Does the presence of abdominal wall adhesions make gynecologic robotic surgery difficult?

This study aimed to assess the status of abdominal wall adhesions resulting from prior surgeries and their impact on the outcomes of robotic surgery. We retrospectively reviewed clinical information, surgical outcomes, and the status of abdominal wall adhesions in patients who underwent gynecologic robotic surgery at Yamanashi Central Hospital, between April 2018 and March 2023. Abdominal wall adhesions were classified into seven locations and their presence was assessed at each site. Among the 768 cases examined, 196 showed the presence of abdominal wall adhesions. Notably, patients with a history of abdominal surgery exhibited a significantly higher incidence of abdominal wall adhesions than those without such surgical history, although no significant difference was observed in the frequency of adhesions in the upper left abdomen. Patients with a history of gynecologic, gastrointestinal, or biliopancreatic surgeries were more likely to have adhesions at the umbilicus or upper abdomen sites where trocars are typically inserted during robotic surgery. Although cases with abdominal wall adhesions experienced longer operative times than those without, there was no significant difference in estimated blood loss. In 13 cases (1.7%), adjustments in trocar placement were necessary due to abdominal wall adhesions, although none of the cases required conversion to open or conventional laparoscopic surgery. Abdominal wall adhesions pose challenges to minimally invasive procedures, emphasizing the importance of predicting these adhesions based on a patient's surgical history to safely perform robotic surgery. These results suggest that the robot's flexibility proves effective in managing abdominal wall adhesions.

Involvement of BCR::ABL1 in laminin adhesion of Philadelphia chromosome-positive acute lymphoblastic leukemia through upregulation of integrin α6.

BACKGROUND: Adhesion of cancer cells to extracellular matrix laminin through the integrin superfamily reportedly induces drug resistance. Heterodimers of integrin α6 (CD49f) with integrin ß1 (CD29) or ß4 (CD104) are major functional receptors for laminin. Higher CD49f expression is reportedly associated with a poorer response to induction therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, a xenograft mouse model transplanted with primary BCP-ALL cells revealed that neutralized antibody against CD49f improved survival after chemotherapy. AIMS: Considering the poor outcomes in Philadelphia chromosome (Ph)-positive ALL treated with conventional chemotherapy without tyrosine kinase inhibitors, we sought to investigate an involvement of the laminin adhesion. METHODS AND RESULTS: Ph-positive ALL cell lines expressed the highest levels of CD49f among the BCP-ALL cell lines with representative translocations, while CD29 and CD104 were ubiquitously expressed in BCP-ALL cell lines. The association of Ph-positive ALL with high levels of CD49f gene expression was also confirmed in two databases of childhood ALL cohorts. Ph-positive ALL cell lines attached to laminin and their laminin-binding properties were disrupted by blocking antibodies against CD49f and CD29 but not CD104. The cell surface expression of CD49f, but not CD29 and CD104, was downregulated by imatinib treatment in Ph-positive ALL cell lines, but not in their T315I-acquired sublines. Consistently, the laminin-binding properties were disrupted by the imatinib pre-treatment in the Ph-positive ALL cell line, but not in its T315I-acquired subline. CONCLUSION: BCR::ABL1 plays an essential role in the laminin adhesion of Ph-positive ALL cells through upregulation of CD49f.

Application of prime editing system to introduce TP53 R248Q hotspot mutation in acute lymphoblastic leukemia cell line.

In childhood acute lymphoblastic leukemia (ALL), TP53 gene mutation is associated with chemoresistance in a certain population of relapsed cases. To directly verify the association of TP53 gene mutation with chemoresistance of relapsed childhood ALL cases and improve their prognosis, the development of appropriate human leukemia models having TP53 mutation in the intrinsic gene is required. Here, we sought to introduce R248Q hotspot mutation into the intrinsic TP53 gene in an ALL cell line, 697, by applying a prime editing (PE) system, which is a versatile genome editing technology. The PE2 system uses an artificial fusion of nickase Cas9 and reverse-transcriptase to directly place new genetic information into a target site through a reverse transcriptase template in the prime editing guide RNA (pegRNA). Moreover, in the advanced PE3b system, single guide RNA (sgRNA) matching the edited sequence is also introduced to improve editing efficiency. The initially obtained MDM2 inhibitor-resistant PE3b-transfected subline revealed disrupted p53 transactivation activity, reduced p53 target gene expression, and acquired resistance to chemotherapeutic agents and irradiation. Although the majority of the subline acquired the designed R248Q and adjacent silent mutations, the insertion of the palindromic sequence in the scaffold hairpin structure of pegRNA and the overlap of the original genomic DNA sequence were frequently observed. Targeted next-generation sequencing reconfirmed frequent edit errors in both PE2 and PE3b-transfected 697 cells, and it revealed frequent successful edits in HEK293T cells. These observations suggest a requirement for further modification of the PE2 and PE3b systems for accurate editing in leukemic cells.

