Racial Disparities in the Diagnostic Evaluation of Multiple Myeloma

Introduction: Multiple myeloma (MM) is the most common hematologic malignancy in Black individuals with a 2-to-3-fold higher incidence rate among Black compared to White individuals. While therapeutic advances have led to significant increases in survival rates in MM across races, there still exist racial disparities in outcomes that have been attributed to inferior access to novel therapies and autologous stem cell transplant among Black patients. Risk stratification is an important strategy that allows clinicians to identify high-risk disease and potentially tailor therapy based on staging to try to abrogate its poor prognosis. Current risk stratification schemata in MM, such as the International Staging System (ISS) and Revised ISS (R-ISS), necessitate serum laboratory data and fluorescence in-situ hybridization (FISH) cytogenetic analysis of bone marrow specimens. To the best of our knowledge, it is unknown if discrepancies exist in the initial diagnostic workup of MM between Blacks and Whites, which ultimately may have treatment and outcome implications. We sought to assess the presence of racial disparities in the diagnostic workup of newly diagnosed MM among Black and White patients. Methods: We performed a retrospective cohort study using the Surveillance, Epidemiology and End Results (SEER)-Medicare linked database, which includes 16,174 MM patients with patient-level demographics, survival data, and health care claims information. The data included patients ≥ 65 years-old with the diagnosis of MM between 2001-2015. Race was documented by SEER registries. Lab and imaging data were collected from 180 days before and after diagnosis to capture an ample and appropriate allotted time for diagnostic workup. CPT codes were queried to determine the frequency of the diagnostic tests of interest. Data were analyzed through R version 4.0.2. Pearson chi-squared tests were used to compare frequency of diagnostic testing between racial groups. Results: A total of 18,267 MM patients were identified in the SEER-Medicare linked database. Of that, 15,360 MM patients (12,645 White and 2,715 Black) were identified with peripheral blood laboratory, bone marrow, and imaging health care claims data available . The remaining 2,907 patients with a documented race other than White or Black were excluded. Complete blood count and comprehensive metabolic panel serum tests were used to evaluate completeness of CPT codes use, which demonstrated that >89% (13,723/15,360) of individuals had both tests performed. Overall, Black patients had lower frequency of nearly all serum and imaging tests completed relative to White patients (Table 1). Only 61% of White patients underwent the testing components necessary to adequately risk-stratify disease by ISS (e.g., beta-2 microglobulin) compared to 50% of Black patients (relative difference 21%). 30% of White individuals underwent the components to determine R-ISS (e.g., FISH cytogenetics). There were low overall rates of FISH cytogenetics obtained in the study population, with White individuals undergoing FISH cytogenetics at a rate of 30% compared to 25% among Black individuals (relative difference 18%). Magnetic resonance imaging (MRI) of the lumbar spine, the most commonly imaged portion of the spine by MRI, was performed more commonly in White vs Black individuals (33% vs 24%, relative difference 35%). PET/CT was performed in only a small percentage of patients (Whites 9% vs Blacks 5%, relative difference 94%). Conclusions: In a real-world analysis of patients with newly diagnosed MM, we found that Black patients were less likely than White patients to undergo a complete initial diagnostic evaluation. While rates of beta-2 microglobulin and FISH cytogenetics testing were low among all patients, Black patients were less likely to undergo testing needed to complete staging for ISS/R-ISS or proper imaging to assess for extramedullary disease (i.e., PET/CT). Whether these differences between the races in the initial diagnostic workup lead to differences in treatment strategies and survival outcomes deserves additional investigation. Further work is needed to improve access to complete diagnostic evaluation among Black patients with newly diagnosed MM. Figure 1 Figure 1. Disclosures Calip: Pfizer: Research Funding; Roche: Current equity holder in publicly-traded company; Flatiron Health: Current Employment. Derman: Sanofi: Membership on an entity's Board of Directors or advisory committees.

