CD30 expression in an emerging group of mesenchymal spindle cell neoplasms with ALK fusion detected by flow cytometry and immunohistochemistry.

An emerging group of spindle cell neoplasms harboring fusions involving NTRK or non-NTRK kinase genes often share characteristic S100 and/or CD34 expression; however, the diagnostic utility of immunohistochemical stains is not well established in this family owing to their lack of specificity. Recently, CD30 expression in spindle cell neoplasms with kinase gene fusions, such as NTRK, BRAF, RAF1, and RET, has been increasingly identified. We herein report a 10-year-old girl with high-grade spindle cell sarcoma of the neck. Prior to histopathological evaluation, flow cytometry (FCM) analysis and touch smear cytology of the tumor tissue revealed CD34+ and dimCD30+ spindle cell populations. Histopathologically, the case was characterized by monomorphic spindle-shaped cytomorphology with CD30, S100, and CD34 positivity and harbored close similarities with spindle cell neoplasms with NTRK or non-NTRK gene fusions. Subsequently, a comprehensive next-generation sequencing sarcoma panel identified a rare PLEKHH2::ALK fusion, and a diagnosis of ALK-rearranged spindle cell neoplasm was made. The patient showed significant tumor response to single-agent treatment with alectinib, an ALK-tyrosine kinase inhibitor. This case supports that CD30 is expressed in an ALK-rearranged mesenchymal neoplasm. The benefit of the early detection of CD30 expression by FCM for a prompt diagnosis and treatment is highlighted in the context of an aggressive clinical course. This case represents a learning experience regarding the need to the check the status of CD30 expression in these tumors and suggests the potential clinical benefits of CD30-targeted therapy.

A phase 1 trial of NY-ESO-1-specific TCR-engineered T-cell therapy combined with a lymph node-targeting nanoparticulate peptide vaccine for the treatment of advanced soft tissue sarcoma.

The efficacy of immune checkpoint inhibitors is limited in refractory solid tumors. T-cell receptor gene-modified T (TCR-T)-cell therapy has attracted attention as a new immunotherapy for refractory cold tumors. We first investigated the preclinical efficacy and mode of action of TCR-T cells combined with the pullulan nanogel:long peptide antigen (LPA) vaccine in a mouse sarcoma model that is resistant to immune checkpoint inhibition. Without lymphodepletion, the pullulan nanogel:LPA vaccine markedly increased the number of TCR-T cells in the draining lymph node and tumor tissue. This change was associated with enhanced CXCR3 expression in TCR-T cells in the draining lymph node. In the phase 1 trial, autologous New York esophageal squamous cell carcinoma 1 (NY-ESO-1)-specific TCR-T cells were infused twice into HLA-matched patients with NY-ESO-1+ soft tissue sarcoma (STS). The pullulan nanogel:LPA vaccine contains an epitope recognized by TCR-T cells, and it was subcutaneously injected 1 day before and 7 days after the infusion of TCR-T cells. Lymphodepletion was not performed. Three patients with refractory synovial sarcoma (SS) were treated. Two out of the three patients developed cytokine release syndrome (CRS) with low-to-moderate cytokine level elevation. We found obvious tumor shrinkage lasting for more than 2 years by tumor imaging and long-term persistence of TCR-T cells in one patient. In conclusion, NY-ESO-1-specific TCR-T-cell therapy plus vaccination with the pullulan nanogel carrying an LPA containing the NY-ESO-1 epitope without lymphodepletion is feasible and can induce promising long-lasting therapeutic effects in refractory SS (Registration ID: JMA-IIA00346).

TP53 Variant in the Blood of a Patient with Gastric Cancer Undergoing Tumor Profiling Tests Diagnosed as Clonal Hematopoiesis.

