Safety of bendamustine for the treatment of indolent non-Hodgkin lymphoma: a UK real-world experience.

ABSTRACT: Bendamustine is among the most effective chemotherapeutics for indolent B-cell non-Hodgkin lymphomas (iNHL), but trial reports of significant toxicity, including opportunistic infections and excess deaths, led to prescriber warnings. We conducted a multicenter observational study evaluating bendamustine toxicity in real-world practice. Patients receiving at least 1 dose of bendamustine with/without rituximab (R) for iNHL were included. Demographics, lymphoma and treatment details, and grade 3 to 5 adverse events (AEs) were analyzed and correlated. In total, 323 patients were enrolled from 9 National Health Service hospitals. Most patients (96%) received bendamustine-R, and 46%, R maintenance. Overall, 21.7% experienced serious AEs (SAE) related to treatment, including infections in 12%, with absolute risk highest during induction (63%), maintenance (20%), and follow-up (17%) and the relative risk highest during maintenance (54%), induction (34%), and follow-up (28%). Toxicity led to permanent treatment discontinuation for 13% of patients, and 2.8% died of bendamustine-related infections (n = 5), myelodysplastic syndrome (n = 3), and cardiac disease (n = 1). More SAEs per patient were reported in patients with mantle cell lymphoma, poor preinduction performance status (PS), poor premaintenance PS, and abnormal preinduction total globulins and in those receiving growth factors. Use of antimicrobial prophylaxis was variable, and 3 of 10 opportunistic infections occurred despite prophylaxis. In this real-world analysis, bendamustine-related deaths and treatment discontinuation were similar to those of trial populations of younger, fitter patients. Poor PS, mantle cell histology, and maintenance R were potential risk factors. Infections, including late onset events, were the most common treatment-related SAE and cause of death, warranting extended antimicrobial prophylaxis and infectious surveillance, especially for maintenance-treated patients.

Real-World Outcomes of Stage IV NSCLC with PD-L1 ≥ 50% Treated with First-Line Pembrolizumab: Uptake of Second-Line Systemic Therapy.

INTRODUCTION: Platinum-based chemotherapy was compared to single-agent pembrolizumab in advanced non-small cell lung cancer (NSCLC) with PDL1 > 50% in KEYNOTE-024. In this trial, it was found that patients who received single-agent pembrolizumab had improved progression-free survival in addition to overall survival (OS). Based on KEYNOTE-024, only 53% of patients treated originally with pembrolizumab received second-line anticancer systemic therapy with an OS of 26.3 months. Based on these results, the objective of this study was to characterize real-world NSCLC patients who received second-line therapy after single-agent pembrolizumab. METHODS: This was a retrospective cohort study considering stage IV NSCLC patients diagnosed with BC Cancer between 2018 and 2021 with PD-L1 ≥ 50% who received first-line single agent pembrolizumab. Patient demographics, cancer history, treatment administered, and survival were collected retrospectively. Descriptive statistics were produced. OS was calculated using Kaplan-Meier curves and compared using the log rank test. A multivariate model evaluated characteristics associated with the receipt of second-line therapy. RESULTS: A total of 718 patients were diagnosed with Stage IV NSCLC and received at least one cycle of pembrolizumab. The median duration of treatment was 4.4 months, and the follow-up duration was 16.0 months. There were 567 (79%) patients who had disease progression, of whom 21% received second-line systemic therapy. Within the subset of patients with disease progression, the median duration of treatment was 3.0 months. It would be found that patients who received second-line therapy had better baseline ECOG performance status, were younger at diagnosis, and had a longer duration of pembrolizumab. Within the full population, the OS from the treatment initiation date was 14.0 months. OS was 5.6 months in patients who did not receive additional therapy after progression and 22.2 months in patients who received subsequent therapy. Baseline ECOG performance status was associated with improved OS in multivariate analysis. CONCLUSION: Based on this real-world Canadian population, 21% of patients received second-line systemic therapy, despite second-line therapy being associated with prolonged survival. In this real-world population, we found that 60% fewer patients received second-line systemic therapy when compared to KEYNOTE-024. Although differences always exist when comparing a clinical and non-clinical trial population, our findings suggest undertreating stage IV NSCLC patients.

