analysis. In conclusion, morphologic features in SLL are not a reliable predictor of underlying genetic status. Thus, we propose a practical, cost-effective approach to the work-up of these cases, which should be driven by clinical necessity.

Analysis of a marsupial MHC region containing two recently duplicated class I loci.

A 37-kb cosmid containing two complete major histocompatibility complex (MHC) class I alpha chain loci from the opossum Monodelphis domestica was isolated, fully sequenced, and characterized. This sequence represents the largest contiguous genomic sequence reported for the MHC region of a nonplacental mammal. Based on particular conserved amino acid residues, and limited expression analyses, the two MHC-I loci, designated ModoUB and ModoUC, appear to encode functional MHC-I molecules. The two coding regions are 98% identical at the nucleotide level; however, their promoter regions differ significantly. Two CpG islands present in the cosmid sequence correspond to the two coding regions. Twelve microsatellites and six retroelements were also present in the cosmid. The retroelements share highest sequence homology to the CORE-SINE family of retroelements. Due to high sequence identity, it is very likely that ModoUB and ModoUC loci are products of recent gene duplication that occurred less than 4 million years ago.

Evaluation of 15 polymerases and phosphorothioate primer modification for detection of UV-induced C:G to T:A mutations by allele-specific PCR.

Allele-specific polymerase chain reaction is based on polymerase extension from primers that contain a 3' end base that is complementary to a specific mutation and inhibition of extension with wild-type DNA due to a 3' end mismatch. Taq polymerase is commonly used for this assay, but because of the high rate of nucleotide extension from primer 3' base mismatches documented for Taq polymerase, high sensitivity is difficult to achieve. To determine whether other polymerases might improve assay sensitivity, 15 polymerases were tested with mutation-specific primers for two ultraviolet-induced mutations in the human 5S ribosomal RNA genes. Of the 15 polymerases tested, six were capable of discriminating these mutations at levels equivalent to or better than Taq polymerase. All primers were phosphorothioate modified on the 3' end to block removal of the critical 3' mutation-specific base by polymerases containing 3' --> 5' exonuclease "proofreading" activity. The effectiveness of phosphorothioate modification was measured in mock polymerase chain reaction reactions and a time course. All six enzymes containing this exonuclease activity showed some ability to digest phosphorothioate-modified primers and could be divided into two groups, showing fast and slow digestion kinetics. Of the three enzymes that showed slow digestion kinetics, two also showed significantly slower digestion kinetics of unmodified primers.

Application of optical trapping for cells grown on plates: optimization of PCR and fidelity of DNA sequencing of p53 gene from a single cell.

BACKGROUND: Optical trapping has traditionally been used to visually select and isolate nonadherent cells grown in suspension because cells grown in monolayers will rapidly reattach to surfaces if suspended in solution. We explored methods to slow cell reattachment that are also compatible with high-fidelity PCR. METHODS: Using HeLa cells grown on plates and suspended after trypsinization, we measured the efficiency of capture by retention and movement of the cell by the laser. Success for removing a captured cell by pipette was determined by PCR amplification of the 5S rRNA gene. After optimizing PCR amplification of a 2049-bp region of the p53 gene, we determined PCR fidelity by DNA sequencing. RESULTS: Addition of bovine serum albumin to suspended cells slowed reattachment from seconds to minutes and allowed efficient trapping. The success rate of removing a cell from the trap by pipette to a PCR tube was 91.5%. The 5S PCR assay also revealed that DNA and RNA that copurify with polymerases could give false-positive results. Sequence analysis of four clones derived from a single cell showed only three polymerase errors in 7200 bp of sequence read and revealed difficulties in reading the correct number in a run of 16 A:T. Comparison of the HeLa and wild-type human sequences revealed several previously unreported base differences and an (A:T)(n) length polymorphism in p53 introns. CONCLUSIONS: These results represent the first use of optical trapping on adherent cells and demonstrate the high accuracy of DNA sequencing that can be achieved from a single cell.