Fish Cytogenetics: Present and Future

Fish is the most species-rich class of vertebrates, including a number of species that correspond to about half of the total vertebrates [...]

Quantitative Approach to Fish Cytogenetics in the Context of Vertebrate Genome Evolution

Our novel Python-based tool EVANGELIST allows the visualization of GC and repeats percentages along chromosomes in sequenced genomes and has enabled us to perform quantitative large-scale analyses on the chromosome level in fish and other vertebrates. This is a different approach from the prevailing analyses, i.e., analyses of GC% in the coding sequences that make up not more than 2% in human. We identified GC content (GC%) elevations in microchromosomes in ancient fish lineages similar to avian microchromosomes and a large variability in the relationship between the chromosome size and their GC% across fish lineages. This raises the question as to what extent does the chromosome size drive GC% as posited by the currently accepted explanation based on the recombination rate. We ascribe the differences found across fishes to varying GC% of repetitive sequences. Generally, our results suggest that the GC% of repeats and proportion of repeats are independent of the chromosome size. This leaves an open space for another mechanism driving the GC evolution in vertebrates.

GC and Repeats Profiling along Chromosomes—The Future of Fish Compositional Cytogenomics

The study of fish cytogenetics has been impeded by the inability to produce G-bands that could assign chromosomes to their homologous pairs. Thus, the majority of karyotypes published have been estimated based on morphological similarities of chromosomes. The reason why chromosome G-banding does not work in fish remains elusive. However, the recent increase in the number of fish genomes assembled to the chromosome level provides a way to analyse this issue. We have developed a Python tool to visualize and quantify GC percentage (GC%) of both repeats and unique DNA along chromosomes using a non-overlapping sliding window approach. Our tool profiles GC% and simultaneously plots the proportion of repeats (rep%) in a color scale (or vice versa). Hence, it is possible to assess the contribution of repeats to the total GC%. The main differences are the GC% of repeats homogenizing the overall GC% along fish chromosomes and a greater range of GC% scattered along fish chromosomes. This may explain the inability to produce G-banding in fish. We also show an occasional banding pattern along the chromosomes in some fish that probably cannot be detected with traditional qualitative cytogenetic methods.

Clinical-Grade Whole Genome Sequencing Reproduces FISH Cytogenetics and Provides Actionable Data in Newly Diagnosed Myeloma - a Pilot Study from the UK 100,000 Genomes Project

