BACKGROUND:This first-in-human clinical study explored lomvastomig, an immunoglobulin G1-based Fc-silenced bispecific antibody that simultaneously blocks the immune checkpoint receptors programmed cell death protein 1 (PD-1) and T-cell immunoglobulin domain and mucin domain-3. METHODS:Lomvastomig was characterized in cell cultures and preclinically in cancer mouse models. The phase 1, open-label, multicenter clinical study of lomvastomig included a dose-escalation part in patients with advanced and/or metastatic solid tumors and an expansion part with four tumor-specific cohorts, which enrolled checkpoint inhibitor (CPI)-experienced patients with melanoma and non-small-cell lung cancer (NSCLC) and CPI-naïve patients with SCLC and esophageal squamous cell carcinoma (ESCC). Primary and secondary objectives included safety/tolerability, maximum tolerated dose (MTD)/recommended dose for expansion (RDE), pharmacokinetics, drug receptor occupancy, and antitumor activity. RESULTS:39 and 95 patients were enrolled in the dose-escalation and expansion parts, respectively. Lomvastomig was well tolerated up to the highest tested dose of 2,100 mg every 2 weeks (Q2W). One dose-limiting toxicity was reported at 1,200 mg (grade 3 troponin T increase). No MTD was reached, and 2,100 mg Q2W was established as the RDE. Linear pharmacokinetics across the studied dose range suggested target saturation. Peripheral blood drug receptor occupancy on CD3+ and CD8+ was saturated at >90% throughout treatment for doses ≥70 mg. Objective responses were observed at 2,100 mg lomvastomig during dose-escalation (21%; n=19), and in the CPI-experienced melanoma (8%, n=38) and CPI-naïve ESCC (20%, n=15) expansion cohorts. CONCLUSIONS:Lomvastomig had a tolerable and manageable safety profile at 2,100 mg Q2W. Clinical activity was limited in CPI-experienced patients with melanoma and NSCLC, while an encouraging signal was observed in CPI-naïve patients with ESCC. TRIAL REGISTRATION NUMBER:NCT03708328 (registration date: 2018-10-09).

Whilst it is recognised that targeting self-renewal is an effective way to functionally impair the quiescent leukaemic stem cells (LSC) that persist as residual disease in chronic myeloid leukaemia (CML), developing therapeutic strategies to achieve this have proved challenging. We demonstrate that the regulatory programmes of quiescent LSC in chronic phase CML are similar to that of embryonic stem cells, pointing to a role for wild type p53 in LSC self-renewal. In support of this, increasing p53 activity in primitive CML cells using an MDM2 inhibitor in combination with a tyrosine kinase inhibitor resulted in reduced CFC outputs and engraftment potential, followed by loss of multilineage priming potential and LSC exhaustion when combination treatment was discontinued. Our work provides evidence that targeting LSC self-renewal is exploitable in the clinic to irreversibly impair quiescent LSC function in CML residual disease - with the potential to enable more CML patients to discontinue therapy and remain in therapy-free remission.

