High-risk Fluorescence In Situ Hybridization Research Articles

Predictors and Impact of Timing of Disease Progression Following Primary Therapy in Multiple Myeloma

In multiple myeloma (MM) significant variation in progression-free survival (PFS) and overall survival (OS) is observed. We examined the outcomes of 1557 MM patients stratified into short (<2 years), medium (between 2 and 5 years) and long (>5 years) PFS. Short PFS occurred in 758 patients (48.7%), medium in 561 patients (36.2%), and long in 238 patients (15.3%). Median post-progression PFS was 9.2 months (95% CI: 8.1-11.0) in the short PFS and 33.1 months (95% CI: 29.0-42.1; P < .001) in the long PFS group. Median post-progression OS was 26.6 months (95% CI: 23.9-29.8) in the short PFS and 87.8 months (95% CI: 71.3- NR; P < .001) in the long PFS. Worse survival in the short PFS was irrespective of high risk (HR) fluorescence in situ hybridization (FISH) features, defined as deletion 17p and/or translocation t(4;14), t(14;16), t(14;20). In a multivariable analysis short PFS was associated with HR FISH, extramedullary plasmacytoma, plasma cell labeling index ≥2% at diagnosis, nonimmunoglobulin G isotype, treatment without autologous stem cell transplantation and achieving less than very good partial remission. In conclusion, the duration of the PFS significantly influences survival, regardless of HR cytogenetic features. Therefore, it should be considered an important parameter for risk stratification in patients experiencing a relapse.

Machine Learning Models Predict Molecular Genetic Subtypes of Multiple Myeloma from Whole-Slide Bone Marrow Aspirate Smears

Multiple myeloma (MM) is a plasma cell neoplasm and the second most common hematologic malignancy. Current guidelines recommend multiple myeloma cases undergo karyotyping and fluorescence in situ hybridization (FISH) analysis on bone marrow biopsy samples to assess for specific recurrent genetic abnormalities. Results of this testing help to predict differences in patient prognosis over time and to guide appropriate selection of therapy. Refinements in risk stratification incorporated additional complex molecular testing which, along with karyotyping and FISH, are costly and not available to all patients. Myeloma cell cytomorphologies have been associated in a subset of cases with more aggressive disease, when plasmablastic, and with a subset of t(11;14) cases, when lymphoplasmacytoid. Nonetheless, cellular features as assessed by manual microscopy have not been shown to identify biologic subtypes of disease reproducibly and broadly, such that these features are not a component of current prognostic systems. Whether accurate risk-stratification information can be extracted from microscopic images of myeloma cells through machine-based approaches is uncertain. Thus, in this study, we tested the feasibility of using machine learning models to predict multiple myeloma molecular genetic subtypes from analysis of neoplastic plasma cell morphology on Wright-stained aspirate smears. We first improved upon a previously developed computational pipeline that identifies and classifies hematopoietic cells from scanned whole-slide images (400x) of bone marrow aspirate smears, including classification of plasma cells with 94% accuracy. Using this pipeline, we obtained images from all plasma cells (up to 68,569 plasma cells per slide) from aspirate smears of 18 patients without plasma cell neoplasms and 96 patients with plasma cell neoplasms (including 20 with t(11;14) and 23 with gain(1q); 44 with standard risk FISH and 22 with high risk FISH (t(4;14), t(14;16), del(17p), or &amp;gt;4 copies 1q+)). We then developed convolutional neural network (CNN)-based multi-instance machine learning models to perform patient-level classifications of disease status and molecular genetic classification from image-level analysis of plasma cell cytomorphology. These models demonstrated strong performance for classifying patients without versus with plasma cell neoplasms (AUROC 0.80), and are capable of making patient-level predictions of multiple myeloma molecular genetic subtypes (t(11;14), AUROC 0.73; gain(1q), AUROC 0.71) as well as prediction of FISH risk level (AUROC 0.68). Finally, by assessing cell attention scores and CNN model weights, this machine learning pipeline can assess which individual plasma cells and which specific morphologic features provided the most utility in the prediction of molecular genetic subtypes for individual patients, hence providing explainability for model output. Importantly, our findings suggest that cytomorphologic features of plasma cells on routine aspirate smear slides contain information, extractable through machine-based approaches, that correlates with molecular genetic subtypes employed for risk stratification. With further refinement these promising computational digital pathology models can potentially yield tools not only for use in low resource settings, but also provide a potential basis for development of multimodal models that incorporate and improve upon results of currently used risk stratification tools.

Impact of cytogenetic abnormalities on the risk of disease progression in solitary bone plasmacytomas

AbstractMost patients with solitary bone plasmacytomas (SBP) progress to multiple myeloma (MM) after definitive radiation therapy as their primary treatment. Whether the presence of high-risk (HR) cytogenetic abnormalities by fluorescence in situ hybridization (FISH) in the clonal plasma cells, obtained either directly from the diagnostic SBP tissue or the corresponding bone marrow examination at the time of diagnosis, is associated with a shorter time to progression (TTP) to MM is unknown. This study evaluated all patients diagnosed with SBP at the Mayo Clinic from January 2012 to July 2022. The presence of del(17p), t(14;16), t(4;14), or +1q (gain or amplification) by FISH in clonal plasma cells was defined as HR. A total of 114 patients were included in this cohort, and baseline FISH was available for 55 patients (48%), of which 22 were classified as HR (40%). The median TTP to MM for patients with SBP and HR FISH was 8 months (95% confidence interval [CI], 6.3-26) compared with 42 months (95% CI, 25-not reached [NR]) in patients with SBP without HR FISH (P < .001). In a multivariate analysis, only HR FISH was a significant predictor for shorter TTP to MM, independent of minimal marrow involvement and an abnormal serum free light chain ratio at diagnosis. Deletion (17p) and gain 1q abnormalities were the most common FISH abnormalities responsible for the short TTP to MM. Thus, assessing for HR FISH abnormalities in clonal plasma cells derived from either the diagnostic SBP tissue or the staging bone marrow examination of patients with newly diagnosed SBP is feasible and prognostic for a shorter TTP to MM.

