Yanjie Hu

Detection of circulating tumor cells and tumor stem cells in patients with breast cancer by using flow cytometry

Circulating tumor stem cells (CTSC), a subpopulation of circulating tumor cells (CTC), may lead to recurrent diseases. The aim of this study was to detect CTC (CD45−EpCAM+) and CTSC (CD45−EpCAM+CD44+CD24−) of breast cancer (BC) patients, as well as to explore their clinical relevance. CTC and CTSC in peripheral blood (PB) of 45 female BC patients were detected by using flow cytometry (FCM). SKBR-3 cells were mixed with MNC of four healthy volunteers at different ratios in order to evaluate the sensitivity of FCM. Real-time quantitative polymerase chain reaction (QRT-PCR) was conducted and compared with FCM. The expression of EPCAM between CTC < 50 and CTC ≥ 50 groups (19.98 ± 23.93 versus 29.46 ± 29.27 × 10−5), and the expression of CD44 between CTSC negative and positive groups (0.85 ± 0.91 versus 0.81 ± 0.75) were statistically the same. FCM had higher specificity than QRT-PCR. Statistical differences were obtained between CTC < 50 and CTC ≥ 50 groups among different TNM stages, histology stages, estrogen receptor (ER) status and progesterone receptor (PR) status (P < 0.05). Statistical differences between CTSC negative and positive groups within different TNM stages and regional lymph node metastasis (RLNM) status (P < 0.05) were also obtained. Moreover, the percentage of CTC on CD45 negative cells (CD45−C) among different clinical pathology was statistically different, P = 0.000. Additionally, the percentage of CTSC on CD45−C with TNM stage was rising (0: 0.00 ± 0.00‰, I: 0.03 ± 0.05‰, II: 0.06 ± 0.14‰, III: 0.10 ± 0.09‰, IV: 0.29 ± 0.35‰, P = 0.034). Statistical difference in the percentage of CTSC on CD45−C among different RLNM status (P = 0.001) was also obtained. FCM to detect CTC and CTSC may be used to diagnose disease at early stage, to guide clinical therapy or to predict prognosis.

A comprehensive cytogenetic classification of 1466 Chinese patients with de novo acute lymphoblastic leukemia.

Cytogenetics and molecular cytogenetics of 1466 Chinese patients with de novo acute lymphoblastic leukemia (ALL) were studied. Cytogenetic results were available in 1175 patients. Cross-correlations of 23 subclasses of cytogenetic abnormalities were described. Childhood cases had higher incidences of normal karyotype, t(1;19), +8, 12q-, +21, +22 and high hyperdiploidy with 51-65 chromosomes, and lower incidences of t(9;22) and -5/5q- than adult ones (all p<0.05). Relationships of cytogenetic subclasses with immunophenotyping subgroups of ALL were studied. Our study presents the cytogenetic characteristics of a large series of Chinese ALL patients.

Analysis of hematogones in bone marrow from acute myeloid leukaemia cases posttherapy

Increased bone marrow (BM) hematogones (HGs) are often observed in patients with marrow regenerating status. Many studies have focused on the role of HGs in acute lymphoblastic leukaemia (ALL), but very little has been done to understand their effects on acute myeloid leukaemia (AML).

Comprehensive profile of cytogenetics in 2308 Chinese children and adults with de novo acute myeloid leukemia.