Improved uptake and survival with systemic treatments for metastatic non-small cell lung cancer: younger versus older adults.

BACKGROUND: Over the past decade, there has been increasing availability of novel therapeutics with improved tolerability and efficacy for advanced non-small cell lung cancer (NSCLC). The study goals were: to compare the uptake of systemic therapy (ST) before and after the availability of targeted tyrosine kinase inhibitors (TKI) and immunotherapy and to examine the changes in overall survival (OS) over time between younger and older adults with advanced NSCLC. METHODS: All patients with advanced NSCLC referred to British Columbia (BC) Cancer in 2009, 2011, 2015 and 2017 were included. One-year time points were based on molecular testing implementation and funded drug availability: baseline (2009), epidermal growth factor receptor TKI (2011), anaplastic lymphoma kinase TKI (2015) and Programed Death-1 (PD-1) inhibitors (2017). Age groups were <70years and ≥70years. Baseline demographics, simplified comorbidity scores (SCS), disease characteristics, and ST details were collected retrospectively. Variables were compared using X2, Fisher's exact tests and logistic-regression analysis. OS was calculated using the Kaplan-Meier method and compared using the log-rank test. RESULTS: 3325 patients were identified. Baseline characteristics were compared between ages < 70 years and ≥ 70 years for each time cohort with significant differences noted in baseline Eastern Cooperative Oncology Group (ECOG) performance status and SCS. The rate of ST delivery trended upwards over time with age <70 years: 2009 44%, 2011 53%, 2015 50% and 2017 52% and age ≥70 years: 22%, 25%, 28% and 29% respectively. Predictors for decreased use of ST for age <70 years: ECOG ≥2, SCS ≥9, year 2011, and smoking history; and age ≥70 years: ECOG ≥2, years 2011 and 2015, and smoking history. The median OS of patients who received ST improved from 2009 to 2017: age <70 years 9.1 m vs. 15.5 m and age ≥70 years 11.4 m vs. 15.0 m. CONCLUSIONS: There was an increased uptake of ST for both age groups with the introduction of novel therapeutics. Although a smaller proportion of older adults received ST, those who received treatment had comparable OS to their young counterpart. The benefit of ST in both age groups was seen across the different types of treatments. With careful assessment and selection of appropriate candidates, older adults with advanced NSCLC appear to benefit from ST.

Stratification of PD-1 blockade response in melanoma using pre- and post-treatment immunophenotyping of peripheral blood.

Efficacy of checkpoint inhibitor therapies in cancer varies greatly, with some patients showing complete responses while others do not respond and experience progressive disease. We aimed to identify correlates of response and progression following PD-1-directed therapy by immunophenotyping peripheral blood samples from 20 patients with advanced malignant melanoma before and after treatment with the PD-1 blocking antibody pembrolizumab. Our data reveal that individuals responding to PD-1 blockade were characterised by increased CD8 T cell proliferation following treatment, while progression was associated with an increase in CTLA-4-expressing Treg. Remarkably, unsupervised clustering analysis of pre-treatment T cell subsets revealed differences in individuals that went on to respond to PD-1 blockade compared to individuals that did not. These differences mapped to expression of the proliferation marker Ki67 and the costimulatory receptor CD28 as well as the inhibitory molecules 2B4 and KLRG1. While these results require validation in larger patient cohorts, they suggest that flow cytometric analysis of a relatively small number of T cell markers in peripheral blood could potentially allow stratification of PD-1 blockade treatment response prior to therapy initiation.

Alternate Pembrolizumab Dosing Interval in Advanced NSCLC with PD-L1 TPS ≥ 50%: 3 Weekly Compared to 6 Weekly Dosing.

