Clinical implications of cancer gene mutations in patients with chronic lymphocytic leukemia treated with lenalidomide.

Lenalidomide is clinically active in chronic lymphocytic leukemia (CLL), but its effectiveness in the context of the CLL mutational landscape is unknown. We performed targeted capture sequencing of 295 cancer genes in specimens from 102 CLL patients with treatment-naïve disease (TN patients) and 186 CLL patients with relapsed/refractory disease (R/R patients) who received lenalidomide-based therapy at our institution. The most frequently mutated gene was SF3B1 (15%), followed by NOTCH1 (14%) and TP53 (14%), with R/R patients having significantly more TP53 mutations than did TN patients. Among all lenalidomide-treated patients, del(17p) (P ≤ .001), del(11q) (P = .032), and complex karyotype (P = .022), along with mutations in TP53 (P ≤ .001), KRAS (P = .034), and DDX3X (P ≤ .001), were associated with worse overall response (OR). R/R patients with SF3B1 and MGA mutations had significantly worse OR (P = .025 and .035, respectively). TN and R/R patients with del(17p) and TP53 mutations had worse overall survival (OS) and progression-free survival (PFS). In R/R patients, complex karyotype and SF3B1 mutations were associated with worse OS and PFS; DDX3X mutations were associated with worse PFS only. Weibull regression multivariate analysis revealed that TP53 aberrations (del(17p), TP53 mutation, or both), along with complex karyotype and SF3B1 mutations, were associated with worse OS in the R/R cohort. Taken together, cancer gene mutations in CLL contribute to the already comprehensive risk stratification and add to prognosis and response to treatment. The related trials were registered at www.clinicaltrials.gov as #NCT00267059, #NCT00535873, #NCT00759603, #NCT01446133, and #NCT01002755.

Clinically relevant doses of tiludronate do not affect bone remodelling in pasture-exercised horses.

BACKGROUND: Bisphosphonates are widely used in equine athletes to reduce lameness associated with skeletal disorders. Widespread off-label use has led to concern regarding potential negative effects on bone healing, but little evidence exists to support or refute this. OBJECTIVES: To investigate the influence of clinically relevant doses of tiludronate on bone remodelling and bone healing. STUDY DESIGN: Randomised, controlled in vivo experiments. METHODS: Each horse had a single tuber coxae biopsied (Day 0), then were divided into a treatment (IV tiludronate) or control (IV saline) group. Treatments were administered 30 and 90 days following initial biopsy. Biopsy of the tuber coxae was repeated on Day 60 to evaluate bone healing following a single treatment. Oxytetracycline was administered on Days 137 and 147 to label bone formation. The contralateral tuber coxae was biopsied on Day 150 to evaluate effects of repeated treatment. Bone biopsies were evaluated with micro-computed tomography and/or dynamic histomorphometry using standard techniques. RESULTS: Nineteen horses completed the study, with no complications following the biopsies and treatments. No significant differences in the trabecular bone parameters or bone formation rate were observed between treatment groups. MAIN LIMITATIONS: The use of a first-generation bisphosphonate may mean some effects of these drugs are underrepresented using this model. The results pertain to the tuber coxae and may not reflect injury or the healing response that occurs in long bones in training or racing. CONCLUSIONS: In this model, tiludronate did not affect normal bone remodelling in the horse, despite repeat dosages.

Cortical porosity is elevated after a single dose of zoledronate in two rodent models of chronic kidney disease.

