Price-Troska, Tammy

Introduction: Despite the improved clinical outcomes with novel therapies in patients with chronic lymphocytic leukemia (CLL), Richter transformation (RT) of CLL remains a major clinical challenge. Our group showed a selective response (~40%) with the PD-1 blocking antibody pembrolizumab in patients with RT, particularly after prior exposure to ibrutinib (Ding et al, Blood, 2017). It has been established that PD-1 inhibits cytotoxic T-cell functions via the interaction with its ligands, PD-L1 and PD-L2. However, the role of PD-1 in tumor B-cells is understudied. Here we show the expression and functional implications of the PD-1 signaling axis in B-cell pathobiology. Methods: CLL-involved lymph node (LN) (n=26) and RT (with diffuse large B-cell lymphoma [DLBCL] histology) -involved LN (n=20) samples were tested for PD-1 expression by immunohistochemistry (IHC) (mouse clone NAT105, Abcam). Then, PD-1 expression was examined in 10 lymphoma cell lines and 1 CLL-like cell line (MEC-1) by both flow cytometry (FC) and Western blot (WB) analysis. PD-1 overexpression using pLEX-lentiviral packaging system (Thermo Scientific) was evaluated for its impact on in vitro cell cycle regulation and in vivo tumor growth. CRISPR/CAS9 mediated PD-1 knockdown evaluated on cell growth in MEC-1 and Mino cells. Role of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Additionally, gene expression signatures were evaluated by RNA-seq using pLEX-lentiviral-PD-1 transfected DLBCL cell lines OCI-LY7 and OCI-LY19. Results: PD-1 expression was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p<0.001) as determined by IHC. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in tumor B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable by both FC and WB analysis, but at very low-levels in DLBCL cell lines OCI-LY7 and OCI-LY19. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold, respectively, compared to original lines after 4 days of culturing of equal number of cells), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% (p<0.05) and 24.5% (p<0.05) in OCI-LY7 and OCI-LY19 cells, respectively. When the luciferase+ PD-1 overexpressing OCI-LY7 cells (1X10 6) were injected intravenously into NSG mice, increased tumor growth (p<0.01) was observed by bioluminescence imaging after 22 days of follow-up. PD-1 knockdown in MEC-1 and Mino cells (high basal PD-1 expression) led to significant inhibition of cell growth (85% and 79.5%, respectively, p<0.05). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and Chk-2 (T68) in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. This finding was further confirmed by WB analyses. In addition, decreased total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. SHP-1 (TPI-1) and SHP-1+SHP-2 (TPI-1+TNO155) inhibitor treatment promoted OCI-LY19 cell death and led to recovery of phosphorylation on ATM, Chk-2 and p53 in these cells. RNA sequencing data in PD-1 overexpressing OCI-LY7 and OCI-LY19 cells revealed increased expression of genes that promote cell cycle progression and DNA replication. Conclusion: Our data showed robust PD-1 expression in patients with Richter transformation from CLL to DLBCL. PD-1 overexpression in DLBCL lymphoma cell lines enhanced cell proliferation in vitro and in vivo. Further investigation identified that PD-1 modified the phosphorylation/function of SHP phosphatases and thereby regulated p53 pathways ( Figure 1). These findings indicate that RT with DLBCL cells have the ability to “hijack” the PD-1 pathway which results in downregulation of p53 mediated DNA repair. This novel finding provides for new strategies to further evaluate the interaction of PD-1 signals with DNA repair pathways and suggest potential novel targets for RT.