BACKGROUND Clonal hematopoiesis is the production of a specific single clonal type of cell in the blood and is often found in cancer genomic profiling tests. When the clone carries a pathogenic variant, it may be important to differentiate between somatic or germline origin. The variant in the blood that has a lower minor allele frequency could reflect heterozygous germline origin, somatic mosaicism, and clonal hematopoiesis. It is important to evaluate suspected variants to determine the course of treatment and follow-up of the patient, depending on the patient's medical condition and family situation. CASE REPORT We report a 53-year-old Japanese man with gastric cancer who underwent a cancer genomic profiling test searching for therapeutic agents. The profiling test detected a variant, TP53 c.559+2T>G minor allele frequencies of 9% (168/1865) in tumor tissue and 29.1% (58/199) in paired blood. Since the TP53 variant has the possibility of Li-Fraumeni syndrome, ancillary testing was performed using fingernails, buccal swab, and blood specimens. The genomic analysis revealed no TP53 variant in his fingernails. The patient had previously received platinum-based chemotherapies, suggesting that the variant reflected treatment-induced clonal hematopoiesis. CONCLUSIONS Identifying clonal hematopoiesis when performing genomic profiling tests for patients with cancer is important. Examining multiple tissues to determine whether a variant arises from clonal hematopoiesis or is of germline origin can provide more accurate genetic information and improve patient follow-up care.

NY-ESO-1-specific redirected T cells with endogenous TCR knockdown mediate tumor response and cytokine release syndrome.

BACKGROUND: Because of the shortage of ideal cell surface antigens, the development of T-cell receptor (TCR)-engineered T cells (TCR-T) that target intracellular antigens such as NY-ESO-1 is a promising approach for treating patients with solid tumors. However, endogenous TCRs in vector-transduced T cells have been suggested to impair cell-surface expression of transduced TCR while generating mispaired TCRs that can become self-reactive. METHODS: We conducted a first-in-human phase I clinical trial with the TCR-transduced T-cell product (TBI-1301) in patients with NY-ESO-1-expressing solid tumors. In manufacturing TCR-T cells, we used a novel affinity-enhanced NY-ESO-1-specific TCR that was transduced by a retroviral vector that enables siRNA (small interfering RNA)-mediated silencing of endogenous TCR. The patients were divided into two cohorts. Cohort 1 was given a dose of 5×108 cells (whole cells including TCR-T cells) preconditioned with 1500 mg/m2 cyclophosphamide. Cohort 2 was given 5× 109 cells preconditioned with 1500 mg/m2 cyclophosphamide. RESULTS: In vitro study showed that both the CD8+ and CD4+ T fractions of TCR-T cells exhibited cytotoxic effects against NY-ESO-1-expressing tumor cells. Three patients and six patients were allocated to cohort 1 and cohort 2, respectively. Three of the six patients who received 5×109 cells showed tumor response, while three patients developed early-onset cytokine release syndrome (CRS). One of the patients developed a grade 3 lung injury associated with the infiltration of the TCR-T cells. No siRNA-related adverse events other than CRS were observed. Cytokines including interleukin 6 I and monocyte chemotactic protein-1/chemokine (C-C motif) ligand (CCL2)increased in the sera of patients with CRS. In vitro analysis showed these cytokines were not secreted from the T cells infused. A significant fraction of the manufactured T cells in patients with CRS was found to express either CD244, CD39, or both at high levels. CONCLUSIONS: The trial showed that endogenous TCR-silenced and affinity-enhanced NY-ESO-1 TCR-T cells were safely administered except for grade 3 lung injury. The TCR-T cell infusion exhibited significant tumor response and early-onset CRS in patients with tumors that express NY-ESO-1 at high levels. The differentiation properties of the manufactured T cells may be prognostic for TCR-T-related CRS. TRIAL REGISTRATION NUMBER: NCT02366546.

Medical guidelines for Li-Fraumeni syndrome 2019, version 1.1.