Accurate assessment of bone marrow fibrosis is central to the diagnosis and assessment of patients with myeloproliferative neoplasms (MPNs).1-3 However, European consensus criteria for fibrosis are subjective, only semiquantitative, and cannot fully capture sample fibrosis heterogeneity.4-6 In response, we have recently demonstrated the potential of machine learning to improve the detection and quantitation of marrow fibrosis in MPN using routinely prepared bone marrow trephine (BMT) samples.7 Such approaches can support accurate MPN classification/risk stratification and provide quantitative analysis of fibrosis heterogeneity, with the potential to support clinical trial teams in the evaluation of current and novel antifibrotic therapies.6 Here, we report evidence of such utility in the context of stage 2 of a phase II study of zinpentraxin alfa in patients diagnosed with primary or secondary myelofibrosis (MF) [ClinicalTrials.gov identifier: NCT01981850]. The primary trial endpoint was bone marrow response (≥1 grade reduction from baseline fibrosis at any timepoint). Secondary endpoints included effects on disease-related anemia, thrombocytopenia, and constitutional symptoms. Zinpentraxin alfa (ZPN; previously PRM-151) is a recombinant form of human pentraxin-2 (PTX2; also known as serum amyloid P component or SAP), a circulating endogenous regulator of the inflammatory response to tissue damage and a natural inhibitor of fibrosis.8-10 In the open-label stage 1 of this phase 2 study, ZPN showed evidence of clinical activity and tolerable safety as monotherapy or in combination with ruxolitinib in patients with primary MF, post-polycythemia vera (PV) MF, or post-essential thrombocythemia (ET) MF.11 A subsequent randomized dose-ranging study (stage 2) evaluated the efficacy and safety of three different doses of ZPN as monotherapy in patients with IPSS intermediate-1, intermediate-2, and high-risk primary MF, post-PV MF, or post-ET MF who were anemic or thrombocytopenic and ineligible for, intolerant of, or had an inadequate prior response to ruxolitinib.12 Patients were randomized to receive 0.3, 3.0, or 10.0 mg/kg ZPN on Days 1, 3, and 5 of cycle 1 and every 4 weeks thereafter for up to nine cycles. Reticulin-stained BMTs from three timepoints (screening, cycle 4 [C4D1], and cycle 9 [C9D29]) were analyzed for a subset of patients enrolled in the stage 2 study for whom digital scanned images were available at all three timepoints (50/97) (Figure 1A,B). Prior manual assessment of marrow fibrosis had been performed as part of a blinded, independent central review by three expert hematopathologists. Quantitative assessment of fibrosis using Continuous Indexing of Fibrosis (CIF) was performed by automated analyses as previously described.7 Briefly, CIF analysis employs a ranking convolutional neural network (CNN) trained on images of reticulin-stained BMT slides to score image tiles for fibrosis severity. These tiles cover the analyzable marrow tissue and are used to generate fibrosis severity maps with output image scores (CIF scores) normalized between 0 and 1. Three sets of features relating to analyzed tiles are extracted from each sample: average tile CIF score, tile score distribution, and heterogeneity of CIF score. Visualization of these outputs into two-dimensional space is performed using principal component analysis (PCA) (Figure 1C). A total of 142/157 (90.4%) BMT samples obtained from 50 patients at three timepoints were evaluable. Overall, there was a moderate correlation between the average sample CIF score and the manually assigned fibrosis grade for all samples (Spearman's rho = 0.39) (Figure 2A). However, there was a marked overlap in the distribution of CIF scores across fibrosis grades, most notably for samples assigned to grades MF-2 and MF-3. Approximately, 38% (n = 16) of MF-2 samples fell within the interquartile range of CIF distribution observed in MF-3, and around 48% of MF-3 (n = 45) samples fell within the interquartile range observed in MF-2. This result is in keeping with the recognized challenge of accurately