Yu Mizutani

NANOG Expression as a Responsive Biomarker during Treatment with Hedgehog Signal Inhibitor in Acute Myeloid Leukemia

Aberrant activation of the Hedgehog (Hh) signaling pathway is involved in the maintenance of leukemic stem cell (LSCs) populations. PF-0444913 (PF-913) is a novel inhibitor that selectively targets Smoothened (SMO), which regulates the Hh pathway. Treatment with PF-913 has shown promising results in an early phase study of acute myeloid leukemia (AML). However, a detailed mode of action for PF-913 and relevant biomarkers remain to be elucidated. In this study, we examined bone marrow samples derived from AML patients under PF-913 monotherapy. Gene set enrichment analysis (GSEA) revealed that PF-913 treatment affected the self-renewal signature and cell-cycle regulation associated with LSC-like properties. We then focused on the expression of a pluripotency factor, NANOG, because previous reports showed that a downstream effector in the Hh pathway, GLI, directly binds to the NANOG promoter and that the GLI-NANOG axis promotes stemness and growth in several cancers. In this study, we found that a change in NANOG transcripts was closely associated with GLI-target genes and NANOG transcripts can be a responsive biomarker during PF-913 therapy. Additionally, the treatment of AML with PF-913 holds promise, possibly through inducing quiescent leukemia stem cells toward cell cycling.

Mixed Phenotype Acute Leukemia with t(12;17)(p13;q21)/TAF15-ZNF384 and Other Chromosome Abnormalities

The t(12;17)(p13;q11∼21) translocation is a very rare but recurrent cytogenetic aberration observed predominantly in early pre-B acute lymphoblastic leukemia (ALL) with CD19+CD10-CD33+ phenotype. This translocation was shown to form a fusion gene between TAF15 at 17q12 and ZNF384 at 12p13. On the other hand, der(1;18)(q10;q10) has been detected as a rare unbalanced whole-arm translocation leading to trisomy 1q in myeloid malignancies. We describe here the first case of mixed phenotype acute leukemia (MPAL) with a t(12;17)(p13;q21)/TAF15-ZNF384, which also had der(1;18)(q10;q10) as an additional abnormality. A 74-year-old woman was diagnosed with MPAL, B/myeloid, because bone marrow blasts were positive for myeloperoxidase, CD19, and CD22. Chromosome analysis showed 46,XX, +1,der(1;18)(q10;q10),t(2;16)(q13;q13),t(12;17)(p13;q21). Expression of the TAF15-ZNF384 fusion transcript was confirmed: TAF15 exon 6 was fused in-frame to ZNF384 exon 3. This type of fusion gene has been reported in 1 acute myeloid leukemia case and 3 ALL cases. Thus, at present, it is difficult to find a specific association between the structure of the TAF15-ZNF384 fusion gene and the leukemia phenotype. The TAF15-ZNF384 fusion may occur in early common progenitor cells that could differentiate into both the myeloid and lymphoid lineages. Furthermore, der(1;18)(q10;q10) might play some role in the appearance of an additional myeloid phenotype.

Distribution of erlotinib in rash and normal skin in cancer patients receiving erlotinib visualized by matrix assisted laser desorption/ionization mass spectrometry imaging

Background The development of skin rashes is the most common adverse event observed in cancer patients treated with epidermal growth factor receptor-tyrosine kinase inhibitors such as erlotinib. However, the pharmacological evidence has not been fully revealed. Results Erlotinib distribution in the rashes was more heterogeneous than that in the normal skin, and the rashes contained statistically higher concentrations of erlotinib than adjacent normal skin in the superficial skin layer (229 ± 192 vs. 120 ± 103 ions/mm2; P = 0.009 in paired t-test). LC-MS/MS confirmed that the concentration of erlotinib in the skin rashes was higher than that in normal skin in the superficial skin layer (1946 ± 1258 vs. 1174 ± 662 ng/cm3; P = 0.028 in paired t-test). The results of MALDI-MSI and LC-MS/MS were well correlated (coefficient of correlation 0.879, P < 0.0001). Conclusions Focal distribution of erlotinib in the skin tissue was visualized using non-labeled MALDI-MSI. Erlotinib concentration in the superficial layer of the skin rashes was higher than that in the adjacent normal skin. Methods We examined patients with advanced pancreatic cancer who developed skin rashes after treatment with erlotinib and gemcitabine. We biopsied both the rash and adjacent normal skin tissues, and visualized and compared the distribution of erlotinib within the skin using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The tissue concentration of erlotinib was also measured by liquid chromatography-tandem mass spectrometry (LC–MS/MS) with laser microdissection.