Li-Fraumeni syndrome (LFS) is a hereditary tumor that exhibits autosomal dominant inheritance. LFS develops in individuals with a pathogenic germline variant of the cancer-suppressor gene, TP53 (individuals with TP53 pathogenic variant). The number of individuals with TP53 pathogenic variant among the general population is said to be 1 in 500 to 20,000. Meanwhile, it is found in 1.6% (median value, range of 0-6.7%) of patients with pediatric cancer and 0.2% of adult patients with cancer. LFS is diagnosed by the presence of germline TP53 pathogenic variants. However, patients can still be diagnosed with LFS even in the absence of a TP53 pathogenic variant if the familial history of cancers fit the classic LFS diagnostic criteria. It is recommended that TP53 genetic testing be promptly performed if LFS is suspected. Chompret criteria are widely used for the TP53 genetic test. However, as there are a certain number of cases of LFS that do not fit the criteria, if LFS is suspected, TP53 genetic testing should be performed regardless of the criteria. The probability of individuals with TP53 pathogenic variant developing cancer in their lifetime (penetrance) is 75% for men and almost 100% for women. The LFS core tumors (breast cancer, osteosarcoma, soft tissue sarcoma, brain tumor, and adrenocortical cancer) constitute the majority of cases; however, various types of cancers, such as hematological malignancy, epithelial cancer, and pediatric cancers, such as neuroblastoma, can also develop. Furthermore, approximately half of the cases develop simultaneous or metachronous multiple cancers. The types of TP53 pathogenic variants and factors that modify the functions of TP53 have an impact on the clinical presentation, although there are currently no definitive findings. There is currently no cancer preventive agent for individuals with TP53 pathogenic variant. Surgical treatments, such as risk-reducing bilateral mastectomy warrant further investigation. Theoretically, exposure to radiation could induce the onset of secondary cancer; therefore, imaging and treatments that use radiation should be avoided as much as possible. As a method to follow-up LFS, routine cancer surveillance comprising whole-body MRI scan, brain MRI scan, breast MRI scan, and abdominal ultrasonography (US) should be performed immediately after the diagnosis. However, the effectiveness of this surveillance is unknown, and there are problems, such as adverse events associated with a high rate of false positives, overdiagnosis, and sedation used during imaging as well as negative psychological impact. The detection rate of cancer through cancer surveillance is extremely high. Many cases are detected at an early stage, and treatments are low intensity; thus, cancer surveillance could contribute to an improvement in QOL, or at least, a reduction in complications associated with treatment. With the widespread use of genomic medicine, the diagnosis of LFS is unavoidable, and a comprehensive medical care system for LFS is necessary. Therefore, clinical trials that verify the feasibility and effectiveness of the program, comprising LFS registry, genetic counseling, and cancer surveillance, need to be prepared.

Successful Alectinib Treatment for Carcinoma of Unknown Primary with EML4-ALK Fusion Gene: A Case Report.

Gene alteration in anaplastic lymphoma kinase (ALK) is rare, and the efficacy of ALK inhibitors in the treatment of carcinoma of unknown primary (CUP) with ALK alteration remains unclear. The patient was a 56-year-old woman who presented with cervical lymph node swelling. Computed tomography revealed paraaortic, perigastric, and cervical lymph node swelling; ascites; a liver lesion; and a left adrenal mass. A cervical lymph node biopsy was performed, and pathological diagnosis of an undifferentiated malignant tumor was conducted. Finally, the patient was diagnosed with CUP and treated with chemotherapy. To evaluate actionable mutations, we performed a multigene analysis, using a next-generation sequencer (FoundationOne® CDx). It revealed that the tumor harbored an echinoderm microtubule-associated protein-like 4 (EML4) and ALK fusion gene. Additionally, immunohistochemistry confirmed ALK protein expression. Alectinib, a potent ALK inhibitor, was recommended for the patient at a molecular oncology conference at our institution. Accordingly, alectinib (600 mg/day) was administered, and the multiple lesions and symptoms rapidly diminished without apparent toxicity. The administration of alectinib continued for a period of 10 months without disease progression. Thus, ALK-tyrosine kinase inhibitors should be considered in patients with CUP harboring the EML4-ALK fusion gene.

Clinical utility of target capture-based panel sequencing in hematological malignancies: A multicenter feasibility study.

Although next-generation sequencing-based panel testing is well practiced in the field of cancer medicine for the identification of target molecules in solid tumors, the clinical utility and clinical issues surrounding panel testing in hematological malignancies have yet to be fully evaluated. We conducted a multicenter prospective clinical sequencing study to verify the feasibility of a panel test for hematological tumors, including acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, and diffuse large B-cell lymphoma. Out of 96 eligible patients, 79 patients (82%) showed potentially actionable findings, based on the clinical sequencing assays. We identified that genetic alterations with a strong clinical significance were found at a higher frequency in terms of diagnosis (n = 60; 63%) and prognosis (n = 61; 64%) than in terms of therapy (n = 8; 8%). Three patients who harbored a germline mutation in either DDX41 (n = 2) or BRCA2 (n = 1) were provided with genetic counseling. At 6 mo after sequencing, clinical actions based on the diagnostic (n = 5) or prognostic (n = 3) findings were reported, but no patients were enrolled in a clinical trial or received targeted therapies based on the sequencing results. These results suggest that panel testing for hematological malignancies would be feasible given the availability of useful diagnostic and prognostic information. This study is registered with the UMIN Clinical Trial Registry (UMIN000029879, multiple myeloma; UMIN000031343, adult acute myeloid leukemia; UMIN000033144, diffuse large B-cell lymphoma; and UMIN000034243, childhood leukemia).

