[A Case of Non-Small Cell Lung Cancer Successfully Treated with Anti-PD-L1 Therapy after Exacerbation Caused by Anti-PD-1 Therapy].

A 52-year-old male was diagnosed with cT4N3M1b, cStage Ⅳ adenocarcinoma of the lung, and underwent 6 courses of pemetrexed(PEM), carboplatin(CBDCA), and bevacizumab(Bev)therapy, followed by 4 courses of PEM/Bev maintenance therapy. Due to the deterioration of his condition, 4 courses of pembrolizumab(Pembro)therapy were administered as second- line therapy, but the treatment was discontinued due to disease progression. Third-line therapy with docetaxel(DTX)/ ramucirumab(Rmab)was administered, resulting in a partial response, but discontinued due to adverse events. Finally, fourth-line therapy with atezolizumab(Atezo)was initiated. After 6 courses of Atezo therapy, partial response was achieved, and the tumor continued to shrink for 3 years. The aim of the treatment strategy for both PD-1 and PD-L1 antibody drugs is to suppress T-cell exhaustion, and if either drug fails, the other drug may theoretically fail. However, in this case, the PD-L1 antibody drug was effective against non-small cell lung cancer that had shown resistance to PD-1 antibody drugs, suggesting that even if a patient becomes resistant to PD-1 antibody drugs, PD-L1 antibody drugs may result in effective outcomes.

[Role of Palliative Care in the Introduction of Chemotherapy in Patients with Poor Performance Status].

Chemotherapy for patients with poor performance status(PS)is not recommended because it increases the risk of death and shortens their survival. We report on 3 cases in which palliative care improved PS and enabled chemotherapy, resulting in a prolonged prognosis. Case 1 involves a 57‒year‒old woman with multiple lung, liver, and bone metastases from breast cancer who was administered celecoxib and oxycodone. She received Mohs paste therapy. Her PS improved from 3 to 2, and paclitaxel and trastuzumab were started; however, the patient died on day 861. Case 2 involves a 53‒year‒old woman with multiple lymph node metastases from carcinoma of an unknown primary cause. She was administered oxycodone, loxoprofen, and dexamethasone. Her PS improved from 4 to 3. The biopsy of her bone marrow showed diffuse large B‒cell lymphoma. The patient is still alive, 6 years and 10 months after the introduction of R‒CHOP. In case 3, a 57‒year‒old man with multiple bone metastases from small cell lung cancer was administered loxoprofen and betamethasone. His PS improved from 4 to 3. Etoposide and carboplatin were administered to him, but the patient died on day 692. Palliative care may enable the introduction of chemotherapy and consequently improve prognosis.

[Advanced Gastric Cancer with Tumor Shrinkage Persisting after the Discontinuation of Nivolumab-A Case Report].

The patient was a 65-year-old man with advanced gastric cancer, cT4bN3aM1, cStage Ⅳ. The SOX therapy was administered as the primary treatment but discontinued after 9 courses because of disease progression. The PTX plus RAM therapy was then administered for 1 courses as the secondary treatment but discontinued because of the development of peritoneal dissemination, increased number of ascites, and increased number of lymph node metastases. The nivolumab(NIV)therapy was initiated as the tertiary treatment, but the patient complained of fatigue and diplopia after 2 courses. Ptosis was observed, and transaminase and creatine kinase levels were elevated. Electrocardiography showed complete right bundle branch block. The patient showed immune-related adverse events and was diagnosed with myocarditis and myasthenia gravis due to NIV. Consequently, systemic steroids were administered. Although 2 course of CPT-11 was administered as the fourth-line treatment, the treatment was discontinued upon the patient's request. Ten months after the discontinuation of chemotherapy, the disease showed no progression. The patient is being followed-up as an outpatient. Here, we reported a case of gastric cancer with tumor shrinkage after the discontinuation of NIV.

Cotransplantation with MSCs improves engraftment of HSCs after autologous intra-bone marrow transplantation in nonhuman primates.

OBJECTIVE: Hematopoietic stem cells (HSCs) reside in the osteoblastic niche, which consists of osteoblasts. Mesenchymal stromal cells (MSCs) have an ability to differentiate into osteoblasts. Here, using nonhuman primates, we investigated the effects of cotransplantation with MSCs on the engraftment of HSCs after autologous intra-bone marrow transplantation. MATERIALS AND METHODS: From three cynomolgus monkeys, CD34-positive cells (as HSCs) and MSCs were obtained. The former were divided into two equal aliquots and each aliquot was genetically marked with a distinctive retroviral vector to track the in vivo fate. Each HSC aliquot with or without MSCs was autologously injected into the bone marrow (BM) cavity of right or left side, enabling the comparison of in vivo fates of the two HSC grafts in the same body. RESULTS: In the three monkeys, CD34(+) cells transplanted with MSCs engrafted 4.4, 6.0, and 1.6 times more efficiently than CD34(+) cells alone, as assessed by BM colony polymerase chain reaction. In addition, virtually all marked cells detected in the peripheral blood were derived from the cotransplantation aliquots. Notably, colony-forming units derived from the cotransplantation aliquots were frequently detected in BM distant sites from the injection site, implying that cotransplantation with MSCs also restored the ability of gene-marked HSCs to migrate and achieve homing in the distant BM. CONCLUSION: Cotransplantation with MSCs would improve the efficacy of transplantation of gene-modified HSCs in primates, with enhanced engraftment in BM as well as increased chimerism in peripheral blood through migration and homing.

Cell and gene therapy using mesenchymal stem cells (MSCs).