Advanced Risk Stratification in Multiple Myeloma Beyond Traditional FISH: The First Prospective Real-World Evidence for SKY92 Gene Expression Profiling

Background The prognosis of multiple myeloma (MM) has been significantly improved in the last decades. However, high-risk (HR) MM still suffers from early disease progression. Therefore, identifying HR MM patients may be a crucial step to improve the prognosis of this patient group. To date, fluorescence in situ hybridization (FISH) is the most commonly used method for risk stratification in MM. In recent years, gene expression profiling (GEP) approaches, e.g. SKY92, have been developed for detection of HR MM patients. Currently, real-world data of MM risk stratification applying SKY92 alone or in addition to traditional FISH are still missing. In this study, we provide the first prospective real-world evidence of the prognostic value of the combined SKY92 and FISH risk stratification in newly diagnosed (ND) and relapsed/refractory (RR) MM. Methods We prospectively collected bone marrow (BM) samples and clinical data of 147 MM patients. Cytogenetics were analyzed on purified CD138 positive MM cells by FISH, and HR cytogenetics was defined applying the Revised International Staging System (del(17p), t(4;14), and t(14;16)) (Palumbo et al, J Clin Oncol. 2015). SKY92 risk classification was determined with MMprofiler gene expression assay (Kuiper et al, Leukemia 2012). In addition, whole genome sequencing (WGS) was performed, and 150 bp paired-end sequences were generated on the NovaSeq6000 sequencing instruments (Illumina) with 100x coverage. Structural variations (SV) were called using Manta, and copy number variations (CNV) calling was performed with GATK4. Strelka2 variant caller was used for single nucleotide variations (SNV). Variants with global population frequencies >1% were excluded. Results We included 147 patients in our study (NDMM: n=51, RRMM: n=96). SKY92 classification was available for 121 (82.3%) patients. The median follow-up time was 344 days. HR SKY92 was significantly enriched in RRMM (40/76) compared with NDMM (6/45) (P<0.0001). RRMM patients with HR SKY92 showed significantly shorter progression free survival (PFS) (P<0.0001) and overall survival (OS) (P=0.0004) than standard-risk (SR). In NDMM, HR SKY92 also indicated a significantly inferior PFS (P=0.001) in comparison with SR. Of note, samples of 26 (17.7%) patients did not meet the SKY92 quality control criteria due to low BM infiltration (median BM infiltration: 25% vs 55% in other patients, P=0.038). We then combined the SKY92 classification with cytogenetic analyses by FISH, which were performed in 99 patients (NDMM: n=33; RRMM: n=66). We noticed a discrepancy between both risk stratification systems, with 44 (44.4%) and 42 (42.4%) patients being classified as HR by SKY92 and FISH, respectively. In total, 24 (36.3%) RRMM patients and 6 (18.2%) NDMM patients displayed HR in both SKY92 and FISH (double-HR) (Figure). Regarding survival outcome in RRMM, double-HR patients showed the worst PFS (P<0.0001) and OS (P<0.0001) when compared to the other patients (Figure). Noteworthy, RRMM patients with only HR SKY92 demonstrated significantly worse PFS than those with only HR FISH (P=0.007). In addition, double-HR presented a negative prognostic factor for PFS in NDMM (P=0.01). To further elucidate the discrepancy between FISH and SKY92 classifications, we performed WGS in 12 patients who exhibited either HR SKY92 only (n=9) or HR FISH only (n=3). Interestingly, 1 patient with bi-allelic TP53 inactivation (deletion + mutation) and 2 patients harbouring t(4;14) or t(14;16) were determined as SR by SKY92 but as HR by FISH; all the 3 patients were still in remission at the time point of data cut-off (July 2022). Moreover, we found abnormalities at chromosome 1 in 7 out of the 9 patients with HR on SKY92 only (gain1q21: n=6; del1p32: n=2) and, notably, gain1q21 and del1p32 were detected only by WGS in 3 and 2 patients, respectively. The remaining 2 patients did not show any known HR SV/CNV/SNVs, suggesting that HR MM may be associated with some other mechanisms, e.g. epigenetic modifications. Conclusion Altogether, risk classification using SKY92 performed well for detecting HR MM in the clinical routine. Here, we provide the first prospective real-world evidence that advanced risk stratification combining SKY92 and FISH may help to identify the "highest-risk" MM, which requires intensive anti-MM therapy and close monitoring in the course of the disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The impact of bortezomib-based induction in newly diagnosed multiple myeloma with chromosome 1q21 gain

