Management of patients with presumed germline pathogenic variant from tumor-only genomic sequencing: A retrospective analysis at a single facility.

Cancer treatment is increasingly evolving toward personalized medicine, which sequences numerous cancer-related genes and identifies therapeutic targets. On the other hand, patients with germline pathogenic variants (GPV) have been identified as secondary findings (SF) and oncologists have been urged to handle them. All SF disclosure considerations for patients are addressed and decided at the molecular tumor boards (MTB) in the facility. In this study, we retrospectively summarized the results of all cases in which comprehensive genomic profiling (CGP) test was conducted at our hospital, and discussed the possibility of presumed germline pathogenic variants (PGPV) at MTB. MTB recommended confirmatory testing for 64 patients. Informed consent was obtained from attending physicians for 53 of them, 30 patients requested testing, and 17 patients tested positive for a confirmatory test. Together with already known variants, 4.5 % of the total confirmed in this cohort. Variants verified in this study were BRCA1 (n = 12), BRCA2 (n = 6), MSH2 (n = 2), MSH6 (n = 2), WT1 (n = 2), TP53, MEN1, CHEK2, MLH1, TSC2, PTEN, RB1, and SMARCB1. There was no difference in the tumor's VAF between confirmed positive and negative cases for variants determined as PGPV by MTB. Current results demonstrate the actual number of cases until confirmatory germline test for patients with PGPV from tumor-only CGP test through the discussion at the MTB. The practical results at this single facility will serve as a guide for the management of the selection and distribution of SF in the genome analysis.

Clinical decisions by the molecular tumor board on comprehensive genomic profiling tests in Japan: A retrospective observational study.

BACKGROUND: A paradigm shift has occurred in cancer chemotherapy from tumor-specific treatment with cytotoxic agents to personalized medicine with molecular-targeted drugs. Thus, it is essential to identify genomic alterations and molecular features to recommend effective targeted molecular medicines regardless of the tumor site. Nevertheless, it takes considerable expertise to identify treatment targets from primary-sequencing data in order to provide drug recommendations. The Molecular Tumor Board (MTB) denotes a platform that integrates clinical and molecular features for clinical decisions. METHODS: This study retrospectively analyses all the cases of discussion and decision at the MTB in Tohoku University Hospital and summarizes genetic alterations and treatment recommendations. RESULTS: The MTB discussed 1003 comprehensive genomic profiling (CGP) tests conducted in patients with solid cancer, and the resulting rate of assessing treatment recommendations was approximately 19%. Among hundreds of genes in the CGP test, only 30 genetic alterations or biomarkers were used to make treatment recommendations. The leading biomarkers that led to treatment recommendations were tumor mutational burden-high (TMB-H) (n = 32), ERBB2 amplification (n = 24), BRAF V600E (n = 16), and BRCA1/2 alterations (n = 32). Thyroid cancer accounted for most cancer cases for which treatment recommendation was provided (81.3%), followed by non-small cell lung cancer (42.4%) and urologic cancer (31.3%). The number of tests performed for gastrointestinal cancers was high (n = 359); however, the treatment recommendations for the same were below average (13%). CONCLUSION: The results of this study may be used to simplify treatment recommendations from the CGP reports and help select patients for testing, thereby increasing the accuracy of personalized medicine.

Development of a national health policy logic model to accelerate the integration of oncology and palliative care: a nationwide Delphi survey in Japan.

BACKGROUND: Despite recommendations to deliver palliative care to cancer patients and their caregivers, their distress has not been alleviated satisfactorily. National health policies play a pivotal role in achieving a comprehensive range of quality palliative care delivery for the public. However, there is no standardised logic model to appraise the efficacy of these policies. This study aimed to develop a logic model of a national health policy to deliver cancer palliative care and to reach consensus towards specific policy proposals. METHODS: A draft version of the logic model and specific policy proposals were formulated by the research team and the internal expert panel, and the independent external expert panel evaluated the policy proposals based on the Delphi survey to reach consensus. RESULTS: The logic model was divided into three major conceptual categories: 'care-delivery at cancer hospitals', 'community care coordination', and 'social awareness of palliative care'. There were 18 and 45 major and minor policy proposals, which were categorised into four groups: requirement of government-designated cancer hospitals; financial support; Basic Plan to Promote Cancer Control Programs; and others. These policy proposals were independently evaluated by 64 external experts and the first to third Delphi round response rates were 96.9-98.4%. Finally, 47 policy proposals reached consensus. The priority of each proposal was evaluated within the four policy groups. CONCLUSIONS: A national health policy logic model was developed to accelerate the provision of cancer palliative care. Further research is warranted to verify the study design to investigate the efficacy of the logic model.

