Nowakowski, Grzegorz S.

Abstract Background: Angiogenesis has been found to be an important regulator in the disease progression of both solid tumors and hematologic malignancies. In solid tumors, the balance of pro and anti-angiogenic factors appears to be a critical factor in tumorigenesis, with disturbance in favor of angiogenesis promoting tumor growth and metastasis. This observation led to the concept of an “angiogenic switch” where pro-angiogenic influences outweigh anti-angiogenic factors. Patients with CLL have detectable levels of plasma and cellular pro and anti-angiogenic cytokines as well as abnormal neovascularization in the marrow and lymph nodes. Limited studies of pro-angiogenic cytokines have suggested that inter-patient variation in serum/plasma and cellular levels of these markers may have prognostic implications. We evaluated pro- and anti-angiogenic cytokines in a large sample of patients with CLL to evaluate the presence of angiogenic switching and its implications for disease progression. Methods: In a prospective, longitudinal study, we analyzed the serum/plasma, intracellular(determined by flow cytometry), and CLL B cell secreted (24 hour medium values) levels of pro-angiogenic cytokines (VEGF, bFGF) and an anti-angiogenic cytokine (Thrombospondin [TSP]) in 311 patients with previously untreated CLL. VEGF:TSP and bFGF:TSP ratios were calculated to evaluate for evidence of angiogenic switching (higher ratio suggests a pro-angiogenic phenotype). The relationship of angiogenic cytokines to Rai stage, IgVH gene mutation status, % CD38 protein expression, and time to treatment (TTT) from the date of sample were evaluated (median follow-up=11 months). Sequential samples were available on >90% of patients and were evaluated to assess if changes in levels of angiogenic cytokines occurred in patients requiring treatment. Results: Serum, cellular, and secreted levels of VEGF, bFGF, and TSP showed no correlation with Rai stage, IgVH gene mutation (continuous variable), or % expression of CD38 (continuous variable). However, among patients with Rai stage 0-II disease, individuals with lower TSP levels (<75 percentile, p=0.007) or higher bFGF:TSP ratios (continuous variable; p=0.003) had shorter TTT (Figure 1). Similar relationships for a shorter TTT were also observed for secreted TSP levels (p=0.007) and the cellular bFGF:TSP ratio (p=0.009). On analysis of sequential samples, no clear difference in serum, cellular, or secreted levels of VEGF, bFGF, TSP, VEGF:TSP, or bFGF:TSP occurred among those who required treatment. Conclusions: Angiogenic cytokines appear to have prognostic significance in patients with previously untreated early stage CLL. Importantly, we report the original observation that the ratio of bFGF (pro-angiogenic) to TSP (anti-angiogenic) correlates with time to treatment as a continuous variable. This result provides further evidence that angiogenic switching relates to clinical progression in CLL. Longer follow-up of this cohort is needed to fully define the prognostic implications of angiogenic cytokines in CLL and determine if integration of these markers with other prognostic parameters improves risk stratification for early stage patients. Survival According to Serum Thrombospondin Level Survival According to Serum Thrombospondin Level

Abstract Background: The presence of circulating myeloma cells (CMC) detected by flow cytometry at the time of diagnosis of multiple myeloma is associated with a shortened response to therapy and reduced overall survival (OS). We hypothesized that the presence of CMC at the time of stem cell collection prior to high dose therapy (HDT) and autologous stem cell transplantation (ASCT) would identifies a cohort of patients with a high risk of rapid progression. Methods: The Mayo Clinic myeloma transplant database was queried for patients who were mobilized using cyclophosphamide and hematopoietic growth factors. CMC was determined using flow cytometry by gating on a population of CD38 bright and CD45 negative cells. The impact of CMC on OS and time to progression (TTP) and its role in the context of established prognostic parameters was evaluated. Results: Of 246 patients with MM undergoing ASCT, 95 had CMC. Patients with CMC had significantly higher plasma cell labeling index, adverse cytogenetics, B2-M and resistant disease. Complete response (CR) rates post transplant were 32% and 36% for patients with and without CMC (p=0.5034). OS was 33.2 and 58.6 months (p=0.0052) while TTP was 14.1 and 22 months respectively (p=0.0005). Figure Figure On multivariate analysis, CMC remained an independent prognostic factor in a model that included cytogenetics and disease status at time of transplant (p=0.0314). A prognostic system based on the presence or absence of CMC and karyotype abnormalities was developed. Patients with neither, one or both parameters had a median, OS of 55, 48 and 21.5 months respectively (p<0.0001) while TTP was 22, 15.4 and 6.5 months for the same groups (p<0.0001). Conclusion: The presence of CMC at the time of HDT and ASCT is an independent prognostic factor. The combination of CMC and cytogenetics provides a simple yet powerful scoring system that stratifies patients and guides their management.