Introduction:Bortezomib has been reported to favourably impact the outcomes of t(4;14) and del(17p) in multiple myeloma (MM), but its impact on gain 1q (+1q) is unknown.Methods:To address this, 250 patients treated with bortezomib-based induction were analysed. All myeloma samples had fluorescence in situ hybridization (FISH) performed on CD138-sorted bone marrow aspirate, and plasma cells were analysed using DNA probes specific for the following chromosomal aberrations: del(13q14), del(17p), t(14;16), t(4;14), and +1q. Presence of +1q was defined as the presence of at least three copies of 1q21 at the cut off level of 20% of bone marrow plasma cells.Results:+1q identified in 167 (66.8%) and associated with t(4;14) and high lactate dehydrogenase (LDH). +1q was not associated with response rate but shorter event-free survival (EFS) (median EFS 35 vs 55 months, p = 0.05) and overall survival (OS) (median OS 74 vs 168 months, p = 0.00025). Copy number and clone size did not impact survival. Multivariate analysis showed +1q was an independent adverse factor for OS together with International Staging System (ISS)3, high LDH, del(17p) and t(4;14). When a risk score of 1 was assigned to each independent adverse factor, OS was shortened incrementally by a risk score from 0 to 4. Post-relapse/progression survival was inferior in those with +1q (median 60 vs 118 months, p = 0.000316). Autologous stem cell transplantation (ASCT) improved OS for those with +1q (median OS 96 vs 49 months, p = 0.000069).Conclusion:+1q is an adverse factor for OS in MM uniformly treated with bortezomib-based induction but was partially mitigated by ASCT. A risk scoring system comprising +1q, LDH, high-risk FISH, and ISS is a potential tool for risk stratification in MM.

DMG26: A Targeted Sequencing Panel for Mutation Profiling to Address Gaps in the Prognostication of Multiple Myeloma

Multiple myeloma presents with numerous primary genomic lesions that broadly dichotomize cases into hyperdiploidy or IgH translocated. Clinically, these large alterations are assessed by fluorescence in situ hybridization (FISH) for risk stratification at diagnosis. Secondary focal events, including indels and single-nucleotide variants, are also reported; however, their clinical correlates are poorly described, and FISH has insufficient resolution to assess many of them. This study examined the exonic sequences of 26 genes reported to be mutated in >1% of patients with myeloma using a custom panel. These exons were sequenced to approximately 1000 times in a cohort of 76 patients from Atlantic Canada with detailed clinical correlates and in four multiple myeloma cell lines. Across the 76 patients, 255 mutations and 33 focal copy number variations were identified. High-severity mutations and mutations predicted by FATHMM-XF to be pathogenic identified patients with significantly reduced progression-free survival. These mutations were mutually exclusive from the Revised International Staging System high-risk FISH markers and were independent of all biochemical parameters of the Revised International Staging System. Applying our panel to patients classified by FISH to be standard risk successfully reclassified patients into high- and standard-risk groups. Furthermore, three patients in our cohort each had two high-risk markers; two of these patients developed plasma cell leukemia, a rare and severe clinical sequela of multiple myeloma.

Gene Expression Profiling (GEP) As a Prognostic Indicator of Multiple Myeloma, Irrespective of Disease Stage, Prior Treatment and Prior Transplant - a Single Center Experience

BackgroundMultiple myeloma (MM) risk is classified using fluorescence in situ hybridization (FISH) routine cytogenetics, and gene expression profiling (GEP). GEP is a clinically validated tool for prognostication of MM in newly diagnosed and relapsed patients (pts), its role in very heavily treated stages is less clear. A study (Nishihori et al, ASH, 2016) concluded that GEP is useful regardless of disease stage. We aim to determine the prognostic significance of GEP heavily pre-treated MM cohort.Methods299 pts with MyPRSTM GEP results at Hackensack University Medical Center (NJ) between 2012 and 2016 were included. High risk GEP was defined with the validated cutoff score of >45.2, high risk FISH was determined according to the mSMART classification as del17p, t(14,16), t(14,20). Chi-square tests were performed on categorical data and Wilcoxon rank sum tests on continuous data. Overall survival (OS) and progression free survival (PFS) were performed using Kaplan-Meier.ResultsMedian age was 63 years (range, 25-91), 170 pts (56%) were men, 81 pts (28%) had International Staging System (ISS) 3 and 191 pts (62%) had Durie-Salmon Staging (DSS) III. 245 pts (81%) had at least one bone marrow transplant (BMT). Median time from myeloma diagnosis to GEP was 30.1 (range 0.1-178.4) months. Most pts had GEP on diagnosis but 24% were heavily pretreated (range, 1-6 lines of treatment), and 13% had a BMT prior to GEP testing. 77 pts (25%) had high-risk disease based on GEP with a median score of 36.5 (range, 9.03-84.05). Molecular subtypes were hyperdiploidy (HY) 31%, cyclin family (CD)-2 20%, low bone disease (LB) 13%, proliferation (PR) 11%, MAF associated (MF) 10%, CD-1 8%, and MMSET associated (MS) 6%. Median OS and PFS were significantly inferior in patients with high-risk by GEP, 25 vs 33 months (p<0.021), 13 vs 21 months (p<0.004) respectively. Molecular subtypes were not individual predictors of OS (p<0.2). When MF, MS and PR subtypes were combined, they had inferior OS compared to other subtypes but this was not statistically significant (p<0.3).ConclusionsGEP can accurately predict risk status in heavily pre-treated and post-transplant MM pts. In conclusion, our data confirm that GEP is a good prognostic indicator in MM pts irrespective of disease stage and prior treatments. Further prospective studies with larger samples and correlating with baseline and intra-treatment GEP are needed. DisclosuresRichter:Janssen: Speakers Bureau; BMS: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Vesole:Takeda: Speakers Bureau; Celgene: Speakers Bureau. Siegel:Celgene, Takeda, Amgen Inc, Novartis and BMS: Consultancy, Speakers Bureau; Merck: Consultancy. Biran:Takeda: Speakers Bureau; Celgene, Amgen: Consultancy, Speakers Bureau.

