MYC protein is a high-risk factor in mantle cell lymphoma and identifies cases beyond morphology, proliferation and TP53/p53 - a Nordic Lymphoma Group study.

The transcription factor MYC is a well-described oncogene with an important role in lymphomagenesis, but its significance for clinical outcome in mantle cell lymphoma (MCL) remains to be determined. We performed an investigation of the expression of MYC protein in a cohort of 251 MCL patients complemented by analyses of structural aberrations and mRNA, in a sub-cohort of patients. Fourteen percent (n=35) of patients showed high MYC protein expression with >20% positive cells (MYChigh), among whom only one translocation was identified, and 86% (n=216) of patients showed low MYC protein expression. Low copy number gains of MYC were detected in ten patients, but with no correlation to MYC protein levels. However, MYC mRNA levels correlated significantly to MYC protein levels with a R2 value of 0.76. Patients with a MYChigh tumor had both an independent inferior overall survival and an inferior progression-free survival (hazard ratio [HR]=2.03, 95% confidence interval [95% CI]: 1.2-3.4 and HR=2.2, 95% CI: 1.04-4.6, respectively) when adjusted for additional high-risk features. Patients with MYChigh tumors also tended to have additional high-risk features and to be older at diagnosis. A subgroup of 13 patients had concomitant MYChigh expression and TP53/p53 alterations and a substantially increased risk of progression (HR=16.9, 95% CI: 7.4-38.3) and death (HR=7.8, 95% CI: 4.4-14.1) with an average overall survival of only 0.9 years. In summary, we found that at diagnosis a subset of MCL patients (14%) overexpressed MYC protein, and had a poor prognosis but that MYC rearrangements were rare. Tumors with concurrent MYC overexpression and TP53/p53 alterations pinpointed MCL patients with a dismal prognosis with a median overall survival of less than 3 years. We propose that MYC needs to be assessed beyond the current high-risk factors in MCL in order to identify cases in need of alternative treatment.

Checkpoint CD47 expression in classical Hodgkin lymphoma.

The glycoprotein CD47 regulates antiphagocytic activity via signal regulatory protein alpha (SIRPa). This study investigated CD47 expression on Hodgkin and Reed-Sternberg (HRS) cells in the classical Hodgkin lymphoma (cHL) tumour microenvironment and its correlation with prognosis, programmed-death (PD) immune markers, and SIRPa+ leukocytes. We conducted immunohistochemistry with CD47 and SIRPa antibodies on diagnostic biopsies (tissue microarrays) from cHL patients from two cohorts (n = 178). In cohort I (n = 136) patients with high expression of CD47 on HRS cells (n = 48) had a significantly inferior event-free survival [hazard ratio (HR) = 5.57; 95% confidence interval (CI), 2.78-11.20; p < 0.001] and overall survival (OS) (HR = 8.54; 95% CI, 3.19-22.90; p < 0.001) compared with patients with low expression (n = 88). The survival results remained statistically significant in multivariable Cox regression adjusted for known prognostic factors. In cohort II (n = 42) high HRS cell CD47 expression also carried shorter event-free survival (EFS) (HR = 5.96; 95% CI, 1.20-29.59; p = 0.029) and OS (HR = 5.61; 95% CI, 0.58-54.15; p = 0.136), although it did not retain statistical significance in the multivariable analysis. Further, high CD47 expression did not correlate with SIRPa+ leukocytes or PD-1, PD-L1 and PD-L2 expression. This study provides a deeper understanding of the role of CD47 in cHL during an era of emerging CD47 therapies.

Superior outcome for splenectomised patients in a population-based study of splenic marginal zone lymphoma in Sweden.