distinguishing between these MF grade categories, although both are consistent with a diagnosis of overt myelofibrosis. Notably, several samples manually assessed as MF-2 had average CIF scores similar to or lower than those graded as MF-0 or MF-1. On review, we suspected this may reflect sample fibrosis heterogeneity; some samples with low average fibrosis (low average sample CIF score) were correctly classified as MF-2 on the basis that ≥30% of the tissue contained more severe fibrosis (high regional CIF score). To investigate this further, we compared the ZPN trial samples taken at screening with an independent cohort of newly diagnosed and untreated MPNs in which PCA was used to combine average tile CIF score, tile score distribution, and heterogeneity of CIF score (Figure 2D). Plotting the ZPN screening samples onto this PCA of MPN "disease space" revealed that while most samples demonstrated such combined fibrosis features typical of primary or secondary myelofibrosis, several displayed features more typically seen in ET, pre-PMF, or PV. Having identified marked variation in both the fibrosis features at screening and average CIF scores of manually assigned MF grades for all samples, we assessed changes in fibrosis from screening to C4 and C9. This revealed an improvement in the average CIF score in 16 of 42 patients (38%) (Figure 3A). Notably, improvements in average CIF score by C9 appeared to be most marked in patients with higher CIF scores at screening, although no obvious ZPN dose-dependent effect was observed. The overall improvement in CIF score was similar to that of manually assessed fibrosis in which 15 of 41 patients (37%) had an improvement of at least one MF grade at either C4 or C9. However, there was notable discordance between manual and quantitative CIF fibrosis assessment for individual cases (Figure 2B,C), with only 6 of 41 cases (15%) demonstrating both an improvement in CIF score and manual MF grade. Next, we sought to correlate changes in CIF score with the secondary trial endpoints. We observed no significant association between a change in average sample CIF score and changes in disease-related anemia, thrombocytopenia, or constitutional symptoms (data not shown). However, we observed a trend toward an association between improving CIF score and best overall response as per modified International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) criteria, with marrows from patients experiencing clinical improvement more likely to have a corresponding improvement in CIF score between screening and C9 (Figure 3B). Finally, logistic regression analysis was used to estimate the association of the treatment group, baseline anemia, or thrombocytopenia and average CIF score at screening with the reduction in average CIF score. This revealed that a higher average CIF score at screening was significantly associated with CIF score reduction for the 42 patients for whom samples were available for all three trial timepoints (Wald's test p < 0.01) (Figure 3C). Our analysis is the first to demonstrate the utility of AI-driven quantitative fibrosis analysis in a multicenter clinical trial of patients with myelofibrosis. Although CIF-based analysis is not designed to specify an MF grade, it provides an objective measure of fibrosis severity and heterogeneity within BMTs, which is beyond conventional manual grading criteria. Moreover, it enables objective comparison across sequential samples from individual patients and allows accurate comparison within trial cohorts. Our results raise important concerns over the subjectivity of conventional fibrosis assessment in myelofibrosis, with marked overlap in CIF scores seen between and within manually assigned MF grades, and poor concordance between manually assessed and CIF-determined fibrosis improvement. Unexpectedly, there was a marked variation in average CIF score at screening in a trial recruiting patients with high-risk primary or secondary MF, supported by our demonstration of striking cohort heterogeneity when compared to a separate cohort of MPN. Indeed, 39% (19/49) of the