The Prognostic Significance of Acquired 1q22 Gain in Multiple Myeloma

The Prognostic Significance of Acquired 1q22 Gain in Multiple Myeloma

Redefining the Prognostic Significance of t(11;14) Multiple Myeloma

Background In the era prior to introduction of novel agents, multiple myeloma (MM) harboring t(11;14) was characterized as standard risk. More recently, its unique biology, predictive ability and the prospect of targeted therapeutic agents have renewed interest in t(11;14) MM. Using a large, contemporary real-world database, we investigated the characteristics and outcomes of t(11;14) MM. Methods We used the Flatiron Health Electronic Health Record (EHR)-derived de-identified database to source patients (pts) with newly diagnosed MM from 1/2011 to 2/2020 with available Fluorescence in situ Hybridization (FISH) results documented within 90 days of diagnosis. We compared characteristics of t(11;14)+ patients [without additional high-risk FISH abnormalities: del(17p), Ch1 abnormality (Ch1a), t(4;14), t(14;16) or t(14;20)] vs. t(11;14)- patients (without additional high risk FISH) vs. del(17p) (irrespective of other abnormality) vs. Ch1a (Ch1a without additional high-risk FISH) vs. high-risk translocations [t(4;14), t(14;16) or t(14;20) without del(17p)]. We subsequently compared real-world progression-free survival (PFS) and overall survival (OS) across these five subsets. Additionally, we assessed the impact of t(11;14) as additional FISH abnormality in patients with del(17p) and in patients with Ch1a. We used Kaplan Meier methods with log-rank test and Cox proportional hazard regression model for survival analysis with date of diagnosis as the index date for follow-up. Results 6039 patients in the database met the inclusion criteria. Overall, 83.6% of patients received initial therapy with immunomodulatory agent (IMiD) and/or proteasome inhibitor (PI); of these 40.3% received combination of IMiD and PI. Overall, 27.1% received autologous hematopoietic cell transplantation. Median follow up was 2.1 years (IQR 0.8-4.0). There were 637 pts in t(11;14)+ group, 3173 in t(11;14)- group, 587 in del(17p), 1205 in Ch1a and 437 with high-risk translocations. The t(11;14)+ group had a higher proportion of men, IgM and light-chain isotype, as well as a higher proportion of patients with serum creatinine ³ 2mg/dl (Table). Patients in t(11;14)+ group had worse PFS (mPFS 3.1 vs. 3.3 years, p=0.02) and worse OS (mOS 5.9 vs. 6.5 years , p=0.04) compared to t(11;14)-, but better PFS and OS than the other three high-risk groups (Figure panels A and B). Worse PFS for t(11;14)+ was demonstrable even after adjustment for sex, age, race/ethnicity, immunoglobulin isotype, stage, comorbidities, and treatment received (adjusted HR=0.87, 95% C.I. 0.77-0.98, P=0.027). We subsequently analyzed the impact of presence of t(11;14) in MM with del(17p) or Ch1a.. The presence of t(11;14) in addition to del(17p) resulted in worse OS compared to del 17p without t(11;14) (mOS 2.8y vs. 3.7y; p=0.04). Indeed, the impact of t(11;14) on del(17p) was comparable to the impact of t(4;14) (Figure, Panel C). There was no difference in survival with concomitant presence of t(11;14) with Ch1a (Figure, Panel D). Conclusion MM with t(11;14) has distinct features at presentation and even when treated with modern therapy carries worse prognosis than otherwise standard-risk MM. The concomitant presence of t(11;14) portends a negative prognostic impact to MM with del(17p) but does not appear to impact MM with Ch1a. When present alongside del17p, t(11;14) behaves like a high-risk translocation and identifies a subset of MM in greatest need of newer therapies. Figure 1 Disclosures Costa: Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; AbbVie: Consultancy; Celgene: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Genentech: Consultancy.

Gene Expression Profiling of Structural and Functional High-Risk Multiple Myeloma

