Expert opinion on NSCLC small specimen biomarker testing - Part 1: Tissue collection and management.

Biomarker testing is crucial for treatment selection in advanced non-small cell lung cancer (NSCLC). However, the quantity of available tissue often presents a key constraint for patients with advanced disease, where minimally invasive tissue biopsy typically returns small samples. In Part 1 of this two-part series, we summarise evidence-based recommendations relating to small sample processing for patients with NSCLC. Generally, tissue biopsy techniques that deliver the greatest quantity and quality of tissue with the least risk to the patient should be selected. Rapid on-site evaluation can help to ensure sufficient sample quality and quantity. Sample processing should be managed according to biomarker testing requirements, because tissue fixation methodology influences downstream nucleic acid, protein and morphological analyses. Accordingly, 10% neutral buffered formalin is recommended as an appropriate fixative, and the duration of fixation is recommended not to exceed 24-48 h. Tissue sparing techniques, including the 'one biopsy per block' approach and small sample cutting protocols, can help preserve tissue. Cytological material (formalin-fixed paraffin-embedded [FFPE] cytology blocks and non-FFPE samples such as smears and touch preparations) can be an excellent source of nucleic acid, providing either primary or supplementary patient material to complete morphological and molecular diagnoses. Considerations on biomarker testing, reporting and quality assessment are discussed in Part 2.

Expert opinion on NSCLC small specimen biomarker testing - Part 2: Analysis, reporting, and quality assessment.

The diagnostic work-up for non-small cell lung cancer (NSCLC) requires biomarker testing to guide therapy choices. This article is the second of a two-part series. In Part 1, we summarised evidence-based recommendations for obtaining and processing small specimen samples (i.e. pre-analytical steps) from patients with advanced NSCLC. Here, in Part 2, we summarise evidence-based recommendations relating to analytical steps of biomarker testing (and associated reporting and quality assessment) of small specimen samples in NSCLC. As the number of biomarkers for actionable (genetic) targets and approved targeted therapies continues to increase, simultaneous testing of multiple actionable oncogenic drivers using next-generation sequencing (NGS) becomes imperative, as set forth in European Society for Medical Oncology guidelines. This is particularly relevant in advanced NSCLC, where tissue specimens are typically limited and NGS may help avoid tissue exhaustion compared with sequential biomarker testing. Despite guideline recommendations, significant discrepancies in access to NGS persist across Europe, primarily due to reimbursement constraints. The use of increasingly complex testing methods also has implications for the reporting of results. Molecular testing reports should include clinical interpretation with additional commentary on sample adequacy as appropriate. Molecular tumour boards are recommended to facilitate the interpretation of complex genetic information arising from NGS, and to collaboratively determine the optimal treatment for patients with NSCLC. Finally, whichever testing modality is employed, it is essential that adequate internal and external validation and quality control measures are implemented.

Paradoxical effects of JZL184, an inhibitor of monoacylglycerol lipase, on bone remodelling in healthy and cancer-bearing mice.

BACKGROUND: Cancer-associated bone disease is a serious complication in bone sarcomas and metastatic carcinomas of breast and prostate origin. Monoacylglycerol lipase (MAGL) is an enzyme of the endocannabinoid system, and is responsible for the degradation of the most abundant endocannabinoid in bone, 2-arachidonoyl glycerol (2AG). METHODS: The effects of the verified MAGL inhibitor on bone remodelling were assessed in healthy mice and in mouse models of bone disease caused by prostate and breast cancers and osteosarcoma. FINDINGS: JZL184 reduced osteolytic bone metastasis in mouse models of breast and prostate cancers, and inhibited skeletal tumour growth, metastasis and the formation of ectopic bone in models of osteosarcoma. Additionally, JZL184 suppressed cachexia and prolonged survival in mice injected with metastatic osteosarcoma and osteotropic cancer cells. Functional and histological analysis revealed that the osteoprotective action of JZL184 in cancer models is predominately due to inhibition of tumour growth and metastasis. In the absence of cancer, however, exposure to JZL184 exerts a paradoxical reduction of bone volume via an effect that is mediated by both Cnr1 and Cnr2 cannabinoid receptors. INTERPRETATION: MAGL inhibitors such as JZL184, or its novel analogues, may be of value in the treatment of bone disease caused by primary bone cancer and bone metastasis, however, activation of the skeletal endocannabinoid system may limit their usefulness as osteoprotective agents.

