Early Detection of Malignant and Premalignant Peripheral Nerve Tumors Using Cell-Free DNA Fragmentomics.

PURPOSE: Early detection of neurofibromatosis type 1 (NF1)-associated peripheral nerve sheath tumors (PNST) informs clinical decision-making, enabling early definitive treatment and potentially averting deadly outcomes. In this study, we describe a cell-free DNA (cfDNA) fragmentomic approach that distinguishes nonmalignant, premalignant, and malignant forms of PNST in the cancer predisposition syndrome, NF1. EXPERIMENTAL DESIGN: cfDNA was isolated from plasma samples of a novel cohort of 101 patients with NF1 and 21 healthy controls and underwent whole-genome sequencing. We investigated diagnosis-specific signatures of copy-number alterations with in silico size selection as well as fragment profiles. Fragmentomics were analyzed using complementary feature types: bin-wise fragment size ratios, end motifs, and fragment non-negative matrix factorization signatures. RESULTS: The novel cohort of patients with NF1 validated that our previous cfDNA copy-number alteration-based approach identifies malignant PNST (MPNST) but cannot distinguish between benign and premalignant states. Fragmentomic methods were able to differentiate premalignant states including atypical neurofibromas (AN). Fragmentomics also adjudicated AN cases suspicious for MPNST, correctly diagnosing samples noninvasively, which could have informed clinical management. CONCLUSIONS: Novel cfDNA fragmentomic signatures distinguish AN from benign plexiform neurofibromas and MPNST, enabling more precise clinical diagnosis and management. This study pioneers the early detection of malignant and premalignant PNST in NF1 and provides a blueprint for decentralizing noninvasive cancer surveillance in hereditary cancer predisposition syndromes.

Molecular profiling of 888 pediatric tumors informs future precision trials and data-sharing initiatives in pediatric cancer.

To inform clinical trial design and real-world precision pediatric oncology practice, we classified diagnoses, assessed the landscape of mutations, and identified genomic variants matching trials in a large unselected institutional cohort of solid tumors patients sequenced at Dana-Farber / Boston Children's Cancer and Blood Disorders Center. Tumors were sequenced with OncoPanel, a targeted next-generation DNA sequencing panel. Diagnoses were classified according to the International Classification of Diseases for Oncology (ICD-O-3.2). Over 6.5 years, 888 pediatric cancer patients with 95 distinct diagnoses had successful tumor sequencing. Overall, 33% (n = 289/888) of patients had at least 1 variant matching a precision oncology trial protocol, and 14% (41/289) were treated with molecularly targeted therapy. This study highlights opportunities to use genomic data from hospital-based sequencing performed either for research or clinical care to inform ongoing and future precision oncology clinical trials. Furthermore, the study results emphasize the importance of data sharing to define the genomic landscape and targeted treatment opportunities for the large group of rare pediatric cancers we encounter in clinical practice.

Off-label prescribing of immune checkpoint inhibitor therapy at a single pediatric cancer center.

BACKGROUND: Immune checkpoint inhibitors (ICI) have improved outcomes in a variety of adult cancers and are prescribed with increasing frequency across oncology. However, patterns of off-label use of ICI in pediatrics remain unclear. METHODS: This is a single-institution, retrospective cohort study evaluating off-label ICI use in pediatric and young adult patients with cancer treated at our institution from 2014 to 2022. Response was based on clinician assessment derived from clinical records. Immune-related adverse events (iRAEs) were classified according to CTCAE v5.0. RESULTS: We identified 50 unique patients treated with off-label ICI (28 with solid tumors, 20 with central nervous system (CNS) tumors, 2 with hematologic malignancies). At time of ICI initiation, only five patients (10%) had localized disease, and all but one patient was treated in the relapsed/refractory setting. All patients were treated with the FDA-approved weight-based dosing recommendations. Overall, there was disease control in 21 patients (42%), with best response including one complete response (melanoma), two partial responses (high-grade glioma, CNS nongerminomatous germ cell tumor), and 18 patients with stable disease. Forty-four patients (88%) eventually experienced disease progression. Among 22 patients (44%) experiencing iRAEs, 10 (20%) had a grade ≥3 irAE, 12 (24%) required corticosteroids, and 14 (28%) required ICI discontinuation. irAE occurrence was associated with significantly improved progression-free survival (HR 0.35; 95% CI: 0.18 to 0.68; p = 0.002) and overall survival (HR 0.33; 95% CI: 0.17 to 0.66; p = 0.002). CONCLUSIONS: At our institution, ICI was most commonly prescribed in the relapsed/refractory setting to patients with metastatic disease. The treatment was generally well-tolerated in the pediatric population. The overall response rate was low, and the majority of patients eventually experienced disease progression. A few patients, however, had durable treatment responses. Further studies are needed to identify which pediatric patients are most likely to benefit from ICI.