Introduction: High-risk multiple myeloma can be defined by the presence of specific cytogenetic abnormalities (structural) or by characteristic changes in bone marrow and peripheral blood biomarkers (functional). While both entities are characterized by therapeutic resistance, frequent disease relapses, and adverse survival outcomes, the underlying molecular mechanisms remain incompletely understood. Methods: We performed gene expression profiling (GEP) using an Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray on CD138+ bone marrow cells from 137 patients diagnosed with multiple myeloma between 2004 and 2012. All patients underwent Fluorescence In-situ Hybridization (FISH) evaluation, plasma cell labeling, International Staging System (ISS) risk stratification, and GEP prior to initiating treatment with novel agents. The presence of del(17p), t(4;14), t(14;16), and t(14;20) on FISH, a plasma cell labeling index (PCLI) &gt; 2%, and ISS stage III were considered high-risk abnormalities: FISH-HR (n = 15, structural high-risk, at least one high-risk FISH lesion), PCLI-HR (n = 20; functional high-risk, PCLI &gt; 2%), and ISS-HR (n = 12; functional high-risk, ISS stage III). For each HR group we sampled standard risk (SR) controls in a 4:1 ratio. After data quality control and normalization, differential gene expression was estimated using limma. Statistical significance was adjusted for multiple comparisons using a false discovery rate-based approach for genome-wide experiments (q-value). We employed PANTHER pathway analysis for the differentially expressed genes in each HR group. We implemented a simple gene expression score (GES) by calculating the sum of quartiles of the normalized gene expression values for genes differentially expressed in more than one HR group (GES = ΣUP(quartile - 1) + ΣDN(4 - quartile)) and externally validated its prognostic significance (UAMS TT2 / TT3, GSE24080). Survival outcomes were analyzed using the methods described by Kaplan, Meier, and Cox. Computation and visualization were performed in R. Results: Median age at diagnosis was 63 years (32 - 87), 53% of the patients were male. High-risk disease was associated with inferior overall survival, regardless of the used definition (left Kaplan-Meier plots): FISH-HR (HR 4.3, 95% CI 1.9 - 9.8, p &lt; 0.001), PCLI-HR (HR 2.7, 95% CI 1.4 - 5.3, p = 0.004), and ISS-HR (HR 2.8, 95% CI 1.2 - 6.5, p = 0.015). There were 59 (FISH-HR), 424 (ISS-HR), and 507 (PCLI-HR) differentially expressed genes (q &lt; 0.050 for all genes, volcano plots). PCLI-HR and FISH-HR demonstrated a predominance of transcriptional up-regulation while ISS-HR had a balanced gene expression profile with a similar number of genes being up- and down-regulated. The involved cellular pathways were different across the HR groups except for anti-apoptotic signaling (bar graphs). All HR groups had distinct gene expression profiles with no complete overlap between all HR groups. There were 71 genes with overlap between two HR groups (69 up-regulated, 2 down-regulated, Venn diagrams). The median GES was 97 (18 - 206, higher numbers indicating higher expression of up-regulated and lower numbers of down-regulated high-risk genes) in 559 patients treated on UAMS TT2 / TT3 (GSE24080). Tertiles of the GES were associated with event-free survival (HR 1.4, 95% CI 1.2 - 1.6, p &lt; 0.001) and remained independently prognostic after adjusting for age, sex, and ISS stage (HR 1.3, 95% CI 1.1 - 1.5, p &lt; 0.001). Conclusions: High-risk multiple myeloma remains associated with inferior overall survival, regardless of the used definition (structural or functional). The subtypes of high-risk disease have distinct gene expression profiles and involve different cellular pathways, providing important clues to the underlying biology. A 71 gene signature derived from the different high-risk subtypes was of prognostic significance in a clinical trial population after adjusting for known prognostic factors. Figure Disclosures Lacy: Celgene: Research Funding. Dispenzieri:Akcea: Consultancy; Intellia: Consultancy; Janssen: Consultancy; Pfizer: Research Funding; Takeda: Research Funding; Celgene: Research Funding; Alnylam: Research Funding. Stewart:Takeda: Consultancy; Seattle Genetics: Consultancy; Roche: Consultancy; Ono: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy; Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy. Bergsagel:Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Kumar:Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding.

Lenalidomide, Adriamycin and Dexamethasone (RAD) Versus Bortezomib, Lenalidomide and Dexamethasone (VRD) in Newly Diagnosed Multiple Myeloma (MM) - Post-Induction Response and MRD Results By Flow Cytometry and NGS from a Phase 3 Randomized Controlled Clinical Trial (RCT)

Lenalidomide, Adriamycin and Dexamethasone (RAD) Versus Bortezomib, Lenalidomide and Dexamethasone (VRD) in Newly Diagnosed Multiple Myeloma (MM) - Post-Induction Response and MRD Results By Flow Cytometry and NGS from a Phase 3 Randomized Controlled Clinical Trial (RCT)

PO-025 Antiproliferative effects of rosemary (rosmarinus officinalis, L.) extract in colon cancer cells lines

IntroductionInfections by oncoviruses are an important risk factor for the development of neoplasms. Particularly, anogenital cancers are strongly associated with persistent infections by high-risk human papillomaviruses (HPV). In the case of cervical cancer the physical state of the virus seems to be an important prognostic marker. Especially the integration of viral DNA into the host cell genome bears the risk of following degenerations. Therefore, the early and sensitive detection is of high interest. Besides Pap-test and PCR, fluorescence in situ hybridization (FISH) is used for a quantitative and qualitative detection of HPV infections and risk assessment.Material and methodsFor analysis high-risk HPV FISH probes should be developed for standardised, type-specific and quantitative detection of HPV nucleic acid in host cells. Due to the short viral DNA sequence, present with just few copies, conventional FISH probes have little sensitivity. Therefore, we tested specific FISH probes targeting different regions of the HPV genome in combination with signal amplification. PCR-produced FISH probes were biotin labelled by direct incorporation of biotinylated nucleotides or nick translation. Our biotinylated HPV probes were tested on HPV-positive and –negative cells and detected using fluorescence labelled streptavidin or subsequent tyramide signal amplification. In addition to FISH, probes were used for chromogenic in situ hybridization. All analysis were performed with our fully automatized multicolor fluorescence imaging platform VideoScan.Results and discussionsThe presence of HPV DNA in cell lines was confirmed by (digital) PCR. The protocol for synthesis of biotinylated probes using HPV sequence specific primers and genomic DNA as template was successful. Biotin incorporation was checked with agarose gel electrophoresis and modified dot blot hybridization. In in situ hybridization experiments, our probes showed high signals in HPV high-copy number CaSki cells and a tissue model, compared to HPV negative samples.ConclusionHighly sensitive and specific FISH probes offer a great potential for the detection of HPV DNA in patient samples and thereby for the assessment of tumour risks. Using our probes, we got positive results in hybridization experiments using chromogenic and fluorescent detection methods with or without further amplification steps. Next, comparative FISH experiments will be performed to check probe sensitivity in cells containing few HPV copies and their practicability in biopsy derived tissue samples.

