Tumor mutational burden and purity adjustment before and after treatment with temozolomide in 27 paired samples of glioblastoma: a prospective study

Treatment of glioblastoma (GBM) remains a challenging task, with limited treatment options, none offering a cure. Immune therapy has proven effective across different cancers with remarkable response rates. Tumor mutational burden (TMB) is a marker of response, but technical and methodological differences in TMB estimates have made a proper assessment and comparison challenging. Here, we analyzed a prospective collection of paired samples from 35 patients with newly diagnosed GBM, all of whom were wild‐type (WT) for isocitrate dehydrogenase, before and after treatment with radiotherapy and temozolomide. Seven patients (20%) had O6‐methylguanine‐DNA methyltransferase‐methylated tumors. Six patients (17%) had two relapse surgeries, and tissue from all three surgeries was collected. We found that accurate evaluation of TMB was confounded by high variability in the cancer cell fraction of relapse samples. To ameliorate this, we developed a model to adjust for tumor purity based on the relative density distribution of variant allele frequencies in each primary–relapse pair. Additionally, we examined the mutation spectra of shared and private mutations. After tumor purity adjustment, we found TMB comparison reliable in tumors with tumor purity between 15% and 40%, resulting in 27/35 patients (77.1%). TMB remained unchanged from 0.65 mutations per megabase (Mb) to 0.67/Mb before and after treatment, respectively. Examination of the mutation spectra revealed a dominance of C > T transitions at CpG sites in both shared and relapse‐private mutations, consistent with cytosine deamination and the clock‐like mutational signature 1. We present and apply a cellularity correction approach that enables more accurate assessment of TMB in paired tumor samples. We did not find a significant increase in TMB after correcting for cancer cell fraction. Our study raises significant concerns when determining TMB. Although a small sample size, corrected TMB can have a clinical significance when stratifying patients to experimental treatment, for example, immune checkpoint therapy.     

Branching clonal evolution patterns predominate mutational landscape in multiple myeloma

Multiple Myeloma (MM) arises from malignant transformation and deregulated proliferation of clonal plasma cells (PCs) harbouring heterogeneous molecular anomalies. The effect of evolving mutations on clone fitness and their cellular prevalence shapes the progressing myeloma genome and impacts clinical outcomes. Although clonal heterogeneity in MM is well established, which subclonal mutations emerge/persist/perish with progression in MM and which of these can be targeted therapeutically remains an open question. In line with this, we have sequenced pairwise whole exomes of 62 MM patients collected at two time points, i.e., at diagnosis and on progression. Somatic variants were called using a novel ensemble approach where a consensus was deduced from four variant callers (Illumina’s Dragen, Strelka2, SomaticSniper and SpeedSeq) and actionable/druggable gene targets were identified. A marked intraclonal heterogeneity was observed. Branching evolution was observed among 72.58% patients, of whom 64.51% had low TMBs (<10) and 61.29% had 2 or more founder clones. The hypermutator patients (with high TMB levels ≥10 to ≤100) showed a significant decrease in their TMBs from diagnosis (median TMB 77.11) to progression (median TMB 31.22). A distinct temporal fall in subclonal driver mutations was identified recurrently across diagnosis to progression e.g., in PABPC1, BRAF, KRAS, CR1, DIS3 and ATM genes in 3 or more patients suggesting such patients could be treated early with target specific drugs like Vemurafenib/Cobimetinib. An analogous rise in driver mutations was observed in KMT2C, FOXD4L1, SP140, NRAS and other genes. A few drivers such as FAT4, IGLL5 and CDKN1A retained consistent distribution patterns at two time points. These findings are clinically relevant and point at consideration of evaluating multi time point subclonal mutational landscapes for designing better risk stratification strategies and tailoring time to time risk adapted combination therapies in future.     

Development of a new risk stratification system for patients with newly diagnosed multiple myeloma using R-ISS and 18F-FDG PET/CT

