Su S. Chen

Application of COLD-PCR for improved detection of KRAS mutations in clinical samples

KRAS mutations have been detected in approximately 30% of all human tumors, and have been shown to predict response to some targeted therapies. The most common KRAS mutation-detection strategy consists of conventional PCR and direct sequencing. This approach has a 10–20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve detection sensitivity, we compared our conventional method with the recently described co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, the critical denaturation temperature is lowered to 80°C (vs 94°C in conventional PCR). The sensitivity of COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens and the formalin-fixed paraffin-embedded (FFPE) tissue of 30 solid tumors. Implementation of COLD-PCR was straightforward and required no additional cost for reagents or instruments. The method was specific and reproducible. COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant-to-wild-type ratio by >4.74-fold, increasing the mutation detection sensitivity to 1.5%. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor-cell percentage in a sample. In conclusion, we validated the utility and superior sensitivity of COLD-PCR for detecting KRAS mutations in a variety of hematopoietic and solid tumors using either fresh or fixed, paraffin-embedded tissue.

Therapy-Related Acute Myeloid Leukemia With t(8;21) (q22;q22) Shares Many Features With De Novo Acute Myeloid Leukemia With t(8;21)(q22;q22) but Does Not Have a Favorable Outcome

To determine if therapy-related acute myeloid leukemia (t-AML) with t(8;21)(q22;q22) [t-AML-t(8;21)] harbors similar characteristic clinicopathologic features as de novo AML-t(8;21) (q22;q22), we studied 13 cases of t-AML-t(8;21) and 38 adult cases of de novo AML-t(8;21) diagnosed and treated at our hospital (1995-2008). Of 13 t-AML-t(8;21) cases, 11 had previously received chemotherapy with or without radiation for malignant neoplasms and 2 received radiation alone. The median latency to t-AML onset was 37 months (range, 11-126 months). Compared with patients with de novo AML-t(8;21), patients with t-AML-t(8;21) were older (P = .001) and had a lower WBC count (P = .039), substantial morphologic dysplasia, and comparable CD19/CD56 expression. The AML1-ETO (RUNX1-RUNX1T1) fusion was demonstrated in all 10 cases assessed. Class I mutations analyzed included FLT3 (0/10 [0%]), RAS (0/10 [0%]), JAK2 V617 (0/11 [0%]), and KIT (4/11 [36%]). With a median follow-up of 13 months, 10 patients with t-AML-t(8;21) died; the overall survival was significantly inferior to that of patients with de novo AML-t(8;21) (19 months vs not reached; P = .002). These findings suggest that t-AML-t(8;21) shares many features with de novo AML-t(8;21)(q22;q22), but affected patients have a worse outcome.

Systemic mastocytosis with associated clonal hematological non‐mast cell lineage disease: Clinical significance and comparison of chomosomal abnormalities in SM and AHNMD components

Some patients with systemic mastocytosis have concurrent hematological neoplasms, designated in the World Health Organization (WHO) classification as systemic mastocytosis with associated clonal hematological non‐mast cell lineage disease (SM‐AHNMD). In this study, we analyzed 29 patients with SM‐AHNMD and compared them to 40 patients with pure SM. The AHNMDs were classified as chronic myelomonocytic leukemia (CMML) (n = 10), myelodysplastic syndrome (MDS) (n = 7), myeloproliferative neoplasms (n = 4), B‐cell lymphoma/leukemia/plasma cell neoplasms (n = 7), and acute myeloid leukemia (n = 1). Patients with SM‐AHNMD were older, more frequently had constitutional symptoms and hematological abnormalities, less often had skin lesions, and had an inferior overall survival compared with pure SM patients (48 months vs. not‐reached, P < 0.001). Karyotypic abnormalities were detected in 9/28 (32%) patients with SM‐AHNMD but not in pure SM patients (P < 0.001). Combined imaging/ fluorescence‐in‐situ hybridization performed in four SM‐AHNMD cases revealed shared abnormal signals in mast cells and myeloid cells in two patients with SM‐CMML and one patient with SM‐MDS, but not in the mast cells of a case SM‐associated with chronic lymphocytic leukemia with ATM‐deletion. Quantitative mutation analysis showed higher levels of mutant KIT D816V in SM‐CMML and SM‐MDS than in pure SM (P < 0.001). Our data indicate that the SM‐AHNMD category in the WHO classification is heterogeneous, including clonally related and unrelated forms of AHNMD. The presentation, treatment, and outcome of patients with SM‐AHNMD is often dictated by the type of AHNMD. Am. J. Hematol. 88:219–224, 2013.