Early detection of malignant and pre-malignant peripheral nerve tumors using cell-free DNA fragmentomics.

Early detection of neurofibromatosis type 1 (NF1) associated peripheral nerve sheath tumors (PNST) informs clinical decision-making, potentially averting deadly outcomes. Here, we describe a cell-free DNA (cfDNA) fragmentomic approach which distinguishes non-malignant, pre-malignant and malignant forms of NF1 PNST. Using plasma samples from a novel cohort of 101 NF1 patients and 21 healthy controls, we validated that our previous cfDNA copy number alteration (CNA)-based approach identifies malignant peripheral nerve sheath tumor (MPNST) but cannot distinguish among benign and premalignant states. We therefore investigated the ability of fragment-based cfDNA features to differentiate NF1-associated tumors including binned genome-wide fragment length ratios, end motif analysis, and non-negative matrix factorization deconvolution of fragment lengths. Fragmentomic methods were able to differentiate pre-malignant states including atypical neurofibromas (AN). Fragmentomics also adjudicated AN cases suspicious for MPNST, correctly diagnosing samples noninvasively, which could have informed clinical management. Overall, this study pioneers the early detection of malignant and premalignant peripheral nerve sheath tumors in NF1 patients using plasma cfDNA fragmentomics. In addition to screening applications, this novel approach distinguishes atypical neurofibromas from benign plexiform neurofibromas and malignant peripheral nerve sheath tumors, enabling more precise clinical diagnosis and management.

A spatial cell atlas of neuroblastoma reveals developmental, epigenetic and spatial axis of tumor heterogeneity.

Neuroblastoma is a pediatric cancer arising from the developing sympathoadrenal lineage with complex inter- and intra-tumoral heterogeneity. To chart this complexity, we generated a comprehensive cell atlas of 55 neuroblastoma patient tumors, collected from two pediatric cancer institutions, spanning a range of clinical, genetic, and histologic features. Our atlas combines single-cell/nucleus RNA-seq (sc/scRNA-seq), bulk RNA-seq, whole exome sequencing, DNA methylation profiling, spatial transcriptomics, and two spatial proteomic methods. Sc/snRNA-seq revealed three malignant cell states with features of sympathoadrenal lineage development. All of the neuroblastomas had malignant cells that resembled sympathoblasts and the more differentiated adrenergic cells. A subset of tumors had malignant cells in a mesenchymal cell state with molecular features of Schwann cell precursors. DNA methylation profiles defined four groupings of patients, which differ in the degree of malignant cell heterogeneity and clinical outcomes. Using spatial proteomics, we found that neuroblastomas are spatially compartmentalized, with malignant tumor cells sequestered away from immune cells. Finally, we identify spatially restricted signaling patterns in immune cells from spatial transcriptomics. To facilitate the visualization and analysis of our atlas as a resource for further research in neuroblastoma, single cell, and spatial-omics, all data are shared through the Human Tumor Atlas Network Data Commons at www.humantumoratlas.org.

Efficacy of Nivolumab in Pediatric Cancers with High Mutation Burden and Mismatch Repair Deficiency.

PURPOSE: Checkpoint inhibitors have limited efficacy for children with unselected solid and brain tumors. We report the first prospective pediatric trial (NCT02992964) using nivolumab exclusively for refractory nonhematologic cancers harboring tumor mutation burden (TMB) ≥5 mutations/megabase (mut/Mb) and/or mismatch repair deficiency (MMRD). PATIENTS AND METHODS: Twenty patients were screened, and 10 were ultimately included in the response cohort of whom nine had TMB >10 mut/Mb (three initially eligible based on MMRD) and one patient had TMB between 5 and 10 mut/Mb. RESULTS: Delayed immune responses contributed to best overall response of 50%, improving on initial objective responses (20%) and leading to 2-year overall survival (OS) of 50% [95% confidence interval (CI), 27-93]. Four children, including three with refractory malignant gliomas are in complete remission at a median follow-up of 37 months (range, 32.4-60), culminating in 2-year OS of 43% (95% CI, 18.2-100). Biomarker analyses confirmed benefit in children with germline MMRD, microsatellite instability, higher activated and lower regulatory circulating T cells. Stochastic mutation accumulation driven by underlying germline MMRD impacted the tumor microenvironment, contributing to delayed responses. No benefit was observed in the single patient with an MMR-proficient tumor and TMB 7.4 mut/Mb. CONCLUSIONS: Nivolumab resulted in durable responses and prolonged survival for the first time in a pediatric trial of refractory hypermutated cancers including malignant gliomas. Novel biomarkers identified here need to be translated rapidly to clinical care to identify children who can benefit from checkpoint inhibitors, including upfront management of cancer. See related commentary by Mardis, p. 4701.