Abstract Background: Solitary plasmacytoma of bone (SBP) is a localized collection of monoclonal plasma cells that is potentially curable with local radiation therapy but associated with a high risk of progression to multiple myeloma. We hypothesized that an abnormal immunoglobulin free light (FLC) ratio at diagnosis may be a prognostic indicator of transformation risk. Methods: We identified a cohort of 133 patients with SBP for whom stored serum taken at the time of diagnosis was available. The diagnosis was ascertained and serum FLC determined in 126 patients. Results: From this cohort, 48 patients have progressed to myeloma and the median time to progression among those who progressed was 1.9 years. On univariate analysis, age (p<0.001), gender (p=0.035), abnormal FLC ratio at diagnosis (p=0.009) and persistence of serum or urine M-protein after therapy (p=0.0070 were all associated with a shorter overall survival (OS) and time to progression to multiple myeloma. Progression by Normal FLC(0.26–1.65) Progression by Normal FLC(0.26–1.65) On multivariate analysis, an abnormal FLC ratio retained its independence in a model that includes age at diagnosis but lost its significance when combined with persistence of the serum or urine M-protein. However, serum or urine M-proteins are not detectable in a significant number of patients with SBP and therefore not informative. Conclusion: The FLC ratio at the time of diagnosis of SBP is a powerful predictor of risk and a useful aid to management of patients with this condition.

Abstract Background: Recent literature suggests that statins may have anticancer potential. This effect is thought to be mediated through 2 primary mechanisms - impairment of protein prenylation and interference with the formation of cholesterol-rich lipid microdomains, or “lipid rafts” within the cell membrane. Both of these processes are critical for signaling activity of numerous proteins important for lymphomagenesis and tumor survival. Recent data, however, suggest that statin use may directly inhibit rituximab binding to CD20 and therefore rituximab efficacy. These findings raised significant concerns about statin use during rituximab treatment. Here we report on statin use and clinical outcome in a cohort of FL and DLBCL treated patients, most of whom were treated with rituximab containing regimens. Methods: 293 newly diagnosed FL patients and 228 newly diagnosed DLBCL patients were prospectively enrolled in our Lymphoma SPORE registry from 9/2002 through 6/2007. Pathology was centrally reviewed. All patients were followed for retreatment, progression-free and overall survival. Statin use at the time of diagnosis and time of initial treatment was abstracted from the medical record. An event was defined by disease progression, retreatment, or death due to any cause. Results: 19% of FL patients and 22% of DLBCL patients were on statins at diagnosis; 16% and 19% were on statins during treatment, respectively. Initial therapy for the FL patients was observation (40%), R-CHOP (19%), R-CVP (12%), rituximab alone (8%), CVP (7%), RT (6%), and other (8%). All DLBCL patients received rituximab with CHOP or a CHOP-like regimen. At a median follow-up of 36 months (range 3–73), 109 (37%) and 65 (29%) of FL and DLBCL patients had an event; 19 (6%) and 46 (20%) of FL and DLBCL patients died, respectively. After adjusting for FLIPI, grade, and initial therapy type, statin use at diagnosis was associated with better event-free survival (HR = 0.57, 95% CI: 0.34–0.95, p=0.03) in FL patients. Statin use during treatment in FL patients was also associated with better event-free survival, though not statistically significant (HR = 0.67, 95% CI: 0.39–1.16, p=0.15). The improvement in EFS for FL patients was consistent across initial therapies, including observation. Statin use was not associated with IPI-adjusted overall survival or event-free survival in DLBCL (all p > 0.50). Conclusions: Statin therapy does not appear to be associated with inferior clinical outcome in DLBCL treated with rituximab and CHOP or CHOP-like therapy. Therefore direct inhibition of rituximab binding to CD20 may have limited clinical significance or/and may be overcome by inhibitory impact of statins on cell signaling. The latter possibility is supported by our observation that statin use is associated with improved event-free survival in follicular lymphoma. Figure Figure Figure Figure