PURPOSE: Patients with chronic kidney disease (CKD) have high risk of fracture in part due to cortical bone deterioration. The goal of this study was to assess the impact of two different bisphosphonates and dosing regimens on cortical microstructure (porosity, thickness, area) and bone mechanical properties in animal models of CKD. METHODS: In experiment 1, Male Cy/+ (CKD) rats were treated with either a single dose or ten fractionated doses of zoledronate at 18 weeks of age. Fractionated animals received 1/10th of single dose given weekly for 10 weeks, with study endpoint at 28 weeks of age. In experiment 2, male C57Bl/6 J mice were given dietary adenine (0.2%) to induce CKD. Bisphosphonate treated groups were given either a single dose of zoledronate or weekly risedronate injections for 4 weeks. Cortical microstructure was assessed via µCT and mechanical parameters evaluated by monotonic bending tests. RESULTS: Exp 1: CKD rats had higher blood urea nitrogen (BUN) and parathyroid hormone (PTH) compared to NL littermate controls. Single dose zoledronate had significantly higher cortical porosity in CKD S.Zol (2.29%) compared to NL control (0.04%) and untreated CKD (0.14%) (p = 0.004). Exp 2: All adenine groups had significantly higher BUN and PTH compared to control mice. Mice treated with single dose zoledronate (Ad + Zol) had the highest porosity (~6%), which was significantly higher compared to either Ad or Ad + Ris (~3%; p < 0.0001) and control mice had the lowest cortical porosity (0.35%). In both experiments, mechanics were minimally affected by any bisphosphonate dosing regimen. CONCLUSION: A single dose of zoledronate leads to higher cortical porosity compared to more frequent dosing of bisphosphonates (fractionated zoledronate or risedronate). Bisphosphonate treatment demonstrated limited effectiveness in preventing cortical bone microstructure deterioration with mechanical parameters remaining compromised due to CKD and/or secondary hyperparathyroidism irrespective of bisphosphonate treatment.

The combination of aging and chronic kidney disease leads to an exacerbated cortical porosity phenotype.

PURPOSE: Chronic kidney disease (CKD) and aging are each independently associated with higher fracture risk. Although CKD is highly prevalent in the aging population, the interaction between these two conditions with respect to bone structure and mechanics is not well understood. The purpose of this study was to examine cortical porosity and mechanical properties in skeletally mature young and aging mice with CKD. METHODS: CKD was induced by feeding 16-week and 78-week male mice 0.2% adenine (AD) for six weeks followed by two weeks of maintenance on a control diet for a total study duration of eight weeks of CKD; control (CON) animals of each age were fed a standard diet. Serum biochemistries, µCT imaging, and mechanical properties via four-point bending were assessed at the endpoint. RESULTS: Phosphorus, parathyroid hormone, and blood urea nitrogen were elevated in both ages of AD mice compared to age-matched CON; aging AD mice had PTH and BUN values higher than all other groups. Femoral cortical porosity was more than four-fold higher in aging AD mice compared to young AD mice and more than two-fold higher compared to age-matched controls. Structural and estimated material mechanical properties were both lower in aging mice, but there were no significant interactions between AD treatment and age. CONCLUSION: These data show an interaction between CKD and aging that produces a more severe biochemical and cortical bone phenotype. This highlights the importance of studying mechanisms and potential interventions in both young and aged animals to translate to a broader spectrum of CKD patients.

Cortical porosity occurs at varying degrees throughout the skeleton in rats with chronic kidney disease.

Cortical porosity develops in chronic kidney disease (CKD) and increases with progressing disease. Cortical porosity is likely a prominent contributor to skeletal fragility/fracture. The degree to which cortical porosity occurs throughout the skeleton is not fully known. In this study, we assessed cortical bone porosity via micro-computed tomography at multiple skeletal sites in rats with progressive chronic kidney disease. We hypothesized that cortical porosity would occur in long bones throughout the body, but to a lesser degree in flat bones and irregular bones. Porosity was measured, using micro-CT, at 17 different skeletal sites in 6 male rats with CKD. Varying degrees of porosity were seen throughout the skeleton with higher porosity in flat and irregular bone (i.e. parietal bone, mandible) vs. long bones (p = 0.01) and in non-weightbearing bones vs. weightbearing bones (p = 0.01). Porosity was also higher in proximal sites vs. distal sites in long bones (p < 0.01 in all comparisons). There was large heterogeneity in porosity within skeletal sites across rats and within the same rat across skeletal sites. Correlations showed cortical porosity of the proximal tibia was positively associated with porosity at the other sites with the strongest correlation to the parietal bone and the weakest to the ulna. Overall, our data demonstrates varying and significant cortical bone porosity across the skeleton of animals with chronic kidney disease. These data point to careful selection of skeletal sites to assess porosity in pre-clinical studies and the potential for fractures at multiple skeletal sites in patients with CKD.