Clinical and molecular biomarkers predicting response to PARP inhibitors in ovarian cancer.

OBJECTIVE: To determine the useful biomarker for predicting the effects of poly-(ADP ribose)-polymerase (PARP) inhibitors in Japanese patients with ovarian cancer. METHODS: We collected clinical information and performed molecular biological analysis on 42 patients with ovarian, fallopian tube, and primary peritoneal carcinomas who received PARP inhibitors. RESULTS: Among the analyzed patients with ovarian cancer, 23.8% had germline BRCA mutation (gBRCAm), 42.9% had homologous recombination repair-related gene mutation (HRRm), and 61.1% had a genomic instability score (GIS) of ≥42. Patients with HRRm had a significantly longer progression-free survival (PFS) than those without HRRm (median PFS 35.6 vs. 7.9 months; p=0.009), with a particularly marked increase in PFS in patients with gBRCAm (median PFS 42.3 months). Similarly, among patients with recurrent ovarian cancer, those with HRRm had a longer PFS than those without HRRm (median PFS 42.3 vs. 7.7 months; p=0.040). Multivariate Cox proportional hazards regression analysis found that performance status and gBRCAm status were independent factors associated with prolonged PFS with PARP inhibitors. In recurrent ovarian cancer, multivariate regression analysis identified platinum-free interval (PFI) in addition to performance status as a significant predictor of PFS. On the contrary, no significant association was observed between PFS and a GIS of ≥42 used in clinical practice. CONCLUSION: We found that HRRm can be a useful biomarker for predicting the effects of PARP inhibitors in treating ovarian cancer and that the PFI can also be useful in recurrent ovarian cancer.

Utility of ASNS gene methylation evaluated with the HPLC method as a pharmacogenomic biomarker to predict asparaginase sensitivity in BCP-ALL.

Asparaginase is an important agent for the treatment of acute lymphoblastic leukaemia (ALL), but it is occasionally associated with severe adverse events. Thus, for safer and more efficacious therapy, a clinical biomarker predicting asparaginase sensitivity is highly anticipated. Asparaginase depletes serum asparagine by deaminating asparagine into aspartic acid, and ALL cells are thought to be sensitive to asparaginase due to reduced asparagine synthetase (ASNS) activity. We have recently shown that allele-specific methylation of the ASNS gene is highly involved in asparaginase sensitivity in B-precursor ALL (BCP-ALL) by using next-generation sequence (NGS) analysis of bisulphite PCR products of the genomic DNA. Here, we sought to confirm the utility of methylation status of the ASNS gene evaluated with high-performance liquid chromatography (HPLC) analysis of bisulphite PCR products for future clinical applications. In the global methylation status of 23 CpG sites at the boundary region of promoter and exon 1 of the ASNS gene, a strong positive correlation was confirmed between the mean percent methylation evaluated with the HPLC method and that with the NGS method in 79 BCP-ALL cell lines (R2 = 0.85, p = 1.3 × 10-33) and in 63 BCP-ALL clinical samples (R2 = 0.84, p = 5.0 × 10-26). Moreover, methylation status of the ASNS gene evaluated with the HPLC method was significantly associated with in vitro asparaginase sensitivities as well as gene and protein expression levels of ASNS. These observations indicated that the ASNS gene methylation status evaluated with the HPLC method is a reliable biomarker for predicting the asparaginase sensitivity of BCP-ALL.

p53 Immunohistochemical Staining and TP53 Gene Mutations in Endometrial Cancer: Does Null Pattern Correlate With Prognosis?