BACKGROUND: A fixed dose of 200 mg of pembrolizumab every 3 weeks (Q3W) is the standard of care for patients with stage IV non-small cell lung cancer (NSCLC) and PDL1 ≥50%. In April 2020, based on pharmacokinetic modeling without formal comparative studies, the FDA approved 400 mg every 6 weeks (Q6W). Pharmacokinetic studies also suggested comparable target engagement with weight-based and flat dosing for the respective schedules. The objective of this study was to determine if overall survival (OS) differs based on the Q3W vs. Q6W dosing schedule of pembrolizumab. METHODS: BC Cancer patients with stage IV NSCLC and PDL1 ≥50% treated with pembrolizumab were retrospectively reviewed. Patients were treated with weight-based dosing, per institution standard, of pembrolizumab 2 mg/kg Q3W or 4 mg/kg Q6W. Patient demographics, treatment and outcome were recorded. Patients were assigned to Q3W or Q6W according to the schedule that was used for the majority of treatment (greater than 50%). RESULTS: 718 patients with NSCLC and PDL1 ≥50% received first-line pembrolizumab between 2017 and2021, Q3W/Q6W dosing 677/41 patients. Baseline characteristics with respect to age, sex, smoking status, histology and performance status (PS) were similar between groups. In the multivariate model, including age, sex, PS and dosing schedule, the hazard ratio for death (HR) for OS Q3W vs. Q6W was 0.759 (p = 0.230). A 2:1 case-matched analysis for OS was performed, controlling for sex, age ± 5 years, PS and duration on pembrolizumab ± 2 months for Q3W vs. Q6W (n = 113) with a HR 0.834 (p = 0.500). CONCLUSIONS: There was no OS difference demonstrated with pembrolizumab dosing Q3W compared to Q6W in a multivariate analysis that included age, sex and PS. A case-matched analysis that controlled for these variables and for duration of treatment confirmed these findings. This study supports the use of Q6W pembrolizumab dosing, allowing for less frequent interactions with the medical system.

The Neoantigen Landscape of the Coding and Noncoding Cancer Genome Space.

Tumor mutation burden (TMB) is a measure to predict patient responsiveness to immune checkpoint immunotherapy because with increased mutation frequency, the likelihood of a greater neoantigen burden is increased. Although neoantigen prediction tools exist, tumor neoantigen burden has not been adopted as a measure to predict immunotherapy response. With both measures, current guidelines are limited to the coding regions, but ectopic expression of sequences in the noncoding space may potentially be a source of neoantigens. A pan-cancer cohort of 574 advanced disease stage patients with whole genome and transcriptome sequencing was analyzed to report mutation burden and neoantigen counts within the coding and noncoding regions. The efficacy of tumor neoantigen burden, reported as tumor neoantigen count (TNC), including neoantigens derived from the expression of noncoding regions, compared with TMB as a predictor of response to immunotherapy for 80 patients who had received treatment, was evaluated. TMB was found to be the best predictor of response to immunotherapy, whereas expression-derived TNC from the noncoding regions did not improve prediction of response. Therefore, there is minimal benefit in extending the calculation of TNC to the noncoding space for the purposes of predicting response. However, it is likely that there is a wealth of neoantigens derived from the noncoding space that may impact patient outcomes and treatments.

Effect of targeted therapy and immunotherapy on advanced nonsmall-cell lung cancer outcomes in the real world.

The evolution of diagnosis and treatment of advanced nonsmall-cell lung cancer (NSCLC) has led to increasing the use of targeted therapy and immune checkpoint inhibitors. The study goal was to assess the effect of molecular testing and the introduction of new therapies on overall survival (OS). All patients with stage IV NSCLC referred to BC Cancer were included in the study. Four 1-year time cohorts were created based on molecular testing implementation and funded drug availability: C1 baseline (2009), C2 EGFR TKI access (2011), C3 ALK inhibitor access (2015), C4 immunotherapy availability (2017). Baseline demographics, disease characteristics, and systemic therapy details were collected retrospectively. OS was calculated using the Kaplan-Meier method and compared using the log-rank test. There were 3421 patients identified with stage IV NSCLC and 1319 (39%) received systemic therapy. In the four 1-year time cohorts C1/C2/C3/C4: driver mutation-targeted treatment increased 1/17/27/34% (of total systemic therapy), as did treatment with any line immunotherapy <1/1/9/38%. Median OS with best supportive care (BSC) was 3.4/3.1/3.2/2.9 m (p = 0.16) and with systemic treatment 9.9/10.9/13.9/15.0 m (p < 0.001). Median OS by treatment exposure was BSC 3.1 m, chemotherapy only 7.3 m, targeted therapy 17.5 m, and immunotherapy 20.7 m. In our real-world study, following the introduction of targeted therapy and immune checkpoint inhibitors, there was a significant improvement in OS in each successive time cohort concordant with advancements in therapeutic options.