A female patient with retinoblastoma and severe intellectual disability carrying an X;13 balanced translocation without rearrangement in the RB1 gene: a case report.

BACKGROUND: Female carriers of a balanced X; autosome translocation generally undergo selective inactivation of the normal X chromosome. This is because inactivation of critical genes within the autosomal region of the derivative translocation chromosome would compromise cellular function. We here report a female patient with bilateral retinoblastoma and a severe intellectual disability who carries a reciprocal X-autosomal translocation. CASE PRESENTATION: Cytogenetic and molecular analyses, a HUMARA (Human androgen receptor) assay, and methylation specific PCR (MSP) and bisulfite sequencing were performed using peripheral blood samples from the patient. The patient's karyotype was 46,X,t(X;13)(q28;q14.1) by G-banding analysis. Further cytogenetic analysis located the entire RB1 gene and its regulatory region on der(X) with no translocation disruption. The X-inactivation pattern in the peripheral blood was highly skewed but not completely selected. MSP and deep sequencing of bisulfite-treated DNA revealed that an extensive 13q region, including the RB1 promoter, was unusually methylated in a subset of cells. CONCLUSIONS: The der(X) region harboring the RB1 gene was inactivated in a subset of somatic cells, including the retinal cells, in the patient subject which acted as the first hit in the development of her retinoblastoma. In addition, the patient's intellectual disability may be attributable to the inactivation of the der(X), leading to a 13q deletion syndrome-like phenotype, or to an active X-linked gene on der (13) leading to Xq28 functional disomy.

[Li-Fraumeni Syndrome-Current Status and Prospects in Clinical Practice].

Li-Fraumeni syndrome(LFS)is a cancer predisposing disorder with early-onset cancers in diverse tissues of origin, caused by germline TP53 mutation. In LFS, 5 core cancers are known: breast cancer, soft tissue sarcoma, osteosarcoma, brain tumor, adrenocortical cancer. Surveillance for LFS must be lifetime long and multitargeted since the patients are susceptible to various cancers from infant to middle-aged and older. Recent investigations have revealed germline TP53 mutations can increase the riskfor radio-induced tumorigenesis and will affect patient's prognosis and treatment in several cancers. The surveillance protocol has developed in Canada and several prospective studies based on the Canadian's one are currently being carried out. We are aware that the surveillance protocol suitable for the current status in Japan is urgently needed to provide the appropriate medical care for LFS patients.

Compound heterozygous TYK2 mutations underlie primary immunodeficiency with T-cell lymphopenia.

Complete tyrosine kinase 2 (TYK2) deficiency has been previously described in patients with primary immunodeficiency diseases. The patients were infected with various pathogens, including mycobacteria and/or viruses, and one of the patients developed hyper-IgE syndrome. A detailed immunological investigation of these patients revealed impaired responses to type I IFN, IL-10, IL-12 and IL-23, which are associated with increased susceptibility to mycobacterial and/or viral infections. Herein, we report a recessive partial TYK2 deficiency in two siblings who presented with T-cell lymphopenia characterized by low naïve CD4+ T-cell counts and who developed Epstein-Barr virus (EBV)-associated B-cell lymphoma. Targeted exome-sequencing of the siblings' genomes demonstrated that both patients carried novel compound heterozygous mutations (c.209_212delGCTT/c.691C > T, p.Cys70Serfs*21/p.Arg231Trp) in the TYK2. The TYK2 protein levels were reduced by 35% in the T cells of the patient. Unlike the response under complete TYK2 deficiency, the patient's T cells responded normally to type I IFN, IL-6, IL-10 and IL-12, whereas the cells displayed an impaired response to IL-23. Furthermore, the level of STAT1 was low in the cells of the patient. These studies reveal a new clinical entity of a primary immunodeficiency with T-cell lymphopenia that is associated with compound heterozygous TYK2 mutations in the patients.