Background Treatment decisions in Multiple Myeloma (MM) have been driven by a patient's age or ability to tolerate therapy, but as yet, not by their tumour genetics, even though understanding of the prognostic utility of genetic features continues to develop. The ability to identify genetic prognostic markers and potential therapeutic targets in a timely fashion within a health service is a vital step in delivering precision medicine to patients. The aims of this study were to assess the implementation of clinical grade whole genome sequencing (WGS) data at diagnosis to replicate, and ultimately replace, standard-of-care Fluorescence In Situ Hybridisation (FISH) cytogenetics; to provide markers for prognostication or MRD; and to identify actionable targets for clinical trials in precision therapy of MM. Methods Bone marrow aspirates from patients with newly-diagnosed myeloma were collected between June 2017 and April 2018 in a single tertiary hospital in the United Kingdom. The population comprised seven male and seven female patients with a mean age of 78 years. From the first-draw of bone marrow aspirate, standard-of-care FISH cytogenetic analyses were performed locally according to criteria from the International Myeloma Working Group (IMWG). From the remnant aspirate samples, CD138-positive plasma cells were enriched by magnetic bead-sorting and genomic DNA was extracted locally for WGS with a success rate of approximately 70%. Fourteen samples underwent successful plasma cell purification (78 - 99% morphological purity) with a yield of at least 0.5 μg DNA. To identify germline genomic variants, DNA was extracted from peripheral blood samples obtained simultaneously. WGS was performed at a centralised facility and mean coverage for germline samples was 35.1x and for plasma cell-enriched samples was 100.9x. Conventional cytogenetic FISH data were compared with genomic data for chromosome-level alterations. Identified somatic variants were automatically cross-referenced against publicly available databases that describe somatic mutations in cancer as well as a virtual panel of potentially actionable therapeutic targets including : NRAS, KRAS, BRAF, CDKN2C, FGFR3 and IDH2. Results In paired samples, WGS replicated all 13 translocation and chromosomal loss/gain events identified by FISH (Figure 1). Furthermore, three translocations involving the IGH locus suggested by FISH analysis were characterised by WGS. Using samples derived from surplus material, fast-track turnaround of 14 days was attainable. Five patients had no identifiable marker by FISH cytogenetics. In these patients, WGS found all five to possess somatic variants could be used as prognostic or potential Measurable Residual Disease (MRD) markers. Nine patients exhibited somatic variants in genes that may be subject to targetable therapy as determined by trials available on ClinicalTrials.gov: five NRAS, two KRAS, one BRAF and one FGFR3 mutation as of July 2019. Conclusion WGS assessment of newly diagnosed myeloma provides accurate, timely and actionable information beyond what is available from standard-of-care FISH. The application of WGS to myeloma diagnostics presents a number of advantages. Firstly, WGS can replicate and exceed existing myeloma FISH assessment of translocation and loss/gain events in a prompt turnaround time. Secondly, germline variants are deducted from tumour variants to provide a gold standard description of the somatic mutational landscape in the patient's disease. Thirdly, virtual panels of known somatic variants can be applied to the data as knowledge accumulates about the role of specific mutations on prognosis or therapeutic response. We demonstrate that centrally provided WGS and its analysis can be incorporated into routine local assessment of newly diagnosed myeloma. Therefore, these observations show that such technology has the potential to be rapidly scalable across existing hospital networks. Disclosures Gooding: Celgene Corporation: Research Funding. Ramasamy:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Grants; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Grants; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Grants; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Grants; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Impact of Clinical Versus Biochemical Progression on Post-Progression Survival in Multiple Myeloma

Background: Overall response rate and response duration in newly diagnosed multiple myeloma (ndMM) has increased in the last decade. However, majority of patients eventually progress and receive multiple lines of therapy. Progression in MM is defined by rise in monoclonal protein and/or clinical manifestations of end-organ damage. However, there is a lack of evidence on the prognostic implication of pattern of progression in the current era. We have hypothesized that patients with clinical manifestations of end-organ damage at first progression have an inferior post-progression overall survival (OS) compared to those with biochemical progression alone. Method: We evaluated all ndMM patients between 1/1/2008 and 12/31/2015 at the Cleveland Clinic. Key inclusion criterion was patients experiencing first progression requiring an additional line of therapy. Patients with primary refractory disease and those in continued 1st remission at latest follow-up were excluded. Progression was categorized into 2 groups: Biochemical Progression (BP) and Clinical Progression (CP; implying CRAB features). Patients with CP were further stratified based on the presence of extramedullary disease (EM): CP+EM and CP-EM. Progression-free survival (PFS) and OS from first progression were estimated with Kaplan-Meier curves and compared among groups with log-rank test. Cox analysis was used to identify prognostic factors for OS and PFS. Potential prognostic factors included progression pattern, age, gender, race, ISS stage at diagnosis, FISH cytogenetics at diagnosis, metaphase cytogenetics at diagnosis, time from diagnosis to first progression, best response at first remission, frontline autologous stem cell transplant (ASCT), and whether progression occurred while on therapy. Results: A total of 527 patients with ndMM were evaluated, among which, 257 experiencing 1st progression were included in our analysis. The median age at progression was 64 years. The median time from diagnosis to first progression was 23 months. An autologous stem cell transplantation (ASCT) after induction therapy in 1st remission was performed in 26% of patients. At 1st progression, BP alone was noted in 52% (n=134), CP -EM in 34% (n=87) and CP+EM in 14% (n=36) of patients. In the CP-EM group, the most common mode of progression was development of new bone lesions (76%) followed by anemia (33%), renal insufficiency (18%) and hypercalcemia (13%), with ≥1 mode in approximately one-third of patients. In the CP+EM group, the most common mode of EM progression was development of new plasmacytomas (89%), followed by emergence of circulating plasma cells (14%), with 1 patient having both. A total of 84% of patients progressed on anti-myeloma therapy, which reflects the contemporary practice of continuous therapy in MM. After first progression, 68% received proteasome inhibitors (PIs), 64% received immunomodulatory drugs (IMiDs) and 11% received monoclonal antibodies (MoAbs). Salvage ASCT in 2nd remission was performed in 12% of patients. A total of 105 patients (41%) were alive at latest follow-up, with the median follow-up of survivors being 26 months from 1st progression. Median time from diagnosis to 1st progression was shorter in the CP+EM (12 months) compared to CP-EM (25 months) and BP (24 months) groups (P<0.001). There was no significant difference in the progression pattern by depth of 1st remission. The 2-year post-progression OS in BP, CP-EM and CP+EM groups was 81%, 40% and 12% respectively (P<0.001; Figure I). The 2-year post-progression PFS in the respective groups were 35%, 8% and 7% respectively (P<0.001). On multivariable analysis for OS, independent prognostic factors included progression pattern, age at 1st progression and high-risk FISH cytogenetics at diagnosis (Table I). Conclusion: In the era of PIs, IMiDs and MoAbs, the pattern of 1st progression in MM has a strong and independent prognostic impact on post-progression OS. Patients progressing with clinical manifestations of end-organ damage or extramedullary disease have an inferior PFS and OS compared to those progressing with biochemical criteria alone. These results should be further validated in large prospective studies with data on FISH cytogenetics at relapse. It has clinical implication at the individual level and can also inform the design of clinical trials in relapsed MM. Disclosures Majhail: Anthem, Inc.: Consultancy; Incyte: Honoraria; Atara: Honoraria.