Genome and Transcriptome Biomarkers of Response to Immune Checkpoint Inhibitors in Advanced Solid Tumors.

PURPOSE: Immune checkpoint inhibitors (ICI) have revolutionized the treatment of solid tumors with dramatic and durable responses seen across multiple tumor types. However, identifying patients who will respond to these drugs remains challenging, particularly in the context of advanced and previously treated cancers. EXPERIMENTAL DESIGN: We characterized fresh tumor biopsies from a heterogeneous pan-cancer cohort of 98 patients with metastatic predominantly pretreated disease through the Personalized OncoGenomics program at BC Cancer (Vancouver, Canada) using whole genome and transcriptome analysis (WGTA). Baseline characteristics and follow-up data were collected retrospectively. RESULTS: We found that tumor mutation burden, independent of mismatch repair status, was the most predictive marker of time to progression (P = 0.007), but immune-related CD8+ T-cell and M1-M2 macrophage ratio scores were more predictive for overall survival (OS; P = 0.0014 and 0.0012, respectively). While CD274 [programmed death-ligand 1 (PD-L1)] gene expression is comparable with protein levels detected by IHC, we did not observe a clinical benefit for patients with this marker. We demonstrate that a combination of markers based on WGTA provides the best stratification of patients (P = 0.00071, OS), and also present a case study of possible acquired resistance to pembrolizumab in a patient with non-small cell lung cancer. CONCLUSIONS: Interpreting the tumor-immune interface to predict ICI efficacy remains challenging. WGTA allows for identification of multiple biomarkers simultaneously that in combination may help to identify responders, particularly in the context of a heterogeneous population of advanced and previously treated cancers, thus precluding tumor type-specific testing.

EGFR circulating tumour DNA testing: identification of predictors of ctDNA detection and implications for survival outcomes.

BACKGROUND: EGFR T790M testing is the standard of care for activating EGFR mutation (EGFRm) non-small cell lung cancer (NSCLC) progressing on 1st/2nd generation TKIs to select patients for osimertinib. Despite sensitive assays, detection of circulating tumour deoxyribonucleic acid (ctDNA) is variable and influenced by clinical factors. The number and location of sites of progressive disease at time of testing were reviewed to explore the effect on EGFR ctDNA detection. The prognostic value of EGFR ctDNA detection on survival outcomes was assessed. METHODS: Following extraction of cell-free DNA from plasma using the QIAamp Circulating Nucleic Acid Kit, custom droplet digital polymerase chair reaction (ddPCR) assays were used to assess EGFR ctDNA using the Bio-Rad QX200 system. The ddPCR assay has a limit of detection of ≤0.15% variant allele fraction. Baseline characteristics and imaging reports at time of EGFR ctDNA testing were reviewed retrospectively for a 1 year period. RESULTS: The study included 177 patients who had an EGFR ctDNA test. Liver (aOR 3.13) or bone (aOR 2.76) progression or 3-5 sites of progression (aOR 2.22) were predictive of EGFR ctDNA detection. The median OS from first ctDNA test after multiple testing iterations was 12.3 m undetectable EGFR ctDNA, 7.6 m for original EGFR mutation only and 24.1 m with T790M (P=0.001). CONCLUSIONS: Patients with liver or bone progression and 3-5 progressing sites are more likely to have informative EGFR ctDNA testing. Detection of EGFR ctDNA is a negative prognostic indicator in the absence of a T790M resistance mutation, potentially reflecting the disease burden in the absence of targeted therapy options.