Derivation and validation of a risk classification tree for patients with synovial sarcoma.

BACKGROUND: Synovial sarcoma (SS) accounts for 8%-10% of all soft-tissue sarcomas. Clinical presentation and outcomes vary, yet discrete risk groups based on validated prognostic indices are not defined for the full spectrum of patients with SS. METHODS: We performed a retrospective cohort study using data from the SEER (surveillance, epidemiology, and end results program) database of SS patients who were <70 years of age at diagnosis. We constructed a recursive partitioning model of overall survival using a training cohort of 1063 patients with variables: Age at diagnosis, sex, race, ethnicity, primary site, tumor size, tumor grade, and stage. Based on this model, we grouped patients into three risk groups and estimated 5-year overall survival for each group. We then applied these groups to a test cohort (n = 1063). RESULTS: Our model identified three prognostic groups with significantly different overall survival: low risk (local/regional stage with either <21 years of age OR tumor <7.5 cm and female sex), intermediate-risk (local/regional stage, age ≥ 21 years with either male sex and tumor <7.5 cm OR any sex with appendicular anatomic location) and high risk (local/regional stage, age ≥ 21 years, tumor size ≥7.5 cm and non-appendicular location OR distant stage). Prognostic groups were applied to the test cohort, showing significantly different survival between groups (p < 0.0001). CONCLUSIONS: Our analysis yields an intuitive risk-classification tree with discrete groups, which may provide useful information for researchers, patients, and clinicians. Prospective validation of this model may inform efforts at risk-stratifying treatment.

Rare FGFR Oncogenic Alterations in Sequenced Pediatric Solid and Brain Tumors Suggest FGFR Is a Relevant Molecular Target in Childhood Cancer.

PURPOSE: Multiple FGFR inhibitors are currently in clinical trials enrolling adults with different solid tumors, while very few enroll pediatric patients. We determined the types and frequency of FGFR alterations (FGFR1-4) in pediatric cancers to inform future clinical trial design. METHODS: Tumors with FGFR alterations were identified from two large cohorts of pediatric solid tumors subjected to targeted DNA sequencing: The Dana-Farber/Boston Children's Profile Study (n = 888) and the multi-institution GAIN/iCAT2 (Genomic Assessment Improves Novel Therapy) Study (n = 571). Data from the combined patient population of 1,395 cases (64 patients were enrolled in both studies) were reviewed and cases in which an FGFR alteration was identified by OncoPanel sequencing were further assessed. RESULTS: We identified 41 patients with tumors harboring an oncogenic FGFR alteration. Median age at diagnosis was 8 years (range, 6 months-26 years). Diagnoses included 11 rhabdomyosarcomas, nine low-grade gliomas, and 17 other tumor types. Alterations included gain-of-function sequence variants (n = 19), amplifications (n = 10), oncogenic fusions (FGFR3::TACC3 [n = 3], FGFR1::TACC1 [n = 1], FGFR1::EBF2 [n = 1], FGFR1::CLIP2 [n = 1], and FGFR2::CTNNA3 [n = 1]), pathogenic-leaning variants of uncertain significance (n = 4), and amplification in combination with a pathogenic-leaning variant of uncertain significance (n = 1). Two novel FGFR1 fusions in two different patients were identified in this cohort, one of whom showed a response to an FGFR inhibitor. CONCLUSION: In summary, activating FGFR alterations were found in approximately 3% (41/1,395) of pediatric solid tumors, identifying a population of children with cancer who may be eligible and good candidates for trials evaluating FGFR-targeted therapy. Importantly, the genomic and clinical data from this study can help inform drug development in accordance with the Research to Accelerate Cures and Equity for Children Act.

PI3K activation allows immune evasion by promoting an inhibitory myeloid tumor microenvironment.