Monitoring AML Disease Status with Next Generation Sequencing

Recent advance in next generation sequencing (NGS) have confirmed that AML is a heterogeneous malignancy harboring may many genetic mutations. These mutations have been studied for leukemia genesis, diagnosis and therapeutic targets. Monitoring minimal residual diseases has also been studied recently. We summarized our experience with NGS in morning AML disease status. NGS data during 2014 and 2016 from patient with newly diagnosed or AML and/or AML follow-up patients along with bone marrow biopsy, FISH/cytogenetics, flow cytometric results were reviewed. Targeted sequencing was performed with customized panel (34 genes) on Ion PGM platform from Life Technology Inc. 41 AML patients with complete bone marrow work-up with bone marrow morphology, flow cytometry, FISH/cytogenetics (MFFC) and NGS were collected. At least one sample with complete work-up for each patient was included. Majority of the patients had several studies (2-8 samples). 15 out of 41 (36.6%) has complete remission based on bone marrow morphology, flow cytometry, FISH/cytogenetic studies. No mutations were detected among these 15 patients. 17 patients (41%) showed concordant result with other technologies, i.e. when the patient was in remission based on MFFC, No mutations were detected. When patient had recurrent AML or residual disease, mutations were detected. It worth to point out that 2 patients showed positive mutation without detectable increase in myeloblasts. These 2 patients had relapsed AML within 3 months. Different subclones were detected at different intervals in 1 patient. 2 (0.5%) patients (1 with newly diagnosed AML and 1 with early recurrent AML) showed no detectable mutations. Mutations were detected in 5 patients (12%) with AML remission by MFFC, additional follow-up is need for these patients. The most common mutations included TET2, ASXL1, DNMT3A, RUNX1, IDH1 and TP53. NGS is valuable to assess the AML status despite of heterogeneous genetic abnormalities. Although the NGS results were concordant with bone marrow morphology, FISH/cytogenetics and flow cytometry in most of the cases (87.5%), persistent mutations may be detectable in cases without detectable residual AML by other modalities, which may be associated with minimal residual disease or early relapse, and need further evaluation. Clonal evaluation may occur at molecular level occasionally. Disclosures No relevant conflicts of interest to declare.