Practice Patterns in Newly Diagnosed Multiple Myeloma in the Prospective Observational Commpass Trial

Introduction:The management of multiple myeloma has become increasingly complex, given late age of onset, underlying co-morbidities, plethora of drugs, and variable clinical presentation and natural history. Practice patterns likely vary based on practice type, physician experience, and geographic distributions. The Multiple Myeloma Research Foundation's (MMRF) CoMMpass Trial (Relating Clinical Outcomes in Multiple Myeloma to Personal Assessment of Genetic Profile) is a prospective, longitudinal, observation trial in NDMM with the primary goal of correlating patient data and response with molecular profiles. Here, we evaluate practice patterns in NDMM the CoMMpass Trial based on staging, high-risk features, and demographics.Methods: Clinical data were derived from MMRF’s CoMMpass IA8, accessed in late July 2016 on https://research.themmrf.org/rp/explore. Independent categorical variables analyzed include International Staging System (ISS), Revised-ISS (R-ISS), LDH (normal vs. above upper limit of normal), Fluorescence-In-Situ Hybridization (FISH) (standard vs. high risk (t(4;14), t(14;16), t(14:20), del17p)), race, performance status (PS) (0-1, 2, 3-4), age (>65, 65-75, 76-80, >80 years), and gender. Dependent variables include use of doublets vs. triplets, the use of triplets using combined immunomodulatory/proteosome inhibition (IMID/PI), receiving or not receiving an autologous stem cell transplantation (ASCT), and timing of transplant (<10 vs. >10 months). For high-risk MM, defined as ISS 3, R-ISS 3, elevated LDH, or high risk FISH, patients receiving doublet therapy without an autologous stem cell transplantation were further analyzed for performance status and age. Descriptive statistics were used. Chi-square testing was used to compare variables, using STATA v14.1.Results: Data on 921 patients has been released and was reviewed. Although men and women had similar upfront therapy, including the use of triplet (57% vs. 61%, p=0.483) and IMID/PI combinations (56% vs. 62%, p=0.181), women were more likely to have an ASCT (44% vs. 34%, p=0.002). When compared to European Americans (EA), African-Americans (AA) were less likely to receive triplets (47% vs. 61%, p=0.004), IMID/PI combination (55% vs. 59%, p=0.001), and ASCT (30% vs. 40%, p=0.034). Patients with high-risk disease were not more likely to be treated more aggressively. Patients with ISS Stage 3 disease were less likely to receive triplets (50% vs. 64%, p=0.002), IMID/PI combinations (51% vs. 66%, p=0.001), or an ASCT (26% vs. 48%, p=<10-3). When comparing standard-risk MM, as defined by LDH or FISH, high-risk patients were not more likely to receive triplet therapy or ASCT. Performance status did not correlate triplet use, but lower rates of IMID/PI combinations and ASCT were noted in those with worse PS.We subsequently reviewed patients with high-risk features who were treated with doublet therapy and without ASCT, specifically looking performance status and age. Of those with elevated LDH, 12 (46%) were younger than 65 years and 12 (46%) had PS<1. Of those with high risk FISH, 20 (49%) were younger than 65 years and 37 (76%) had PS<1. Of those with ISS 3, 24 (26%) were younger than 65 years and 49 (56%) had PS<1. Of those with R-ISS 3, 12 (44%) were younger than 65 years and 17 (63%) had PS<1.Conclusions: The MMRF CoMMpass trial allowed assessment of practice patterns in the United States both inside and outside of academic medical centers. AA appear to be treated less aggressively, possibly explaining shorter survival despite more favorable cytogenetics. Second, high-risk disease does not appear to be treated more aggressively, even in younger patients with excellent performance status. This presented data must be interpreted with caution since this trial does not capture the treating physician's decision-making, nor survival data. DisclosuresGirnius:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau.

Occurrence and Prognostic Significance of Cytogenetic Evolution in Patients with Multiple Myeloma