Abstract BACKGROUND: Karyotype analysis of metaphase cells for discovering genome-wide cytogenetic abnormalities has played an important role in prognosis and counseling of patients with chronic lymphocytic leukemia (CLL). Due to the low proliferative rate of CLL B cells, karyotype analysis has been of limited clinical utility in this disease. Thus, fluorescence in situ hybridization (FISH) on interphase cells has replaced karyotype analysis for the assessment of recurrent genetic changes in CLL. Recently, immunostimulatory CpG-oligodeoxynucleotides (CpG) have been shown to induce primary CLL B cell proliferation via engagement of toll-like receptor 9 (TLR9), thereby rendering karyotype analysis feasible and providing for a useful and reliable method to discover new cytogenetic abnormalities in CLL B cells. However, we and others have shown that CpG stimulation of CLL and normal B cells also induces robust expression of activation induced cytidine deaminase (AID), a mutagenic enzyme that is linked to the development of cytogenetic abnormalities. It is therefore plausible that cytogenetic abnormalities observed following CpG stimulation of CLL B cells may not reflect de novo abnormalities but rather reflect CpG induced-AID mediated cytogenetic alterations. We began to address this possibility by assessing the effects of CpG stimulation on the karyotypes of normal blood B cells. HYPOTHESIS: Since CpG stimulation induces AID expression in CLL and normal B cells, we hypothesize that: robust B cell activation by CpG could result in cytogenetic abnormalities thereby contributing to lymphomagenesis; and if CpG induces cytogenetic abnormalities that are linked to disease initiation and/or progression, such information would be of great value in the interpretation of cytogenetic data obtained from CpG-stimulated CLL B cells. METHODS: We performed interphase FISH analysis on freshly isolated blood B cells, and metaphase karyotype analysis on CpG stimulated B cells from 17 healthy blood donors. Pure (>98%) B cells obtained by negative selection, were hybridized at isolation with a probe set to identify deletion 6q, 11q, 13q, and 17p, trisomy 12, and IGH gene rearrangements, and cells were also stimulated for 5 days with 10 μg/ml CpG (CpG 2006, synthesized in-house) plus IL-2 and IL-15 for metaphase analysis. At least 20 post-CpG metaphase spreads were analyzed for each sample and abnormalities were considered clonal using the accepted convention (gains or translocations are present in two or more cells and loss present in at least 3 cells). The expression of AID in the B cells pre- and post-CpG stimulation was assessed by RT-PCR. RESULTS: FISH analysis of freshly isolated B cells showed that all 17 subjects were normal. After CpG stimulation, all samples examined exhibited strong AID expression as revealed by RT-PCR. By karyotype analysis, none of the 17 CpG stimulated samples showed evidence of clonal abnormalities. However, one or more nonclonal chromosomal abnormalities were observed in 7 out of 17 (41.2%) samples. Among those 7 samples, 4 samples had 1 abnormal metaphase out of a minimum of 20 examined while the other 3 samples exhibited two or more cells, each with distinct abnormalities. Some of those nonclonal abnormalities have also been observed in CLL (i.e., trisomy 3, trisomy 12 and 14q32 rearrangements). This is a much higher incidence of nonclonal abnormalities than has been seen with peripheral blood cells cultured by other methods. CONCLUSION: Our data have significance for the biology of CLL and for clinical practice. First, our data suggest that signaling through TLR9, and perhaps other receptors that likewise induce robust B cell AID expression, may represent a natural mechanism by which B lineage cells acquire chromosomal abnormalities during normal immune responses that may predispose toward neoplastic transformation. Second, given that none of the CpG-induced metaphases in normal B cells resulted in clonal abnormalities in the 5 day timeframe, our data also validate the clinical utility of CpG as a valuable tool for the cytogenetic analysis of CLL B cells since only clonal abnormalities are currently scored. However, it remains possible that non-clonal abnormalities seen in CpG-stimulated CLL B cells may be relevant to the disease process if future work shows that CpG stimulation uncovers recurring non-clonal defects of chromosomes/genes associated with tumorigenic events.

