Adam Sperling

Heterogeneity of genomic evolution and mutational profiles in multiple myeloma

Multiple myeloma is an incurable plasma cell malignancy with a complex and incompletely understood molecular pathogenesis. Here we use whole-exome sequencing, copy-number profiling and cytogenetics to analyse 84 myeloma samples. Most cases have a complex subclonal structure and show clusters of subclonal variants, including subclonal driver mutations. Serial sampling reveals diverse patterns of clonal evolution, including linear evolution, differential clonal response and branching evolution. Diverse processes contribute to the mutational repertoire, including kataegis and somatic hypermutation, and their relative contribution changes over time. We find heterogeneity of mutational spectrum across samples, with few recurrent genes. We identify new candidate genes, including truncations of SP140, LTB, ROBO1 and clustered missense mutations in EGR1. The myeloma genome is heterogeneous across the cohort, and exhibits diversity in clonal admixture and in dynamics of evolution, which may impact prognostic stratification, therapeutic approaches and assessment of disease response to treatment.

The genetics of myelodysplastic syndrome: from clonal haematopoiesis to secondary leukaemia

Myelodysplastic syndrome (MDS) is a clonal disease that arises from the expansion of mutated haematopoietic stem cells. In a spectrum of myeloid disorders ranging from clonal haematopoiesis of indeterminate potential (CHIP) to secondary acute myeloid leukaemia (sAML), MDS is distinguished by the presence of peripheral blood cytopenias, dysplastic haematopoietic differentiation and the absence of features that define acute leukaemia. More than 50 recurrently mutated genes are involved in the pathogenesis of MDS, including genes that encode proteins involved in pre-mRNA splicing, epigenetic regulation and transcription. In this Review we discuss the molecular processes that lead to CHIP and further clonal evolution to MDS and sAML. We also highlight the ways in which these insights are shaping the clinical management of MDS, including classification schemata, prognostic scoring systems and therapeutic approaches.

Differential and limited expression of mutant alleles in multiple myeloma

Recent work has delineated mutational profiles in multiple myeloma and reported a median of 52 mutations per patient, as well as a set of commonly mutated genes across multiple patients. In this study, we have used deep sequencing of RNA from a subset of these patients to evaluate the proportion of expressed mutations. We find that the majority of previously identified mutations occur within genes with very low or no detectable expression. On average, 27% (range, 11% to 47%) of mutated alleles are found to be expressed, and among mutated genes that are expressed, there often is allele-specific expression where either the mutant or wild-type allele is suppressed. Even in the absence of an overall change in gene expression, the presence of differential allelic expression within malignant cells highlights the important contribution of RNA-sequencing in identifying clinically significant mutational changes relevant to our understanding of myeloma biology and also for therapeutic applications.

A DNA target-enrichment approach to detect mutations, copy number changes and immunoglobulin translocations in multiple myeloma

Genomic lesions are not investigated during routine diagnostic workup for multiple myeloma (MM). Cytogenetic studies are performed to assess prognosis but with limited impact on therapeutic decisions. Recently, several recurrently mutated genes have been described, but their clinical value remains to be defined. Therefore, clinical-grade strategies to investigate the genomic landscape of myeloma samples are needed to integrate new and old prognostic markers. We developed a target-enrichment strategy followed by next-generation sequencing (NGS) to streamline simultaneous analysis of gene mutations, copy number changes and immunoglobulin heavy chain (IGH) translocations in MM in a high-throughput manner, and validated it in a panel of cell lines. We identified 548 likely oncogenic mutations in 182 genes. By integrating published data sets of NGS in MM, we retrieved a list of genes with significant relevance to myeloma and found that the mutational spectrum of primary samples and MM cell lines is partially overlapping. Gains and losses of chromosomes, chromosomal segments and gene loci were identified with accuracy comparable to conventional arrays, allowing identification of lesions with known prognostic significance. Furthermore, we identified IGH translocations with high positive and negative predictive value. Our approach could allow the identification of novel biomarkers with clinical relevance in myeloma.