screening samples analyzed in this study demonstrated fibrotic features (average severity and heterogeneity) more typical of MPNs other than primary or secondary myelofibrosis (i.e., ET, PV, and pre-PMF). However, it should be noted that most patients recruited to this trial had high-risk disease (39/50 with IPSS Int-2/high risk) and 39/50 patients had received prior JAK2 inhibition. By contrast, our previously analyzed cohort of MPN included only newly diagnosed patients with no significant pretreatment. It remains unclear to what extent the inclusion of MPN patient samples with longstanding disease and/or significant pretreatment will influence our existing description of bone marrow fibrosis state in ongoing studies. Notwithstanding this caveat, our analysis suggests that variation in manual fibrosis assessment could adversely influence the accuracy and consistency of trials aiming to evaluate therapeutics targeting MF, and alternative methods for quantifying and defining fibrosis changes following therapy are indicated. This is particularly important given recent work questioning the role of marrow fibrosis assessment in evaluating outcomes in JAK inhibitor-naïve patients treated with momelotinib or ruxolitinib, particularly as the authors relied upon local fibrosis grading with no central review.13 Although we could not demonstrate evidence for a significant association between CIF score improvement and the secondary clinical endpoints, we had access to WSI from only 50 of the 97 recruited patients. This reflects challenges in collecting such data as part of post hoc analytical studies and highlights the value of including such analysis in the study protocols of future clinical trials looking to evaluate bone marrow morphological response. Our observation of a trend toward an association between improving CIF score and the best overall response as per IWG-MRT criteria warrants further evaluation of quantitative fibrosis analysis as a surrogate for clinical response in MPN trials aiming to stabilize or reverse marrow fibrosis.14 Conception and design: Daniel Royston, Kerstin Trunzer, Korsuk Sirinukunwattana, Hosuk Ryou, Alan Aberdeen & Jens Rittscher. Collection and assembly of data: Kerstin Trunzer, Frank Peale, Brian Higgins, Pontus Lundberg, Claire N. Harrison, Olga K. Weinberg, Robert Hasserjian & Olga Pozdnyakova. Data analysis and interpretation: Hosuk Ryou, Korsuk Sirinukunwattana, Ruby Wood, Alan Aberdeen & Daniel Royston. Manuscript writing: Daniel Royston, Korsuk Sirinukunwattana, Kerstin Trunzer, Pontus Lundberg & Alan Aberdeen. Final approval of manuscript: All authors. Korsuk Sirinukunwattana, Alan Aberdeen, and Jens Rittscher are cofounders and equity holders of Ground Truth Labs Ltd. Daniel Royston provides consulting services to Ground Truth Labs Ltd. and Johnson & Johnson. Kerstin Trunzer and Pontus Lundberg are employees of F. Hoffmann-La Roche and have stock ownership. Brian Higgins is an employee of F. Hoffmann-La Roche and Genentech and has stock ownership. Frank Peale is an employee of Genentech and has stock ownership. Claire N. Harrison has received consulting fees from AbbVie, AOP, BMS, Constellation Pharmaceuticals, CTI BioPharma, Galecto, GSK, Karyopharm, Keros, MorphoSys, Novartis, Promedior, and Roche; honoraria from AbbVie, BMS, GSK, and Novartis; has advisory roles for Galecto and Keros; has received support from Novartis for attending meetings; and has a leadership or fiduciary role with the European Hematology Association and MPN Voice; and is an Editor of HemaSphere. The remaining authors declare no conflict of interest. This study was supported by F. Hoffmann-La Roche, Ltd.; Blood Cancer UK, Grant/Award Number: 23012; Cancer Research UK, Grant/Award Number: EDDPJT-May23/100034; EPSRC-funded Seebibyte programme (EP/M013774/1); and Ludwig Institute for Cancer Research, Oxford Branch. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supplementary Table Legends 1-2 from Characterization of a Novel Mitogen-Activated Protein Kinase Kinase 1/2 Inhibitor with a Unique Mechanism of Action for Cancer Therapy