In multiple myeloma (MM), a high number of focal lesions (FL) detected using positron emission tomography/computed tomography (PET/CT) was found to be associated with adverse prognosis. To design a new risk stratification system that combines the Revised International Staging System (R-ISS) with FL, we analyzed the data of 380 patients with newly diagnosed MM (NDMM) who underwent ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) PET/CT upon diagnosis. The K-adaptive partitioning algorithm was adopted to define subgroups with homogeneous survival. The combined R-ISS with PET/CT classified NDMM patients into four groups: R-ISS/PET stage I (n = 31; R-ISS I with FL ≤ 3), stage II (n = 156; R-ISS I with FL > 3 and R-ISS II with FL ≤ 3), stage III (n = 162; R-ISS II with FL > 3 and R-ISS III with FL ≤ 3), and stage IV (n = 31; R-ISS III with FL > 3). The 2-year overall survival rates for stages I, II, III, and IV were 96.7%, 89.8%, 74.7%, and 50.3%. The 2-year progression-free survival rates were 84.1%, 64.7%, 40.8%, and 17.1%, respectively. The new R-ISS/PET was successfully validated in an external cohort. This new system had a remarkable prognostic power for estimating the survival outcomes of patients with NDMM. This system helps discriminate patients with a good prognosis from those with a poor prognosis more precisely.Subject terms: Myeloma, Risk factors     

Clinical and prognostic significance of bone marrow abnormalities in the appendicular skeleton detected by low-dose whole-body multidetector computed tomography in patients with multiple myeloma

Clinical significance of medullary abnormalities in the appendicular skeleton (AS) detected by low-dose whole-body multidetector computed tomography (MDCT) in patients with multiple myeloma (MM) was investigated. A total of 172 patients with monoclonal gammopathy of undetermined significance (MGUS) (n=17), smoldering MM (n=47) and symptomatic MM (n=108) underwent low-dose MDCT. CT values (CTv) of medullary density of AS⩾0 Hounsfield unit (HU) was considered as abnormal. Percentage of medullary abnormalities and the mean CTv of AS in patients with MGUS, smoldering MM and symptomatic MM were 18, 55 and 62% and −44.5 , −20.3 and 11.2 HU, respectively (P<0.001 and P<0.001). Disease progression of MM was independently associated with high CTv on multivariate analysis. In symptomatic MM, the presence of abnormal medullary lesions was associated with increased incidence of high-risk cytogenetic abnormalities (34.4% vs 7.7% P=0.002) and extramedullary disease (10.4% vs 0% P=0.032). It was also an independent poor prognostic predictor (hazard ratio 3.546, P=0.04). This study showed that CTv of AS by MDCT is correlated with disease progression of MM, and the presence of abnormal medullary lesions is a predictor for poor survival.     

Serum BCMA levels predict outcomes in MGUS and smoldering myeloma patients

Soluble BCMA (sBCMA) levels are elevated in monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). However, the association between sBCMA levels and prognosis in MGUS and SMM has not been studied. We retrospectively analyzed sBCMA levels in stored samples from 99 MGUS and 184 SMM patients. Baseline sBCMA levels were significantly higher in MGUS and SMM patients progressing to MM during clinical follow up. When stratified according to the median baseline sBCMA level for each cohort, higher levels were associated with a shorter PFS for MGUS (HR 3.44 comparing sBCMA ≥77 vs <77 ng/mL [95% CI 2.07–5.73, p < 0.001] and SMM (HR 2.0 comparing sBCMA ≥128 vs <128 ng/mL, 95% 1.45–2.76, p < 0.001) patients. The effect of sBCMA on PFS was similar even after adjusting for the baseline MGUS or SMM risk stratification. We evaluated paired serum samples and found that sBCMA increased significantly in MGUS and SMM patients who eventually progressed to MM, whereas among MGUS non-progressors the sBCMA level remained stable. While our results require independent validation, they suggest that sBCMA may be a useful biomarker to identify MGUS and SMM patients at increased risk of progression to MM independent of the established risk models.Subject terms: Myeloma, Tumour immunology, Prognosis     

Polyclonal serum IgM level identifies a subgroup of multiple myeloma patients with low-risk clinicobiological features and superior survival

Normal plasma cells (PCs) are either undetectable or outnumbered by the myelomatous PC compartment in bone marrow of multiple myeloma (MM). However, residual normal PCs have been detected in a minority of symptomatic MM patients with superior survival. The number of normal PCs is also an important factor to identify monoclonal gammopathy of undetermined significance (MGUS)-like MM. We speculate that the polyclonal serum IgM level in non-IgM myelomas may reflect the number of residual normal PCs. Here we investigated the prognostic relevance of polyclonal serum IgM level in a series of 485 newly diagnosed symptomatic MM (NDMM) patients. Our results showed that symptomatic MM patients with polyclonal IgM more than 0.5 g/L displayed a favorable baseline clinical feature, together with a significantly lower frequency of high-risk cytogenetic abnormalities. This group of patients had a significantly prolonged progression-free survival (PFS) and overall survival (OS) regardless of thalidomide or bortezomib therapy. Furthermore, the superior outcome was independent of the depth of response. Our findings suggest that polyclonal IgM level is capable of identifying a group of symptomatic MM patients with distinct clinicobiological characteristics and favorable survival, similar with MGUS-like MM.     