Intratumoral heterogeneity in programmed death-ligand 1 immunoreactivity is associated with variation in non-small cell lung carcinoma histotype.

AIMS: Accurate assessment of programmed death-ligand 1 (PD-L1) levels in non-small cell lung carcinoma (NSCLC) samples is complicated by intratumoral heterogeneity. We aimed to: (i) establish whether intratumoral PD-L1 variation is associated with differences in local histotype; (ii) identify histotypes associated with a tendency for there to be higher or lower PD-L1 scores; and (iii) estimate the frequency of clinically significant discordance in PD-L1 levels between intratumoral histotype areas. METHODS AND RESULTS: We reviewed 166 NSCLC resection specimens clinically tested for PD-L1 with the 22C3 pharmDx assay. Multiple histotypes were present in 55% (68/123) of non-mucinous adenocarcinoma samples. Solid histotypes had significantly higher PD-L1 levels than other histotypes, both when samples were grouped by predominant histotype, and when histotype areas within a tumour were compared (P < 0.02). Lepidic areas had significantly lower PD-L1 levels than other histotype areas within the same tumour (P < 0.02). Discordance between intratumoral histotype areas at a clinically relevant threshold (PD-L1 tumour proportion score of 1% or 50%) was present in 32% (22/68) of non-mucinous adenocarcinoma specimens with multiple histotype areas. The lepidic histotype was most frequently involved in discordance. CONCLUSIONS: Intratumoral heterogeneity in PD-L1 is associated with variation in histotype. Over-representation of solid areas may increase the PD-L1 score assigned to a tumour, whereas over-representation of lepidic areas may decrease the PD-L1 score. Evaluation of how histotype representation impacts on the predictive value of PD-L1 testing is warranted.

Optimising Cancer Vaccine Design in Sarcoma.

Immunotherapeutics are increasingly recognized as a key tool in the armamentarium against malignancy. The success of immune checkpoint-targeting drugs and adoptive cell therapy has refocused attention on the potential anti-cancer effect of eliciting a tumour-specific immunological response. Sarcomas are a rare and diverse group of tumours with a limited prognosis in advanced disease despite systemic therapeutics. Various vaccine strategies including peptide vaccines against cancer testis antigens, dendritic cell vaccines, and viral vectors have been trialled in sarcoma with growing evidence of efficacy. Here, we review the principles of successful vaccine development and how these have been applied thus far to the treatment of sarcoma.

Poor treatment outcomes with palliative gemcitabine and docetaxel chemotherapy in advanced and metastatic synovial sarcoma.

The outcome for patients with unresectable or metastatic soft tissue sarcoma remains poor with few treatment options. Synovial sarcoma is a rare type of sarcoma, predominantly affecting adolescents and young adults. Following failure of first-line anthracycline-based chemotherapy, several salvage options are available. We reviewed the safety and efficacy of gemcitabine/docetaxel chemotherapy in two tertiary oncology centres. We identified patients treated with gemcitabine/docetaxel between 2004 and 2016 in a UK and a US oncology centre using retrospective pharmacy and medical records. Treatment response, toxicity and outcome data were collected. Twenty one patients were treated with gemcitabine/docetaxel, the majority as a second- or third-line treatment for metastatic disease. The response rate was 5% with a median progression-free survival of 2 months (95% CI 1.3-3.7). Toxicities reported were as expected for this chemotherapy combination. Treatment was not discontinued due to toxicity. Gemcitabine/docetaxel chemotherapy shows little efficacy in synovial sarcoma and should not be offered to this patient group outside a clinical trial context.

Olaratumab: a platelet-derived growth factor receptor-α-blocking antibody for the treatment of soft tissue sarcoma.