Supplementary Figure 1 from RG7204 (PLX4032), a Selective BRAFV600E Inhibitor, Displays Potent Antitumor Activity in Preclinical Melanoma Models

Supplementary Methods, Tables 1-3, Figure Legend from RG7204 (PLX4032), a Selective BRAFV600E Inhibitor, Displays Potent Antitumor Activity in Preclinical Melanoma Models

Background Acute myeloid leukemia (AML) is a heterogeneous disease with poor outcomes, thus there remains a need to integrate molecular information to identify patients most likely to respond to drugs and combinations. Disease segmentation based on transcriptomics has provided valuable insight into disease risk and the likelihood of response to targeted compounds. IL2RA(CD25) is a receptor expressed on both AML leukemic and immune cells, and has shown initial promise as a potential dual target of leukemic blasts and regulatory T (Treg) cells in pre-clinical models (Pousse et al. Front. Oncol. 2023). We used a systems approach based on a recently described transcriptomic classifier for AML (Hamidi et al. ASH 2021) and other molecular tools to characterize the association between IL2RA expression levels and AML genomic markers, clinical features and patient outcomes. In addition, we evaluated IL2RA in relation to ex vivo drug sensitivity, to identify patient segments who are most likely to benefit from CD25-targeting drugs. Methods BEAT-AML (NCT01728402) RNAseq data from patient samples, associated ex vivo drug sensitivity data (N=283), was VOOM normalized. Patients were binned into quartiles based on IL2RA expression. Gene signature scoring was performed using xCell cell type enrichment algorithm and GSVA for Hallmark pathways and scRNAseq signature based on Van Galen et al. Cell 2019. Associations were performed with clinical outcome (log-rank test), gene signatures (Spearman correlation), mutations (Wilcoxon test) and ex vivo drug sensitivity (Kruskal Wallis test). Results We characterized the BeatAML dataset and found that elevated IL2RA expression associates with inferior overall survival (p=0.014) and high-risk features. In addition, we identified a strong association between IL2RA expression and FLT3-ITD status (Figure), as well as other genetic alterations. Using correlation analyses, we established an association between IL2RA expression levels and “primitive” AML signatures (leukemic stem cell (LSC), R=0.4, p=6.8x10-12, hematopoietic stem cell (HSC)-like, R=0.43, p=6.8x10-14) and Tregs (R=0.47, p=1.7x10-15), and an anti-correlation with promonocytic signatures (R=-0.23, p=1.1x10-4). Interestingly, HSC-like and Treg signatures were also correlated (R=0.19, p=0.00087), consistent with an association between stem cell abundance and a repressive immune microenvironment. Finally, using a multivariate model adjusting for the effect of Tregs and LSCs, the prognostic value of IL2RA remained significant (p=0.029). We previously used unsupervised machine learning clustering based on consensus non-negative factorization (cNMF) to discover novel transcription-based classification (Hamidi ASH 2021). Using this methodology, we identified a strong correlation between IL2RA expression in cNMF subtypes (p=1.6x10-10). Moreover, this method uncovered patient subtypes in which IL2RA correlates with LSC (cNMF 6.3), Treg (6.4, 6.6) or both signatures (6.1, 6.2). This differential association was independent of prognostic category, maturation state or venetoclax sensitivity (Table). Transcription-based classifiers have been shown to be highly predictive of ex vivo drug sensitivity. Consistent with an association between IL2RA expression and FLT3-ITD status, we identified strong correlations between IL2RA and sensitivity to FLT-3 and other tyrosine kinase inhibitors, providing a rationale for combining these compounds with CD25-targeting therapeutics. In contrast, there was no correlation between IL2RA levels and venetoclax AUC (p=0.25), while the cNMF classification system similarly revealed that patient segments with increased IL2RA expression had heterogeneous venetoclax sensitivity (Table), supporting CD25 targeting in patients who may not respond to venetoclax. Conclusion AML patients with elevated IL2RA expression have inferior prognosis, enrichment of stem-like and Treg signatures and FLT3-ITD alterations. Transcriptomics-based clustering could be used to guide combination therapies based on potential impact of CD25 targeting on Tregs, leukemic cells, or both populations. Ex vivo drug sensitivity analyses support combinations of CD25-targeting agents with FLT3 inhibitors, as well as venetoclax. Additional work to evaluate these and other combinations using functional assays is warranted.

Supplementary Table 1 from Characterization of a Novel Mitogen-Activated Protein Kinase Kinase 1/2 Inhibitor with a Unique Mechanism of Action for Cancer Therapy

Supplementary Figure 1 from RG7204 (PLX4032), a Selective BRAF^V600E Inhibitor, Displays Potent Antitumor Activity in Preclinical Melanoma Models

Supplementary Figures 1-2 from Preclinical In vivo Evaluation of Efficacy, Pharmacokinetics, and Pharmacodynamics of a Novel MEK1/2 Kinase Inhibitor RO5068760 in Multiple Tumor Models