CD117 expression is a sensitive but nonspecific predictor of FLT3 mutation in T acute lymphoblastic leukemia and T/myeloid acute leukemia

Others have suggested that CD117, or an immunophenotypic profile including CD117, can serve as surrogate for FLT3 mutation in T acute lymphoblastic leukemia (ALL), thereby guiding targeted therapy. We report the results of flow cytometry immunophenotypic analysis in 42 cases of T-ALL and T/myeloid acute leukemia also assessed for FLT3 mutation. CD117 was expressed in 21 (50%), and FLT3 was mutated in 8 cases (19%; 1 T-ALL and 7 T/myeloid). FLT3-mutated cases were terminal deoxynucleotidyl transferase (TdT)+/CD2+ (7/8), cytoplasmic CD3+/CD5+ (5/8), CD7+/CD13+/CD15+ (4/6), CD33+ (4/8), CD34+, and CD117+ (bright). Cytochemistry showed myeloperoxidase-positive cells in all T/myeloid acute leukemias (3%-50%). We conclude that FLT3 mutation is rare in T-ALL, and its presence supports T/myeloid lineage. CD117 expression alone is sensitive but not specific for FLT3 mutation. The immunophenotypic profile of TdT, CD7, CD13, CD34, and CD117 (bright) is helpful for predicting FLT3 mutation, with a sensitivity of 100% and specificity of 94%.

Flow cytometric detection of peripheral blood involvement by mycosis fungoides and Sézary syndrome using T-cell receptor VΒ chain antibodies and its application in blood staging

Peripheral blood involvement has been recognized as an adverse prognostic factor in patients with mycosis fungoides and Sézary syndrome. However, accurate identification and enumeration of the neoplastic cells in these diseases can be challenging. We assessed the clinical utility of flow cytometric immunophenotypic analysis of T-cell receptor Vβ expression in 82 mycosis fungoides and 6 Sézary syndrome patients, with an atypical T-cell immunophenotype, or abnormal CD4:CD8 ratio, identified from peripheral blood specimens of 723 patients submitted for routine mycosis fungoides/Sézary syndrome blood staging. To improve detection sensitivity, Vβ expression was analyzed on gated CD3+CD4+ T cells or T cells with an aberrant immunophenotype, if present. The flow cytometric results were compared with traditional morphologic assessment (n=88) and molecular methods to assess the T-cell receptor γ or β genes (n=41 tested in parallel). Flow cytometric immunophenotyping yielded a clonal Vβ pattern in 60/82 mycosis fungoides and 6/6 Sézary syndrome patients. By contrast, flow cytometric Vβ was negative in all 10 healthy donors and 18 control patients, showing a specificity of 100% and concordance with molecular testing of 86%. Using flow cytometric Vβ results instead of morphologic assessment, 12 patients were upstaged from B1 to B2, and 20 patients from B0 to B1 (P<0.0001). The 12 upstaged B2 patients had no morphologic evidence of involvement, but had an aggressive clinical course similar to those staged by traditional morphologic assessment (median survival 27 vs 41 months, log-rank P=0.701). In 30/44 patients with a tumor-associated Vβ expression, a single Vβ tube was used to monitor treatment response. In conclusion, flow cytometric Vβ analysis is rapid and convenient, can assess T-cell clonality and tumor quantity simultaneously, and is useful both in initial blood staging and monitoring tumor burden during therapy in patients with mycosis fungoides or Sézary syndrome.

De novo acute myeloid leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2): A clinicopathologic and cytogenetic study of an entity recently added to the WHO classification

Acute myeloid leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) is a rare type of leukemia recently added to the World Health Organization (WHO) classification scheme. In this study, we analyzed the clinicopathologic and cytogenetic features of 30 cases of de novo acute myeloid leukemia with inv(3)/t(3;3). The median patient age was 53 years (range, 27–77 years). The platelet count was variable (range, 21–597 × 109/l, median: 128 × 109/l), and two (6.7%) patients presented with thrombocytosis (>450 × 109/l). Morphologically, these neoplasms showed a spectrum of findings. Myelomonocytic differentiation was most common in 11 (37%) cases. Morphological evidence of dysplasia was observed in at least one lineage in 23 of 25 (92%) cases in which maturing elements could be assessed. In all, 5 (17%) patients had isolated inv(3) or t(3;3) and 25 (83%) patients had additional cytogenetic abnormalities, most often monosomy 7 (40%). Eleven (37%) patients had a complex karyotype (≥3 additional abnormalities). FLT3 gene mutation by internal tandem duplication was identified in 2 of 23 (9%) cases assessed. No clinical, pathological, or cytogenetic features independent of inv(3) or t(3;3) correlated with a worse outcome. However, patients treated with allogeneic stem cell transplantation (n=11) had a significantly better survival than did those treated with chemotherapy alone (n=17) (13.8 vs 8.0 months, P=0.041). We conclude that de novo acute myeloid leukemia associated with inv(3)/t(3;3) is an aggressive type of leukemia regardless of morphological or karyotypic findings, supporting the inclusion of this disease as a specific entity defined by inv(3)/t(3;3) in the WHO classification. Allogeneic stem cell transplantation seems to improve outcome in patients with this disease.