An mTORC1/2 kinase inhibitor enhances the cytotoxicity of gemtuzumab ozogamicin by activation of lysosomal function

Gemtuzumab ozogamicin (GO), the first antibody-drug conjugate (ADC), has attracted the interest of hematologists because more than 90% of acute myeloid leukemia (AML) blasts express its target, CD33. Although GO and subsequently developed ADCs depend on lysosomes for activation, lysosome number and activity in tumor cells has not been well elucidated. In this study, we investigated whether an mTORC1/2 kinase inhibitor, PP242, which was reported to activate lysosomal function, potentiates the cytotoxicity of GO in AML cells. Eight AML cell lines (U937, THP-1, SKM-1, SKK-1, SKNO-1, HL-60, MARIMO and KO52) were treated with GO and PP242. The cytotoxic effect of GO was enhanced by concurrent treatment with a non-cytotoxic concentration (500 nM) of PP242 in most cell lines, except MARIMO and KO52 cells. We then used LysoTracker to label acidic lysosomes in U937, THP-1, SKM-1, MARIMO and KO52 cells. LysoTracker fluorescence was dramatically increased by treatment with PP242 in U937, THP-1 and SKM-1 cells, and the intensified fluorescence was retained with PP242 + GO. In contrast, PP242 did not induce a significant increase in fluorescence in MARIMO cells, consistent with the lack of combinatory cytotoxicity. LysoTracker fluorescence was also increased by PP242 in KO52 cells, which have been reported to strongly express multidrug resistance (MDR). Further, PP242 suppressed GO-induced Chk1 activation and G2/M cell cycle arrest, which in turn triggered cell cycle promotion and cell death. These results indicate that inhibition of mTORC1/2 kinase by PP242 enhanced the cytotoxicity of GO by increasing lysosomal compartments and promoting the cell cycle via suppression of GO-induced Chk1 activation. This combination may represent an attractive new therapeutic strategy for the treatment of leukemia.

A Case of Classical Hodgkin Lymphoma with Total Lymph Node Infarction

Lymph node infarction is very rare, and is frequently associated with neoplasms, such as malignant lymphoma and non-neoplastic disease, or interventions such as fine-needle aspiration (FNA). A 76-year-old-man presented with cervical lymph node swelling. Although FNA was performed, the findings were insufficient for a definitive diagnosis. Consequently, surgical biopsy of the cervical lymph node was performed, which revealed total infarction; a diagnosis of classical Hodgkin lymphoma was made later. Both lymphoma itself and FNA may cause total lymph node infarction, which makes diagnosis confusing. Therefore, it is important to repeat the biopsy rather than repeat FNA to correctly diagnose malignant lymphoma, including Hodgkin lymphoma.

Invasive Scopulariopsis alboflavescens infection in patient with acute myeloid leukemia

Scopulariopsis alboflavescens is a soil saprophyte that is widely distributed in nature. Recently, there have been increasing number of reports of invasive infections with Scopulariopsis species in immunocompromised patients. In this report, we described an adult woman with acute myeloid leukemia and who developed S. alboflavescens pneumonia. Liposomal amphotericin B and voriconazole combination therapy was unsuccessful and the patient died because of pneumonia. Scopulariopsis is highly resistant to available antifungal agents and almost invariably fatal. This case report should alert clinicians to the importance of listing Scopulariopsis as a pathogenic fungus in immunocompromised patients.

MYC Amplification in the Form of Ring Chromosomes 8 in Acute Myeloid Leukemia with t(11;16)(q13;p11.2)

Oncogene amplification is uncommon in acute myeloid leukemia (AML). Cytogenetically, it is primarily found as double minute chromosomes (dmin) or homogeneously staining regions (hsr). A 62-year-old woman was admitted to our hospital because of anemia and thrombocytopenia. Her bone marrow was hypercellular with 78.6% myeloperoxidase- positive blasts. Some had micronuclei. The patient was diagnosed with AML M2 and remains in complete remission (CR) after induction therapy. G-banding at diagnosis showed 51,XX,t(11;16)(q13;p11.2),+r1,+mar1×4. Spectral karyotyping confirmed t(11;16) and revealed that the ring and the marker chromosomes were derived from multiple copies of ring chromosome 8. Fluorescence in situ hybridization (FISH) with a MYC probe at 8q24 detected amplified MYC signals on 1 large and 4 small ring chromosomes 8. One MYC signal was deleted from one of the 2 chromosomes 8. FISH with a FUS probe at 16p11.2 showed monoallelic deletion of FUS. Immunohistochemistry demonstrated MYC protein overexpression at diagnosis and almost negative expression in CR. These results indicate that MYC amplification could occur in ring chromosomes without dmin. A cryptic MYC deletion suggests that an episome model could be applicable to MYC amplification in ring chromosomes as observed for dmin and hsr. Furthermore, considering 2 further reported cases, t(11;16)(q13;p11) may be a very rare but recurrent translocation in AML.