Splenic marginal zone lymphoma (SMZL) is a rare low-grade B-cell lymphoma where associations with viral hepatitis and autoimmune and inflammatory diseases (AID) have been indicated. We aimed at assessing the prevalence of viral hepatitis and AID at SMZL diagnosis and outcome by treatment in a Swedish population-based study. A total of 277 SMZL patients registered in the Swedish Lymphoma Register in 2007-2017 were included. A history of viral hepatitis was reported in five (2%) patients and AID prior to SMZL in 72/240 (30%) patients. Treatment was given up front for 207 (75%) patients. Splenectomy with or without systemic treatment was performed in 119 (57%) and was associated with statistically significantly better overall survival [hazard ratio, HR = 0·47 (95% confidence interval, CI: 0·23-0·93), P = 0·03] and progression-free survival (HR = 0·55, 95% CI: 0·35-0·86, P = 0·008) compared to non-splenectomised patients in multivariable analyses. The up-front splenectomised group was younger and generally had a lower Ann Arbor stage, but also more frequently B symptoms and high lactate dehydrogenase than the non-splenectomised group. Viral hepatitis and AID history did not affect SMZL outcome. We report high incidence of AIDs and low incidence of viral hepatitis in this population-based study of SMZL. Splenectomy up front was associated with a favourable outcome.

Infiltration of CD163-, PD-L1- and FoxP3-positive cells adversely affects outcome in patients with mantle cell lymphoma independent of established risk factors.

We characterised patients with mantle cell lymphoma (MCL) with poor prognosis based on differences in immune infiltration. Different expressions of the tumour cell markers Cyclin D1 and sex-determining region Y-box transcription factor 11 (SOX11), and the immune markers cluster of differentiation 3 (CD3), CD4, CD8, CD25, forkhead box protein P3 (FoxP3), T-box transcription factor TBX21 (T-bet), programmed cell death protein 1 (PD-1), programmed-death ligand 1 (PD-L1) and CD163 were investigated for all-cause mortality in 282 patients with MCL and time-to-progression (TTP) in 106 clinical trial patients. With increasing age, a significantly lower infiltration of CD3+ T lymphocytes was seen. T-cell infiltration was independent of cellular tumour antigen p53 (p53) expression, Ki-67, morphology and frequency of tumour cells. The all-cause mortality was higher in patients with PD-L1-expression above cut-off [hazard ratio (HR) 1·97, 95% confidence interval (CI) 1·18-3·25, adjusted for sex and MCL International Prognostic Index (MIPI)] and a higher frequency of CD163+ cells (continuously, HR 1·51, 95% CI 1·03-2·23, adjusting for age, sex, morphology, Ki-67 and p53). In patients treated within the Nordic Lymphoma Group MCL2/3 trials, TTP was shorter in patients with a higher frequency of FoxP3+ cells (HR 3·22, 95% CI 1·40-7·43) and CD163+ cells (HR 6·09, 95% CI 1·84-20·21), independent of sex and MIPI. When combined a higher frequency of CD163+ macrophages and PD-L1+ cells or high CD163+ macrophages and FoxP3+ regulatory T cells indicated worse outcome independent of established risk factors. The T-cell infiltrate was in turn independent of molecular characteristics of the malignant cells and decreased with age.

PD-L1 and IDO1 are potential targets for treatment in patients with primary diffuse large B-cell lymphoma of the CNS.

BACKGROUND: Programmed cell death 1 (PD-1) and its ligands PD-L1 and PD-L2, as well as Indoleamine 2,3-deoxygenase (IDO1) can be expressed both by tumor and microenvironmental cells and are crucial for tumor immune escape. We aimed to evaluate the role of PD-1, its ligands and IDO1 in a cohort of patients with primary diffuse large B-cell lymphoma of the CNS (PCNSL). MATERIAL AND METHODS: Tissue microarrays (TMAs) were constructed in 45 PCNSL cases. RNA extraction from whole tissue sections and RNA sequencing were successfully performed in 33 cases. Immunohistochemical stainings for PD-1, PD-L1/paired box protein 5 (PAX-5), PD-L2/PAX-5 and IDO1, and Epstein-Barr virus encoding RNA (EBER) in situ hybridization were analyzed. RESULTS: High proportions of PD-L1 and PD-L2 positive tumor cells were observed in 11% and 9% of cases, respectively. High proportions of PD-L1 and PD-L2 positive leukocytes were observed in 55% and 51% of cases, respectively. RNA sequencing revealed that gene expression of IDO1 was high in patients with high proportion of PD-L1 positive leukocytes (p = .01). Protein expression of IDO1 in leukocytes was detected in 14/45 cases, in 79% of these cases a high proportion of PD-L1 positive leukocytes was observed. Gene expression of IDO1 was high in EBER-positive cases (p = .0009) and protein expression of IDO1 was detected in five of six EBER-positive cases. CONCLUSION: Our study shows a significant association between gene and protein expression of IDO1 and protein expression of PD-L1 in the tumor microenvironment of PCNSL, possibly of importance for prediction of response to immunotherapies.