Dynamic modulation of thymidylate synthase gene expression and fluorouracil sensitivity in human colorectal cancer cells.

Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori experimental approaches are needed. Thymidylate synthase (TS) expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU) treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an in vitro system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our in vitro data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained in vitro. Thus, our in vitro and in vivo observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.

NF-κB signaling mediates acquired resistance after PARP inhibition.

PARP inhibitors are a class of promising anti-cancer drugs, with proven activity in BRCA mutant cancers. However, as with other targeted agents, treatment with PARP inhibitors generates acquired resistance within these tumors. The mechanism of this acquired resistance is poorly understood. We established cell lines that are resistant to PARP inhibitor by continuous treatment with the drug, and then used RNA sequencing to compare gene expression. Pathway analysis on the RNA sequencing data indicates that NF-κB signaling is preferentially up-regulated in PARP inhibitor-resistant cells, and that knockdown of core components in NF-κB signaling reverses the sensitivity to PARP inhibitor in resistant cells. Of therapeutic relevance, we show that PARP inhibitor-resistant cells are sensitive to an NF-κB inhibitor in comparison to their parental controls. Malignancies with up-regulation of NF-κB are sensitive to bortezomib, a proteasome inhibitor that is currently used in the clinic. We also show that treatment with bortezomib results in cell death in the PARP inhibitor-resistant cells, but not in parental cells. Therefore we propose that up-regulation of NF-κB signaling is a key mechanism underlying acquired resistance to PARP inhibition, and that NF-κB inhibition, or bortezomib are potentially effective anti-cancer agents after the acquisition of resistance to PARP inhibitors.

p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response.

The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4-24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients.

A cancer-associated BRCA2 mutation reveals masked nuclear export signals controlling localization.

Germline missense mutations affecting a single BRCA2 allele predispose humans to cancer. Here we identify a protein-targeting mechanism that is disrupted by the cancer-associated mutation, BRCA2(D2723H), and that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mislocalization of mutant BRCA2 inhibits the nuclear retention of RAD51 by exposing a similar NES in RAD51 that is usually obscured by the BRCA2-RAD51 interaction. Thus, a series of NES-masking interactions localizes BRCA2 and RAD51 in the nucleus. Notably, BRCA2(D2723H) decreases RAD51 nuclear retention even when wild-type BRCA2 is also present. Our findings suggest a mechanism for the regulation of the nucleocytoplasmic distribution of BRCA2 and RAD51 and its impairment by a heterozygous disease-associated mutation.

A DNA-damage selective role for BRCA1 E3 ligase in claspin ubiquitylation, CHK1 activation, and DNA repair.

BACKGROUND: The breast and ovarian cancer suppressor BRCA1 is essential for cellular responses to DNA damage. It heterodimerizes with BARD1 to acquire an E3 ubiquitin (Ub) ligase activity that is often compromised by cancer-associated mutations. Neither the significance of this activity to damage responses, nor a relevant in vivo substrate, is clear. RESULTS: We have separated DNA-damage responses requiring the BRCA1 E3 ligase from those independent of it, using a gene-targeted point mutation in vertebrate DT40 cells that abrogates BRCA1's catalytic activity without perturbing BARD1 binding. We show that BRCA1 ubiquitylates claspin, an essential coactivator of the CHK1 checkpoint kinase, after topoisomerase inhibition, but not DNA crosslinking by mitomycin C. BRCA1 E3 inactivation decreases chromatin-bound claspin levels and impairs homology-directed DNA repair by interrupting signal transduction from the damage-activated ATR kinase to its effector, CHK1. CONCLUSIONS: Our findings identify claspin as an in vivo substrate for the BRCA1 E3 ligase and suggest that its modification selectively triggers CHK1 activation for the homology-directed repair of a subset of genotoxic lesions. This mechanism unexpectedly defines an essential but selective function for BRCA1 E3 ligase activity in cellular responses to DNA damage.

Context dependence of checkpoint kinase 1 as a therapeutic target for pancreatic cancers deficient in the BRCA2 tumor suppressor.

