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Background: GRP78 (78-kDa glucose-regulated protein) is a molecular chaperone that is upregulated during cellular stress. It has been well demonstrated that GRP78 upregulation is associated with chemoresistance and metastasis in solid tumours. GRP78 has not been widely explored in multiple myeloma (MM), however, we and others have shown that GRP78 is much more overexpressed in myeloma cell lines compared to other cell lines. To assess the clinical relevance of GRP78 overexpression in MM, we investigated the association of plasma cell GRP78 expression on primary bone marrow (BM) trephines to clinical outcome in patients with MM, and correlate this finding to concurrent in vitro studies to investigate the potential usefulness of targeting GRP78 for the treatment of MM. Method: The degree of GRP78 expression within CD138+ plasma cells was assessed by immunohistochemistry (IHC) on archived bone marrow trephines of patients with newly diagnosed MM, who underwent autologous stem cell transplant (ASCT) at St.Vincent's Hospital Melbourne. Independent assessment of GRP78 was performed by 3 hematopathologists, who underwent initial calibration. The degree of GRP78 expression within plasma cells was assigned as low, medium or high. Clinical data was abstracted from medical records of the corresponding patients with respect to baseline demographics, treatment-response, progression free survival (PFS), time to next treatment (TTNT) and overall survival (OS). The association GRP78 expression to each of these clinical parameters was assessed using Kaplan-Meier product limit method and the Mantel-Cox logrank test. In vitro, GRP78 expression was also quantified in various myeloma cell lines by RT-PCR and western blot. The association of GRP78 expression to MM-cell survival and drug resistance was assessed in vitro. The impact of GRP78 inhibition on reversal of drug resistance and myeloma-cell viability was investigated. Result: Between the years 2000 to 2014, a total of 243 patients with newly diagnosed MM underwent ASCT as part of initial therapy, and were included in the study. Baseline bone marrow trephine was available for CD138 and GRP78 staining for 91 patients. Of these, 20, 42 and 34% of patients had low, medium and high expression of GRP78 within BM plasma cells, respectively. Low GRP78 expression was associated with a shorter PFS (HR 2.4, p=0.0006) and shorter TTNT (HR 2.5, p=0.008) compared to intermediate or high GRP78 expression. No significant difference was seen in OS. High GRP78 correlated with a higher probability of achieving CR (p=0.03). In vitro, inhibition of GRP78 resulted in decreased myeloma cell viability, and sensitized myeloma cells to various antimyeloma agents. As a result, synergistic anti-myeloma activity was seen when GRP78 inhibition was combined with melphalan (synergy quotient (SQ) 1.2), dexamethasone (SQ 1.97) and especially bortezomib (SQ 2.06). Conclusion: In contrast to what is reported for solid tumours in the literature, higher GRP78 expression appeared to predict for a more favorable clinical outcome in patients with MM. In vitro, GRP78 inhibition resulted in significant anti-myeloma effects and increased the antimyeloma activity of various agents especially bortezomib. Together, these findings suggest that GRP78 is potentially a useful biomarker and therapeutic target that warrants further investigation in patients with MM. Disclosures Quach: Celgene Corp, ONYX, Janssen, Takeda, Novartis, BMS: Honoraria, Research Funding.

Background: Disease relapse remains the main cause of mortality after allogeneic stem cell transplantation (allo-SCT) for patients with myeloid malignancies. Loss of donor chimerism (DC) is commonly used as a biomarker of impending relapse, which allows the initiation of pre-emptive therapies such as withdrawal of immunosuppression or donor lymphocyte infusion (DLI). Surprisingly, there are few if any direct comparisons of peripheral blood CD34+ and CD3+ DC as biomarkers to predict relapse. We hypothesized that loss of CD34+ DC may be a more direct measure of impending relapse given most myeloid malignancies express CD34. Methods: We prospectively measured peripheral blood CD34+ and CD3+ DC on days 30, 60, 90, 120 and 180 following allo-SCT for patients with AML (n=113) or MDS (n=23) transplanted at a single centre between July 2011 and November 2019. Chimerism analysis was performed using purified cell subsets isolated from 60 mL peripheral blood using PCR-based amplification of short tandem repeats (STRs). The goal of this retrospective analysis was to compare the value of CD3+ and CD34+ DC for predicting relapse. Institutional practice for CD34+ DC below 80% included a bone marrow biopsy to identify morphologic relapse and donor lymphocyte infusion. Statistical analysis was performed with R 3.5.2 (The R project for Statistical computing) or GraphPad (v8.2.0). Results: Overall, 41 of 136 (30%) patients had morphologic relapse at a median time of 153 days after allo-SCT (range 50-1742). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of CD3+ and CD34+ donor chimerism for morphologic relapse is shown in Figure 1A. CD34+ DC outperformed CD3+ DC in all criteria, irrespective of the percentage chosen. Furthermore, a concurrent reduction in CD34+ DC was seen in almost all patients (14/16 at the 80% level) that had a fall in CD3+ DC. A fall in CD34+ DC below 80% was highly predictive for relapse, with a 5 year relapse free survival of only 17% compared with 80% for those that maintained DC > 80% for the first 180 days (Figure 1B). To determine the clinical utility of CD34+ DC, we measured the time to relapse in those patients that had a fall in DC without morphologic relapse at the time of DC measurement (Figure 1A). Based upon our institutional trigger of CD34+ DC < 80%, the median time to relapse was 49 days in 22 patients. In contrast, a CD34+ DC < 90% had a much longer time to relapse (71 days). However, this longer lead time would come at the price of unnecessary intervention in almost half of all patients as the PPV for CD34+ DC < 90% was only 55%. Loss of CD3+ DC had the longest lead time (> 70 days), however it was only useful up to 10 (24%) of all relapses. Finally, DLI administered for CD34+ DC < 80% maintained durable remission (> 12 months) in only 2 of 19 patients. Conclusion: This is the largest comparison of peripheral blood CD34+ and CD3+ DC following allo-SCT. Our results show that monthly monitoring of CD34+ DC in the first 6 months after allo-SCT is a more useful biomarker than CD3+ DC for predicting relapse in patients allografted for AML or MDS. The level of CD34+ DC chosen to trigger intervention should be guided by characteristics of the intervention such as toxicity and expected response time. Given the relative ineffectiveness of DLI for CD34+ < 80%, we suggest that 90% may provide greater time for immunologic responses. Figure Legend (A) Characteristics of different levels of CD3 and CD34 DC at any time in the first 180 days post-allo-SCT. Number of patients that fulfil the level in the total cohort of 136 patients. Number of patients before relapse indicates the number that do not have morphologic relapse at the time of DC measurement. Median days before relapse is shown for those patients. (B) Relapse free survival of patients according to CD34+ DC > 80% (blue line) or < 80% (yellow line) in the first 180 days. Disclosures Spencer: AbbVie, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Consultancy; Celgene, Janssen and Takeda: Speakers Bureau; Amgen, Celgene, Haemalogix, Janssen, Servier and Takeda: Research Funding; AbbVie, Amgen, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Honoraria. Wei:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; MacroGenics: Consultancy, Honoraria; Servier: Consultancy, Honoraria, Research Funding; Walter and Eliza Hall Institute: Other: former employee and receives a fraction of its royalty stream related to venetoclax; Pfizer: Honoraria; Genentech: Honoraria; Astra Zeneca: Honoraria, Research Funding; AbbVie Inc.: Consultancy, Honoraria, Research Funding.

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