Prognostic impact of abdominal lymph node involvement in diffuse large B-cell lymphoma.

OBJECTIVE: The prognostic value of site of nodal involvement in diffuse large B-cell lymphomas (DLBCL) is mainly unknown. We aimed to determine the prognostic significance of nodal abdominal involvement in relation to tumour cell markers and clinical characteristics of 249 DLBCL patients in a retrospective single-centre study. METHODS: Contrast-enhanced computed tomography (CT) of the abdomen and thorax revealed pathologically enlarged abdominal lymph nodes in 156 patients, while in 93 patients there were no pathologically enlarged lymph nodes in the abdomen. In 81 cases, the diagnosis of DLBCL was verified by histopathological biopsy obtained from abdominal lymph node. RESULTS: Patients with abdominal nodal disease had inferior lymphoma-specific survival (P = .04) and presented with higher age-adjusted IPI (P < .001), lactate dehydrogenase (P < .001) and more often advanced stage (P < .001), bulky disease (P < .001), B symptoms (P < .001), and double expression of MYC and BCL2 (P = .02) compared to patients without nodal abdominal involvement, but less often extranodal involvement (P < .02). The worst outcome was observed in those where the abdominal nodal involvement was verified by histopathological biopsy. CONCLUSION: Diffuse large B-cell lymphomas patients with abdominal nodal disease had inferior outcome and more aggressive behaviour, reflected both in clinical and biological characteristics.

Cell-of-origin determined by both gene expression profiling and immunohistochemistry is the strongest predictor of survival in patients with diffuse large B-cell lymphoma.

The tumor cells in diffuse large B-cell lymphomas (DLBCL) are considered to originate from germinal center derived B-cells (GCB) or activated B-cells (ABC). Gene expression profiling (GEP) is preferably used to determine the cell of origin (COO). However, GEP is not widely applied in clinical practice and consequently, several algorithms based on immunohistochemistry (IHC) have been developed. Our aim was to evaluate the concordance of COO assignment between the Lymph2Cx GEP assay and the IHC-based Hans algorithm, to decide which model is the best survival predictor. Both GEP and IHC were performed in 359 homogenously treated Swedish and Danish DLBCL patients, in a retrospective multicenter cohort. The overall concordance between GEP and IHC algorithm was 72%; GEP classified 85% of cases assigned as GCB by IHC, as GCB, while 58% classified as non-GCB by IHC, were categorized as ABC by GEP. There were significant survival differences (overall survival and progression-free survival) if cases were classified by GEP, whereas if cases were categorized by IHC only progression-free survival differed significantly. Importantly, patients assigned as non-GCB/ABC both by IHC and GEP had the worst prognosis, which was also significant in multivariate analyses. Double expression of MYC and BCL2 was more common in ABC cases and was associated with a dismal outcome. In conclusion, to determine COO both by IHC and GEP is the strongest outcome predictor to identify DLBCL patients with the worst outcome.

CD30 expression and survival in posttransplant lymphoproliferative disorders.