Pharmacological inhibition of the IKKε/TBK-1 axis potentiates the anti-tumour and anti-metastatic effects of Docetaxel in mouse models of breast cancer.

IκB kinase subunit epsilon (IKKε), a key component of NFκB and interferon signalling, has been identified as a breast cancer oncogene. Here we report that the IKKε/TBK1 axis plays a role in the initiation and progression of breast cancer osteolytic metastasis. Cancer-specific knockdown of IKKε in the human MDA-MB-231-BT cells and treatment with the verified IKKε/TBK1 inhibitor Amlexanox reduced skeletal tumour growth and osteolysis in mice. In addition, combined administration of Amlexanox with Docetaxel reduced mammary tumour growth of syngeneic 4T1 cells, inhibited metastases and improved survival in mice after removal of the primary tumour. Functional and mechanistic studies in breast cancer cells, osteoclasts and osteoblasts revealed that IKKε inhibition reduces the ability of breast cancer cells to grow, move and enhance osteoclastogenesis by engaging both IRF and NFκB signalling pathways. Thus, therapeutic targeting of the IKKε/TBK1 axis may be of value in the treatment of advanced triple negative breast cancer.

2D and 3D In Vitro Co-Culture for Cancer and Bone Cell Interaction Studies.

Co-culture assays are used to study the mutual interaction between cells in vitro. This chapter describes 2D and 3D co-culture systems used to study cell-cell signaling crosstalk between cancer cells and bone marrow adipocytes, osteoblasts, osteoclasts, and osteocytes. The chapter provides a step-by-step guide to the most commonly used cell culture techniques, functional assays, and gene expression.

Bidirectional regulation of bone formation by exogenous and osteosarcoma-derived Sema3A.

Semaphorin 3A (Sema3A), a secreted member of the Semaphorin family, increases osteoblast differentiation, stimulates bone formation and enhances fracture healing. Here, we report a previously unknown role of Sema3A in the regulation of ectopic bone formation and osteolysis related to osteosarcoma. Human recombinant (exogenous) Sema3A promoted the expression of osteoblastic phenotype in a panel of human osteosarcoma cell lines and inhibited the ability of these cells to migrate and enhance osteoclastogenesis in vitro. In vivo, administration of exogenous Sema3A in mice after paratibial inoculation of KHOS cells increased bone volume in non-inoculated and tumour-bearing legs. In contrast, Sema3A overexpression reduced the ability of KHOS cells to cause ectopic bone formation in mice and to increase bone nodule formation by engaging DKK1/ß-catenin signalling. Thus, Sema3A is of potential therapeutic efficacy in osteosarcoma. However, inhibition of bone formation associated with continuous exposure to Sema3A may limit its long-term usefulness as therapeutic agent.

TRAF2 in osteotropic breast cancer cells enhances skeletal tumour growth and promotes osteolysis.

NFκB plays an important role in inflammation and bone remodelling. Tumour necrosis factor receptor associated factor 2 (TRAF2), a key component of NFκB signalling, has been identified as an oncogene, but its role in the regulation of breast cancer osteolytic metastasis remains unknown. Here, we report that stable overexpression of TRAF2 in parental and osteotropic sub-clones of human MDA-MB-231 (MDA-231) breast cancer cells increased cell growth and motility in vitro, whereas TRAF2 knockdown was inhibitory. In vivo, TRAF2 overexpression in the parental MDA-231-P cells enhanced tumour growth after orthotopic injection into the mammary fat pad of mice but failed to promote the metastasis of these cells to bone. In contrast, overexpression of TRAF2 in osteotropic MDA-231-BT cells increased skeletal tumour growth, enhanced osteoclast formation and worsened osteolytic bone loss after intra-tibial injection in mice. Mechanistic and functional studies in osteotropic MDA-231-BT and osteoclasts revealed that upregulation of TRAF2 increased the ability of osteotropic MDA-231-BT cells to migrate and to enhance osteoclastogenesis by a mechanism dependent, at least in part, on NFκB activation. Thus, the TRAF2/NFκB axis is implicated in the regulation of skeletal tumour burden and osteolysis associated with advanced breast cancer.