Abstract Background: Normal and tumor stem cells are characterized by high activity of multidrug resistance (MDR) transporters. One of these, ABCG2 (ATP-binding cassette, sub-family G member 2 protein), is an ATP dependent transporter and putative stem cell marker responsible for verapamil sensitive Hoechst efflux. While ABCG2 is known to be expressed in normal and leukemic stem cells, as well as a small population of normal lymphocytes and some B-cell malignancies, its expression in chronic lymphocytic leukemia (CLL) is unknown. It has been postulated that leukemic stem cells due to their quiescent nature and expression of MDR transporters represent a population resistant to therapy and that this residual population is critical for tumor persistence and recurrence. Hypothesis: We hypothesized that ABCG2 is expressed in a small percentage of primary CLL B cells; gene expression profiles of ABCG2 positive versus ABCG2 negative CLL B cells differ in respect to expression of self renewal and lymphoid development genes; the frequency of ABCG2+ CLL B cells increases after treatment in patients responding to therapy. Methods: We analyzed ABCG2 expression by primary CD5+, CD19+ CLL-B cells from untreated CLL patients of all Rai stages by flow cytometry. In a subset of patients we used fluorescence activated cell sorting (FACS) to sort CD19+, CD5+ ABCG2+ and CD19+, CD5+ ABCG2- cells. Gene expression profiling was then performed using the U133 plus 2.0 Affymetrix microarray platform. In a separate cohort of patients treated in a clinical trial of pentostatin, cyclophosphamide and rituximab (PCR), the percentage of ABCG2+, CD19+, CD5+, CD79b dim cells at baseline and then two months after completion of 6 cycles of PCR therapy where patients had minimal residual disease (MRD) was assessed and correlated with clinical response. Results: ABCG2+ CD19+, CD5+ detectable populations were seen in all 20 CLL assessed patients (median percentage 0.6%; range 0.08%–3.8%). There was no difference in percentage of ABCG2+ cells based on Rai stage, IGVH mutational status, Zap70 or CD38 expression. Preliminary analysis of the gene expression profiling of ABCG2 positive versus negative CLL B cells from four randomly selected patients revealed significantly higher expression of genes associated with self-renewal, cell cycle and early B-cell development including: cyclin-dependent kinase inhibitor 1C (CDKN1C, p=0.034), transcription factor 7-like 2 (TCF7L2, involved in WNT pathway regulation, p=0.016), beta-catenin (p=0.034) and pre-B-cell colony enhancing factor 1 (PBEF-1, p=0.037). Flow based assessment of the levels of ABCG2 positive populations at baseline and after therapy with PCR in patients with minimal residual disease showed a dramatic increase in frequency of ABCG2 positive CLL B cells. The percentage of ABCG2+ cells went from a median level of 0.19% (range 0.04%–0.19%) prior to therapy to a median level of 10.93% (range 0.15%–25.12%), p<0.001. In contrast two patients who did not reach MRD (partial responses by NCI-WG criteria) had no significant increase in percentage of ABCG2 positive cells (0.14%; 0.23% and 0.16%; 0.21% prior and after therapy, respectively, p=0.68). Conclusion: Our data indicate that ABCG2 positive CLL B-cells constitute 0.1–3.8% of circulating CLL B-cells in untreated patients. The frequency of ABCG2+ CLL B-cells appears to dramatically increase after therapy in the MRD state; this could be related to their relative resistance to therapy and/or a shift from extravascular compartments post therapy. Since ABCG2 positive CLL B-cells demonstrate expression of early B-cell development and self-renewal genes we believe that that this population could represent a putative self renewing CLL B-cell compartment. Further studies to characterize features of ABCG2 CLL-B –cells in relation to their capacity to be self renewing and resistance to therapy are warranted.