Comparison of the quality of death between primary malignant brain tumor patients and other cancer patients: results from a nationwide bereavement survey in Japan.

PURPOSE: No studies have investigated the association between malignant brain tumor and the quality of dying, which is an important outcome in end-of-life care. This study aimed to clarify whether the quality of dying and related factors in patients with malignant brain tumor differ from those in patients with other malignant diseases. METHODS: This was a secondary analysis of data collected by two nationwide, multicenter, bereavement surveys of palliative care units in Japan. This analysis included 14,171 bereaved family members (160 patients with malignant brain tumors). The quality of dying was examined using the good death inventory (GDI), a validated tool widely used in palliative care settings. RESULTS: Patients with malignant brain tumors were younger (p < 0.0001) and had a longer palliative care unit stay during their end-of-life (p < 0.0001) than others. The total GDI score was significantly lower in patients with malignant brain tumors than others (p < 0.0001). Five GDI items were significantly lower in the malignant brain tumor group than other cancer group: "Being able to stay in one's favorite place" (p = 0.03); "Trusting the physician" (p = 0.003); "Not being a burden to others" (p = 0.01); "Being independent in daily activities" (p = 0.01); and "Feeling that one's life is worth living" (p = 0.001). CONCLUSION: This study showed that the quality of dying of patients with malignant brain tumors was lower compared to other cancers, suggesting the need to review care for patients with malignant brain tumors based on their characteristics.

TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation.

Microtubules (MTs) play critical roles in various cellular events, including cell migration. End-binding proteins (EBs) accumulate at the ends of growing MTs and regulate MT end dynamics by recruiting other plus end-tracking proteins (+TIPs). However, how EBs contribute to MT dynamics through +TIPs remains elusive. We focused on tau-tubulin kinase 2 (TTBK2) as an EB1/3-binding kinase and confirmed that TTBK2 acted as a +TIP. We identified MT-depolymerizing kinesin KIF2A as a novel substrate of TTBK2. TTBK2 phosphorylated KIF2A at S135 in intact cells in an EB1/3-dependent fashion and inactivated its MT-depolymerizing activity in vitro. TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion. Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration. These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration.

PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity.

The organization of the Golgi apparatus is essential for cell polarization and its maintenance. The polarity regulator PAR complex (PAR3, PAR6, and aPKC) plays critical roles in several processes of cell polarization. However, how the PAR complex participates in regulating the organization of the Golgi remains largely unknown. Here we demonstrate the functional cross-talk of the PAR complex with CLASP2, which is a microtubule plus-end-tracking protein and is involved in organizing the Golgi ribbon. CLASP2 directly interacted with PAR3 and was phosphorylated by aPKC. In epithelial cells, knockdown of either PAR3 or aPKC induced the aberrant accumulation of CLASP2 at the trans-Golgi network (TGN) concomitantly with disruption of the Golgi ribbon organization. The expression of a CLASP2 mutant that inhibited the PAR3-CLASP2 interaction disrupted the organization of the Golgi ribbon. CLASP2 is known to localize to the TGN through its interaction with the TGN protein GCC185. This interaction was inhibited by the aPKC-mediated phosphorylation of CLASP2. Furthermore, the nonphosphorylatable mutant enhanced the colocalization of CLASP2 with GCC185, thereby perturbing the Golgi organization. On the basis of these observations, we propose that PAR3 and aPKC control the organization of the Golgi through CLASP2 phosphorylation.

Plk1 phosphorylates CLIP-170 and regulates its binding to microtubules for chromosome alignment.