Whether immunohistochemistry (IHC) of p53 accurately reflects the TP53 mutational status of endometrial carcinoma (EC) has not yet been established. This study aimed to clarify the relationship between p53 IHC and TP53 mutations in EC and to examine whether p53 IHC can be a more convenient prognostic marker than TP53 mutation in EC. We performed p53 IHC staining of EC samples obtained via surgery and genetic analyses using next-generation sequencing. p53 IHC results showed that of the 101 cases, 71 (70%) were wild-type (WT), 12 (12%) were overexpression (OE), and 18 (18%) were in the null group. Missense mutations were found in 9 cases (47.4%) in OE, 2 (10.5%) in null, and 8 (42.1%) in the WT group. Truncating mutations were found in 1 case (8.3%) in OE, 6 (50%) in null, and 5 (41.7%) in the WT group. The 5-year progression-free survival was 0% in OE, 74.8% in null, and 79.0% in the WT group. In the prognosis for each type of TP53 mutation, the 5-year progression-free survival was missense (32.2%), truncating (65.6%), and WT (79.7%). These survival comparisons showed that the p53 IHC OE had the poorest prognosis. These results suggest that the p53 IHC OE is an independent poor prognostic factor for EC and can be used as a simple and rapid surrogate marker for TP53 mutations. Contrastingly, the complete absence of p53 IHC-the null staining pattern-may not accurately predict a TP53 mutation in EC, and it is necessary to be more careful in making the diagnosis of "abnormal."

Comparison of surgical outcomes between robot-assisted and conventional laparoscopic hysterectomy for large uterus.

We compared the effectiveness of conventional total laparoscopic hysterectomy (TLH) against robot-assisted total hysterectomy (RAH) in patients with a large uterus. According to the subtype of minimally invasive hysterectomy performed for benign indications, the patients (n = 843) were grouped as follows: TLH (n = 340) and RAH (n = 503). The median operative time (OT) for TLH was 98 min (47-406 min), and the estimated blood loss (EBL) was 50 mL (5-1800 mL). The median OT for RAH was 90 min (43-251 min), and the EBL was 5 mL (5-850 mL), with a significantly shorter OT and a lower EBL in RAH than in TLH. Uterine weight was categorized into four groups in increments of 250 g. The number of cases in each group was 163 (< 250 g), 116 (250-500 g), 41 (500-750 g), and 20 (≥ 750 g) for TLH, and 308 (< 250 g), 137 (250-500 g), 33 (500-750 g), and 25 (≥ 750 g) for RAH. In patients with a uterus < 250 g, there was no significant difference in OT between TLH and RAH, but in patients with a uterus ≥ 250 g, OT tended to be shorter with RAH, which was also true for a uterus ≥ 750 g. The EBL was significantly lower with RAH compared to TLH, regardless of uterine weight. In patients with a large uterus, the advantages of robotic surgery can be utilized, which may lead to a shorter OT and less EBL.

CRISPR/Cas9-Mediated Induction of Relapse-Specific NT5C2 and PRPS1 Mutations Confers Thiopurine Resistance as a Relapsed Lymphoid Leukemia Model.

6-Mercaptopurine (6-MP) is a key component in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). Recent next-generation sequencing analysis of childhood ALL clarified the emergence of the relapse-specific mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism. In this scenario, minor clones of leukemia cells could acquire the 6-MP-resistant phenotype as a result of the NT5C2 or PRPS1 mutation during chemotherapy (including 6-MP treatment) and confer disease relapse after selective expansion. Thus, to establish new therapeutic modalities overcoming 6-MP resistance in relapsed ALL, human leukemia models with NT5C2 and PRPS1 mutations in the intrinsic genes are urgently required. Here, mimicking the initiation process of the above clinical course, we sought to induce two relapse-specific hotspot mutations (R39Q mutation of the NT5C2 gene and S103N mutation of the PRPS1 gene) into a human lymphoid leukemia cell line by homologous recombination (HR) using the CRISPR/Cas9 system. After 6-MP selection of the cells transfected with Cas9 combined with single-guide RNA and donor DNA templates specific for either of those two mutations, we obtained the sublines with the intended NT5C2-R39Q and PRPS1-S103N mutation as a result of HR. Moreover, diverse in-frame small insertion/deletions were also confirmed in the 6-MP-resistant sublines at the target sites of the NT5C2 and PRPS1 genes as a result of nonhomologous end joining. These sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations in the 6-MP sensitivity and development of therapy overcoming the thiopurine resistance of leukemia cells. SIGNIFICANCE STATEMENT: Mimicking the initiation process of relapse-specific mutations of the NT5C2 and PRPS1 genes in childhood acute lymphoblastic leukemia treated with 6-mercaptopurine (6-MP), this study sought to introduce NT5C2-R39Q and PRPS1-S103N mutations into a human lymphoid leukemia cell line by homologous recombination using the CRISPR/Cas9 system. In the resultant 6-MP-resistant sublines, the intended mutations and diverse in-frame small insertions/deletions were confirmed, indicating that the obtained sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations.