Widespread intronic polyadenylation diversifies immune cell transcriptomes

Alternative cleavage and polyadenylation (ApA) is known to alter untranslated region (3ʹUTR) length but can also recognize intronic polyadenylation (IpA) signals to generate transcripts that lose part or all of the coding region. We analyzed 46 3ʹ-seq and RNA-seq profiles from normal human tissues, primary immune cells, and multiple myeloma (MM) samples and created an atlas of 4927 high-confidence IpA events represented in these cell types. IpA isoforms are widely expressed in immune cells, differentially used during B-cell development or in different cellular environments, and can generate truncated proteins lacking C-terminal functional domains. This can mimic ectodomain shedding through loss of transmembrane domains or alter the binding specificity of proteins with DNA-binding or protein–protein interaction domains. MM cells display a striking loss of IpA isoforms expressed in plasma cells, associated with shorter progression-free survival and impacting key genes in MM biology and response to lenalidomide.Recognition of intronic polyadenylation (IpA) signals can lead to expression of truncated proteins lacking C terminal domains. Analysis of 3ʹ -seq and RNA-seq shows that IpA is widespread in circulating immune cells, while multiple myeloma cells show loss of IpA isoforms that are normally expressed in plasma cells, impacting key genes in the disease.

A Phase I Study of Lenalidomide plus Chemotherapy with Mitoxantrone, Etoposide, and Cytarabine for the Reinduction of Patients with Acute Myeloid Leukemia

Patients with relapsed AML have a poor prognosis and limited responses to standard chemotherapy. Lenalidomide is an immunomodulatory drug that may modulate anti‐tumor immunity. We performed a study to evaluate the safety and tolerability of lenalidomide with mitoxantrone, etoposide and cytarabine (MEC) in relapsed/refractory AML. Adult patients with relapsed/refractory AML were eligible for this phase I dose‐escalation study. We enrolled 35 patients using a “3 + 3” design, with a 10 patient expansion cohort at the maximum tolerated dose (MTD). Lenalidomide was initially given days 1‐14 and MEC days 4‐8; due to delayed count recovery, the protocol was amended to administer lenalidomide days 1‐10. The dose of lenalidomide was then escalated starting at 5 mg/d (5‐10‐25‐50). The primary objective was tolerability and MTD determination, with secondary outcomes including overall survival (OS). The MTD of lenalidomide combined with MEC was 50 mg/d days 1‐10. Among the 35 enrolled patients, 12 achieved complete remission (CR) (34%, 90%CI 21‐50%); 30‐day mortality was 6% and 60‐day mortality 13%. The median OS for all patients was 11.5 months. Among 17 patients treated at the MTD, 7 attained CR (41%); the median OS was not reached while 12‐month OS was 61%. Following therapy with MEC and lenalidomide, patient CD4+ and CD8+ T‐cells demonstrated increased inflammatory responses to autologous tumor lysate. The combination of MEC and lenalidomide is tolerable with an RP2D of lenalidomide 50 mg/d days 1‐10, yielding encouraging response rates. Further studies are planned to explore the potential immunomodulatory effect of lenalidomide and MEC.