Reversing cortical porosity: Cortical pore infilling in preclinical models of chronic kidney disease.

PURPOSE: Chronic kidney disease (CKD) patients have a high incidence of fracture due in part to cortical porosity. The goal of this study was to study cortical pore infilling utilizing two rodent models of progressive CKD. METHODS: Exp 1: Female C57Bl/6J mice (16-week-old) were given dietary adenine (0.2%) to induce CKD for 10 weeks after which calcium water supplementation (Ca-H2O; 1.5% and 3%) was given to suppress PTH for another 4 weeks. Exp 2: Male Cy/+ rats were aged to ~30 weeks with baseline porosity assessed using in vivo µCT. A second in vivo scan followed 5-weeks of Ca-H2O (3%) supplementation. RESULTS: Exp 1: Untreated adenine mice had elevated blood urea nitrogen (BUN), parathyroid hormone (PTH), and cortical porosity (~2.6% porosity) while Ca-H2O lowered PTH and cortical porosity (0.5-0.8% porosity). Exp 2: Male Cy/+ rats at baseline had variable porosity (0.5%-10%), but after PTH suppression via Ca-H2O, cortical porosity in all rats was lower than 0.5%. Individual pore dynamics measured via a custom MATLAB code demonstrated that 85% of pores infilled while 12% contracted in size. CONCLUSION: Ca-H2O supplementation causes net cortical pore infilling over time and imparted mechanical benefits. While calcium supplementation is not a viable clinical treatment for CKD, these data demonstrate pore infilling is possible and further research is required to examine clinically relevant therapeutics that may cause net pore infilling in CKD.

Age and sex effects on FGF23-mediated response to mild phosphate challenge.

BACKGROUND: During aging, there is a normal and mild loss in kidney function that leads to abnormalities of the kidney-bone metabolic axis. In the setting of increased phosphorus intake, hyperphosphatemia can occur despite increased concentrations of the phosphaturic hormone FGF23. This is likely from decreased expression of the FGF23 co-receptor Klotho (KL) with age; however, the roles of age and sex in the homeostatic responses to mild phosphate challenges remain unclear. METHODS: Male and female 16-week and 78-week mice were placed on either normal grain-based chow or casein (higher bioavailable phosphate) diets for 8 weeks. Gene expression, serum biochemistries, micro-computed tomography, and skeletal mechanics were used to assess the impact of mild phosphate challenge on multiple organ systems. Cell culture of differentiated osteoblast/osteocytes was used to test mechanisms driving key outcomes. RESULTS: Aging female mice responded to phosphate challenge by significantly elevating serum intact FGF23 (iFGF23) versus control diet; males did not show this response. Male mice, regardless of age, exhibited higher kidney KL mRNA with similar phosphate levels across both sexes. However, males and females had similar blood phosphate, calcium, and creatinine levels irrespective of age, suggesting that female mice upregulated FGF23 to maintain blood phosphorus, and compromised renal function could not explain the increased serum iFGF23. The 17ß-estradiol levels were not different between groups, and in vivo bone steroid receptor (estrogen receptor 1 [Esr1], estrogen receptor 2 [Esr2], androgen receptor [Ar]) expression was not different by age, sex, or diet. Trabecular bone volume was higher in males but decreased with both age and phosphate challenge in both sexes. Cortical porosity increased with age in males but not females. In vitro studies demonstrated that 17ß-estradiol treatment upregulated FGF23 and Esr2 mRNAs in a dose-dependent manner. CONCLUSIONS: Our study demonstrates that aging female mice upregulate FGF23 to a greater degree during a mild phosphate challenge to maintain blood phosphorus versus young female and young/old male mice, potentially due to direct estradiol effects on osteocytes. Thus, the control of phosphate intake during aging could have modifiable outcomes for FGF23-related phenotypes.