Distinct patterns of cytogenetic and clinical progression in chronic myeloproliferative neoplasms with or without JAK2 or MPL mutations

Chronic myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET) and primary myelofibrosis (PMF), result from interactions between initiating growth factor mutations and secondary genomic changes. Codon 617 mutation of the JAK2 kinase is found in 40-50% of ET/PMF, whereas the mutation of codon 515 in the JAK2-linked thrombopoietin receptor MPL is found in approximately 20% of JAK2-unmutated cases of ET and PMF. Using quantitative mutation assays, we compared patterns of clinical and cytogenetic progression in MPL-mutated MPN (n=21) to those with JAK2 V617F mutation (n=383) or neither mutation (n=109). Among patients with MPL mutations, ET was seen in 9 and PMF in 12. Median mutation levels in pretreatment ET samples were significantly higher for MPL-mutated cases (60%) than for JAK2-mutated cases (24%; P=0.01), as was presentation with anemia. Differential genomic changes included +9 in JAK2-mutated cases and chromosome 1 alterations in MPL-mutated ones, implicating dosage effects related to gene copy number. Decreases in the levels of MPL mutation were seen in sequential marrow samples from some patients under treatment with biologic therapies, but not in those treated with kinase inhibitors, consistent with selective response of the MPL-mutated clone similar to the responses seen in JAK2-mutated MPN.

Chromosome 20q deletion: A recurrent cytogenetic abnormality in patients with chronic myelogenous leukemia in remission

del(20q) can be observed in hematologic neoplasms, including chronic myelogenous leukemia (CML), and has been reported in patients undergoing blast transformation. We describe 10 patients with CML in hematologic and cytogenetic remission with del(20q) detected by conventional cytogenetics. There were 6 men and 4 women with a median age of 56 years. All patients initially had BCR-ABL1 and t(9;22) (q34;q11.2) and achieved morphologic and cytogenetic remission after therapy. del(20q) was identified before (2/10 [20%]), at the time of (3/10 [30%]), or after (5/10 [50%]) cytogenetic remission and was not associated with morphologic evidence of dysplasia. At last follow-up, no patients had a myelodysplastic syndrome (MDS). Leukocyte and platelet counts were normal; 4 of 10 patients had mild anemia. Nine patients have remained in morphologic and cytogenetic remission with stable del(20q). BCR-ABL1 fusion transcript levels were absent or low (median, 0.01%). Recently, in 1 patient, recurrent CML developed and del(20q) was lost. We conclude that del(20q) in the setting of CML in remission is not predictive of MDS or blast transformation.

Monitoring of Minimal Residual Disease in Acute Myeloid Leukemia: Methods and Best Applications

Posttherapy minimal residual disease assessment in acute myeloid leukemia is an important prognostic indicator as well as a potential early surrogate measure of therapeutic effectiveness. The goal of this brief review is to discuss the broad classes of targets for molecular diagnostic assays, with specific examples of each, and to describe the analytic approach to flow cytometry testing for minimal residual disease in acute myeloid leukemia. Several of the most significant recent contributions to this field are highlighted.

A pathway-based gene signature correlates with therapeutic response in adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia

Biomarkers to predict response to therapy in adults with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) are not yet established. In this study, we performed a meta-analysis of earlier genome-wide gene expression studies to identify pathway-based genes that are associated with therapeutic response. The predictive power of these genes was validated by transcript profiling in diagnostic bone marrow samples from Ph+ ALL patients using a quantitative real-time PCR array. Gene expression was correlated with cytogenetic and molecular characteristics, including presence of ABL1 mutations and IKZF1 deletion. A total of 43 de novo Ph+ ALL patients treated uniformly with tyrosine kinase inhibitors combined with chemotherapy were selected to validate 46 identified genes. A 9-gene signature was established to distinguish optimal responders from patients with persistent residual disease and early molecular recurrence. The signature was subsequently validated with 87% predictive accuracy in an independent validation set of patients. When initially optimal responders relapsed, their gene expression patterns also shifted. Optimal responders showed upregulation of genes involved in proliferation and apoptosis pathways, whereas poor responders had higher expression of genes that facilitate tumor cell survival in hypoxic conditions as well as development of drug resistance. This unique 9-gene signature may better enable stratification of patients to proper therapeutic regimens and provides new insights into mechanisms of Ph+ ALL response to therapy.