Diagnostic cytogenetic and molecular analysis is recognized as the most valuable prognostic factor in acute myeloid leukemia (AML). Among 2516 consecutive Chinese patients with de novo AML, 2308 patients had successful cytogenetic results including 61 subclasses of cytogenetic abnormalities and 27 kinds of additional cytogenetic abnormalities. The incidence of t(15;17)(q22;q12) was highest (16.7% of 2308 patients), followed by t(8;21)(q22;q22) (15.1%), trisomy 8 (5.5%), loss of Y (4.5%), trisomy 21 (2.4%), inv(16)(p13q22) or t(16;16)(p13;q22) (2.1%), etc. In comparison to children, adults had higher incidence of normal karyotype (41.5% vs. 29.1%, P<0.001) and lower incidences of t(8;21)(q22;q22) (13.4% vs. 25.8%, P<0.001), t(9;11)(p22;q23) (0.2% vs. 1.2%, P=0.001) and other 11q23 rearrangements (1.0% vs. 3.4%, P<0.001). Among 349 AML patients with t(8;21)(q22;q22), 310 (35.5%) were found in 873 patients with M2. The t(15;17)(q22;q12) was exclusively observed in 386 (71.0%) of 544 patients with M3. In 48 AML patients with inv(16)(p13q22) or t(16;16)(p13;q22), 42 (15.2%) were detected in 276 patients with M4. Our study displayed the cytogenetic characteristics in a large series of Chinese patients with de novo AML. Our results revealed the similarities and differences of cytogenetic abnormalities existing between Chinese and western AML patients.

Chronic myeloid leukemia and BCR/ABL signal pathways are not associated with AKT1 pleckstrin homology domain (E17K) mutations.

To the Editor: The v-akt murine thymoma viral oncogene homologues (AKT) is the major downstream mediator of growth factor receptors that signal through phosphatidylinositol-3 kinase (PI3K) (1). Mounting evidence suggests that aberrant activation of AKT proteins is involved in the development of human malignancies. The activation of AKT proteins has frequently been attributed to the presence of activating PIK3CA mutation and the lack of PTEN (phosphatase and tensin homology deleted on chromosome 10) activity in tumor cells (2, 3). Recently, Carpten et al. (4) described a somatic missense mutation of AKT1 in human cancers. The substitution of an amino acid (E17K) in pleckstrin homology domain of AKT1 Lys 17 resulted in an increased level of AKT phosphorylation on Thr 308 and Ser 473. Moreover, E17K mutation induces leukemia in mice, suggesting the oncogenic potential of this AKT1 activating mutation. Previous screening studies found E17K mutation in lung, breast, colorectal, ovarian and urothelial cancers but not in acute myeloid leukemia, B-cell-derived lymphoid leukemia, glioblastomas or medulloblastomas, promoting further studies to investigate the presence of this mutation in other malignancies (5–9). Chronic myeloid leukemia (CML) is a malignant clonal hematopoietic stem cell disorder characterized by the translocation of 9q34 to 22q11 (Ph), generating the breakpoint cluster region (BCR)-Abelson (ABL) fusion gene (10, 11). The BCR-ABL oncogenic protein has constitutive tyrosine kinase activity to enhance proliferative and anti-apoptotic signals. Increasing evidences demonstrate that PI3K ⁄AKT is one of major downstream signal pathways of BCR-ABL oncogene and plays essential role in BCR-ABL-mediated transformation. More recently, it has been shown that increased PI3K ⁄AKT activity in CML contributes to the development of drug resistance of malignant cells and the combination of PI3K ⁄AKT pathway inhibitor and rapamycin can efficiently kill imatinib (IM)-resistant BCR-ABL-expressing cells, highlighting the potential of PI3K ⁄AKT as the new targets for CML therapy (12). To study the frequency of AKT1 E17K mutation in Chinese CML patients as well as its possible relation to IM sensitivity, genome DNA was extracted from the peripheral blood mononuclear cell (PBMNC) or bone marrow mononuclear cell of 86 CML patients whose karotype showed the presence of Philadelphia chromosome at diagnosis. At the molecular level, B2a3, B3a3, B2a2 and B3a2 were identified by RT-PCR in 2, 1, 41 and 42 cases, respectively. The complete cytogenetic response (CCyR), major molecular response (MMR) and complete molecular response (CMR) were achieved in 74, 39 and 27 cases at 3–12 months after IM administration (Table 1). Direct PCR product sequencing analysis was performed as previously described (9). No AKT1 E17K mutation was found in all patients. The absence of AKT1 E17K mutation in our study suggests the pre-existence of AKT1 E17K mutation plays, if any, minor role in determining the clinical response to IM as well as for the leukemogenesis of CML. Nevertheless, because of the low counts of leukemic cells in most patients, we did not exam the presence of AKT1 E17K mutant in patients after IM exposure. Thus, the possibility remains that the acquisition of AKT1 E17K mutation after IM administration may lead to the failure of achieving MMR in some patients. Further studies with more sensitive techniques are required to clarify this possibility. Acquisition of additional genetic alternations has been considered as a major reason for the occurrence of CML blast crisis. We tested the possibility that AKT1 E17K mutation may contribute to the blast crisis of CML because oncogenic AKT1 E17K mutation has been shown to induce the development acute leukemia in transgenic