Background: Myeloproliferative neoplasms (MPNs) are clonal disorders of the hematopoietic stem cell (HSC), caused by somatic mutations in JAK2, MPL or CALR . Myelofibrosis, characterized by increased deposition of reticulin and/or collagen fibers, is found in advanced stages of MPN. Pentraxin-2 (PTX2, serum amyloid P component/SAP) belongs to the family of short pentraxins and acts as an inhibitor of fibrocyte differentiation and modulator of macrophage polarization. Zinpentraxin alfa (PRM-151, ZPN), a recombinant form of human PTX2, was reported to reduce myelofibrosis in a retroviral model of MPN driven by MPL-W515L (Verstovsek S et al, J Exp Med 2016). ZPN has also been investigated as monotherapy and in combination with ruxolitinib (RUX) in a phase 2 clinical study in patients with myelofibrosis (NCT01981850; Verstovsek S et al, Haematologica 2023). Evidence of clinical activity and tolerable safety as monotherapy and in combination with RUX was shown in that open-label, non-randomized trial. Here, we examined the effects of ZPN alone or in combination with RUX in a mouse model of MPN driven by Cre-inducible expression of human JAK2-V617F (Tiedt R et al, Blood 2008). Methods: To obtain sufficient numbers of mice for drug testing, bone marrow cells from JAK2-V617F mice were transplanted into lethally irradiated C57BL/6 recipients (Kubovcakova L et al., Blood 2013), and groups of 6 mice were sacrificed at 16, 20, and 24 weeks to determine the histological grade of reticulin fibrosis. 24 weeks post transplantation, grade 1-2 fibrosis was confirmed in these satellite mice and treatment was initiated. During treatment, weight and complete blood counts were monitored (n=8 mice per group). At terminal work-up, spleen weight and fibrosis grade were determined, along with flow cytometry of bone marrow and peripheral blood, single cell RNA sequencing of bulk bone marrow, and proteomics analysis by mass spectrometry of bone marrow and plasma. Additionally, we characterized the plasma pharmacokinetics (PK) in these mice following single IP (10 mg/kg) administration of ZPN. Results: Systemic exposure after IP administration was confirmed in each animal (T max at 4 h, C max 27.9 ug/mL,AUC 0-48 381 h*ug/mL). No weight loss or mortality were observed in ZPN monotherapy cohorts, and the slight weight loss observed in RUX treated animals was not potentiated by the addition of ZPN. A trend towards lower platelet, monocyte and total leukocyte counts was observed for ZPN treatment groups compared to vehicle controls (Figure 1A). RUX alone largely normalized hemoglobin values, while ZPN alone or in combination with RUX had less effect on hemoglobin. Terminal work-up showed grade 1-2 fibrosis in the vehicle group, whereas reticulin fibrosis decreased in the majority of mice with ZPN monotherapy or in combination with RUX (Figure 1B). ZPN monotherapy did not reduce spleen weight. Single cell RNA sequencing and proteomics analyses are currently being analyzed and data will be shown. Conclusion: ZPN treatment in a JAK2-V617F mouse model of MPN with myelofibrosis was well tolerated as monotherapy and in combination with RUX. A reduction in the grade of myelofibrosis was observed in all ZPN treatment groups. ZPN showed promising trends in reducing platelet and monocyte counts, while the decrease in hemoglobin by RUX was in part prevented in combination with ZPN.