Erythropoietin and a hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHDi) lowers FGF23 in a model of chronic kidney disease (CKD).

Iron-deficiency anemia is a potent stimulator of the phosphaturic hormone Fibroblast growth factor-23 (FGF23). Anemia, elevated FGF23, and elevated serum phosphate are significant mortality risk factors for patients with chronic kidney disease (CKD). However, the contribution of anemia to overall circulating FGF23 levels in CKD is not understood. Our goal was to investigate the normalization of iron handling in a CKD model using the erythropoiesis stimulating agents (ESAs) Erythropoietin (EPO) and the hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHDi) FG-4592, on the production of, and outcomes associated with, changes in bioactive, intact FGF23 ("iFGF23"). Our hypothesis was that rescuing the prevailing anemia in a model of CKD would reduce circulating FGF23. Wild-type mice were fed an adenine-containing diet to induce CKD, then injected with EPO or FG-4592. The mice with CKD were anemic, and EPO improved red blood cell indices, whereas FG-4592 increased serum EPO and bone marrow erythroferrone (Erfe), and decreased liver ferritin, bone morphogenic protein-6 (Bmp-6), and hepcidin mRNAs. In the mice with CKD, iFGF23 was markedly elevated in control mice but was attenuated by >70% after delivery of either ESA, with no changes in serum phosphate. ESA treatment also reduced renal fibrosis markers, as well as increased Cyp27b1 and reduced Cyp24a1 mRNA expression. Thus, improvement of iron utilization in a CKD model using EPO and a HIF-PHDi significantly reduced iFGF23, demonstrating that anemia is a primary driver of FGF23, and that management of iron utilization in patients with CKD may translate to modifiable outcomes in mineral metabolism.

Distinct Immunophenotypes of T Cells in Bronchoalveolar Lavage Fluid From Leukemia Patients With Immune Checkpoint Inhibitors-Related Pulmonary Complications.

Patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) treated with immune checkpoint inhibitors (ICIs) are at risk of pneumonitis as well as pneumonia (combined henceforth as ICI-related pulmonary complications). Little is known about the cellular and molecular mechanisms underlying ICI-related pulmonary complications. We characterized lymphocytes from bronchoalveolar lavage (BAL) fluid and peripheral blood from seven AML/MDS patients with pulmonary symptoms after ICI-based therapy (ICI group) and four ICI-naïve AML/MDS patients with extracellular bacterial or fungal pneumonias (controls). BAL T cells in the ICI group were clonally expanded, and BAL IFNγ+ IL-17- CD8+ T and CXCR3+ CCR6+ Th17/Th1 cells were enriched in the ICI group. Our data suggest that these cells may play a critical role in the pathophysiology of ICI-related pulmonary complications. Understanding of these cell populations may also provide predictive and diagnostic biomarkers of ICI-related pulmonary complications, eventually enabling differentiation of pneumonitis from pneumonia in AML/MDS patients receiving ICI-based therapies.

Spatially resolved analyses link genomic and immune diversity and reveal unfavorable neutrophil activation in melanoma.

Complex tumor microenvironmental (TME) features influence the outcome of cancer immunotherapy (IO). Here we perform immunogenomic analyses on 67 intratumor sub-regions of a PD-1 inhibitor-resistant melanoma tumor and 2 additional metastases arising over 8 years, to characterize TME interactions. We identify spatially distinct evolution of copy number alterations influencing local immune composition. Sub-regions with chromosome 7 gain display a relative lack of leukocyte infiltrate but evidence of neutrophil activation, recapitulated in The Cancer Genome Atlas (TCGA) samples, and associated with lack of response to IO across three clinical cohorts. Whether neutrophil activation represents cause or consequence of local tumor necrosis requires further study. Analyses of T-cell clonotypes reveal the presence of recurrent priming events manifesting in a dominant T-cell clonotype over many years. Our findings highlight the links between marked levels of genomic and immune heterogeneity within the physical space of a tumor, with implications for biomarker evaluation and immunotherapy response.