Abstract T cell Ig and ITIM domain (TIGIT) is an immune checkpoint molecule and its ligation delivers an inhibitory signal to T cells that negatively regulates anti-tumor responses. However, the expression and biological relevance of TIGIT in B-cell non-Hodgkin lymphoma (NHL) is completely unknown. To phenotypically characterize TIGIT+ T cell subsets, we profiled TIGIT-expressing CD4+ and CD8+ T cells from biopsy specimens of B-cell NHL. TIGIT expression was not detectable on T cells from peripheral blood, however, TIGIT is constitutively expressed on CD4+ T cells from biopsy specimens of B-cell NHL with a median of 44.8% (range: 25.9–62.2, n=8) of CD4+ T cells expressing TIGIT. By profiling the CD4+TIGIT+ T cells, we observed that CD25 was highly expressed on intratumoral CD4+TIGIT+ T cells. Intracellular staining revealed that Foxp3 was expressed by intratumoral CD4+TIGIT+ T cells. These intratumoral CD4+TIGIT+ T cells expressed high level of chemokine receptor CCR4 and were absent of IL-7 receptor-α (CD127). Functionally, CD4+TIGIT+ T cells displayed reduced cytokine production, as the number of IFN-γ- and TNF-α-producing cells was lower in the TIGIT+ population than in TIGIT− T cells. While TIGIT expression can be induced by TCR activation, treatment of T cells with IL-2 and TGF-β inhibited TCR-mediated TIGIT induction. Furthermore, lymphoma B cells were involved in TIGIT induction as in vitro depletion of lymphoma B cells altered upregulation of TIGIT on CD4+ T cells in B-cell NHL. Taken together, these results indicated that constitutive expression of TIGIT defines a CD4+ population with similar phenotype to regulatory T cells. Inhibition of TIGIT signaling may be an additional mechanism to prevent T-cell suppression in B-cell NHL

Abstract Although extensively studied, the exact mechanism by which TGF-β mediates T-cell inhibition is not entirely clear. When we examined the effect of TGF-β on the phenotype and function of T cells, we noted that CD70 was significantly up-regulated by TGF-β. This surprising finding led us to explore the underlying mechanism by which TGF-β upregulates CD70 expression on T cells and to define the function of CD70+ cells in B-cell NHL. Interestingly, we observed that TGF-β preferentially induced CD70 expression on effector memory T cells while it predominantly up-regulated Foxp3 on naïve T cells. CD70 induction is Smad3-dependent and involves Stat5 signaling. CD70+ T cells were highly susceptible to TGF-β-mediated apoptosis and that transfection of Jurkat cells with the CD70 gene led to a significantly reduced survival. TGF-β-induced CD70+ T cells had a diminished response to immune stimulation associated with downregulation of co-stimulatory molecules CD27 and CD28. CD70-expressing T cells from biopsy specimens of patients with B-cell NHL failed to produce cytokines and displayed no signal transduction when stimulated. CD70-expressing T cells were highly represented in follicular NHL biopsy specimens and increased numbers of CD70+ cells correlated with an inferior patient outcome. These novel findings not only revealed a biological role for a TGF-β/CD70 axis in mediating immune inhibition, but also may lead to therapeutic approaches in patients with B-cell NHL.

ABSTRACT Introduction: Little is known regarding peripheral blood mononuclear cell telomere length (PBMC-TL) and response to traumatic injury. The objective of this study was to characterize the role of PBMC-TL in coagulation and clinical outcomes after injury. Methods: Plasma and buffy coats were prospectively collected from trauma patients and healthy volunteers. DNA was purified and PBMC-TL quantified by quantitative polymerase chain reaction. Thrombin generation kinetics were expressed as lag time (in minutes), peak height (in nanometers), time to peak (in minutes), and endogenous thrombin potential (in nM × min). Results are in median and quartiles [Q1, Q3]. P < 0.05 was considered significant (Wilcoxon rank sum testing). Results: Forty-two younger patients (21 [20, 22] years, 69% were male) and 39 older patients (62 [61, 64] years, 79% were male) were included. There was no significant difference in Clinical Frailty Scores between groups. Younger patients had longer total PBMC-TL (0.40 Mb [0.30, 0.49] vs. 0.29 Mb [0.23, 0.33], P < 0.001) and longer average PBMC-TL per chromosome (4.3 kb [3.3, 5.3] vs. 3.2 kb [2.5, 3.7], P < 0.001). When older patients were stratified by 50th percentile of PBMC-TL, there were no differences in thrombin generation; however, those with shorter telomeres were less likely to be discharged home (29% vs. 77%, P = 0.004). Older patients in the bottom quartile of PBMC-TL had shorter lag time (2.78 min [2.33, 3.00] vs. 3.33 min [3.24, 3.89], P = 0.030) and were less likely to be discharged home (22% vs. 90%, P = 0.006) than those in the top quartile of PBMC-TL. Multivariable logistic regression models revealed both increased age and shorter PBMC-TL to be independent predictors of discharge disposition other than home. Conclusion: In older trauma patients, shorter PBMC-TL is associated with accelerated initiation of thrombin generation and lower likelihood of being discharged to home.