Pentraxin 2 (PTX-2; serum amyloid P component), a circulating endogenous regulator of the inflammatory response to tissue injury and fibrosis, is reduced in patients with myelofibrosis (MF). Zinpentraxin alfa (RO7490677, PRM-151) is a recombinant form of PTX-2 that has shown preclinical antifibrotic activity and no dose-limiting toxicities in phase I trials. We report results from stage 1 of a phase II trial of zinpentraxin alfa in patients with intermediate-1/2 or high-risk MF. Patients (n=27) received intravenous zinpentraxin α weekly (QW) or every 4 weeks (Q4W), as monotherapy or an additional therapy for patients on stable-dose ruxolitinib. The primary endpoint was overall response rate (ORR; investigatorassessed) adapted from International Working Group-Myeloproliferative Neoplasms Research and Treatment criteria. Secondary endpoints included modified Myeloproliferative Neoplasm-Symptom Assessment Form Total Symptom Score (MPN-SAF TSS) change, bone marrow (BM) MF grade reduction, pharmacokinetics, and safety. ORR at week 24 was 33% (n=9/27) and varied across individual cohorts (QW: 38% [3/8]; Q4W: 14% [1/7]; QW+ruxolitinib: 33% [2/6]; Q4W+ruxolitinib: 50% [3/6]). Five of 18 evaluable patients (28%) experienced a ≥50% reduction in MPN-SAF TSS, and six of 17 evaluable patients (35%) had a ≥1 grade improvement from baseline in BM fibrosis at week 24. Most treatment-emergent adverse events (AE) were grade 1–2, most commonly fatigue. Among others, anemia and thrombocytopenia were infrequent (n=3 and n=1, respectively). Treatment-related serious AE occurred in four patients (15%). Overall, zinpentraxin alfa showed evidence of clinical activity and tolerable safety as monotherapy and in combination with ruxolitinib in this open-label, non-randomized trial (clinicaltrials gov. Identifier: NCT01981850).

PDF file - 311KB, Table S1. Results of proliferation assay. Table S2. Estimated PKPD population parameters. Fig. S1. Schematic view of the PK/PD model. Fig S2. Observed PK parameter and model predicted. Fig S3. Visual predictive check.

Supplementary Figures 1-2 from Identification of the MEK1(F129L) Activating Mutation as a Potential Mechanism of Acquired Resistance to MEK Inhibition in Human Cancers Carrying the B-RafV600E Mutation

Supplementary Methods, Tables 1-3, Figure Legend from RG7204 (PLX4032), a Selective BRAFV600E Inhibitor, Displays Potent Antitumor Activity in Preclinical Melanoma Models

Quantitative image analysis has potential to transform the interpretation of bone marrow trephine (BMT) samples in myeloproliferative neoplasms (MPN) and improve the evaluation of anti-fibrotic therapies. To investigate the utility of recently developed algorithms evaluating reticulin fibrosis and megakaryocyte features in myelofibrosis (MF), we analysed samples from a multi-center, phase II study of zinpentraxin alfa (ZPN, PRM-151). ZPN is a recombinant form of human pentraxin-2 (PTX2) that has shown clinical activity as monotherapy and in combination with ruxolitinib (RUX) in a phase II trial in patients with Int-1/-2 or high risk MF (NCT01981850). In stage 2 of this study, patients ineligible for, intolerant of, or with an inadequate response to RUX were randomised to receive 0.3, 3.0, or 10.0 mg/kg ZPN on Days 1, 3, and 5 of Cycle 1, and every 4 weeks thereafter for up to 9 cycles. We demonstrate the potential of quantitative image analysis to augment and refine conventional expert histological assessment of MF. Reticulin and H+E-stained BMTs from three timepoints (screening, C4D1 and C9D29) were analysed from 50/97 patients enrolled. Manual assessment (MA) of marrow fibrosis and megakaryocyte features was performed by blinded, independent central review. Assessment of fibrosis using the Continuous Indexing of Fibrosis (CIF) score and analysis of megakaryocytes was performed by automated analyses ( Ryou H, Leukaemia, 2022). There was a moderate correlation between the automated average CIF score and manual fibrosis grade (Spearman's rho = 0.39). However, there was significant overlap in the distribution of CIF scores between samples manually assigned to MF-2 and MF-3. Approximately 38% of MF-2 samples fell within the interquartile range of CIF distribution observed in MF-3, and around 48% of MF-3 samples fell within the interquartile range observed in MF-2. Next, we visualised the screening BMTs in fibrosis PCA (principal component analysis) space to capture both average CIF score and Shannon entropy (‘unevenness‘) of CIF score for each sample. This revealed significant heterogeneity in reticulin fibrosis at screening when compared to an independent reference cohort of 130 MPN samples ( Ryou H, Leukaemia, 2022), likely reflecting the effects of pre-treatment [ Figure 1]. To track fibrosis across all timepoints we plotted the average CIF score for each sample. This revealed striking variability in fibrotic response within the trial cohort, with 15 of 42 patients (36%) demonstrating improvement in average CIF score [ Figure 2]. Notably, improvements in average CIF score by cycle 9 appeared to be most marked in patients with higher CIF scores at screening. No significant ZPN dose-dependent effect on fibrosis was observed. We next measured megakaryocyte density (megakaryocytes per unit area of intertrabecular space) and compared it to MA. We found a strong correlation (Spearman's rho = 0.81) between automated and MA of megakaryocyte density, with no significant change across any of the treatment arms. For megakaryocyte clustering (defined as ≥ 3 megakaryocytes in direct contact) we observed a moderate to strong correlation (Spearman's rho = 0.62) between MA and automated assessment, with no significant change across any of the treatment arms. Finally, we sought to compare the sample megakaryocyte cell features (cytomorphology and topology) at screening with those of an independent reference cohort of 88 MPN samples ( Sirinukunwattana, Blood Adv., 2020). Strikingly, not only was there marked heterogeneity in megakaryocyte features between patient samples at screening, but the observed features were significantly different to those of newly diagnosed MF. No significant changes in megakaryocyte cytomorphology or topology were seen across any of the treatment arms. In summary, our findings reveal considerable BMT morphological heterogeneity in patients participating in a Phase II trial of ZPN. This likely reflects poorly understood and under-recognised variation in morphological features encountered in patients with longstanding and/or pre-treated MF when compared to newly diagnosed patients. This highlights the potential of such variability to confound the evaluation of novel therapeutics in MPN, and emphasises the utility of robust quantitative methods to analyse and visualise morphological features in clinical study samples that can complement conventional manual assessment.