Abstract Acquired pure red cell aplasia (APRCA) is associated with a number of conditions including malignancies, infections and autoimmune disorders. APRCA also occurs in patients with T-LGLL. It is postulated that suppression of erythropoiesis in T-LGLL is related to the immune destruction of erythroid precursors by the LGL clone. We hypothesize that APRCA can be associated with clonal T-cell expansion without pathological evidence of T-LGLL. Methods: We identified patients who were diagnosed with APRCA and had clonal TCR gene rearrangements. Clinical course, bone marrow pathology and flow cytometry data were reviewed. Immunohistochemical studies were performed on available bone marrow biopsies using antibodies to CD3, CD8, CD20 and the cytotoxic granule proteins TIA-1 and granzyme B. Clonal TCR gene rearrangements were detected by PCR and Southern blot. The diagnosis of APRCA was based on the absence of erythroid progenitors, with intact granulo- and thrombopoiesis. A pathologic diagnosis of T-LGL leukemia was established by the presence of clonal TCR gene rearrangements and an immunophenotypically distinct CD8 positive T-cell population as previously described. The diagnostic flow cytometric features included abnormal patterns of T-cell antigen expression and aberrant co-expression of NK-cell antigens. Immunoperoxidase features supportive of a diagnosis of LGL were the presence of interstitial clusters or intravascular staining of cells positive for CD8 and TIA-1 and/or granzyme B. Results: We identified 28 patients with APRCA and the presence of clonal TCR gene rearrangements seen at Mayo Clinic Rochester between 7/1/1991 to 6/1/03. All patients presented with a transfusion dependent anemia (median hemoglobin 7.8 g/dL, range 3.6–10.3). Tissue was available for immunohistochemical studies for 21 cases. 13 of 21 patients (61%) had an immunophenotypically abnormal T-cell population which, in conjunction with the clonal TCR gene rearrangements were considered diagnostic of T-LGL. 8 patients (39%) had no pathological evidence of T-LGLL despite clonal TCR gene rearrangement studies. The most commonly used treatment consisted of oral cyclophosphamide which was used in 14 of 21 patients (8 and 6 in patients with and without pathological evidence of T-LGLL respectively). A normalization of hemoglobin was seen in 11 patients (6 and 5 in patients with and without evidence of T-LGLL respectively). Median duration of response was 30 months (range 22–120). None of 8 patients with APRCA and clonal TCR gene rearrangements without evidence of LGLL at the the time of initial diagnosis developed overt T-LGLL during follow up (median 64 months, range 30–118). Discussion: 39 % of patients with APRCA and evidence of clonal TCR gene rearrangements did not meet pathological criteria for diagnosis of T-LGLL. There was no apparent difference in disease presentation and response to therapy in patients with or without pathological evidence of LGLL. The significance of the presence of clonal TCR gene rearrangements in the absence of a detectable immunophenotypically abnormal T-cell population is unclear. It is possible that T-LGL was present which could not be detected by our analyses. Alternatively, the clonal TCR gene rearrangements could be indicative of a pertubation of the T-cell compartment related to an autoimmune disorder causing the APRCA. The fact that none of the patients developed progression to overt LGLL supports the latter possibility.