A CD22-reactive TCR from the T-cell allorepertoire for the treatment of acute lymphoblastic leukemia by TCR gene transfer.

CD22 is currently evaluated as a target-antigen for the treatment of B-cell malignancies using chimeric antigen receptor (CAR)-engineered T-cells or monoclonal antibodies (mAbs). CAR- and mAbs-based immunotherapies have been successfully applied targeting other antigens, however, occurrence of refractory disease to these interventions urges the identification of additional strategies. Here, we identified a TCR recognizing the CD22-derived peptide RPFPPHIQL (CD22RPF) presented in human leukocyte antigen (HLA)-B*07:02. To overcome tolerance to self-antigens such as CD22, we exploited the immunogenicity of allogeneic HLA. CD22RPF-specific T-cell clone 9D4 was isolated from a healthy HLA-B*07:02neg individual, efficiently produced cytokines upon stimulation with primary acute lymphoblastic leukemia and healthy B-cells, but did not react towards healthy hematopoietic and nonhematopoietic cell subsets, including dendritic cells (DCs) and macrophages expressing low levels of CD22. Gene transfer of TCR-9D4 installed potent CD22-specificity onto recipient CD8+ T-cells that recognized and lysed primary B-cell leukemia. TCR-transduced T-cells spared healthy CD22neg hematopoietic cell subsets but weakly lysed CD22low-expressing DCs and macrophages. CD22-specific TCR-engineered T-cells could form an additional immunotherapeutic strategy with a complementary role to CAR- and antibody-based interventions in the treatment of B-cell malignancies. However, CD22 expression on non-B-cells may limit the attractiveness of CD22 as target-antigen in cellular immunotherapy.

Generation of CD20-specific TCRs for TCR gene therapy of CD20low B-cell malignancies insusceptible to CD20-targeting antibodies.

Immunotherapy of B-cell leukemia and lymphoma with CD20-targeting monoclonal antibodies (mAbs) has demonstrated clinical efficacy. However, the emergence of unresponsive disease due to low or absent cell surface CD20 urges the need to develop additional strategies. In contrast to mAbs, T-cells via their T-cell receptor (TCR) can recognize not only extracellular but also intracellular antigens in the context of HLA molecules. We hypothesized that T-cells equipped with high affinity CD20-targeting TCRs would be able to recognize B-cell malignancies even in the absence of extracellular CD20. We isolated CD8+ T-cell clones binding to peptide-MHC-tetramers composed of HLA-A*02:01 and CD20-derived peptide SLFLGILSV (CD20SLF) from HLA-A*02:01neg healthy individuals to overcome tolerance towards self-antigens such as CD20. High avidity T-cell clones were identified that readily recognized and lysed primary HLA-A2pos B-cell leukemia and lymphoma in the absence of reactivity against CD20-negative but HLA-A2pos healthy hematopoietic and nonhematopoietic cells. The T-cell clone with highest avidity efficiently lysed malignant cell-lines that had insufficient extracellular CD20 to be targeted by CD20 mAbs. Transfer of this TCR installed potent CD20-specificity onto recipient T-cells and led to lysis of CD20low malignant cell-lines. Moreover, our approach facilitates the generation of an off-the-shelf TCR library with broad applicability by targeting various HLA alleles. Using the same methodology, we isolated a T-cell clone that efficiently lysed primary HLA-B*07:02pos B-cell malignancies by targeting another CD20-derived peptide. TCR gene transfer of high affinity CD20-specific TCRs can be a valuable addition to current treatment options for patients suffering from CD20low B-cell malignancies.