Idasanutlin, an MDM2 antagonist, showed clinical activity and a rapid reduction in JAK2 V617F allele burden in patients with polycythemia vera (PV) in a phase 1 study. This open-label phase 2 study evaluated idasanutlin in patients with hydroxyurea (HU)-resistant/-intolerant PV, per the European LeukemiaNet criteria, and phlebotomy dependence; prior ruxolitinib exposure was permitted. Idasanutlin was administered once daily on days 1 through 5 of each 28-day cycle. The primary end point was composite response (hematocrit control and spleen volume reduction > 35%) in patients with splenomegaly and hematocrit control in patients without splenomegaly at week 32. Key secondary end points included safety, complete hematologic response (CHR), patient-reported outcomes, and molecular responses. All patients (n = 27) received idasanutlin; 16 had response assessment (week 32). Among responders with baseline splenomegaly (n = 13), 9 (69%) attained any spleen volume reduction, and 1 achieved composite response. Nine patients (56%) achieved hematocrit control, and 8 patients (50%) achieved CHR. Overall, 43% of evaluable patients (6/14) showed a ≥50% reduction in the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (week 32). Nausea (93%), diarrhea (78%), and vomiting (41%) were the most common adverse events, with grade ≥ 3 nausea or vomiting experienced by 3 patients (11%) and 1 patient (4%), respectively. Reduced JAK2 V617F allele burden occurred early (after 3 cycles), with a median reduction of 76%, and was associated with achieving CHR and hematocrit control. Overall, the idasanutlin dosing regimen showed clinical activity and rapidly reduced JAK2 allele burden in patients with HU-resistant/- intolerant PV but was associated with low-grade gastrointestinal toxicity, leading to poor long-term tolerability. This trial was registered at www.clinincaltrials.gov as #NCT03287245.

Although it has been recognized for many years that cancer stem cells and embryonic stem cells (ESC) share molecular features, identifying ways to exploit this therapeutically has proved challenging. To date, these shared features have not been examined in the leukemic stem cells (LSC) found in patients with chronic myeloid leukemia (CML).