Background: Post-transplant lymphoproliferative disorder (PTLD) is a rare but life-threatening complication of transplantation. For refractory and relapsed PTLD new therapies are needed, such as the antibody-drug conjugate brentuximab vedotin that targets CD30. There is limited knowledge of CD30 expression in various subtypes of PTLD and its correlation to clinicopathological features. Therefore, we studied the expression of CD30 in PTLD following solid organ transplantation and correlated CD30 expression to PTLD subtype, Epstein-Barr virus (EBV)-status, intratumoral regulatory T-cells (Tregs), clinical features, and outcome.Methods: We included 50 cases of PTLD from a nation-wide study of PTLDs following solid organ transplantation in Sweden. The tumor biopsies were reevaluated, and clinical data were collected. CD30 expression on tumor cells was analyzed by immunohistochemistry with the clone Ber-H2. Thirty-one cases were stained with clone 236 A/E7 for detection of forkhead box protein 3 (FoxP3, a Treg biomarker).Results: The case series consisted of 6% polymorphic, 88% monomorphic, and 6% Hodgkin lymphoma-like PTLDs and 53% of the cases were EBV+. Overall, 70% (35/50) of the PTLDs were CD30+ (≥1% CD30+ tumor cells) and 30% (15/50) were CD30-. All polymorphic PTLDs (n = 3) and Hodgkin lymphomas (n = 3), 88% (14/16) of non-germinal center type of diffuse large B-cell lymphoma (DLBCL), and 75% (9/12) of T-cell PTLDs were CD30+ whereas all germinal center-type of DLBCL (n = 5) and Burkitt type PTLD (n = 2) were CD30-. CD30+ PTLD tended to be EBV+ more frequently (p = .07) and occurred earlier posttransplant (2.1 vs. 8.2 years, p = .01) than CD30- PTLD. Type of transplant and localization of the tumor did not differ between the groups except that CNS engagement was more common in CD30- PTLD (p = .02). CD30-status was not associated with presence of intratumoral Tregs or overall survival.Conclusion: Expression of CD30 varied with PTLD subtype. There was no association between CD30 and survival, regardless of subtype.

High tumour plasma cell infiltration reflects an important microenvironmental component in classic Hodgkin lymphoma linked to presence of B-symptoms.

Plasma cells are important prognostic actors in different malignancies. The tumour microenvironmental composition in classic Hodgkin lymphoma (cHL) is a major prognostic key element; however, clinicopathological studies regarding plasma cells in cHL are lacking. The aim of this study was to investigate CD138+ (also termed SDC1+) plasma cell and IgG4 producing (IgG4+) plasma cells infiltration in the microenvironment of cHL. Immunohistochemistry with anti-CD138 and IgG4 antibodies was performed on diagnostic tumour biopsies from 124 patients with cHL, on tissue micro array (TMA). In 120 cases, CD138+ plasma cell-infiltration was associated with the presence of B-symptoms (P = 0·028) and advanced stage, IIB-IVB (P = 0·009). In multivariate analysis, CD138+ plasma cells correlated with eosinophil infiltration (P = 0·013). The subgroup of IgG4+ plasma cells was analysed in 122 cases and only correlated to CD138+ plasma cells (P = 0·004). Patients with high proportion of tumour infiltrating CD138+ plasma cells (defined as ≥10%), had a more inferior event-free survival (P = 0·007) and overall survival (P = 0·004) than patients with a low proportion of infiltrating CD138+ plasma cells (<10%), although significance was not maintained in multivariate analysis. In summary, a high proportion of tumour-associated plasma cells in cHL reflect an important component in the microenvironment of cHL.

An anergic immune signature in the tumor microenvironment of classical Hodgkin lymphoma is associated with inferior outcome.

OBJECTIVE: The classical Hodgkin lymphoma (cHL) tumor microenvironment shows an ongoing inflammatory response consisting of varying degrees of infiltrating eosinophils, mast cells, macrophages, regulatory T lymphocytes (Tregs), and activated lymphocytes surrounding the malignant cells. Herein, different immune signatures are characterized and correlated with treatment outcome. METHODS: Tumor-infiltrating leukocytes were phenotyped in biopsies from 459 patients with cHL. Time to progression (TTP) (primary progression, relapse, or death from cHL) and overall survival were analyzed using Cox proportional hazards regression. RESULTS: The leukocyte infiltration in the microenvironment was highly diverse between patients and was categorized in 4 immune signatures (active, anergic, innate, or mixed). A high proportion of Tregs (anergic) resulted in shorter TTP (median 12.9-year follow-up) in age-adjusted analyses (hazard ratio = 1.82; 95% confidence interval 1.05-3-15). Epstein-Barr virus (EBV)-positive cases had higher proportions of macrophages and activated lymphocytes than EBV negative, but neither of those leukocytes predicted prognosis. CONCLUSIONS: Abundant Tregs (anergic signature) indicate a shorter TTP, particularly in younger patients. This is probably due to a reduced ability of the immune system to attack the tumor cells. Our data warrant further investigation if these suggested immune signatures could predict outcome of immunotherapy such as immune checkpoint inhibitors.

Autoimmune and Atopic Disorders and Risk of Classical Hodgkin Lymphoma.