Abstract The role and immunological constitution of the tumor microenvironment (TME) in splenic marginal zone lymphoma (sMZL) is largely unknown and studies on the phenotype and subset composition of intratumoral T cells are lacking. In the present study, we evaluated 36 sMZL spleen specimens by single cell analysis to gain a better understanding of the TME in sMZL. Using mass cytometry (CyTOF), we observed that the TME in sMZL is distinct from that of control non-malignant reactive spleen (rSP). We found that the number of TFH cells varied greatly in sMZL, ICOS+ TFH cells were more abundant in sMZL than rSP, and TFH cells positively correlated with increased numbers of memory B cells. Treg cell analysis revealed that TIGIT+ Treg cells are enriched in sMZL and correlate with suppression of TH17 and TH22 cells. Intratumoral CD8+ T cells were comprised of subsets of short-lived, exhausted and late-stage differentiated cells, thereby functionally impaired. We observed that T-cell exhaustion was present in sMZL and TIM-3 expression on PD-1low cells identified cells with severe immune dysfunction. Gene expression profiling by CITE-seq analysis validated this finding. Taken together, our data sugget that the TME as a whole, and T-cell population specifically, are heterogenous in sMZL and immune exhaustion is one of the major factors impairing T-cell function.

Introduction: PD-1 inhibits the cytotoxic T-cell functions via the interaction with its ligands (PDL1 and PDL2). Recently, our group showed a selective response (~40%) with PD-1 blocking antibody pembrolizumab in patients with Richter transformation (RT), particularly after prior exposure to ibrutinib (Wei Ding et al, Blood, 2017). Additionally, PD-1 showed an increased expression in tumor B-cells of patients with RT (Rong He et al, AJSP, 2018). Here we investigated the functional implications of the PD-1 signaling axis in lymphoma B-cell pathobiology. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN samples were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). Then, we checked PD-1 expression in 10 lymphoma cell lines and 1 CLL cell line (MEC-1) by both, flow cytometry and Western blot (WB) analysis. Over-expression of PD-1 using pLEX-lentiviral system (Thermo Scientific) was evaluated for in vitro cell cycle regulation and in vivo tumor growth. Overexpression of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Gene expression signatures in CLL and RT patients were also identified by Illumina-based RNA sequencing using Formalin-Fixed Paraffin-embedded (FFPE)-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p &lt; 0.001) [Fig 1A]. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in lymphoma B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable, but at very low-levels in lymphoma lines OCI-LY7 and OCI-LY19 (data not shown). Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold compared to original lines on day 4, respectively, Fig 1BI-II), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% and 24.53% in OCI-LY7 and OCI-LY19 cells (Fig 1B III), respectively. When the luciferase + PD-1 expressing OCI-LY7 cells were injected intravenously (1X106 cells) into NSG mice, increased tumor growth was observed at 22 days of follow up by bioluminescence imaging (p&lt;0.01, Fig 1C). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and CHK2 (T68) (data not shown). This finding was further confirmed by WB analyses (Fig1DI). In addition, decreased of total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY19 and OCI-LY17 cell lines (Fig 1DI). Immunoprecipitation experiments with anti-PD1 antibody showed that PD-1 was in the same complex with SHP-1 and SHP-2 in OCI-LY7 cells but only with SHP-2 in OCI-LY19 cells (Fig 1DII). In parallel, mRNA sequencing was performed on 11 nodal tissues from either RT (n=8) or progressive CLL (n=3) after they developed clinical progression. The expression of PD-1 (PDCD1) was positively correlated with the expression of SHP-1 (PTPN6, r=0.56) and Cyclin E1 (CCNE1, r=0.62), implicating a direct role of PD-1 in regulating cellular proliferation and the induction in phosphatase expression in RT and CLL. Conclusion: Our data support the notion that PD-1 overexpression in lymphoma cells modifies cell proliferation in vitro and in vivo and regulates the function of phosphatase SHP and p53- pathways. These findings also indicate that aggressive lymphoma or CLL leukemic cells have the ability to hijack the PD-1 pathway and may result in downregulation of the p53 mediated DNA repair. This novel information provide for new strategies to further evaluate the interaction of checkpoint signals with DNA repair pathway, and possibly provide novel targets for Richter's transformation. Disclosures Ding: alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; MEI Pharma: Membership on an entity's Board of Directors or advisory committees. Sakemura:Humanigen: Patents & Royalties. Parikh:Janssen: Honoraria, Research Funding; Merck: Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding; GlaxoSmithKline: Honoraria; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Verastem Oncology: Honoraria; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; TG Therapeutics: Research Funding. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Liu:Eisal: Research Funding; Genentech: Research Funding; GRAIL: Research Funding; Menarini Silicon Biosystems: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Tesaro: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ansell:Seattle Genetics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding; ADC Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Bristol Myers Squibb: Research Funding. Kenderian:Sunesis: Research Funding; Tolero: Research Funding; Torque: Consultancy; BMS: Research Funding; Gilead: Research Funding; Kite: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Novartis: Patents & Royalties, Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding; Juno: Research Funding. Kay:Acerta Pharma: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; MEI Pharma: Research Funding; Sunesis: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees.