The microtubule (MT) cytoskeleton is essential for cellular morphogenesis, cell migration, and cell division. MT organization is primarily mediated by a variety of MT-associated proteins. Among these proteins, plus-end-tracking proteins (+TIPs) are evolutionarily conserved factors that selectively accumulate at growing MT plus ends. Cytoplasmic linker protein (CLIP)-170 is a +TIP that associates with diverse proteins to determine the behavior of MT ends and their linkage to intracellular structures, including mitotic chromosomes. However, how CLIP-170 activity is spatially and temporally controlled is largely unknown. Here, we show that phosphorylation at Ser312 in the third serine-rich region of CLIP-170 is increased during mitosis. Polo-like kinase 1 (Plk1) is responsible for this phosphorylation during the mitotic phase of dividing cells. In vitro analysis using a purified CLIP-170 N-terminal fragment showed that phosphorylation by Plk1 diminishes CLIP-170 binding to the MT ends and lattice without affecting binding to EB3. Furthermore, we demonstrate that during mitosis, stable kinetochore/MT attachment and subsequent chromosome alignment require CLIP-170 and a proper phosphorylation/dephosphorylation cycle at Ser312. We propose that CLIP-170 phosphorylation by Plk1 regulates proper chromosome alignment by modulating the interaction between CLIP-170 and MTs in mitotic cells and that CLIP-170 activity is stringently controlled by its phosphorylation state, which depends on the cellular context.

Tiam1 interaction with the PAR complex promotes talin-mediated Rac1 activation during polarized cell migration.

Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration.

Numb controls E-cadherin endocytosis through p120 catenin with aPKC.

Cadherin trafficking controls tissue morphogenesis and cell polarity. The endocytic adaptor Numb participates in apicobasal polarity by acting on intercellular adhesions in epithelial cells. However, it remains largely unknown how Numb controls cadherin-based adhesion. Here, we found that Numb directly interacted with p120 catenin (p120), which is known to interact with E-cadherin and prevent its internalization. Numb accumulated at intercellular adhesion sites and the apical membrane in epithelial cells. Depletion of Numb impaired E-cadherin internalization, whereas depletion of p120 accelerated internalization. Expression of the Numb-binding fragment of p120 inhibited E-cadherin internalization in a dominant-negative fashion, indicating that Numb interacts with the E-cadherin/p120 complex and promotes E-cadherin endocytosis. Impairment of Numb induced mislocalization of E-cadherin from the lateral membrane to the apical membrane. Atypical protein kinase C (aPKC), a member of the PAR complex, phosphorylated Numb and inhibited its association with p120 and α-adaptin. Depletion or inhibition of aPKC accelerated E-cadherin internalization. Wild-type Numb restored E-cadherin internalization in the Numb-depleted cells, whereas a phosphomimetic mutant or a mutant with defective α-adaptin-binding ability did not restore the internalization. Thus, we propose that aPKC phosphorylates Numb to prevent its binding to p120 and α-adaptin, thereby attenuating E-cadherin endocytosis to maintain apicobasal polarity.

EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells.

Highly conserved EB1 family proteins bind to the growing ends of microtubules, recruit multiple cargo proteins, and are critical for making dynamic microtubules in vivo. However, it is unclear how these master regulators of microtubule plus ends promote microtubule dynamics. In this paper, we identify a novel EB1 cargo protein, Sentin. Sentin depletion in Drosophila melanogaster S2 cells, similar to EB1 depletion, resulted in an increase in microtubule pausing and led to the formation of shorter spindles, without displacing EB1 from growing microtubules. We demonstrate that Sentin's association with EB1 was critical for its plus end localization and function. Furthermore, the EB1 phenotype was rescued by expressing an EBN-Sentin fusion protein in which the C-terminal cargo-binding region of EB1 is replaced with Sentin. Knockdown of Sentin attenuated plus end accumulation of Msps (mini spindles), the orthologue of XMAP215 microtubule polymerase. These results indicate that EB1 promotes dynamic microtubule behavior by recruiting the cargo protein Sentin and possibly also a microtubule polymerase to the microtubule tip.

Functional roles of the tissue inhibitor of metalloproteinase 3 (TIMP-3) during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells.

The tissue inhibitor of the metalloproteinase-3 (TIMP-3) gene was isolated as a gene involved in the process of ascorbate-induced differentiation of mouse MC3T3-E1 cells by the differential display method. The functional roles of TIMP-3 were characterized by establishing stable cell lines, which constitutively expressed the TIMP-3 gene. The TIMP-3 transfectants produced type I collagen at the same level as that of normal cells in response to ascorbic acid 2-phosphate (AscP). However, the expression of the other osteoblastic marker proteins such as alkaline phosphatase (ALPase), osteopontin (OP), osteocalcin (OC), osteonectin (ON) and matrix metalloproteinases (MMPs) remained at a low level even in the presence of AscP. Furthermore, no mineralization of the extracellular matrix (ECM) occurred with the transfectants. Remodeling ECM through TIMPs and MMPs is concluded to be required for osteoblastic differentiation.