Creation of Philadelphia chromosome by CRISPR/Cas9-mediated double cleavages on BCR and ABL1 genes as a model for initial event in leukemogenesis.

The Philadelphia (Ph) chromosome was the first translocation identified in leukemia. It is supposed to be generated by aberrant ligation between two DNA double-strand breaks (DSBs) at the BCR gene located on chromosome 9q34 and the ABL1 gene located on chromosome 22q11. Thus, mimicking the initiation process of translocation, we induced CRISPR/Cas9-mediated DSBs simultaneously at the breakpoints of the BCR and ABL1 genes in a granulocyte-macrophage colony-stimulating factor (GM-CSF) dependent human leukemia cell line. After transfection of two single guide RNAs (sgRNAs) targeting intron 13 of the BCR gene and intron 1 of the ABL1 gene, a factor-independent subline was obtained. In the subline, p210 BCR::ABL1 and its reciprocal ABL1::BCR fusions were generated as a result of balanced translocation corresponding to the Ph chromosome. Another set of sgRNAs targeting intron 1 of the BCR gene and intron 1 of the ABL1 gene induced a factor-independent subline expressing p190 BCR::ABL1. Both p210 and p190 BCR::ABL1 induced factor-independent growth by constitutively activating intracellular signaling pathways for transcriptional regulation of cell cycle progression and cell survival that are usually regulated by GM-CSF. These observations suggested that simultaneous DSBs at the BCR and ABL1 gene breakpoints are initiation events for oncogenesis in Ph+ leukemia. (200/200 words).

Comparison of transvaginal mesh surgery and robot-assisted sacrocolpopexy for pelvic organ prolapse.

BACKGROUND: Pelvic organ prolapse (POP) is greatly affecting the quality of life (QOL) of women. There are some surgical techniques for POP repair, for example, transvaginal mesh surgery (TVM), laparoscopic sacrocolpopexy (LSC), and robot-assisted sacrocolpopexy (RSC). In the United States and Europe, the number of TVM has rapidly decreased since 2011 due to complications and safety concerns and has shifted to LSC/RSC. In Japan, RSC has increased after the insurance coverage of RSC in 2020. Therefore, we compared the surgical outcomes of TVM and RSC in POP surgery. METHODS: We retrospectively collected POP surgery underwent TVM or RSC at our hospital and compared the operative time, blood loss, postoperative hospital stay, postoperative complications, and preoperative and postoperative stress urinary incontinence (SUI) of two groups. Preoperative and postoperative SUI were classified into 3 groups: "improved preoperative SUI", "persistent preoperative SUI" and "de novo SUI", which occurred for the first time in patients with no preoperative SUI, and compared incidence rate. The Mann-Whitney U test and Fisher's exact test were used to compare the two groups, and P < 0.05 was considered statistically significant. RESULTS: From August 2011 to July 2021, 76 POP surgery was performed and they were classified into two groups: TVM group (n = 39) and RSC group (n = 37). There was no difference in patient age and BMI between the TVM and RSC groups. The median of operative time was 78.0 vs. 111.0 min (p = 0.06), blood loss was 20.0 ml vs. 5.0 ml (p < 0.05), and postoperative hospital stay was 4.0 days vs. 3.0 days (p < 0.05), with less blood loss and shorter postoperative hospital stay in the RSC group. There was no difference in postoperative complications between the TVM and RSC groups (17.9% vs. 16.2%, p = 1.00). De novo SUI was 25.6% vs. 5.4% (p < 0.05) in the TVM and RSC groups, of which 23.1% vs. 5.4% (p < 0.05) occurred within 3 months of surgery. CONCLUSION: RSC is more beneficial and less invasive for patients with pelvic organ prolapse than TVM. In addition, de novo SUI as postoperative complication of RSC was lower than of TVM.

Molecular analysis of ascitic fluid cytology reflects genetic changes of malignancies of the ovary equivalent to surgically resected specimens.