Abstract Background: Multiple mechanisms exist for the positive selection of genes involved in oncogenesis such as gene amplification and transcriptional deregulation associated with chromosome translocations. Here we describe the case of a myeloma cell line that over time acquired extra copies of a derivative chromosome 4 involved in the t(4;14). These changes occurred over time both in vitro (in the generated JMW myeloma cell lines) as well as in vivo from extramedullary myeloma samples collected from the patient at various time points. In the case of the t(4;14)(p16;q32) 30% can be unbalanced translocations, always with loss of the der(14) and consequent loss of the FGFR3 gene expression, suggesting an indispensable role for MMSET located in the der(4). Results: We performed serial analysis on patient samples obtained at 9, 10 and 12 mos from diagnosis, along with a time course analysis of the cell line. At diagnosis the patient tumor cells displayed a complex clinical karyotype subsequently characterized by hypodiploidy, a t(4;14)(p16;q32), 13 monosomy and 17p13.1-. The remaining chromosome 13 and 17 were involved in an unbalanced reciprocal translocation. Notably, over time the cell line duplicated the total chromosome number becoming near-tetraploid. At the time of diagnosis, FISH analysis was performed on the patient sample revealing the translocation t(4;14)(p16.3:q32) in nearly all cells. The t(4;14) was also present in the stable cell line. It was noted that the patient cells exhibited more than one fusion signal per cell for the t(4;14) by FISH. As the disease progresses both in vitro and in vivo, the number of t(4;14)(p16.3:q32) fusion signals detected per cell increased with time suggesting a positive selection of the derivative chromosomes of the translocation. We further analyzed the t(4;14) in the JMW cells by FISH using MMSET/VH, and FGFR3/CH probe combinations. The full-length (MMSET/FGFR3+VH/CH) probe was used to detect the total number of fusions in the JMW cells. At the time of analysis (cell line passage #23), the majority of the cells (69%) had 6 fusion signals per cell while 23% had 5 fusions and 8% of the cells had 4 fusions. We found that most of the cells (88%) displayed 4 fusion signals detected using the MMSET/VH probes. In parallel studies using the FGFR3/CH probes, 61% of the cells had 2 fusion signals and 39% with 1 fusion signal. It is evident that the tumor cells display positive selection of the derivative chromosome der(4) of the t(4;14)(p16.3;q32), accompanied by an increased dosage of the target gene (MMSET) upregulated by the translocation. Conclusion: We present evidence for the amplification of the translocated chromosome in multiple myeloma, which we have termed “trans-amplicons”. Trans-amplicons may provide a selective growth and survival advantage to the myeloma tumor cells in an analogous manner to amplicons of DNA segments, but with much more powerful transcriptional consequences. Importantly, the evolution of genomic instability exemplified by the “trans-amplicons” in the patient cells was paralleled in vitro in the JMW cells.

