ALBERT

Description: Genetic makers such as TP53, ATM mutations and IgH somatic hypermutation status have been used to predict clinical behaviour and response to therapy in Chronic Lymphocytic Leukaemia (CLL) for the last decade. Recently identified genetic markers including splicing factor 3 B1 (SF3B1), NOTCH and BirC3 are also significant but their role cannot be fully evaluated until a robust assay is available for a routine molecular diagnostic laboratory The cell spliceosome is involved in the precise excision of introns from pre-mRNA and is composed of five subunits of which SF3B1 is a core component. Patient samples with SF3B1 mutations display enhanced intron retention in a small subset of transcripts involved in cancer related regulation such as cell cycle control, apoptosis and angiogenesis. Studies of CLL patient samples has shown that those with mutations in SF3B1 have been associated with shorter treatment free and overall survival. The screening methodology for SF3B1 mutations through deep sequencing, though very sensitive, is not available in routine diagnostic laboratories. Approximately 90% of CLL-associated SF3B1 mutations occur is 336bp region of the cDNA exome amenable to PCR amplification. We designed primers flanking this region and following reverse transcription (RT) PCR amplification, detected the products on standard agarose gels. Following bidirectional Sanger sequencing, we compared the sequences obtained with reference SF3B1 cDNA/amino acid code. We assessed the impact of the mutations by the polyphen database and compared this to the immunophenotype and cytogenetic data as well as response to treatment through minimal residual disease (MRD) tracking. We subsequently designed an assay for detection of sequence variants in the SF3B1 region of interest by High Resolution Melt (HRM) curve analysis. This has enabled us to identify genetic variants of the SF3B1 region prior to sequencing. The application of this HRM pre-sequencing screening assay has resulted in a significant reduction in the number of samples requiring sequencing. The patient cohort consisted of 52 patients enrolled on a Phase II, multi centre study of fludarabine, cyclophosphamide and rituximab (FCR) and included patients 〈 65 yrs, WHO status of 0, 1 or 2 without a deletion 17p. All samples were collected and tested prior to treatment, at the end of treatment (after 4 or 6 cycles when MRD negative), 6 monthly post treatment for 5 years for minimal residual disease and then annually by 6 colour immunophenotyping. Patients (51/52, no RNA available for 1 patient) were screened prior to treatment for the presence of SF3B1 mutations. Five of 51 (9.8 %) patients had SF3B1 mutations detectable by this methodology shown in the table below. Table 1. Pt. Nucleotide change Amino acid change Polyphen IGHV M=mutatedU=unmutated Cytogenetics Immunophenotyping MRD 1 c.2146A〉G p.K700E Damaging U V4-6*01 Normal CD5 -ve Not tracked due to neg CD5 2 c.2179G〉C p.A711P Damaging U V1-69*01 Normal Neg @ 3 mts Pos @ 16 mts 3 c.2267G〉A p.G740E Damaging M V3-74*01 del 13q CD5 weak +ve CD38 +ve Neg @ 3 mts Pos @ 12 mts 4 c.2146A〉G p.K700E Damaging U V1-69*01 del 13q Never MRD neg 5 c.2032C〉T p.H662Y Damaging U V3-11*01 del 13q CD38 +ve Withdrawn Patients with mutations in SF3B1 all had favourable cytogenetics (normal or del 13q) and the majority (4/5) had unmutated IGHV genes. We detected two patients with a mutation at c.2146A〉G, p.K700E and one at c.2267G〉A, pG740E which have been described previously and two novel mutations at c.2179G〉C, p.A711P and c.2032C〉T, p.H662Y. All mutations detected were damaging as per the polyphen database. Two patients have weak or no expression of CD5. One patient has never achieved MRD negativity, and two patients who became MRD negative at 3 months reverted to MRD positivity 9 and 13 months later respectively. One patient's MRD levels could not be tracked due to negative CD5 expression and was followed by a less sensitive IgH clonality assay (sensitivity approx. 1%) and one patient was withdrawn from the study. In summary, we have developed a simple PCR based technique to detect SF3B1 mutations in CLL patient samples with high disease burden which can be applied in routine molecular diagnostic laboratories without access to next generation sequencing. The use of HRM technology resulted in a 〉 90% reduction in the number of samples requiring sequencing. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Patients (pts) with newly diagnosed multiple myeloma (MM) are commonly treated with the standard of care combination of lenalidomide (Len), bortezomib (Bz), and dexamethasone (Dex), also known as RVD. A recent randomized phase 3 study found that the addition of Bz to Len and Dex significantly increased median overall and progression free survival as well as response rate (Durie et al. Lancet 2017). Mild to moderate peripheral neuropathy (PN) is commonly reported with Bz use, although lower rates of PN have been reported with subcutaneous (SC) administration of single agent Bz compared with IV Bz (Moreau et al. Lancet Oncol 2011). Here we present preliminary results of a multi-center, open-label, single arm phase II trial of Len, SC Bz, and Dex in pts with newly diagnosed MM. Maintenance was risk-stratified, with high risk patients (defined as those with high risk cytogenetics (del17p, t(4:14), t(14;16)) or ISS stage II or III) receiving Bz in addition to Len. Primary endpoints included 1) overall response rate (ORR) after 4 induction cycles, 2) best response to induction therapy, and 3) rate and severity of PN during induction therapy. Methods: Patients enrolled in this study were newly diagnosed with active MM as defined by the revised IMWG criteria (Rajkumar et al. Lancet Oncol 2014). Protocol specified induction treatment consisted of 21-day cycles with Len 25 mg on days 1-14, SQ Bz 1.3 mg/m2 days 1, 4, 8, and 11, and Dex 20 mg on days 1, 2, 4, 5, 8, 9, 11, and 12. Stem cell mobilization followed induction cycle 4 and patients subsequently proceeded to either high dose melphalan and autologous stem cell transplant (ASCT) or 4 additional cycles of induction therapy based on patient preference with provider input. Following ASCT or completion of the 8th induction cycle pts proceeded to risk-stratified maintenance therapy. Maintenance consisted of 28-day cycles of therapy with Len on days 1-21 for all patients, while those pts defined as high-risk also received SC bortezomib Bz on days 1 and 15. Patients remained on maintenance therapy until progression, unacceptable toxicity, or withdrawal from protocol-directed treatment. Response was based on the IMWG uniform criteria (Rajkumar et al. Lancet Oncol 2011) and toxicities were graded based on the NCI-CTCAE V4. Correlative samples of blood and bone marrow for genomics and proteomics were collected from baseline and then throughout the study, and are currently being analyzed. Results: Forty-five pts were enrolled across 8 US sites between December 2015 and June 2017. Median age at enrollment was 61 years (range: 43 to 79) and 60% of the patients were male, 40% female. FISH cytogenetics found del 17p in 8% of pts tested, t(14;16) in 9%, and t(4;14) in 14%. At baseline, 60% of pts were ISS II/III. High risk pts comprised 62% of the study population overall. 80% of pts (36/45) collected stem cells and 31% of pts (14/45) continued to ASCT. The median number of CD34+ stem cells collected was 9.67 x 10^6. The median number of induction cycles completed was 8 (1 to 8 cycles) and 43 of 45 pts were evaluable for the primary endpoint of response after 4 induction cycles, with preliminary results indicating an ORR of 91% (39/43). Three pts did not reach the end of cycle 4 and 1 patient had stable disease. ORR at any point up to the beginning of maintenance was 98% (42/43). Any grade PN was reported by 80% of patients, including 38% with grade 1 and 36% with grade 2 PN. There were two cases of Grade 3 PN and one case of Grade 4 PN. Among the three patients with Grade ≥ 3 PN, symptoms improved to Grade ≤ 2 with dose reduction, modification of treatment schedule, or discontinuation of Bz. Importantly, given the higher than expected rate of all and high-grade PN, hydration with IV normal saline 500-1000 ccs prior to Bz administration as part of supportive care in selected patients was instituted and a comprehensive evaluation of the impact of this intervention on PN is in process. Conclusions: The combination of RVD with SC Bz is a highly effective treatment regimen for patients with newly diagnosed MM, including high risk pts. However, rates of all- and high-grade PN were greater than expected despite the use of SC Bz. Prompt dose reduction and/or change in schedule of Bz administration to weekly administration is recommended, with careful attention to supportive care in order to further improve tolerability. Disclosures Rosenblatt: Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding. Matous:Celgene: Consultancy, Honoraria, Speakers Bureau. Farber:Charles M. Farber, MD, PhD, LLC-Medical legal consulting: Consultancy; Gilead: Honoraria; Genentech: Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Research Funding; ummit Medical Group-MD Anderson Cancer Center: Employment; BeiGene: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Speakers Bureau; Acerta: Research Funding. Ghobrial:Celgene: Consultancy; BMS: Consultancy; Takeda: Consultancy; Janssen: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Fludarabine, cyclophosphamide and rituximab (FCR) therapy results in a complete remission (CR) rate of 72% and a median progression free survival (PFS) of 80 months in non-del (17P) CLL1. Achieving an MRD negative (MRD-ve) CR after completing therapy is an early surrogate marker for overall survival (OS) and PFS2. Specific genetic CLL subtypes determined by fluorescent in-situ hybridisation (FISH) analysis, immunoglobulin mutation IgVH, NOTCH1 and SF3B1 status determine response to chemotherapy3,4. We completed a multi-centre prospective study between 2008 and 2012, with a median follow up of 62.6 months using MRD status to determine length of therapy. Patients who achieved an MRD-veCR after 4 courses of FCR received no further therapy and the remaining patients completed 6 cycles of FCR. MRD status was tracked 6 monthly in patients who became MRD-veuntil MRD was detected. The genetic subtype was also analysed but did not influence treatment. Fifty-two patients {35M;17F, median age 61years (range 37-73)} were enrolled. Forty-six patients completed the MRD assessment after 4 cycles. Eleven patients discontinued assigned FCR therapy for the following reasons: prolonged cytopenia (4); non-compliance (1); autoimmune haemolytic anaemia (2); renal impairment (1); pleural effusion (1); not recorded (2). Eighteen (34.6%) patients achieved an MRD-veCR after 4 cycles and a further11 after 6cycles resulting in 29/52 (55.8%) MRD-veCRs in total. The median PFS was 72.3 months (95% Confidence Interval 61.3-84.1 months) and the median OS has not been reached. Patients who attained an MRD-veversusMRD+vestatus had a prolonged PFS (81.1 vs 46.2 months, p

Description: Introduction: Lenalidomide, bortezomib and dexamethasone (RVD) is considered a new standard of care regimen for patients with newly diagnosed multiple myeloma. A previous phase I/II study of RVD in front-line myeloma enrolled 66 patients and achieved a partial response rate or better of 100%, overall and a CR/nCR rate of 52% in the phase 2 portion of the study with encouraging tolerability, but high rates of peripheral neuropathy (PN), albeit mainly mild to moderate grade (Richardson et al, Blood 2010). Subcutaneous (SQ) administration of single agent bortezomib has been shown to be non-inferior to IV bortezomib and led to lower rates of PN, a common treatment-related toxicity (Moreau et al, Lancet Oncol 2011). Herein we present preliminary results of the RsqVD Study, a multi-center, open-label single arm phase II trial, incorporating SQ bortezomib with lenalidomide and dexamethasone and including patients who were considered either transplant eligible or ineligible. All patients subsequently received maintenance therapy with lenalidomide until progression, plus the addition of subcutaneous bortezomib twice monthly in high risk patients (ISS stage II or III and/or high risk cytogenetics features, t(4;14, t(14;16) and del17p). The primary endpoint was overall response rate (ORR) after 4 cycles of induction therapy (PR or better). Secondary endpoints include: rate and severity of PN, safety, time to progression, progression-free survival, duration of response and overall survival. Methods: Planned treatment was 4 cycles of lenalidomide 25 mg/day on days 1-14 and dexamethasone 20/mg/day on days 1, 2, 4, 5, 8, 9, 11 and 12 plus bortezomib 1.3 mg/m2as SQ injection on days 1, 4, 8 and 11 of a 21-day cycle. Thromboprophylaxis with aspirin 75 mg/day or higher was mandatory and HSV prophylaxis was as per institutional standard. Following 4 cycles, patients were planned to proceed with stem cell mobilization and autologous stem cell transplant (ASCT) or further induction therapy up to a total of 8 cycles. Following completion of ASCT or induction therapy, all patients were scheduled to receive lenalidomide maintenance in 28 - day cycle until progression, unacceptable toxicity or withdrawal of consent. Patients with high-risk features received SQ bortezomib on days 1 and 15 during maintenance phase. Response was investigator-assessed as per IMWG criteria. Sample size (n=42) was determined to provide 80% power to test an acceptable ORR of 〉70% versus an unacceptable ORR of