Mesenchymal stem cells (MSCs) are considered to be a promising platform for cell and gene therapy for a variety of diseases. First, in the field of hematopoietic stem cell transplantation, there are two applications of MSCs: 1) the improvement of stem cell engrafting and the acceleration of hematopoietic reconstitution based on the hematopoiesis-supporting ability; and 2) the treatment of severe graft-versus-host disease (GVHD) based on the immunomodulatory ability. Regarding the immunosuppressive ability, we found that nitric oxide (NO) is involved in the MSC-mediated suppression of T cell proliferation. Second, tumor-bearing nude mice were injected with luciferase-expressing MSCs. An in vivo imaging analysis showed the significant accumulation of the MSCs at the site of tumors. The findings suggest that MSCs can be utilized to target metastatic tumors and to deliver anti-cancer molecules locally. As the third application, MSCs may be utilized as a cellular vehicle for protein-supplement gene therapy. When long-term transgene expression is needed, a therapeutic gene should be introduced with a minimal risk of insertional mutagenesis. To this end, site-specific integration into the AAVS1 locus on the chromosome 19 (19q13.4) by using the integration machinery of adeno-associated virus (AAV) would be particularly valuable. There will be wide-ranging applications of MSCs to frontier medical treatments in the near future.

Pleocytosis after hemopoietic stem cell transplantation.

Frequency and clinical significance of cerebrospinal fluid (CSF) pleocytosis in hemopoietic stem cell (HSC) transplantation were surveyed. Cyclosporine (CSA)- or tacrolimus (FK506)-based regimens were used as graft-vs-host disease (GVHD) prophylaxis in allogeneic HSC transplantation. CSF pleocytosis with or without neurologic symptoms was detected in 12 of 25 patients receiving allogeneic HSC transplants but in none of 11 patients receiving autologous HSC transplants. Of the 12 patients with CSF pleocytosis, only one patient developed leukoencephalopathy later. There was a correlation between CSF cell numbers and trough levels of CSA but not with those of FK506. In patients receiving allogeneic HSC transplants, CSF pleocytosis may be relatively common and may reflect neurologic damage associated with calcineurin inhibitors.

Removal of empty capsids from type 1 adeno-associated virus vector stocks by anion-exchange chromatography potentiates transgene expression.

Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), virus protein shells without the vector genome. The contaminating VLPs would interfere with transduction by competing for cell-surface receptors and, when administered in vivo, contribute to antigen load, which may elicit a stronger immune response. Density-gradient ultracentrifugation provides a means to separate VLPs from rAAV particles, but is not feasible for large-scale preparations of vectors. Since the compositions of the VLP and vector differ by the single-stranded DNA genome, we hypothesized that the isoelectric point of the vector may differ from that of the VLP. In an attempt to separate type 1 rAAV particles from VLPs by ion-exchange chromatography, we tested a number of buffer systems and found that trimethylammonium sulfate, or [(CH3)4N]2SO4, effectively separated rAAV1 particles from VLPs. The rAAV1-GFP chromatographically separated from VLPs induced stronger GFP expression in HEK293 cells than rAAV1-GFP contaminated with VLPs. The transduction of mouse muscles with rAAV1-SEAP (secreted form of alkaline phosphatase) isolated from VLPs also showed higher serum SEAP levels than rAAV1-SEAP with VLPs. These results suggest that chromatographic separation of rAAV1 from empty capsids increased the efficacy of rAAV1.

Scalable generation of high-titer recombinant adeno-associated virus type 5 in insect cells.

We established a method for production of recombinant adeno-associated virus type 5 (rAAV5) in insect cells by use of baculovirus expression vectors. One baculovirus harbors a transgene between the inverted terminal repeat sequences of type 5, and the second expresses Rep78 and Rep52. Interestingly, the replacement of type 5 Rep52 with type 1 Rep52 generated four times more rAAV5 particles. We replaced the N-terminal portion of type 5 VP1 with the equivalent portion of type 2 to generate infectious AAV5 particles. The rAAV5 with the modified VP1 required alpha2-3 sialic acid for transduction, as revealed by a competition experiment with an analog of alpha2-3 sialic acid. rAAV5-GFP/Neo with a 4.4-kb vector genome produced in HEK293 cells or Sf9 cells transduced COS cells with similar efficiencies. Surprisingly, Sf9-produced humanized Renilla green fluorescent protein (hGFP) vector with a 2.4-kb vector genome induced stronger GFP expression than the 293-produced one. Transduction of murine skeletal muscles with Sf9-generated rAAV5 with a 3.4-kb vector genome carrying a human secreted alkaline phosphatase (SEAP) expression cassette induced levels of SEAP more than 30 times higher than those for 293-produced vector 1 week after injection. Analysis of virion DNA revealed that in addition to a 2.4- or 3.4-kb single-stranded vector genome, Sf9-rAAV5 had more-abundant forms of approximately 4.7 kb, which appeared to correspond to the monomer duplex form of hGFP vector or truncated monomer duplex SEAP vector DNA. These results indicated that rAAV5 can be generated in insect cells, although the difference in incorporated virion DNA may induce different expression patterns of the transgene.

[Pseudomonas sepsis with ecthyma gangrenosum in an acute myeloid leukemia patient].

A 56-year-old woman with acute myeloid leukemia had two rapidly growing necrotizing nodules with ulcer formation on her head after the first course of consolidation therapy. Clinical features corresponding to sepsis (e.g., fever) appeared following the development of the skin lesion. Pseudomonas aeruginosa was isolated from the blood as well as pus of the lesion. Based on these findings, a diagnosis of ecthyma gangrenosum was made. Treatment with ciprofloxacin and gamma-globulin dramatically improved the patient's clinical features. Since Pseudomonas sepsis with ecthyma gangrenosum is associated with a high mortality rate, it is important to start immediate treatment with appropriate antibiotics.