Results from previous investigations have shown associations between the risk of Hodgkin lymphoma (HL) and a history of autoimmune and atopic diseases, but it remains unknown whether these associations apply to all types of HL or only to specific subtypes. We investigated immune diseases and the risk of classical HL in a population-based case-control study that included 585 patients and 3,187 controls recruited from October 1999 through August 2002. We collected information on immune diseases through telephone interviews and performed serological analyses of specific immunoglobulin E reactivity. Tumor Epstein-Barr virus (EBV) status was determined for 498 patients. Odds ratios with 95% confidence intervals were calculated using logistic regression analysis. Rheumatoid arthritis was associated with a higher risk of HL (odds ratio (OR) = 2.63; 95% confidence interval (CI): 1.47, 4.70), especially EBV-positive HL (OR = 3.18; 95% CI: 1.23, 8.17), and with mixed-cellularity HL (OR = 4.25; 95% CI: 1.66, 10.90). HL risk was higher when we used proxies of severe rheumatoid arthritis, such as ever having received daily rheumatoid arthritis medication (OR = 3.98; 95% CI: 2.08, 7.62), rheumatoid arthritis duration of 6-20 years (OR = 3.80; 95% CI: 1.72, 8.41), or ever having been hospitalized for rheumatoid arthritis (OR = 7.36; 95% CI: 2.95, 18.38). Atopic diseases were not associated with the risk of HL. EBV replication induced by chronic inflammation in patients with autoimmune diseases might explain the higher risk of EBV-positive HL.

CD163+ macrophages in mantle cell lymphoma induce activation of prosurvival pathways and immune suppression.

ABSTRACT: Mantle cell lymphoma (MCL) is dependent on a supportive tumor immune microenvironment (TIME) in which infiltration of CD163+ macrophages has a negative prognostic impact. This study explores how abundance and spatial localization of CD163+ cells are associated with the biology of MCL, using spatial multiomic investigations of tumor and infiltrating CD163+ and CD3+ cells. A total of 63 proteins were measured using GeoMx digital spatial profiling in tissue microarrays from 100 diagnostic MCL tissues. Regions of interest were selected in tumor-rich and tumor-sparse tissue regions. Molecular profiling of CD163+ macrophages, CD20+ MCL cells, and CD3+ T-cells was performed. To validate protein profiles, 1811 messenger RNAs were measured in CD20+ cells and 2 subsets of T cells. Image analysis was used to extract the phenotype and position of each targeted cell, thereby allowing the exploration of cell frequencies and cellular neighborhoods. Proteomic investigations revealed that CD163+ cells modulate their immune profile depending on their localization and that the immune inhibitory molecules, V-domain immunoglobulin suppressor of T-cell activation and B7 homolog 3, have higher expression in tumor-sparse than in tumor-rich tissue regions and that targeting should be explored. We showed that MCL tissues with more abundant infiltration of CD163+ cells have a higher proteomic and transcriptional expression of key components of the MAPK pathway. Thus, the MAPK pathway may be a feasible therapeutic target in patients with MCL with CD163+ cell infiltration. We further showed the independent and combined prognostic values of CD11c and CD163 beyond established risk factors.

Expression of IDO1 and PD-L2 in Patients with Benign Lymphadenopathies and Association with Autoimmune Diseases.

The expression patterns of IDO1 and PD-L2 have not been thoroughly investigated in benign lymphadenopathies. The aim with this study was to elucidate how IDO1 and PD-L2 are expressed in benign lymphadenopathies in patients with autoimmune diseases (AD) compared to patients without AD. Formalin-fixed paraffin-embedded lymph nodes from 22 patients with AD and 57 patients without AD were immunohistochemically stained to detect IDO1 and PD-L2. The material was previously stained with EBER in situ hybridization to detect cells harboring the Epstein-Barr virus (EBV). IDO1 and PD-L2 were generally expressed by leukocytes to low degrees, while follicular IDO1+ cells were very rare. IDO1+ cells in single germinal centers were detected in five patients, and there was a high co-occurrence of follicular EBV+ cells in these cases (three of five patients). There were also significant correlations between interfollicular EBV+ cells and interfollicular IDO1+ cells (Spearman rho = 0.32, p = 0.004) and follicular IDO1+ cells (Spearman rho = 0.34, p = 0.004). High or low amounts of IDO1+ or PD-L2+ cells were not statistically significantly associated with patients with AD. However, the lymphadenopathy with the highest amount of interfollicular IDO1+ cells, which was also the only lymphadenopathy in which endothelial cells expressed IDO1, was in a patient with sarcoidosis. This study further supports that the EBV induces the expression of IDO1 and our findings should be recognized by future studies on IDO1 and PD-L2 in inflammatory and malignant conditions.