CLL: A Prognostic Model Comprising Only Two Biomarkers (IGHV Mutational Status and FISH-Cytogenetics) Separates Patients with Different Prognosis and Simplifies the CLL-IPI.

Background: Survival of pts with chronic lymphocytic leukemia (CLL) ranges from a few years to a normal lifespan. Clinical stages (i.e. Rai, Binet) are the basis for CLL prognostication but do not reflect the complex biology of CLL, which ultimately shapes the disease heterogeneity. Recently, a CLL-International Prognostic Index (CLL-IPI) which includes five clinical and biological variables (i.e. age, IGHV mutational status, del(17p), beta2-microglobulin [B2M] and Rai or Binet stages) and stratifies pts into four different categories has been proposed. This prognostic index is obtained by the sum of the score given to each parameter and includes dichotomized continuous variables. The aim of this study was to determine whether a prognostic model based only on biomarkers could separate CLL patients with different outcomes and simplify the CLL-IPI. Material and Methods: Five hundred twenty-four CLL pts from the Hospital Clínic, Barcelona, in whom information at diagnosis included age, clinical stage (Rai and Binet), IGHV mutational status, B2M, and FISH-cytogenetics were analyzed. For validation purposes a cohort of 417 pts from the Brno Hospital, Czech Republic was used. The two series included patients as seen in clinical practice. Primary endpoints were overall survival (OS) and time to first treatment (TTFT). The internal validity of the models was evaluated using bootstrapping, and the discriminatory value by c-statistics. Double sided P values < .05 were considered significant. Results: First, we confirmed that all five covariates included in the CLL-IPI were independently predictive of OS. Both sets of five covariates (age + B2M + IGHV + FISH + Rai or Binet) were incorporated into the regression models. All four patient subgroups had a significantly different survival (c-statistic: 0.72), although the high- and very-high risk groups overlapped and the number of patients in the very-high-risk group was small. We then evaluated all possible combinations of these five covariates to identify the simplest model with robust discriminatory value. A prognostic model based on IGHV mutational status + FISH [del(17p) and/or del(11q)] separated three subgroups of pts [i.e, good-biomarkers (mutated IGHV + no poor FISH cytogenetics), intermediate-biomarkers (either unmutated IGHV or poor FISH cytogenetics), and poor biomarkers (unmutated IGHV + poor FISH cytogenetics)] with different prognosis (c-statistic: 0.68). In the Barcelona series, the good-biomarkers category identified around 50% of pts from the whole series whose survival did not differ from the general population; patients with intermediate-biomarkers had a projected 10-year survival of 68%. Finally, the poor-biomarkers category captured pts with a projected 10-year survival of 17%. The corresponding TTFTs at 3 years from diagnosis were 16%, 50% and 63%, respectively (Figure). Furthermore, it separated patients with different outcome within clinical stages, notably Binet A or Rai 0, and across all age groups. This model was fully validated in the Brno series. Conclusions: The biomarkers-only CLL prognostic model presented here is based on the two most important CLL biomarkers (i.e. IGHV mutational status and FISH cytogenetics) which are included as a backbone in the CLL-IPI and other CLL prognostic systems. This model is simple, easy to apply and to remember; it separates three groups of pts rather than four; it does not contain continuous variables; it can be applied to younger and older pts, and has clinical implications. This prognostic model could be useful in CLL prognostication either alone or in combination with clinical stages and warrants prospective validation, including in patients receiving targeted therapies. Figure Figure. Disclosures Montserrat: Pharmacyclics: Consultancy; Vivia Biotech: Equity Ownership; Janssen: Honoraria, Other: travel, accommodations, expenses; Gilead: Consultancy, Other: Expert Testimony; Morphosys: Other: Expert Testimony.