BACKGROUND: Oncogenes act in a cell-intrinsic way to promote tumorigenesis. Whether oncogenes also have a cell-extrinsic effect on suppressing the immune response to cancer is less well understood. METHODS: We use an in vivo expression screen of known cancer-associated somatic mutations in mouse syngeneic tumor models treated with checkpoint blockade to identify oncogenes that promote immune evasion. We then validated candidates from this screen in vivo and analyzed the tumor immune microenvironment of tumors expressing mutant protein to identify mechanisms of immune evasion. RESULTS: We found that expression of a catalytically active mutation in phospho-inositol 3 kinase (PI3K), PIK3CA c.3140A>G (H1047R) confers a selective growth advantage to tumors treated with immunotherapy that is reversed by pharmacological PI3K inhibition. PIK3CA H1047R-expression in tumors decreased the number of CD8+ T cells but increased the number of inhibitory myeloid cells following immunotherapy. Inhibition of myeloid infiltration by pharmacological or genetic modulation of Ccl2 in PIK3CA H1047R tumors restored sensitivity to programmed cell death protein 1 (PD-1) checkpoint blockade. CONCLUSIONS: PI3K activation enables tumor immune evasion by promoting an inhibitory myeloid microenvironment. Activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy. Our data suggest that some oncogenes promote tumorigenesis by enabling tumor cells to avoid clearance by the immune system. Identification of those mechanisms can advance rational combination strategies to increase the efficacy of immunotherapy.

Molecular Characterization of Inflammatory Tumors Facilitates Initiation of Effective Therapy.

Inflammatory myofibroblastic tumor (IMT) is a rare, mesenchymal tumor that has an increased incidence in childhood. Tumors are usually isolated to the chest, abdomen, and retroperitoneum, but metastatic presentations can be seen. Presenting symptoms are nonspecific and include fever, weight loss, pain, shortness of breath, and cough. Approximately 85% of IMTs harbor actionable kinase fusions. The diagnosis can be delayed because of overlapping features with inflammatory disorders, such as elevated inflammatory markers, increased immunoglobin G levels, fever, weight loss, and morphologic similarity with nonmalignant conditions. We present a girl aged 11 years with a TFG-ROS1 fusion-positive tumor of the lung that was initially diagnosed as an immunoglobin G4-related inflammatory pseudotumor. She underwent complete left-sided pneumonectomy and later recurred with widely metastatic disease. We then report the case of a boy aged 9 years with widely metastatic TFG-ROS1 fusion-positive IMT with rapid molecular diagnosis. In both children, there was an excellent response to oral targeted therapy. These cases reveal that rapid molecular testing of inflammatory tumors is not only important for diagnosis but also reveals therapeutic opportunities. Targeted inhibitors produce significant radiologic responses, enabling potentially curative treatment approaches for metastatic ROS1 fusion IMT with previously limited treatment options. Primary care pediatricians and pediatric subspecialists have a crucial role in the early consultation of a pediatric oncology center experienced in molecular diagnostics to facilitate a comprehensive evaluation for children with inflammatory tumors.

Role of Surgery in Rhabdomyosarcoma of the Head and Neck in Children.

OBJECTIVES: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The goal of this research is to analyze the role of surgery in the management of pediatric parameningeal (PM) and non-PM head and neck RMS (HNRMS). STUDY DESIGN: Retrospective review. METHODS: Retrospective chart review of patients <20 years of age treated for HNRMS between 1970 and 2015. Clinical presentation, tumor characteristics, treatment, recurrence, follow-up, and outcome data were collected. RESULTS: Of 97 patients with HNRMS, 56% were male. Overall median (IQR: interquartile range) age at diagnosis was 5.8 (3.3-9.8) years. Sixty-five patients (67%) had PM tumors. Of 75 patients with histologic subtype identified, 51 (53%) had embryonal and 20 (21%) alveolar RMS. Almost all patients received chemotherapy (99%) and radiotherapy (95%). Forty-four patients (45%) underwent surgery. Surgery was more likely to be conducted in patients with lesions of a non-PM site. Median follow-up time was 3.4 years (IQR: 1.1-10.8). In 5 years of follow-up, 20% (17 of 85) died and 29% (20 of 70) had recurrence. The estimated 5-year survival rate was 72% (95% CI, 57.8, 81.5%). Surgery was associated with a reduced risk of mortality after accounting for TNM stage 4 and tumor site (adjusted HR 0.24; 95% CI, 0.07, 0.79; P = .02). The association between surgery and risk of mortality was similar in PM and non-PM tumors. CONCLUSION: A multimodal protocol for treatment including chemotherapy, surgery, and radiotherapy is the mainstay for management of children with HNRMS. While surgery is more commonly used to treat non-PM HNRMS, patients who are able to undergo surgery have significantly higher 5-year survival. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:E984-E992, 2021.