Abstract Background: Cytogenetic evaluation, especially using fluorescence in situ hybridization (FISH), at the time of diagnosis is essential for initial risk stratification and the employment of risk-adapted treatment strategies in multiple myeloma. Little is known about the occurrence and prognostic significance of cytogenetic evolution during follow up. Methods: We studied 433 patients who were diagnosed with multiple myeloma between January 2000 and December 2011 and had at least two FISH evaluations at Mayo Clinic Rochester, including the diagnostic specimen. Bone marrow aspirates were evaluated for deletions, monosomies, trisomies, and tetrasomies using chromosome- or centromere-specific FISH probes. IGH rearrangements were evaluated using an IGH break-apart probe and up to five potential partners (FGFR3, CCND1, CCND3, MAF, and MAFB). Cytogenetic evolution was defined as a new deletion, monosomy, trisomy, tetrasomy, or translocation during follow up. Multivariable-adjusted logistic regression models were used to assess the associations between the parameters of interest and the presence of cytogenetic evolution in follow-up specimens. Multivariable-adjusted Cox proportional hazards models were used to assess the effect of cytogenetic evolution on overall survival. All models were adjusted for sex, age, the presence of high-risk FISH abnormalities, and the number of abnormalities at the time of diagnosis. Likelihood ratio tests were used to assess the goodness of fit of nested models. The χ2 or Fisher's exact test was used to assess the distribution of cytogenetic abnormalities in subgroups. Results: The median age at diagnosis was 60 years (32 - 82), 264 (61%) of the patients were male. The median overall survival for the entire cohort was 7.0 years (6.2 - 7.8). At the time of diagnosis, 150 (35%) and 57 (13%) of the 433 patients presented with a hyperdiploid karyotype and cytogenetic high-risk abnormalities, respectively. Independent of each other, the presence of a translocation at the time of diagnosis was associated with decreased odds of cytogenetic evolution during follow up (OR 0.39, 95% CI 0.24 - 0.63, p &lt; 0.001) while the presence of at least one trisomy or tetrasomy at the time of diagnosis was associated with increased odds (OR 2.53, 95% CI 1.37 - 4.70, p = 0.003). A greater proportion of patients presenting with a hyperdiploid karyotype experienced cytogenetic evolution during follow up. Those patients more frequently evolved additional trisomies and tetrasomies, while translocations were more common in those presenting with a non-hyperdiploid karyotype (Table 1). The development of additional abnormalities during the three years following diagnosis (compared to no new abnormalities) was associated with increased subsequent mortality in those who survived at least three years (HR 3.22, 95% CI 1.82 - 5.68, p &lt; 0.001). Including the time between first and last cytogenetic evaluation as a covariate did not significantly change the parameter estimates or improve model fit (p = 0.727). Conclusions: Demographics, risk profile, and overall survival of this cohort reflect the fact that patients had to survive long enough to undergo repeated cytogenetic evaluation. Hyperdiploid and non-hyperdiploid genotypes were associated with distinct behavior regarding cytogenetic evolution during follow up. The identification of cytogenetic evolution was an adverse prognostic factor in those who survived at least three years after diagnosis. These findings emphasize the importance of the dynamics of the underlying clonal disease process for accurate risk assessment and suggest that selected subgroups of patients may benefit from risk stratification during follow up. Table 1. Cytogenetic evolution during follow up in 433 patients with multiple myeloma stratified by karyotype at the time of diagnosis Hyperdiploid (n = 150) Non-hyperdiploid (n = 283) p New abnormality 76 (51%) 108 (38%) 0.012 New monosomy 8 (5%) 24 (8%) 0.243 New trisomy 48 (32%) 55 (19%) 0.004 New tetrasomy 37 (25%) 27 (10%) &lt; 0.001 New deletion 17 (11%) 27 (10%) 0.557 New translocation 1 (1%) 11 (4%) 0.065 Most common new abnormality [type (percent of type in each group)] Monosomy mono(13) (75%) mono(13) (62%) Trisomy tri(11) (22%) tri(3) (22%) Tetrasomy tetra(15) (48%) tetra(15) (30%) Deletion del(17p) (79%) del(17p) (63%) Translocation t(11;14) (100%) t(11;14) (36%) Data are given as count (percent) unless denoted otherwise. Disclosures Binder: American Society of Hematology: Research Funding. Kumar:AbbVie: Research Funding; Onyx: Research Funding; Sanofi: Research Funding; Celgene, Millenium, Sanofi, Skyline, BMS, Onyx, Noxxon,: Other: Consultant, no compensation,; Skyline, Noxxon: Honoraria; Millenium/Takeda: Research Funding; Janssen: Research Funding; Celgene: Research Funding.

An abnormal nonhyperdiploid karyotype is a significant adverse prognostic factor for multiple myeloma in the bortezomib era

Multiple myeloma is clinically heterogeneous and risk stratification is vital for prognostication and informing treatment decisions. As bortezomib is able to overcome several high-risk features of myeloma, the validity of conventional risk-stratification and prognostication systems needs to be reevaluated. We study the survival data of 261 previously untreated myeloma patients managed at our institution, where bortezomib became available from 2004 for the treatment of relapse disease. Patient and disease characteristics, and survival data were evaluated overall, and with respect to bortezomib exposure. Overall, the international staging system (ISS), metaphase karyotyping and interphase fluorescence in situ hybridization (FISH) were discerning of survival outcomes, where the median for the entire cohort was 5.2 years. However, when stratified by bortezomib exposure, only metaphase karyotyping was still discriminating of long-term prognosis. The presence of an abnormal nonhyperdiploid karyotype overrides all other clinical and laboratory parameters in predicting for a worse outcome on multivariate analysis (median survival 2.6 years, P = 0.001), suggesting that bortezomib used at relapse is better able to overcome adverse risk related to high tumor burden (as measured by the ISS) than adverse cytogenetics on conventional karyotyping. Metaphase karyotyping provides additional prognostic information on tumor kinetics where the presence of a normal diploid karyotype in the absence of any high-risk FISH markers correlated with superior survival and could act as a surrogate for lower plasma cell proliferation.

MicroRNA Expression Profiling of CLL B Cells From Relapsed/Refractory CLL Patients Reveals a Signature Group of Genes That Are Associated with Clinical Responses: Results of An ECOG Trial E2903.