Abstract 2872: Quantitative mass spectrometry imaging of erlotinib in skin rashes of cancer patients receiving erlotinib

Background The development of rashes is the most common adverse event observed in cancer patients treated with epidermal growth factor receptor-targeted tyrosine kinase inhibitors (EGFR-TKI) such as erlotinib. A significant relationship exists between the severity of a rash and survival in various cancers. However, whether erlotinib becomes distributed to the skin and whether its concentration is higher in a rash than in normal skin of treated cancer patients remains unknown. Here, using quantitative mass spectrometry imaging (qMSI), we successfully visualized the distribution of erlotinib in rashes and normal skin of patients with advanced pancreatic cancer receiving this drug. Methods We studied five patients with advanced pancreatic cancer who developed rashes after treatment with gemcitabine (1000 mg/m² by intravenous infusion for 30 minutes on days 1, 8, and 15 every 4 weeks) and erlotinib (given orally at 100 mg/day). We biopsied both rashes and normal skin, and compared the distribution of erlotinib using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). The plasma concentration of erlotinib was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results Erlotinib concentrations in five patients were 2.55 ± 1.46 ng/mm3 in normal skin and 3.18 ± 1.53 ng/mm3 (mean ± SD) in rashes (p=0.123). In four out of five patients, the erlotinib concentration in rashes was higher than that in normal skin. The epidermis showed the strongest expression of EGFR in skin as judged by immunohistological staining. Erlotinib showed a greater tendency for distribution to the epidermis than subcutaneous tissue. There was no correlation between erlotinib plasma concentrations vs. concentrations in rashes or normal skin. Conclusions Using qMSI, we, for the first time, visualized the distribution of erlotinib in skin tissue of pancreatic cancer patients receiving erlotinib. We found a tendency for a higher concentration of erlotinib in rashes than in normal skin. A greater distribution of erlotinib to the epidermis than subcutaneous tissue suggests erlotinib may directly bind EGFR expressed in the epidermis. Citation Format: Meiko Nishimura, Hiroaki Aikawa, Mitsuhiro Hayashi, Yu Mizutani, Kei Takenaka, Yoshinori Imamura, Naoko Chayahara, Masanori Toyoda, Naomi Kiyota, Toru Mukohara, Akinobu Hamada, Hironobu Minami. Quantitative mass spectrometry imaging of erlotinib in skin rashes of cancer patients receiving erlotinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2872. doi:10.1158/1538-7445.AM2017-2872

Coexpression of NUP98/TOP1 and TOP1/NUP98 in de novo Acute Myeloid Leukemia with t(11;20)(p15;q12) and t(2;5)(q33;q31)

The t(11;20)(p15;q11∼12) translocation is a very rare but recurrent cytogenetic aberration that occurs in myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). This translocation was shown to form a fusion gene between NUP98 at 11p15 and TOP1 at 20q12. Here, we describe a new case of de novo AML M2 with t(11;20) which was associated with another balanced translocation. An 81-year-old man was admitted to undergo salvage therapy for relapsed AML. G-banding and spectral karyotyping showed 46,XY,t(2;5)(q33;q31),t(11;20)(p15;q12)[20]. Expression of the NUP98/TOP1 fusion transcript was confirmed: NUP98 exon 13 was in-frame fused with TOP1 exon 8. The reciprocal TOP1/NUP98 fusion transcript was also detected: TOP1 exon 7 was fused with NUP98 exon 14. After achieving hematological complete remission, the karyotype converted to 46,XY,t(2;5)(q33;q31)[19]/46,sl,t(11;20)(p15;q12)[1]. FISH analysis demonstrated that the 5q31 breakpoint of t(2;5) was centromeric to EGR1. In all 10 cases described in the literature, the NUP98 exon 13/TOP1 exon 8 fusion transcript was expressed, indicating that it may be responsible for the pathogenesis of MDS/AML with t(11;20). On the other hand, the TOP1/NUP98 transcript was coexpressed in 4 cases of de novo AML, but not in 3 cases of therapy-related MDS. Thus, this reciprocal fusion may be associated with progression to AML.