BACKGROUND: The objective of this study was to identify the clinical utility of genomic analysis of ascitic fluid cytology (AC) in patients with epithelial ovarian cancer. METHODS: Targeted next-generation sequencing was used to analyze 66 samples from 33 patients who had ovarian (n = 23), fallopian tube (n = 2), and peritoneal (n = 8) carcinoma, and the concordance rate of molecular profiles was compared between surgically resected, formalin-fixed, paraffin-embedded (FFPE) tissues and AC samples. RESULTS: In total, 159 mutations were identified (54 oncogenic mutations and 105 nononcogenic mutations) in 66 DNA samples (33 FFPE tissues and 33 AC samples) from 33 patients. Of the 159 mutations, 57 (35.8%) were shared between surgically resected FFPE tissues and AC samples. However, the concordance rate of the molecular profiles between the 2 was significantly higher for oncogenic mutations compared with nononcogenic mutations (85.1% vs 10.5%; P < .01). Indeed, the AC samples covered all oncogenic mutations (n = 46) that were detected in surgically resected specimens and identified additional mutations (n = 8). CONCLUSIONS: The current results indicated that genomic analysis of AC can identify all of the genetic changes associated with epithelial ovarian cancer to understand tumor characteristics without interventional surgery or biopsy and may play an important role in developing personalized precision medicine.

Introduction of the T315I gatekeeper mutation of BCR/ABL1 into a Philadelphia chromosome-positive lymphoid leukemia cell line using the CRISPR/Cas9 system.

Imatinib and second-generation tyrosine kinase inhibitors (TKIs) have dramatically improved the prognosis of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). However, overcoming TKI resistance due to the T315I gatekeeper mutation of BCR/ABL1 is crucial for further improving the prognosis. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is appropriate for establishing a human model of Ph+ ALL with the T315I mutation, because it can induce specific mutations via homologous recombination (HR) repair in cells with intact endogenous HR pathway. Here we used CRISPR/Cas9 to introduce the T315I mutation into the Ph+ lymphoid leukemia cell line KOPN55bi, which appeared to have an active HR pathway based on its resistance to a poly (ADP-Ribose) polymerase-1 inhibitor. Single-guide RNA targeting at codon 315 and single-strand oligodeoxynucleotide containing ACT to ATT nucleotide transition at codon 315 were electroporated with recombinant Cas9 protein. Dasatinib-resistant sublines were obtained after one-month selection with the therapeutic concentration of dasatinib, leading to T315I mutation acquisition through HR. T315I-acquired sublines were highly resistant to imatinib and second-generation TKIs but moderately sensitive to the therapeutic concentration of ponatinib. This authentic human model is helpful for developing new therapeutic strategies overcoming TKI resistance in Ph+ ALL due to T315I mutation.

Glucocorticoid receptor gene mutations confer glucocorticoid resistance in B-cell precursor acute lymphoblastic leukemia.

Glucocorticoid (GC) is a key drug in the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and the initial GC response is an important prognostic factor. GC receptors play an essential role in GC sensitivity, and somatic mutations of the GC receptor gene, NR3C1, are reportedly identified in some BCP-ALL cases, particularly at relapse. Moreover, associations of somatic mutations of the CREB-binding protein (CREBBP) and Wolf-Hirschhorn syndrome candidate 1 (WHSC1) genes with the GC-resistance of ALL have been suggested. However, the significance of these mutations in the GC sensitivity of BCP-ALL remains to be clarified in the intrinsic genes. In the present study, we sequenced NR3C1, WHSC1, and CREBBP genes in 99 BCP-ALL and 22 T-ALL cell lines (32 and 67 cell lines were known to be established at diagnosis and at relapse, respectively), and detected their mutations in 19 (2 cell lines at diagnosis and 15 cell lines at relapse), 26 (6 and 15), and 38 (11 and 15) cell lines, respectively. Of note, 14 BCP-ALL cell lines with the NR3C1 mutations were significantly more resistant to GC than those without mutations. In contrast, WHSC1 and CREBBP mutations were not associated with GC resistance. However, among the NR3C1 unmutated BCP-ALL cell lines, WHSC1 mutations tended to be associated with GC resistance and lower NR3C1 gene expression. Finally, we successfully established GC-resistant sublines of the GC-sensitive BCP-ALL cell line (697) by disrupting ligand binding and DNA binding domains of the NR3C1 gene using the CRISPR/Cas9 system. These observations demonstrated that somatic mutations of the NR3C1 gene, and possibly the WHSC1 gene, confer GC resistance in BCP-ALL.