Abstract Background: The p53 tumor suppressor gene has a common sequence polymorphism that results in either proline or arginine at amino-acid position 72. The Pro72 and Arg72 variants have been reported to differ in its functional activity. The Arg72 variant induces apoptosis more efficiently than Pro72 variant by its greater ability to localize to the mitochondria and increase the release of cytochrome c into the cytoplasm. In addition, recent studies have demonstrated that Arg72 variant was a better suppressor of cellular transformation, an activity believed to rely on the pro-apoptotic function of p53. Moreover, the Pro72 variant has been associated with a higher incidence of tumors. Aim: To evaluate the effect of the polymorphic variants of codon 72 and clinical outcome in patients with plasma cell malignancies. Patients and Methods: We analyzed 79 patients with plasma cell malignancies seen at Mayo Clinic between 2000 and 2003. Genomic DNA was extracted from CD138+ sorted plasma cell from bone marrow aspirates. PCR amplification of codon 72 and posterior sequencing was used for the analysis. Results: A total of 79 patients were analyzed. Thirty-four patients (34%) were heterozygous for p53, 57 (59%) homozygous for Arginine and 6 (6%) homozygous for Proline. The distribution among disorders was as follow, 52 patients had MM, 14 patients with SMM and 13 with MGUS. Among patient with MM 25% were Pro/Arg heterozygous, 71 % were Arg/Arg homozygous and 3.8% Pro/Pro homozygous. Survival data among MM patients and genotype were analyzed to Kaplan- Meier method. No significant differences were found among patients who carried homozygous (72R) or heterozygous genotype and overall survival. We exclude the analysis in MGUS and SMM for a low number of patients available. Conclusions: These findings indicate that codon 72 arginine p53 may not be associated with a prolonged survival in patients with Multiple Myeloma, but further study is needed to assess whether this polymorphism is associated with progression of MGUS to MM, age of onset, clinical manifestations, response to treatment etc.

Abstract Background: Plasma cell leukemia (PCL) is a rare plasma cell (PC) dyscrasia which may be designated as primary (PPCL) or de novo PCL when recognized at the time of diagnosis and secondary (SPCL) when there is leukemic transformation of a previously recognized multiple myeloma (MM). PCL has been reported to exhibit distinct clinical and immunophenotypic features that distinguish it from MM, but little is known about the specific genetics of this disease. To better understand the genetic features of the clonal PC from PPCL and SCPL we assessed in these patients the molecular and cytogenetic abnormalities most commonly found in MM. Patients and Methods: In our study we analyzed 3 groups of patients; 18 with PPCL, 23 with SPCL and a control group of 345 newly diagnosed MM previously published. Diagnostic criteria for PCL followed the presence of &gt;2 x103/L PC in PB (Kyle et al. Arch Intern Med1974; 133–813–8). Cytogenetic abnormalities were assessed by the c-Ig FISH method; mutational analysis was performed using Conformational-sensitive gel electrophoresis (CSGE). Methylation was analyzed by methyl specific PCR (MSP) after bisulfite genomic DNA modification. Results: Translocation of immunoglobulin heavy chain with Cyclin D1 (t (11; 14)) was present in 76.9% (10 of 13) of PPCL, 71% (5 of 7) of SPCL, in contrast to 15.8% (53 of 336) of MM cases. Translocations’ involving the FGFR3-MMSET genes (4p16.3 locus) and c-MAF (16q32) genes were not observed among 10 patients studied with PPCL, but were present in 12.7 % each in SPCL. The t (4;14)(p16;q32) was present in 12.7% cases of MM and the t(14;16)(q32;q23) was present in 4.6 % of MM cases. Deletion of 17p13.1 (p53 locus) was found in 10% (37 of 345) of MM cases, 53.8% (7 of 13) for PPCL and 42.8% (3 of 7) for SPCL. Deletion of 13q was present in 54.2% (176 of 325) of MM, 76.9 % (10 of 13) of PPCL and 57.1% of SPCL. Ras mutations were found in 15% (2/13) of PPCL, located in codon 1 and 2 of the K-ras gene. In addition, 23% for SPCL presented mutations in the N-ras gene. Two mutations were located in codon 2 of N-ras and 1 patients in showed a mutation in codon 1 of K-ras gene. Ras mutations were present in 27% of MM cases. Mutations for p53 gene were present in 30.7% (4/13) of PPCL, 17% (3/17) for SPCL and 5% for MM. Methylation specific PCR for p16 gene was found in 23% (3/13) of PPCL, 29% of SPCL and 33.9% (149/439) of MM. Hypermethylation of p14 was not detected in PPCL but a 23 % was found in SPCL. Conclusions: In this study we demonstrate that PCs from PPCL more frequently carry the t (11; 14). Comparing SPCL and MM, PC’s from de novo PPCL do not show t (4; 14) or (16; 14). Deletion of 13q was more prevalent among patients with PPCL. Ras mutations appear to be as frequent in MM as in PPCL and SPCL. Mutations in p53 appeared to be more prevalent in PPCL than in SPCL and MM. Hypermethylation of p16 does not differ while hypermethylation of p14 is more frequent in SPCL than PPCL. These characteristics may lead to a different onset or disease evolution of PPCL compared to MM and secondary plasma cell leukemia and this may be important for diagnostic and treatment.