Characterisation of clonal Philadelphia‐negative cytogenetic abnormalities in a large cohort of chronic myeloid leukaemia

Clonal Philadelphia (Ph)‐negative cytogenetic abnormalities (CPCA) have been reported in chronic myeloid leukaemia (CML) patients treated with either interferon or tyrosine kinase inhibitor (TKI). However, the incidences and types of these cytogenetic abnormalities after treatment vary due to the limited populations enroled.

Detection of Circulating Tumor Cells and Circulating Tumor Stem Cells in Breast Cancer by Using Flow Cytometry

We demonstrated the value of multiparameter flowcytometry in detecting human tumor cells of breast cancer in peripheral blood, which had a sensitivity limit of 10-5 and higher specificity compares with real‐time polymerase chain reaction (RT‐PCR). It was also found that circulating tumor cell (CTC) number was related with TNM stage, metastasis and the overall survival of patients. CTC level was one of the important factors for patients’ prognosis. At the same time, we also verified the circulating tumor stem cell (CTSC) was connected with TNM stage by multiparameter cytometry. The detection of CTC and CTSC by multiparameter flowcytometry may be used to diagnose disease at early stage to guide clinical therapy or to predict prognosis. Multiparame‐ ter flowcytometry has the potential to be a valuable tool for prognosis assessment among patients with breast cancer in clinical situation in China.

A Pediatric Acute Promyelocytic Leukemia With a Rare Karyotype of ider(17)(q10)t(15;17) and Favorable Outcome: A Case Report.

AbstractAcute promyelocytic leukemia (APL) is a specific malignant hematological disorder with a diagnostic hallmark of chromosome translocation t(15;17)(q22;q21). As a very rare secondary cytogenetic aberration in pediatric APL, ider(17q) (q10)t(15;17) was suggested to be a poor prognostic factor based on previous case reports.Here, we report a pediatric APL case with a rare karyotype of ider(17)(q10)t(15;17). Bone marrow aspiration, immunophenotyping, molecular biology, cytogenetic, and fluorescence in situ hybridization (FISH) analyses were performed at initial diagnosis and during the treatment.A 6-year-old boy was brought to our hospital with the chief complaint of bleeding gums twice and intermittent fever for 3 days in January 2013. He was diagnosed as low-risk APL according to the 2012 NCCN guideline on APL, with the expression of PML-RARA (bcr3 subtype) and the karyotype of 46,XY, der(15)t(15;17)(q22;q21),ider(17)(q10)t(15;17), which was further verified by FISH. The patient was treated through combination all-trans retinoic acid (ATRA) and arsenic with daunorubicin according to the 2012 NCCN guideline for APL. Continuous hematological completed remission (HCR) and major molecular remission (MMR) were achieved with normal karyotype for >28 months after induction chemotherapy.Different from previously reported cases, this pediatric APL patient with ider(17)(q10)t(15;17) displays favorable clinical outcomes, which might be related to the low-risk classification and arsenic treatment during the treatment. It suggests that ider(17)(q10)t(15;17) may not be the sole determinant for worse outcomes in pediatric APL and implies that more contributed factors should be considered for pediatric APL prognosis.