Abstract Background: Cytotoxic T-cell infiltrates are a nearly universal finding in the bone marrow of patients with multiple myeloma. It has been postulated that presence of T-cells in the bone marrow of multiple myeloma (MM) patients represents an immune response against the tumor and therefore, might be associated with an improved prognosis. However, the impact of bone marrow T-cells on the prognosis of multiple myeloma patients has not been studied systematically. Methods: Bone marrow biopsies of patients with newly diagnosed multiple myeloma were stained by immnohistochemistry for the CD8 antigen and reviewed by a blinded hematopathologist. Three high power fields are reviewed for each biopsy and the total number of CD8 positive cells counted and reported. For patients with more than 300 cells per 3 fields, results were reported as >300. The number of bone marrow CD8 positive cells was then correlated with patients’ clinical data, including other prognostic factors and overall survival. Results: Bone marrow biopsy specimens from 100 patients, performed within the week of a diagnosis of multiple myeloma and collected between May 1998 and January 2001 were evaluated. The median number of CD8 positive cells was 270 (33 – >300). Patients’ characteristics are shown in Table 1. Median follow up was 30 months (0–80). The number of cytotoxic T-cells as a continuous variable was a risk factor for shortened overall survival, HR 1.86 (95% CI 1.11–3.35). Using minimal p value approach, the cutoff of 270 cells (the median) risk stratified patients into two groups: the median survival of patients with > 270 CD8 positive cells was 16 months vs. 48 months in patients with ≤270 cells, p=0.005 (Figure). In multivariate analysis including age, B2M, albumin, CRP, bone marrow plasma cell percentage and plasma cell labeling index, the number of cytotoxic T-cells was an independent predictor of overall survival was HR 3.1, p=0.0017. Conclusion: We show that the number of cytotoxic T-cells in the bone marrow is a strong and independent prognostic factor in patients with newly diagnosed multiple myeloma. Our observation does not contradict the hypothesis that cytotoxic T-cells participate in an immune response against the tumor since our findings may represent a higher level of immune response associated with baseline aggressive disease biology. However, our study suggests for the first time that increased marrow cytotoxic T-cells have an adverse effect on outcome in myeloma, and suggest that these cells may have a direct facilitating effect on tumor growth and on the marrow microenvironment. Further studies of the biology of behind this observation are warranted. Characteristic N Median (range) Gender male 61 CRP 81 0.4mg/L (0.01–11.2) Albumin 99 3.6 g/dL (2.6–5.4) B2microglobulin 94 4.0 (0.9–28) μg/mL Marrow PC% 90 45% (11–99) PC labeling index 90 high (>1%) 36 BM CD8 cells 100 270 (33 – >300) ISS 94 1 19 2 41 3 34 Figure Figure

TPS7569 Background: Progress in genome technology allows analysis of previously completed trials to identify patients subgroups potentially benefiting from therapy. Enzastaurin is a potent inhibitor of protein kinase C beta (PKC- b) and suppresses the phosphoinositide 3-kinase (PI3K)/AKT pathway. The safety and efficacy of Enzastaurin has been tested in more than 60 clinical trials including 2 major studies in DLBCL: (1) PRELUDE (A phase III maintenance trial of Enzastaurin vs Placebo, N=758) ( Crump, 2016), and (2) S028 (A randomized phase II study of Enzastaurin/R-CHOP vs R-CHOP in frontline intermediate/high-risk DLBCL, N=101) ( Hainsworth, 2016). DNA samples extracted from blood of patients from PRELUDE were retrospectively genotyped using whole genome SNP arrays. From the genome wide screening a novel genetic biomarker, DGM1, was identified showing high correlation with response to Enzastaurin treatment ( Luo, ASH 2018). Importantly, these findings were replicated in the phase II S028 study. In the S028 study the hazard ratio (HR) for OS in high-risk (IPI ≥ 3) DGM1 positive (+) patients who received Enzastaurin/R-CHOP was 0.28 (0.1-0.81) when compared to subjects who received R-CHOP, a benefit favoring Enzastaurin (p=0.018). These data suggest that addition of Enzastaurin to R-CHOP may significantly improve outcome in frontline high-risk DGM1 (+) DLBCL. The ENGINE study was initiated to validate this finding in a prospective study. Methods: Adult patients must have untreated CD20+ DLBCL, IPI ≥ 3. Patients are randomized 1:1 to Enzastaurin/R-CHOP or Placebo/R-CHOP for 6 cycles during combination phase. Each subject’s treatment assignment will be unblinded after response assessment at the end of the combination phase. Subjects randomized to the investigational arm who have a complete or partial response will have the option to continue in the single agent phase to receive Enzastaurin for up to 2 additional years. The study intends to enroll approximately 235 patients with primary endpoint of OS in DGM1 (+) patients. The study is ongoing with 51 sites open in the US and China. As of 22 Jan 2019, 70 patients have been randomized. Clinical trial information: NCT03263026.