Blood-based risk stratification for pre-malignant and symptomatic plasma cell neoplasms to improve patient management

Standard risk stratification (sRisk) guides clinical management in monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM) and multiple myeloma (MM). Nonetheless, clinical results are considerably heterogeneous among patients with similar risk status. Blood and bone marrow samples from 276 MGUS, 56 SMM and 242 MM in regular clinical practice were analyzed at diagnosis by flow cytometry. Higher levels of aberrant circulating plasma cells (cPC) (> 0.0035% of leukocytes), combined with albumin, beta2-microglobuline and lactate-dehydrogenase levels, offered minimally-invasive risk stratification (RcPC) with results comparable to sRisk. RcPC and sRisk 10-year progression-free-survival (10y-PFS) rates were: 93.8% vs. 95.1% for low-risk, 78.4% vs. 81.7% for intermediate-risk and 50.0% vs. 47.8% for high-risk MGUS; 58.3% vs. 57.8% low-risk, 44.4% vs. 45.8% intermediate-risk and 8.9% vs. 15.0% high-risk SMM; and 44.4% vs. 44.4% low-risk, 36.1% vs. 36.8% intermediate-risk, and 13.3% vs. 16.2% high-risk MM. Circulating-PC > 0.0035% vs. cPC<0.0035% was an independent prognostic factor for PFS (HR=4.389, P=1.2×10^-15, Harrell C-statistic =0.7705±0.0190) and over-all survival (OS, HR=4.286, 2.3×10^-9, Harrell C-statistic =0.8225±0.0197) that complemented sRisk in patients with low-sRisk (10y-PFS rates 48.1% vs. 87.3%, P=1.2×10^-8) and intermediate-sRisk (10y-PFS rates 28.9% vs. 74.1%, P=8.6×10^-12). Patients with high cPCs values are associated with higher proliferation and lower apoptosis rates of PC. Circulating-PC > 0.0035% identified MGUS, SMM and MM patients at higher risk of progression or death and predicted a cohort of patients that after relapse from stringent complete response showed shorter OS. These patients could benefit from early consolidation therapy, tandem ASCT or intensive maintenance.     

Serum thymidine kinase and beta-2 microglobulin in monoclonal gammopathies

We evaluated the serum thymidine kinase (TK) and beta-2 microglobulin (beta-2) levels of 22 patients with monoclonal gammopathy of undetermined significance (MGUS) and of 29 patients with multiple myeloma (MM). Both parameters were significantly lower in MGUS than in MM patients and in early (stage I + II) than in advanced (stage III) MM. TK was also lower in MGUS than in stage I MM (p less than 0.025). A seven-fold increase of TK level was documented in one patient who developed a full blown picture of MM 6 years after a diagnosis of MGUS. In 3 patients with stage III MM, a sharp decrease in TK (40-77%) and in beta-2 (29-53%) levels at remission was evident with respect to the levels measured at diagnosis. Patients with high levels of TK or beta-2 had a shorter survival than those with low levels; however, this was statistically significant only for beta-2 levels (p less than 0.02). Serum TK as well as beta-2 levels appear to be of clinical value in monoclonal gammopathies and related to the course of the disease.     

Association of Tumor Mutational Burden With DNA Repair Mutations and Response to Anti–PD-1/PD-L1 Therapy in Non–Small-Cell Lung Cancer

PurposeTo examine clinical predictors of tumor mutational burden (TMB), to explore the association between TMB and DNA repair mutations, and to analyze TMB as a biomarker for response to immune checkpoint blockade in non–small-cell lung cancer.Patients and MethodsTMB scores were determined retrospectively for 72 consecutive patients at our institution with next-generation sequencing comprehensive genomic profiling testing by Foundation Medicine. TMB scores were correlated with a number of clinical variables and presence of DNA repair mutations. Thirty-four patients were treated with anti–programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) therapies, and survival analyses based on TMB score were performed. In addition, tissue immunohistochemical analysis was performed for a subset of patients.ResultsHistory of smoking, but not other clinical variables, including prior treatment lines, stage of disease, and number of metastatic sites, predicted higher TMB score. Higher TMB score was significantly associated with greater number of DNA repair mutations. In the subset of patients treated with immune checkpoint blockade, higher TMB score significantly predicted overall survival, but not progression-free survival (hazard ratio = 0.10, P = .003; hazard ratio 1.1, P = .84, respectively). In a small subset of patients, PD-1/PD-L1 staining did not independently predict progression-free survival or overall survival.ConclusionTissue TMB was significantly associated with smoking history and number of DNA repair mutations. TMB is a promising biomarker for response to anti–PD-1/PD-L1 therapy, with higher TMB score predicting longer overall survival.     

Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways,druggable mutations and mutated epitopes for targeted therapy

We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of “general” immune checkpoint drugs in this subset of patients.     

Improvement in renal function and its impact on survival in patients with newly diagnosed multiple myeloma

Renal impairment (RI) is seen in over a quarter of patients with newly diagnosed multiple myeloma (NDMM). It is not clear if reversal of RI improves the outcome to that expected for NDMM patients without RI. We evaluated 1135 consecutive patients with NDMM seen at the Mayo Clinic between January 2003 and December 2012. RI was defined as having a creatinine clearance (CrCl) <40ml/min. The median overall survival (OS) for patients with RI at diagnosis receiving and not receiving novel agent induction therapy was not reached vs 46 months (P<0.001). The median OS for patients with CrCl ⩾40 ml/min at diagnosis, CrCl <40 ml/min at diagnosis but improved to ⩾40 ml/min and CrCl <40 ml/min at diagnosis and remained <40 ml/min, were 112, 56 and 33 months, respectively (P<0.001). The complete renal response rate for patients with RI at diagnosis receiving novel agent induction therapy compared to the rest was 40 vs 16% (P<0.001). In conclusion, patients with reversal of RI have improved outcomes, but it remains inferior to patients with normal renal function at diagnosis. These results have implications for identifying early treatment strategies for patients at risk of developing renal insufficiency.     

Survival analysis of transplant-eligible newly-diagnosed multiple myeloma patients harboring t(4;14), t(14;16), and/or del(17p) in the real-world setting

Cytogenetic abnormalities (CA) such as t(4;14), t(14;16), and del(17p), are associated with a poor prognosis in Multiple Myeloma (MM) patients. However, there is scarce information regarding the Latin-American population. This study aims to analyze the impact of t(4;14), t(14;16), and del(17p) on the progression-free survival (PFS) and overall survival (OS) of transplant-eligible newly-diagnosed MM (NDMM) patients in Latin America. Retrospective survival analysis based on the Grupo de Estudio Latinoamericano de MM (GELAMM) registry, including all adult patients with NDMM harboring CA t(4;14), t(14;16), and/or del(17p). Fifty-nine patients were included; the median age was 57 years, 55.9% males, 22% ISS-I, 25.4% ISS-II, and 47.5% ISS-III. The majority (89.8%) had one alteration, whereas 10.2% had del(17p) and t(4;14). The frequencies of CA were del(17p) in 61.0%, t(4;14) in 25.4%, and t(14;16) in 3,4%. Autologous stem cell transplantation was performed in 36 cases, 20 patients did not use this consolidative strategy, and this data was missed in three cases.Five-year OS for the entire cohort was 60.8% and 5-year PFS was 28.1%. Bortezomib-based induction regimen (BBR) (p=0.029), consolidation with ASCT (p<0.001), and maintenance therapy (p=0.004) were associated with an improved 5-year OS. In the multivariate analysis, ASCT was the only variable with a positive impact on OS (HR 0.11, 95% CI 0.033 to 0.34, p<0.001). The median PFS presented a non-statistically significant benefit in BBR, ASCT, and maintenance therapy groups. BBR induction, ASCT, and maintenance therapy were associated with improved OS in high-risk NDMM patients.     

伴1q21扩增初治多发性骨髓瘤患者疗效及预后的回顾性分析

目的 探讨1q21扩增（1q21+）与初治多发性骨髓瘤（newly diagnosed multiple myeloma,NDMM）患者的治疗疗效及预后的关系,以及1q21+在MM危险分层中的优势。方法 回顾性分析选择2015年11月至2021年11月于首都医科大学附属北京潞河医院就诊的NDMM患者作为研究对象,采用iFISH技术检测细胞遗传学异常情况,分析1q21+与以硼替佐米为基础的诱导化疗及自体干细胞移植（autologous stem cell transplant,ASCT）疗效及预后的关系,比较MM R2-ISS与R-ISS的分层优势。结果 本研究共纳入159例NDMM患者,1q21+患者79例,非1q21+患者80例,其中41例患者接受RVD方案治疗,118例患者接受非RVD方案治疗。在1q21+的患者中,RVD组的完全缓解（complete remission,CR）率高于非RVD组（P<0.001）;RVD组中,1q21+的CR率低于非1q21+的患者（P=0.004）。多因素分析显示,1q21+是NDMM患者无进展生存期（progression-free su...     