The outcome of patients with unresectable or metastatic soft tissue sarcoma (STS) remains poor with few treatment options. A number of randomized trials in the first-line setting have shown no difference in overall survival between combination anthracycline schedules and single-agent doxorubicin. A Phase Ib/randomized Phase II trial of doxorubicin with or without the platelet-derived growth factor receptor-α (PDGFRα)-blocking antibody, olaratumab, demonstrated a significant difference in median overall survival in favor of the olaratumab arm. The results of this trial led to the approval of olaratumab in combination with doxorubicin in adult anthracycline-naïve unresectable STS. In this review, we discuss the potential role of PDGFRα signaling, early clinical data with olaratumab in sarcomas, the Phase Ib/II trial and ongoing trials with olaratumab in sarcomas.

Olaratumab for the treatment of soft-tissue sarcoma.

The outcome for patients with unresectable/metastatic soft tissue sarcoma remains poor with few treatment options. In the first line setting, a number of randomized trials have shown no difference in overall survival between combination anthracycline schedules and single agent doxorubicin. A Phase Ib/randomized Phase II trial of doxorubicin with or without the monoclonal antibody to PDGFR-α, olaratumab, demonstrated a significant difference in median overall survival in favor of the olaratumab arm. The results of this trial led to approval of olaratumab in combination with doxorubicin in adult anthracycline-naive unresectable soft tissue sarcoma. In this review, we describe some of the preclinical and early clinical data of olaratumab in sarcomas, the Phase Ib/II trial and ongoing trials with olaratumab in sarcomas.

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.

Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.

The efficacy of crizotinib in patients with ALK-positive nonsmall cell lung cancer.

Molecular profiling of nonsmall cell lung cancer (NSCLC) contributes to better understanding the different molecular subtypes of this heterogeneous group of diseases. The discovery of oncogenic ALK rearrangements in NSCLC and the subsequent success in their therapeutic targeting with crizotinib reinforces the benefits of a precision approach to systemic anticancer therapy. In addition, the rapid development of crizotinib from first discovery thorough accelerated US Food and Drug Administration approval, and late stage confirmatory clinical trials, exemplifies the success of the drug development strategy of close collaboration between clinicians, industry and regulatory authorities. In this review we describe the identification of ALK rearranged NSCLC, clinical characteristics of such patients, and clinical outcomes when treated with crizotinib.

Recruitment of the earliest component of the bacterial flagellum to the old cell division pole by a membrane-associated signal recognition particle family GTP-binding protein.

The specialised signal recognition particle family guanosine 5c-triphosphate (GTP)-binding protein FlhF is required for the correct localisation of flagella in several bacterial species. Here, we characterise the regions of Vibrio cholerae FlhF that are required for its function and targeting to the old cell pole, and we present evidence for a mechanism by which FlhF establishes flagellum polar localisation. Substitution of residues in FlhF nucleotide-binding motifs reduced GTP binding and the efficiency of flagellum biogenesis, and caused flagellum mislocalisation. However, replacement of conserved putative catalytic residues (D(321), R(324), and Q(330)) had no effect, suggesting that while GTP binding influences FlhF function, GTPase activity might not be essential. FlhF associated with the inner membrane in the absence of other flagellar proteins, and a functional FlhF-green fluorescent protein fusion was targeted to the old cell pole where the flagellum is localised. FlhF targeting to the pole was intrinsic, as no other flagellar proteins were needed. Neither the FlhF C-terminal GTP-binding region nor the N-terminal 166-residue B-region was required for polar localisation, though they were essential for FlhF function. Deletion of the central 108-residue N-region of FlhF, comprising alpha-helices N1-N4, did however severely reduce the efficiency of FlhF polar targeting, as well as FlhF function. The intrinsic localisation of FlhF to the old cell pole membrane suggested that FlhF might function at an early stage of flagellum assembly; to test this, we assessed the effect of FlhF on the localisation of the earliest flagellar structural component, the membrane-supramembrane ring protein FliF. Recruitment of FliF to the pole required only FlhF and no other flagellar proteins. FliF polar targeting was abolished in the absence of FlhF and by deletion of the FlhF B-domain or GTP-binding region. Our data indicate that FlhF establishes the site of flagellum assembly at the old cell pole membrane by recruiting the earliest flagellar structural component FliF.