No difference in component migration at five years between the cemented cruciate-retaining ATTUNE and PFC-Sigma knee prosthesis: an update of a randomized clinical radiostereometry trial.

Aims: Conflicting clinical results are reported for the ATTUNE Total Knee Arthroplasty (TKA). This randomized controlled trial (RCT) evaluated five-year follow-up results comparing cemented ATTUNE and PFC-Sigma cruciate retaining TKAs, analyzing component migration as measured by radiostereometric analysis (RSA), clinical outcomes, patient-reported outcome measures (PROMs), and radiological outcomes. Methods: A total of 74 primary TKAs were included in this single-blind RCT. RSA examinations were performed, and PROMs and clinical outcomes were collected immediate postoperatively, and at three, six, 12, 24, and 60 months' follow-up. Radiolucent lines (RLLs) were measured in standard anteroposterior radiographs at six weeks, and 12 and 60 months postoperatively. Results: At five-year follow-up, RSA data from 61 patients were available and the mean maximum total point motion (MTPM) of the femoral components were: ATTUNE: 0.96 mm (95% confidence interval (CI) 0.79 to 1.14) and PFC-Sigma 1.37 mm (95% CI 1.18 to 1.59) (p < 0.001). The PFC-Sigma femoral component migrated more in the first postoperative year, but stabilized thereafter. MPTM of the tibial components were comparable at five-year follow-up: ATTUNE 1.12 mm (95% CI 0.95 to 1.31) and PFC-Sigma 1.25 mm (95% CI 1.07 to 1.44) (p = 0.438). RLL at the medial tibial implant-cement interface remained more prevalent for the ATTUNE at five-year follow-up compared to the PFC-Sigma (20% vs 3%). RLL did not progress over time, and varied between patients at different timepoints for both TKA systems. Clinical outcomes and PROMs improved compared with preoperative scores, and were not different between groups. Conclusion: MTPM migration at five-year follow-up of the femoral and tibial component of the ATTUNE were similar and as low as that of the PFC-Sigma. MTPM migration of both knee implants did not significantly change from one year post-surgery, indicating stable fixation. Long-term ATTUNE performance may be expected to be comparable to the clinically well-performing PFC-Sigma. We have not found evidence of increased tibial component migration as measured by RSA to support concerns about cement debonding and a higher risk of aseptic loosening with the ATTUNE TKA.

Soluble CD163 predicts outcome in both chemoimmunotherapy and targeted therapy-treated mantle cell lymphoma.

The outcome for patients with mantle cell lymphoma (MCL) has drastically improved with new treatments directed toward the tumor immune microenvironment, where macrophages play an important role. In MCL, the presence of M2 macrophages defined by CD163 expression in diagnostic biopsies has been associated with a worse prognosis. An alternative way to assess the abundance of M2 macrophages is by measuring the level of soluble CD163 in serum (sCD163). We aimed to investigate the prognostic value of sCD163 in 131 patients with MCL. We found that high sCD163 at diagnosis was associated with shorter progression-free survival (PFS) and shorter overall survival (OS) in 81 patients who were newly diagnosed and subsequently treated with chemoimmunotherapy. The same was seen in a cohort of 50 patients with relapsed MCL that were mainly treated within the phase 2 Philemon-trial with rituximab, ibrutinib, and lenalidomide. In patients who were newly diagnosed and had low levels of sCD163, 5-year survival was 97%. There was a moderate correlation between sCD163 and tissue CD163. The association with a poor prognosis was independent of MCL international prognostic index, Ki67, p53 status, and blastoid morphology, as assessed in a multivariable Cox proportional hazards model. In this study, high sCD163 was associated with both shorter PFS and shorter OS, showing that high levels of the M2 macrophage marker sCD163 is an independent negative prognostic factor in MCL, both in the chemoimmunotherapy and ibrutinib/lenalidomide era. In addition, low sCD163 levels identify patients with MCL with a very good prognosis.