Inherited mutations in the tumor suppressor BRCA2 are predisposed to pancreatic adenocarcinomas, which carry activating mutations in the KRAS oncogene in more than 95% of cases, as well as frequent TP53 inactivation. Here, we have established an RNA interference (RNAi) screen to identify genes whose depletion selectively inhibits the growth of cells lacking BRCA2, and then studied the effects of the genetic depletion or pharmacologic inhibition of 1 candidate, the checkpoint kinase 1 (CHK1), in the context of pancreatic cancer. Pharmacologic inhibition of CHK1 using small-molecule inhibitors (CHK1i) reduced cell growth in several cell lines depleted of BRCA2. Unexpectedly, these drugs did not suppress the growth of BRCA2-deficient pancreatic cancer cell lines from humans or gene-targeted mice expressing active Kras and trans-dominant inhibitory mutant Trp53. Remarkably, the expression of KRAS(G12V) and TP53(G154V) in BRCA2-depleted HEK293 cells was sufficient to render them resistant to CHK1i (but not to mitomycin C or inhibitors of PARP1). CHK1i sensitivity was restored by gemcitabine, an S-phase genotoxin used to treat pancreatic adenocarcinoma. Thus, the growth-suppressive effect of CHK1 inhibition in BRCA2-mutant tumors can be opposed by concurrent KRAS activation and TP53 mutations typical of pancreatic adenocarcinoma, and CHK1i resistance in this setting can be overcome by gemcitabine. Our findings show that approaches that use potential therapeutic targets for cancer identified in synthetic lethal RNAi screens are affected by the genetic context of specific malignancies and combination therapy with other agents. This concept should be taken into account in the ongoing and future development of targeted cancer therapies.

DNA damage regulates the mobility of Brca2 within the nucleoplasm of living cells.

How the biochemical reactions that lead to the repair of DNA damage are controlled by the diffusion and availability of protein reactants within the nucleoplasm is poorly understood. Here, we use gene targeting to replace Brca2 (a cancer suppressor protein essential for DNA repair) with a functional enhanced green fluorescent protein (EGFP)-tagged form, followed by fluorescence correlation spectroscopy to measure Brca2-EGFP diffusion in the nucleoplasm of living cells exposed to DNA breakage. Before damage, nucleoplasmic Brca2 molecules exhibit complex states of mobility, with long dwell times within a sub-fL observation volume, indicative of restricted motion. DNA damage significantly enhances the mobility of Brca2 molecules in the S/G2 phases of the cell cycle, via signaling through damage-activated protein kinases. Brca2 mobilization is accompanied by increased binding within the nucleoplasm to its cargo, the Rad51 recombinase, measured by fluorescence cross-correlation spectroscopy. Together, these results suggest that DNA breakage triggers the redistribution of soluble nucleoplasmic Brca2 molecules from a state of restricted diffusion, into a mobile fraction available for Rad51 binding. Our findings identify signal-regulated changes in nucleoplasmic protein diffusion as a means to control biochemical reactions in the cell nucleus.

Immunotherapy with autologous dendritic cells and tumor antigens for children with refractory malignant solid tumors.

Immature DCs were generated from the peripheral blood monocytes from five children with refractory solid tumors (Ewing sarcoma, synovial sarcoma, neuroblastoma) using GM-CSF and IL-4. These DCs were then pulsed with tumor-specific synthetic peptides or tumor lysates in the presence of the immunogenic protein KLH for 12 h. Pulsed DCs were administered subcutaneously every one or two weeks in an outpatient setting without any toxicity. In one patient with Ewing sarcoma, the residual tumor disappeared following autologous PBSCT and DC therapy, and a complete remission has been maintained for 77 months. In two patients with synovial sarcoma or with neuroblastoma, growth of the tumors was temporally suppressed for one and 10 months, respectively, followed by their exacerbation. A DTH response was detected against KLH in all five patients and against the tumor lysate in one patient. In the patients with a possible DC-mediated anti-tumor effect, the number of CD8(+) HLA-DR(+) lymphocytes and INF-gamma(+)CD8(+) lymphocytes increased and an elevation of the NK cell cytotoxic activity was observed during and/or after DC therapy. DC-based immunotherapy may therefore be a feasible, well-tolerated and promising approach in the treatment of children with refractory malignant tumors.