Comprehensive T cell repertoire characterization of non-small cell lung cancer.

Immunotherapy targeting T cells is increasingly utilized to treat solid tumors including non-small cell lung cancer (NSCLC). This requires a better understanding of the T cells in the lungs of patients with NSCLC. Here, we report T cell repertoire analysis in a cohort of 236 early-stage NSCLC patients. T cell repertoire attributes are associated with clinicopathologic features, mutational and immune landscape. A considerable proportion of the most prevalent T cells in tumors are also prevalent in the uninvolved tumor-adjacent lungs and appear specific to shared background mutations or viral infections. Patients with higher T cell repertoire homology between the tumor and uninvolved tumor-adjacent lung, suggesting a less tumor-focused T cell response, exhibit inferior survival. These findings indicate that a concise understanding of antigens and T cells in NSCLC is needed to improve therapeutic efficacy and reduce toxicity with immunotherapy, particularly adoptive T cell therapy.

Multi-region exome sequencing reveals genomic evolution from preneoplasia to lung adenocarcinoma.

There has been a dramatic increase in the detection of lung nodules, many of which are preneoplasia atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) or invasive adenocarcinoma (ADC). The molecular landscape and the evolutionary trajectory of lung preneoplasia have not been well defined. Here, we perform multi-region exome sequencing of 116 resected lung nodules including AAH (n = 22), AIS (n = 27), MIA (n = 54) and synchronous ADC (n = 13). Comparing AAH to AIS, MIA and ADC, we observe progressive genomic evolution at the single nucleotide level and demarcated evolution at the chromosomal level supporting the early lung carcinogenesis model from AAH to AIS, MIA and ADC. Subclonal analyses reveal a higher proportion of clonal mutations in AIS/MIA/ADC than AAH suggesting neoplastic transformation of lung preneoplasia is predominantly associated with a selective sweep of unfit subclones. Analysis of multifocal pulmonary nodules from the same patients reveal evidence of convergent evolution.

KMT2D/MLL2 inactivation is associated with recurrence in adult-type granulosa cell tumors of the ovary.

Adult-type granulosa cell tumors of the ovary (aGCTs) are rare gynecologic malignancies that exhibit a high frequency of somatic FOXL2 c.C402G (p.Cys134Trp) mutation. Treatment of relapsed aGCT remains a significant clinical challenge. Here we show, using whole-exome and cancer gene panel sequencing of 79 aGCTs from two independent cohorts, that truncating mutation of the histone lysine methyltransferase gene KMT2D (also known as MLL2) is a recurrent somatic event in aGCT. Mono-allelic KMT2D-truncating mutations are more frequent in recurrent (10/44, 23%) compared with primary (1/35, 3%) aGCTs (p = 0.02, two-sided Fisher's exact test). IHC detects additional non-KMT2D-mutated aGCTs with loss of nuclear KMT2D expression, suggesting that non-genetic KMT2D inactivation may occur in this tumor type. These findings identify KMT2D inactivation as a novel driver event in aGCTs and suggest that mutation of this gene may increase the risk of disease recurrence.

Integrative genomic analysis of adult mixed phenotype acute leukemia delineates lineage associated molecular subtypes.