LymphoTrack Is Equally Sensitive as PCR GeneScan and Sanger Sequencing for Detection of Clonal Rearrangements in ALL Patients.

Monoclonal rearrangements of immunoglobulin (Ig) genes and T-cell receptor (TCR) genes are used for minimal measurable disease in acute lymphoblastic leukemia (ALL). The golden standard for screening of gene rearrangements in ALL has been PCR GeneScan and Sanger sequencing, which are laborsome and time-consuming methods. More rapid next-generation sequencing methods, such as LymphoTrack could possibly replace PCR GeneScan and Sanger sequencing for clonality assessment. Our aim was to evaluate to what extent LymphoTrack can replace PCR GeneScan and Sanger sequencing concerning sensitivity and quantifiability in clonality assessment in 78 ALL samples. With LymphoTrack, clonality assessment was based on the %Total reads, where ≥10% was used as cut off for clonal rearrangements. The patients displayed 0 to 4 clonal rearrangements per assay. The detection rate (rearrangements detected with PCR GeneScan and/or Sanger sequencing, also detected with LymphoTrack) was 85/85 (100%) for IGH, 64/67 (96%) for IGK, 91/93 (98%) for TCRG and 34/35 (97%) for TCRB. Our findings demonstrate that LymphoTrack was equally sensitive in detecting clonal rearrangements as PCR GeneScan and Sanger Sequencing. The LymphoTrack assay is reliable and therefore applicable for clonal assessment in ALL patients in clinical laboratories.

Expression of PD-1, PD-L1 and PD-L2 in Lymphomas in Patients with Pre-Existing Rheumatic Diseases-A Possible Association with High Rheumatoid Arthritis Disease Activity.

Current research seeks to identify subgroups of non-Hodgkin lymphoma (NHL) patients responsive to PD-1 blocking agents. Whether patients with pre-existing rheumatic diseases might constitute such a subgroup is unknown. We determined intratumoral expression of PD-1 and its ligands in lymphoma patients with pre-existing rheumatic diseases. We included 215 patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) or Sjögren's syndrome with subsequent lymphoma and 74 diffuse large B-cell lymphoma (DLBCL) controls without rheumatic disease. PD-1 and PD-ligand immunohistochemical markers were applied on tumor tissue microarrays. The number of PD-1+ tumor infiltrating leukocytes (TILs) and proportions of PD-L1+ and PD-L2+ tumor cells and TILs were calculated and correlated with clinical data. Expression of PD-L1 in tumor cells and TILs was highest in classical Hodgkin lymphoma and DLBCL. In DLBCLs, expression of PD-1 in TILs and PD-L1 in tumor cells was similar in RA, SLE and controls. In RA-DLBCL, high expression of PD-L1 in tumor cells was significantly more common in patients with the most severe RA disease and was associated with inferior overall survival in multivariable analysis.

Patients with autoimmune diseases have an altered activity of the PD-1 pathway and proportions of Epstein-Barr virus infected cells in benign lymphadenopathies.

Patients with autoimmune diseases (AD) have an increased risk to develop benign lymphadenopathies compared to patients without AD. The aim with this study was to determine the role of the PD-1 pathway and the number of cells harboring Epstein-Barr virus (EBV) in benign lymphadenopathies in patients with AD (cases) compared to patients without AD (controls). Pathology registries were screened to identify patients with biopsies diagnosed as benign lymphadenopathy and medical journals were reviewed for information on AD. Immunohistochemical stainings (PD-1 and PD-L1) and EBER in situ hybridization for EBV were applied on lymph node biopsies in patients with AD (n = 22) and patients without AD (n = 57). The case group was compared with the control group with Wilcoxon-signed rank, chi-square and Fischers exact test. There was a statistically significantly higher proportion of PD-1+ cells and a tendency for a lower prevalence of PD-L1+ and EBV+ cells in cases compared to controls. Apparently, patients with AD have an altered immune response as revealed in benign lymphadenopathies compared to patients without AD. If this association might be a piece of the puzzle for the increased risk of development of lymphomas in patients with AD remains to be determined.