Factoring in the missing link

A 51-year-old woman with a history of uterine fibroids presented with 2 weeks of persistent heavy vaginal bleeding. These symptoms developed in the setting of irregular menstrual cycles over the previous year. After 2 months without menstruation, she noted heavy, painless vaginal bleeding. She reported no abdominal pain, dysuria, nausea, vomiting, vaginal discharge, fever, chills, hot flashes, or night sweats. Menorrhagia can be related to uterine fibroids, bleeding diatheses (most commonly von Willebrand disease), hypothyroidism, or advanced liver disease. Intermenstrual causes of vaginal bleeding include pregnancy, perimenopause, medications, intrauterine devices, infections (e.g., pelvic inflammatory disease), or structural abnormalities, the most concerning of which is malignancy. The presence of uterine fibroids could account for vaginal bleeding, but the persistence and excessive volume suggests an additional process. Endometrial cancer occurs in a significant number of perimenopausal and postmenopausal women, often presenting as irregular painless vaginal bleeding. Rarely, uterine fibroids undergo sarcomatous degeneration, which may present as vaginal bleeding. Past medical history included hypertension, nephrolithiasis, hyperlipidemia, and cholecystitis for which she underwent an uncomplicated cholecystectomy. Her Papanicolaou smears were normal and up-to-date. She had no history of sexually transmitted diseases. Her medications included metoprolol, iron supplementation, vitamin D, and multivitamins. She worked as a nurse in a methadone clinic. She was in a monogamous relationship with her husband and had three uncomplicated pregnancies. She never smoked tobacco or used illicit drugs and did not regularly consume alcohol. The absence of prolonged bleeding during her prior cholecystectomy decreases the likelihood of an inherited bleeding disorder, which would be expected to manifest itself earlier. Her medications are not associated with an increased risk of bleeding. Occupational hazards for individuals within the health profession can include substance abuse and self-inflicted disease (e.g., surreptitious use of warfarin). Certain herbal supplements such as ginkgo can alter estrogen levels or clotting factors leading to irregular menstrual cycles. On physical examination, the patient appeared well and in no distress. She was afebrile, with a pulse of 88 beats per minute, blood pressure 110/66 mmHg, and respiratory rate 18 breathes per minute. There was no conjunctival pallor. No rash, lymphadenopathy, or hepatosplenomegaly was detected. The cardiac, abdominal, and lung exams were normal. Soft tissue bruising and petechiae were noted on her upper extremities. There was moderate bilateral pitting edema of the lower extremities to the mid thighs. She had normal appearing external female genitalia. There was clotted blood visualized within the vaginal vault without evidence of active bleeding. No lesions or purulent discharge was observed in the vulva, vagina, or cervix. No cervical motion tenderness or pelvic mass was appreciated. Bilateral lower extremity edema may result from increased intravascular hydrostatic pressure as seen in volume overload conditions or decreased intravascular oncotic pressure related to hypoalbuminemic states. The patient lacked evidence of volume overload. Hypoalbuminemia can result from malnutrition, defective protein synthesis, or excessive loss from protein-losing enteropathies or nephropathies. The patient did not have physical exam findings consistent with advanced cirrhosis. A protein-losing enteropathy is less likely given the absence of diarrhea. Protein-losing renal disease remains a possibility as it cannot be easily assessed by history or physical exam. Bilateral lower extremity edema can be caused by hypothyroidism and medications (e.g., amlodipine). Polycystic ovarian syndrome is unlikely in the absence of obesity, hirsutism, or acne. An adnexal mass was not detected, decreasing the possibility of an ovarian tumor, ectopic pregnancy, or cyst. The absence of pelvic pain, fever, and cervical motion tenderness would argue against pelvic inflammatory disease. The urine human chorionic gonadotropin assay was negative. Electrolyte and aminotransferase levels were normal. The white blood cell count (WBC) was 9,600 per cubic millimeter, hematocrit 24.3%, mean corpuscular volume 86 femtoliters (normal range, 80–95), and the platelet count 441,000 per cubic millimeter. The prothrombin time (PT) was 34 sec (normal range, 12–14.4), international normalized ratio 3.5 (normal range, 0.8–1.2), and partial thromboplastin time (PTT) 58 sec (normal range, 23.8–36.6). Albumin was 2.3 grams per deciliter, total protein 4.3 grams per deciliter (normal range, 6–8.3), and globulin 2 g per deciliter (normal range, 2.3–3.5). Urinalysis revealed 31 protein, 1 WBC, and 28 red blood cells. Twenty-four hours urine protein and creatinine was 12,012 mg (normal value, <150 mg per day) and 926 mg (normal range, 8–170 mg per day), respectively. Fibrinogen level was 434 mg per deciliter (normal range, 200–400). A chest x-ray was clear without evidence of pleural effusions.