Association of allele-specific methylation of the ASNS gene with asparaginase sensitivity and prognosis in T-ALL.

Asparaginase therapy is a key component of chemotherapy for patients with T-cell acute lymphoblastic leukemia (T-ALL). Asparaginase depletes serum asparagine by deamination into aspartic acid. Normal hematopoietic cells can survive due to asparagine synthetase (ASNS) activity, whereas leukemia cells are supposed to undergo apoptosis due to silencing of the ASNS gene. Because the ASNS gene has a typical CpG island in its promoter, its methylation status in T-ALL cells may be associated with asparaginase sensitivity. Thus, we investigated the significance of ASNS methylation status in asparaginase sensitivity of T-ALL cell lines and prognosis of childhood T-ALL. Sequencing of bisulfite polymerase chain reaction products using next-generation sequencing technology in 22 T-ALL cell lines revealed a stepwise allele-specific methylation of the ASNS gene, in association with an aberrant methylation of a 7q21 imprinted gene cluster. T-ALL cell lines with ASNS hypermethylation status showed significantly higher in vitro l-asparaginase sensitivity in association with insufficient asparaginase-induced upregulation of ASNS gene expression and lower basal ASNS protein expression. A comprehensive analysis of diagnostic samples from pediatric patients with T-ALL in Japanese cohorts (N = 77) revealed that methylation of the ASNS gene was associated with an aberrant methylation of the 7q21 imprinted gene cluster. In pediatric T-ALL patients in Japanese cohorts (n = 75), ASNS hypomethylation status was significantly associated with poor therapeutic outcome, and all cases with poor prognostic SPI1 fusion exclusively exhibited ASNS hypomethylation status. These observations show that ASNS hypomethylation status is associated with asparaginase resistance and is a poor prognostic biomarker in childhood T-ALL.

NUDT15 polymorphism and NT5C2 and PRPS1 mutations influence thiopurine sensitivity in acute lymphoblastic leukaemia cells.

In chemotherapy for childhood acute lymphoblastic leukaemia (ALL), maintenance therapy consisting of oral daily mercaptopurine and weekly methotrexate is important. NUDT15 variant genotype is reportedly highly associated with severe myelosuppression during maintenance therapy, particularly in Asian and Hispanic populations. It has also been demonstrated that acquired somatic mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism, are detectable in a portion of relapsed childhood ALL. To directly confirm the significance of the NUDT15 variant genotype and NT5C2 and PRPS1 mutations in thiopurine sensitivity of leukaemia cells in the intrinsic genes, we investigated 84 B-cell precursor-ALL (BCP-ALL) cell lines. Three and 14 cell lines had homozygous and heterozygous variant diplotypes of the NUDT15 gene, respectively, while 4 and 2 cell lines that were exclusively established from the samples at relapse had the NT5C2 and PRPS1 mutations, respectively. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with DNA-incorporated thioguanine levels after exposure to thioguanine at therapeutic concentration. Considering the continuous exposure during the maintenance therapy, we evaluated in vitro mercaptopurine sensitivity after 7-day exposure. Mercaptopurine concentrations lethal to 50% of the leukaemia cells were comparable to therapeutic serum concentration of mercaptopurine. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with mercaptopurine sensitivity in 83 BCP-ALL and 23 T-ALL cell lines. The present study provides direct evidence to support the general principle showing that both inherited genotype and somatically acquired mutation are crucially implicated in the drug sensitivity of leukaemia cells.

Epigenetic Modification of Death Receptor Genes for TRAIL and TRAIL Resistance in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia.

