TOP2A expression predicts responsiveness to carfilzomib in myeloma and informs novel combinatorial strategies for enhanced proteasome inhibitor cell killing.

Microarray was utilized to determine if a genetic signature associated with resistance to carfilzomib (CFZ) could be identified. Twelve human myeloma (MM) cell lines (HMCLs) were treated with CFZ and a cell-viability profile was assessed categorizing HMCLs as sensitive or resistant to CFZ. The gene expression profiles (GEP) of untreated resistant versus sensitive HMCLs revealed 29 differentially expressed genes. TOP2A, an enzyme involved in cell cycle and proliferation, was overexpressed in carfilzomib-resistant HMCLs. TOP2A protein expression levels, evaluated utilizing trephine biopsy specimens acquired prior to treatment with proteasome inhibitors, were higher in patients failing to achieve a response when compared to responding patients. Logistic-regression analysis confirmed that TOP2A protein expression was a highly significant predictor of response to PIs (AUC 0.738). Further, the combination of CFZ with TOP2A inhibitors, demonstrated synergistic cytotoxic effects in vitro, providing a rationale for combining topoisomerase inhibitors with CFZ to overcome resistance in MM.

DNA-Repair Gene Mutations Are Highly Prevalent in Circulating Tumour DNA from Multiple Myeloma Patients.

Mutational characterisation utilising plasma (PL)-derived circulating tumour DNA (ctDNA) in multiple myeloma (MM) has been recently described. Mutational analyses of paired bone marrow (BM) MM cell DNA and ctDNA from 76 patients (n = 24, new diagnosis (ND), n = 52, relapsed/refractory (RR)) for (ras/raf signaling pathway) and tumour protein p53 (TP53) mutations using the OnTarget™ Mutation Detection (OMD) platform was performed. The total number and proportions of mutations in each of the compartments (BM-specific, PL-specific or shared) was significantly higher in RR patients compared to ND patients (p = 0.0002 and p < 0.0001, respectively). Patients with > 2 mutations or > 1% fractional abundance (FA) in the PL had significantly shorter overall survival (OS) (p = 0.04 and p = 0.0006, respectively). Patients with PL-specific TP53 mutations had significantly shorter OS compared to patients with no PL-TP53 mutations (p = 0.003), while no differences were observed in patients with (K-ras) KRAS mutations. Targeted deep amplicon sequencing (TAS) of matched PL and BM samples from 36 MM patients for DNA-repair and RAS-RAF pathway genes found that DNA-repair genes were present at significantly higher levels in the PL when compared to RAS-RAF mutations (p = 0.0095). We conclude that ctDNA analysis identifies a higher prevalence of potentially actionable DNA-repair gene mutated subclones than BM analysis.

Monitoring tumour burden and therapeutic response through analysis of circulating tumour DNA and extracellular RNA in multiple myeloma patients.

Monitoring tumour burden and therapeutic response through analyses of circulating cell-free tumour DNA (ctDNA) and extracellular RNA (exRNA) in multiple myeloma (MM) patients were performed in a Phase Ib trial of 24 relapsed/refractory patients receiving oral azacitidine in combination with lenalidomide and dexamethasone. Mutational characterisation of paired BM and PL samples at study entry identified that patients with a higher number of mutations or a higher mutational fractional abundance in PL had significantly shorter overall survival (OS) (p = 0.005 and p = 0.018, respectively). A decrease in ctDNA levels at day 5 of cycle 1 of treatment (C1D5) correlated with superior progression-free survival (PFS) (p = 0.017). Evaluation of exRNA transcripts of candidate biomarkers indicated that high CRBN levels coupled with low levels of SPARC at baseline were associated with shorter OS (p = 0.000003). IKZF1 fold-change <0.05 at C1D5 was associated with shorter PFS (p = 0.0051) and OS (p = 0.0001). Furthermore, patients with high baseline CRBN coupled with low fold-change at C1D5 were at the highest risk of progression (p = 0.0001). In conclusion, this exploratory analysis has provided the first demonstration in MM of ctDNA for predicting disease outcome and of the utility of exRNA as a biomarker of therapeutic response.

Liquid biopsies for liquid tumors: emerging potential of circulating free nucleic acid evaluation for the management of hematologic malignancies.

Circulating free nucleic acids; cell free DNA and circulating micro-RNA, are found in the plasma of patients with hematologic and solid malignancies at levels higher than that of healthy individuals. In patients with hematologic malignancy cell free DNA reflects the underlying tumor mutational profile, whilst micro-RNAs reflect genetic interference mechanisms within a tumor and potentially the surrounding microenvironment and immune effector cells. These circulating nucleic acids offer a potentially simple, non-invasive, repeatable analysis that can aid in diagnosis, prognosis and therapeutic decisions in cancer treatment.

Excellent outcomes for adolescents and adults with acute lymphoblastic leukemia and lymphoma without allogeneic stem cell transplant: the FRALLE-93 pediatric protocol.

Adolescents and adults with acute lymphoblastic leukemia/lymphoma (ALL) have better outcomes when treated using pediatric protocols compared with treatment using adult protocols. We reviewed the progress and outcomes of 40 adolescents and adults up to 45 years of age, from three Australian centers, treated on the intensive French group for childhood ALL (FRALLE)-93 pediatric protocol. All except one patient achieved a morphologic complete remission following induction chemotherapy. Three-year overall survival for all-risk and standard-risk disease was 70% and 75%, respectively. The treatment protocol was generally well tolerated with no treatment related mortality. The FRALLE-93 pediatric protocol showed excellent overall survival for patients with standard-risk disease, without the need for allogeneic hematopoietic stem cell transplant in first remission.

Fludarabine, cytarabine, granulocyte-colony stimulating factor and amsacrine: an effective salvage therapy option for acute myeloid leukemia at first relapse.

Improved therapeutic options for relapsing patients with acute myeloid leukemia (AML) are urgently needed. Poor outcomes following salvage therapy have been reported in those with short initial remission duration, adverse risk karyotype, prior allograft, older age, FLT3-internal tandem duplication (ITD) AML and prior high-dose cytarabine (HiDAC) induction therapy. We present a cohort of 58 patients (aged 18-70) treated with fludarabine, cytarabine, granulocyte-colony stimulating factor (G-CSF) and amsacrine (FLAG-amsacrine) as salvage chemotherapy for AML at first relapse. 83% had received prior HiDAC-based therapy. The overall complete remission (CR/CR with incomplete blood count recovery [CRi]) rate was 59%, with median event-free survival (EFS) and overall survival (OS) of 6.9 and 10.6 months, respectively. FLAG-amsacrine was an effective bridge to allogeneic transplant with 38% successfully transplanted with excellent outcomes (median OS not reached). FLAG-amsacrine was also effective in elderly patients (≥ 60 years), with 61% achieving second remission. The regimen was well tolerated, with 30- and 42-day treatment-related mortality of 3.4% and 13.8%, respectively. Outcomes remained poor in those with short initial remission duration (<6 months). We conclude that FLAG-amsacrine is a useful salvage option for AML at first relapse.