Impact of high tumor mutational burden in solid tumors and challenges for biomarker application

Accurate identification of patients with solid tumors likely to respond to immunotherapy is crucial. Tumor mutational burden (TMB) measures the number of somatic mutations in a tumor and is an emerging prognostic and predictive biomarker for anti-programmed cell death (PD) 1/anti-PD-ligand 1 therapy and other immunotherapeutic agents. Tumor mutational burden is assessed optimally by whole exome sequencing, but next generation sequencing provides TMB estimates in a more timely and cost-effective manner. Blood-based measurement of TMB in plasma offers an alternative to the need for adequate tumor tissue for molecular testing, and has demonstrated the ability to identify patients who derive benefit from immunotherapy. Tumor mutational burden has diverse prognostic impact in different solid tumor types and also has a demonstrated role in predicting improved survival in patients receiving immunotherapy. There are challenges to TMB adoption into standard clinical practice, including variations in its definition, with the mutational number defining TMB-high appearing to vary across cancer types. The magnitude of TMB also varies across different tumor types, with the highest levels reported in melanoma and other skin cancers (where ultraviolet light is the dominant mutational process), followed by non-small cell lung cancer and other squamous carcinomas. Concerns regarding inter-laboratory and inter-platform variations in analysis methods have been raised, highlighting the need for standardization. Integration of other genomic or pathological biomarkers with TMB may increase its prognostic and predictive capabilities and validation of individual or combination models in prospective trials is warranted.     

Clinical features and outcome of multiple myeloma arising from the transformation of a monoclonal gammapathy of undetermined significance.

Patients with a monoclonal gammapathy of undetermined significance (MGUS) are usually submitted to a periodical clinical follow-up, but it is not known if this surveillance can ameliorate the prognosis of a plasma cell malignancy that will be eventually detected. We compared the clinical and laboratory characteristics at onset, the response to chemotherapy and the survival, of 21 cases of newly diagnosed multiple myeloma (MM) arising from the malignant transformation of MGUS and 41 cases without a previous history of MGUS, recruited to the same first-line treatments over a 3-years period. The former group showed a significant lower frequency of advanced stages as well as other several prognostic factors of high risk including anemia, renal failure, bone lesions and increase of beta2 microglobulin and C-reactive protein levels. Despite a similar response to treatment of the two groups, MM arising from MGUS showed a significantly longer median survival than MM without prior MGUS. This was particularly true for stage I, while stages II and III behaved similarly. We conclude that the regular clinical monitoring of MGUS patients allowed the identification of earlier malignant transformation, when tumor burden is lower, as indicated by lower beta2 microglobulin levels and marrow plasmacytosis of stage I MM arising from MGUS. Moreover, a slower proliferation rate of myeloma cells, as suggested by lower C-reactive protein levels, may be considered so as to explain the longer survival of these patients.     

RAS mutations are uncommon in multiple myeloma and other monoclonal gammopathies

Monoclonal gammopathies are a group of diseases characterised by the proliferation of a single clone of plasma cells that produce a homogeneous monoclonal protein (M protein or myeloma protein) that consist of two heavy polypeptide chains of the same class and subclass and two light polypeptide chains of the same type. Multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) are the most common monoclonal gammopathies. Despite advances in systemic and supportive therapies, MM is an incurable hematological malignancy with a median survival of between two and three years. Point mutations in the Ras genes can be detected in a variety of human malignancies, indicating that ras activation represents a widespread oncogenic event. Several studies have analysed the incidence of Ras mutation in MM and MGUS with great differences in their results. To date, the etiopathogenesis of these diseases is still unknown and the relevance of Ras mutation to the clinical and biological behaviour of monoclonal gammopathies remains to be elucidated. In this study, we have analysed K-ras codon 12 and N-ras codon 61 mutations on anti-CD138 sorted bone marrow plasma cell samples of 44 cases of monoclonal gammopathies: 30 MM, 13 MGUS and 1 plasma cell leukaemia, using polymerase chain reaction. No mutations within either codon 12 of K-ras or codon 61 of N-ras have been found in any of the samples. These results indicate that Ras mutations do not play a significant role in the pathogenesis of MM in the Spanish population.