Abstract 4377 IntroductionMicroRNA (miRNA) profiling in CLL has revealed a leukemia specific gene signature in CLL patients. Our earlier work found a unique signature of 13 deregulated microRNAs from 190 previously untreated CLL patients. This signature included some of the most frequently deregulated miRNAs in different types of hematological malignancies (miRNA-15-a/16-1, miRNA-29 family, miRNA-155, miRNA-221) and correlation with CLL prognostic factors (ZAP-70 expression and immunoglobulin heavy chain variable region (IGVH) mutations). Since these miRNAs have both diagnostic and prognostic implications, we wished to extend our observations to a cohort of relapsed/refractory CLL patients entered on the ECOG trial E2903. This trial is a chemoimmunotherapy protocol that uses 6 cycles of pentostatin, cyclophosphamide and rituximab to induce responses followed by consolidation with Alemtuzumab. MethodsTo assess miRNA status, we used our previously described method which employs a bead-based profiling that interrogates 649 microRNA genes. We used the Trizol extracted RNA from the CLL B-cells of 33 patients entered onto the E2903 clinical trial. All patients were also characterized by determining their CD38, ZAP-70, fluorescence in situ hybridization (FISH) and IGVH status. All microRNA and prognostic parameters were done on baseline CLL B-cells obtained from the patients prior to going on study. ResultsThe demographics of this CLL patient population included 28 males with median age of 61 (range; 47-76), 1 Rai stage 3 and 24 Rai stage 4. The median number of prior treatments was 2 (range;1-5). To date with PCR treatment only assessed, there were 15 partial responses (PR), 15 stable disease, and 3 unevaluable patients. Because of a batch effect, we restricted our analysis in this report to only 24 patients. We decided to initially compare the microRNA profiles for CLL patients segregated on the following; responders (CR/PR) vs. non responders (SD or less), ZAP-70+ vs. ZAP-70- (20 % cutoff), CD38+ vs. CD38– (30% cutoff), Low-risk FISH (normal, 13q- or trisomy 12) vs. High-Risk FISH (17p- or 11q- or complex) and age '65 vs. ≥ 65. In addition, we did combine some of the prognostic parameters or responses together for comparison of microRNA status (ZAP-70 with either response, IGVH status, or CD38 status). This analysis revealed a set of 10 microRNA genes with a median false discovery rate of 20% that were aberrantly expressed when we compared the ZAP-70- responders to the ZAP-70+ non-responders. In addition, we found 15 differentially expressed miRNAs when we compared ZAP-70- to ZAP-70+ cohorts. We then evaluated the association of the 15 genes found in the ZAP-70 comparison with the overall survival and progression free survival (PFS). We found that miR-625* was significantly associated with PFS (p=0.01). The expression level of miR-625* was more than 2-fold increased in ZAP-70- compared to ZAP-70+. miR-625*, located at 14q23.3, has more than 60 putative target genes, including nuclear and cell cycle, and cell adhesion targets. ConclusionInterrogation of the genes that are impacted by the deregulated miRNAs uncovered in our analysis of this previously treated CLL cohort may provide valuable information regarding the mechanism of resistance commonly seen in this cohort of heavily pretreated CLL patients. Disclosures:Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees.

Quantitative molecular urinary cytology by fluorescence in situ hybridization: a tool for tailoring surveillance of patients with superficial bladder cancer?

To determine whether it is possible to stratify patients with superficial bladder cancer into low- and high-risk groups for tumour recurrence/progression based on the chromosomal pattern detected by fluorescence in situ hybridization (FISH) in one urine cytology specimen used for follow-up testing. Voided urine samples from 47 consecutive patients with urinary tract neoplasms (13 with no history of urothelial malignancy and 34 under follow-up after complete transurethral resection of superficial urothelial carcinoma of the bladder) were evaluated by liquid-based cytology (ThinPrep(R), CYTYC Corp., Boxborough, MA, USA) and UroVysion FISH (Vysis-Abbott, Downers Grove, IL). Of the 34 patients under surveillance, the UroVysion test was negative in four, 17 had loss of 9p21 sequences either alone or combined with low-frequency trisomy/ies or tetrasomy/ies of chromosomes 3, 7 and 17 in single cells (low-risk FISH), and 13 also had complex aneusomies of the remaining chromosomes (high-risk FISH). One of the four FISH-negative neoplasms, four of the 17 low-risk FISH cases and five of the 11 informative high-risk FISH-positive patients developed recurrence. Progression occurred only in patients with high-risk FISH results, showing high-frequency complex chromosomal polysomies (four of 11). The results from this pilot study indicate that the UroVysion FISH test may help to individually assess the clinical behaviour of superficial bladder cancer, based on the chromosomal pattern of exfoliated tumour cells in follow-up urinary cytology. It might be of use to identify those patients likely to progress at earlier and curable stages of disease, and lengthen the surveillance period in those with persistent or recurrent low-risk disease.

Chromosome anomalies detected by interphase fluorescence in situ hybridization: correlation with significant biological features of B-cell chronic lymphocytic leukaemia.

Fluorescence in situ hybridization (FISH) was used to detect 6q-, 11q-, +12, 13q-, 17p- and translocations involving 14q32 in interphase nuclei from blood and/or bone marrow from 113 patients with B-cell chronic lymphocytic leukaemia (B-CLL). A total of 87 patients (77%) had a FISH anomaly: 13q- x 1 was most frequent (64%) followed by 13q- x 2 (28%), +12 (25%), 11q- (15%), 17p- (8%) and 6q- (0%). FISH results for blood and bone marrow cells in 38 patients were similar. Purified CD5+/CD19+ cells from blood were studied in eight patients and results indicate that in some patients not all B cells have FISH anomalies. We used a defined set of hierarchical FISH risk categories to compare FISH results by stable versus progressive disease, age, sex, Rai stage, CD38+ expression and IgVH mutational status. Significant differences in FISH risk distributions were associated with Rai stage, disease status and CD38+, but not by age, sex or IgVH mutational status. To look for baseline factors associated with high-risk disease, multivariate analysis of age, sex, Rai stage, CD38+ and disease status versus FISH risk category was performed. Importantly, only CD38+ was significantly associated with high-risk FISH categories (+12, 11q- and 17p-) after adjustment for the effects of other variables.