The use of interval-compressed chemotherapy with the addition of vincristine, irinotecan, and temozolomide for pediatric patients with newly diagnosed desmoplastic small round cell tumor.

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is a rare aggressive sarcoma that affects children and young adults, and portends poor outcomes despite intensive multimodal treatment approaches. We report toxicity, response, and outcomes of patients with DSRCT treated with the addition of vincristine, irinotecan, and temozolomide (VIT) to interval-compressed chemotherapy as per Children's Oncology Group ARST08P1. METHODS: All newly diagnosed pediatric patients with DSRCT treated at Dana-Farber Cancer Institute and Boston Children's Hospital between 2014 and 2019 as per ARST08P1, Arm P2 with replacement of VAC cycles with VIT, were identified. Medical records were reviewed for clinical and disease characteristics, and treatment response and outcomes. RESULTS: Six patients were treated as per the above regimen. Median age at diagnosis was 15.1 years (range 3.2-16.4) and five patients were male. Five patients had abdominal primary tumors, of which one had exclusively intraabdominal and four had extraabdominal metastases. Two initial cycles of VIT were well tolerated with nausea, vomiting, diarrhea, and constipation as the most common adverse events. Overall response rate defined as partial or complete response after two initial cycles of VIT was 50%. For local control, all patients had surgical resection followed by radiotherapy, and two patients received hyperthermic intraperitoneal chemotherapy at the time of surgery. Of the four patients who have completed therapy to date, three remain disease-free with median follow-up time of 46.7 months. CONCLUSIONS: The addition of VIT to interval-compressed chemotherapy is tolerable and active in DSRCT, with activity warranting additional investigation.

Author Correction: A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.

Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

Binding of FANCI-FANCD2 Complex to RNA and R-Loops Stimulates Robust FANCD2 Monoubiquitination.

Fanconi anemia (FA) is characterized by developmental abnormalities, bone marrow failure, and cancer predisposition. FA cells are hypersensitive to DNA replicative stress and accumulate co-transcriptional R-loops. Here, we use the Damage At RNA Transcription assay to reveal colocalization of FANCD2 with R-loops in a highly transcribed genomic locus upon DNA damage. We further demonstrate that highly purified human FANCI-FANCD2 (ID2) complex binds synthetic single-stranded RNA (ssRNA) and R-loop substrates with high affinity, preferring guanine-rich sequences. Importantly, we elucidate that human ID2 binds an R-loop structure via recognition of the displaced ssDNA and ssRNA but not the RNA:DNA hybrids. Finally, a series of RNA and R-loop substrates are found to strongly stimulate ID2 monoubiquitination, with activity corresponding to their binding affinity. In summary, our results support a mechanism whereby the ID2 complex suppresses the formation of pathogenic R-loops by binding ssRNA and ssDNA species, thereby activating the FA pathway.

Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade.

Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and MDA5; this results in growth inhibition and tumour inflammation, respectively. Loss of ADAR1 overcomes resistance to PD-1 checkpoint blockade caused by inactivation of antigen presentation by tumour cells. Thus, effective anti-tumour immunity is constrained by inhibitory checkpoints such as ADAR1 that limit the sensing of innate ligands. The induction of sufficient inflammation in tumours that are sensitized to interferon can bypass the therapeutic requirement for CD8+ T cell recognition of cancer cells and may provide a general strategy to overcome immunotherapy resistance.

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target.

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function.

Previous work has shown several proteins defective in Fanconi anemia (FA) are phosphorylated in a functionally critical manner. FANCA is phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation are unknown. Mass spectrometry of FANCA revealed one phosphopeptide, phosphorylated on serine 1449. Serine 1449 phosphorylation was induced after DNA damage but not during S phase, in contrast to other posttranslational modifications of FA proteins. Furthermore, the S1449A mutant failed to completely correct a variety of FA-associated phenotypes. The DNA damage response is coordinated by phosphorylation events initiated by apical kinases ATM (ataxia telangectasia mutated) and ATR (ATM and Rad3-related), and ATR is essential for proper FA pathway function. Serine 1449 is in a consensus ATM/ATR site, phosphorylation in vivo is dependent on ATR, and ATR phosphorylated FANCA on serine 1449 in vitro. Phosphorylation of FANCA on serine 1449 is a DNA damage-specific event that is downstream of ATR and is functionally important in the FA pathway.