Mixed phenotype acute leukemia (MPAL) is a rare subtype of acute leukemia characterized by leukemic blasts presenting myeloid and lymphoid markers. Here we report data from integrated genomic analysis on 31 MPAL samples and compare molecular profiling with that from acute myeloid leukemia (AML), B cell acute lymphoblastic leukemia (B-ALL), and T cell acute lymphoblastic leukemia (T-ALL). Consistent with the mixed immunophenotype, both AML-type and ALL-type mutations are detected in MPAL. Myeloid-B and myeloid-T MPAL show distinct mutation and methylation signatures that are associated with differences in lineage-commitment gene expressions. Genome-wide methylation comparison among MPAL, AML, B-ALL, and T-ALL sub-classifies MPAL into AML-type and ALL-type MPAL, which is associated with better clinical response when lineage-matched therapy is given. These results elucidate the genetic and epigenetic heterogeneity of MPAL and its genetic distinction from AML, B-ALL, and T-ALL and further provide proof of concept for a molecularly guided precision therapy approach in MPAL.

Clearance of Somatic Mutations at Remission and the Risk of Relapse in Acute Myeloid Leukemia.

Purpose The aim of the current study was to determine whether the degree of mutation clearance at remission predicts the risk of relapse in patients with acute myeloid leukemia (AML). Patients and Methods One hundred thirty-one previously untreated patients with AML who received intensive induction chemotherapy and attained morphologic complete remission (CR) at day 30 were studied. Pretreatment and CR bone marrow were analyzed using targeted capture DNA sequencing. We analyzed the association between mutation clearance (MC) on the basis of variant allele frequency (VAF) at CR (MC2.5: if the VAF of residual mutations was < 2.5%; MC1.0: if the VAF was < 1%; and complete MC [CMC]: if no detectable residual mutations) and event-free survival, overall survival (OS), and cumulative incidence of relapse (CIR). Results MC1.0 and CMC were associated with significantly better OS (2-year OS: 75% v 61% in MC1.0 v non-MC1.0; P = .0465; 2-year OS: 77% v 60% in CMC v non-CMC; P = .0303) and lower CIR (2-year CIR: 26% v 46% in MC1.0 v non-MC 1.0; P = .0349; 2 year-CIR: 24% v 46% in CMC v non-CMC; P = .03), whereas there was no significant difference in any of the above outcomes by MC2.5. Multivariable analysis adjusting for age, cytogenetic risk, allogeneic stem-cell transplantation, and flow cytometry-based minimal residual disease revealed that patients with CMC had significantly better event-free survival (hazard ratio [HR], 0.43; P = .0083), OS (HR, 0.47; P = .04), and CIR (HR, 0.27; P < .001) than did patients without CMC. These prognostic associations were stronger when preleukemic mutations, such as DNMT3A, TET2, and ASXL1, were removed from the analysis. Conclusion Clearance of somatic mutation at CR, particularly in nonpreleukemic genes, was associated with significantly better survival and less risk of relapse. Somatic mutations in nonpreleukemic genes may function as a molecular minimal residual disease marker in AML.

Copy number alterations detected as clonal hematopoiesis of indeterminate potential.

Recent studies have revealed that clonal hematopoiesis of indeterminate potential (CHIP) is an important risk factor for therapy-related myeloid neoplasms (t-MNs). CHIP is currently defined as a clonal hematopoietic population carrying somatic point mutations in 1 of the leukemia-associated genes. Patients with t-MNs often present with chromosomal abnormalities in addition to somatic point mutations. It remains unclear whether chromosomal abnormalities can cooccur with point mutations as part of CHIP. Here we report that 3 of 14 patients with t-MNs had low amplitude but detectable chromosome arm-level copy number alterations (CNAs) in the peripheral blood samples that were taken at the time of their primary cancer diagnosis and before exposure to therapy. These CNAs were the same CNAs seen in t-MN bone marrow samples and affected the same allele, suggesting the same clonal origin. These data suggest that not only somatic point mutations but also chromosome arm-level CNAs are detectable as CHIP and preexist before patients' exposure to chemotherapy and/or radiation therapy. These data suggest that screening of both somatic point mutations and CNAs might allow more complete ascertainment of CHIP.