Immunotherapies specific for B-cell precursor acute lymphoblastic leukemia (BCP-ALL), such as anti-CD19 chimeric antigen receptor (CAR) T-cells and blinatumomab, have dramatically improved the therapeutic outcome in refractory cases. In the anti-leukemic activity of those immunotherapies, TNF-related apoptosis-inducing ligand (TRAIL) on cytotoxic T-cells plays an essential role by inducing apoptosis of the target leukemia cells through its death receptors (DR4 and DR5). Since there are CpG islands in the promoter regions, hypermethylation of the DR4 and DR5 genes may be involved in resistance of leukemia cells to immunotherapies due to TRAIL-resistance. We analyzed the DR4 and DR5 methylation status in 32 BCP-ALL cell lines by sequencing their bisulfite PCR products with a next-generation sequencer. The DR4 and DR5 methylation status was significantly associated with the gene and cell-surface expression levels and the TRAIL-sensitivities. In the clinical samples at diagnosis (459 cases in the NOPHO study), both DR4 and DR5 genes were unmethylated in the majority of cases, whereas methylated in several cases with dic(9;20), MLL-rearrangement, and hypodiploidy, suggesting that evaluation of methylation status of the DR4 and DR5 genes might be clinically informative to predict efficacy of immunotherapy in certain cases with such unfavorable karyotypes. These observations provide an epigenetic rational for clinical efficacy of immunotherapy in the vast majority of BCP-ALL cases.

IMiDs uniquely synergize with TKIs to upregulate apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressing a dominant-negative IKZF1 isoform.

The long-term prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) is still unsatisfactory even after the emergence of tyrosine kinase inhibitors (TKIs) against chimeric BCR-ABL, and this is associated with the high incidence of genetic alterations of Ikaros family zinc finger 1 (IKZF1), most frequently the hemi-allelic loss of exons 4-7 expressing a dominant-negative isoform Ik6. We found that lenalidomide (LEN), a representative of immunomodulatory drugs (IMiDs), which have been long used for the treatment of multiple myeloma, specifically induced accumulation of Ik6 with the disappearance of functional isoforms within 24 h (i.e., abrupt and complete shut-down of the IKZF1 activity) in Ik6-positive Ph+ALL cells in a neddylation-dependent manner. The functional IKZF3 isoforms expression was also abruptly and markedly downregulated. The LEN treatment specifically suppressed proliferation of Ik6-positive-Ph+ALL cells by inducing cell cycle arrest via downregulation of cyclins D3 and E and CDK2, and of importance, markedly upregulated their apoptosis in synergy with the TKI imatinib (IM). Apoptosis of IM-resistant Ph+ALL cells with T315I mutation of BCR-ABL was also upregulated by LEN in the presence of the newly developed TKI ponatinib. Analyses of flow cytometry, western blot, and oligonucleotide array revealed that apoptosis was caspase-/p53-dependent and associated with upregulation of pro-apoptotic Bax/Bim, enhanced dephosphorylation of BCR-ABL/Akt, and downregulation of oncogenic helicase genes HILLS, CDC6, and MCMs4 and 8. Further, the synergism of LEN with IM was clearly documented as a significant prolongation of survival in the xenograft mice model. Because this synergism was further potentiated in vitro by dexamethasone, a key drug for ALL treatment, the strategy of repositioning IMiDs for the treatment of Ik6-positive Ph+ALL patients certainly shed new light on an outpatient-based treatment option for achieving their long-term durable remission and higher QOL, particularly for those who are not tolerable to intensified therapeutic approaches.

Inherited genetic variants associated with glucocorticoid sensitivity in leukaemia cells.

Identification of genetic variants associated with glucocorticoids (GC) sensitivity of leukaemia cells may provide insight into potential drug targets and tailored therapy. In the present study, within 72 leukaemic cell lines derived from Japanese patients with B-cell precursor acute lymphoblastic leukaemia (ALL), we conducted genome-wide genotyping of single nucleotide polymorphisms (SNP) and attempted to identify genetic variants associated with GC sensitivity and NR3C1 (GC receptor) gene expression. IC50 measures for prednisolone (Pred) and dexamethasone (Dex) were available using an alamarBlue cell viability assay. IC50 values of Pred showed the strongest association with rs904419 (P = 4.34 × 10-8 ), located between the FRMD4B and MITF genes. The median IC50 values of prednisolone for cell lines with rs904419 AA (n = 13), AG (n = 31) and GG (n = 28) genotypes were 0.089, 0.139 and 297 µmol/L, respectively. For dexamethasone sensitivity, suggestive association was observed for SNP rs2306888 (P = 1.43 × 10-6 ), a synonymous SNP of the TGFBR3 gene. For NR3C1 gene expression, suggestive association was observed for SNP rs11982167 (P = 6.44 × 10-8 ), located in the PLEKHA8 gene. These genetic variants may affect GC sensitivity of ALL cells and may give rise to opportunities in personalized medicine for effective and safe chemotherapy in ALL patients.