EBV-Positive and EBV-Negative Posttransplant Diffuse Large B Cell Lymphomas Have Distinct Genomic and Transcriptomic Features.

The molecular pathogenesis of posttransplant diffuse large B cell lymphoma (PT-DLBCL) is largely unknown. We have recently shown that Epstein-Barr virus-positive (EBV(+)) and -negative (EBV(-)) PT-DLBCL have distinct gene expression profiles, and the transcriptomic profile of EBV(-) PT-DLBCL is similar to that of DLBCL in immunocompetent individuals (IC-DLBCL). To validate these observations at the genomic level, we performed array-comparative genome hybridization (aCGH) analysis of 21 EBV(+) PT-DLBCL, 6 EBV(-) PT-DLBCL, and 11 control IC-DLBCL, and subsequently combined genomic and transcriptomic data. The analysis showed that EBV(+) and EBV(-) PT-DLBCL have distinct aCGH profiles and shared only one recurrent imbalance. EBV(-) PT-DLBCL, however, displayed at least 10 aberrations recurrent in IC-DLBCL, among which characteristic gain of 3/3q and 18q, and loss of 6q23/TNFAIP3 as well as 9p21/CDKN2A. The most prevalent aberration in EBV(+) PT-DLBCL was gain/amplification of 9p24.1 targeting PDCD1LG2/PDL2. Our data indicate that the FOXP1 oncogene and the tumor suppressor CDKNA2 implicated in EBV(-) DLBCL, do not play a critical role in the pathogenesis of EBV(+) PT-DLBCL. Altogether, genomic profiling of PT-/IC-DLBCL confirms that EBV(-) and EBV(+) PT-DLBCL are distinct entities, while EBV(-) PT-DLBCL has features in common with IC-DLBCL. These findings support the hypothesis that EBV(-) PT-DLBCL are de novo lymphomas in transplant recipients.

Mycobacterium genavense infection in a solid organ recipient: a diagnostic and therapeutic challenge.

Renal transplant recipients are highly susceptible to infections caused by uncommon pathogens because of their immunocompromised state. We report a case of disseminated Mycobacterium genavense infection in a patient with a combined renal and cardiac transplant. Diagnosing M. genavense infections remains a challenge because of the absence of specific clinical symptoms in combination with the difficulties of culturing the organism using standard mycobacterial culture procedures. This clinical case demonstrates the importance of molecular techniques as part of the initial work-up in order to rapidly establish the diagnosis.

Microsatellite instable vs stable colon carcinomas: analysis of tumour heterogeneity, inflammation and angiogenesis.

BACKGROUND: Microsatellite instability (MSI) accounts for 15% of all colorectal tumours. Several specific clinicopathologicals (e.g., preference for the proximal colon over the distal colon, improved prognosis and altered response to chemotherapeutics) are described for this subset of tumours. This study aimed to analyse morphological, inflammatory and angiogenic features of MSI vs microsatellite stable (MSS) tumours. METHODS: Twenty-seven MSS and 29 MSI, TNM stage matched, colorectal tumours were selected from the archive of the Department of Pathology, UZ Leuven. Morphology was analysed on haematoxylin-eosin sections. Immunohistochemistry for CD3, CD4, CD8, CD20 and CD68 was used to map tumour infiltration in both a digital and traditional microscope-based manner for all distinct morphological components of the tumour. CD31 immunostains were performed to assess angiogenesis. RESULTS: Morphological tumour heterogeneity was a marked feature of MSI tumours, occurring in 53% of the cases as compared with 11% of the MSS tumours (P<0.001). Digital immune quantification showed an increased number of tumour-infiltrating cytotoxic T-lymphocytes (CD8+) in MSI compared with MSS tumours for both the tumour (P=0.02) and peritumoural area (P=0.03). Traditional microscope-based quantification confirmed these results (P<0.001 for both) and, in addition, revealed large numbers of CD68+ macrophages in the peritumoural area of MSI cancers (P=0.001). Moreover, traditional microscope-based analysis was able to distinguish between lymphocytes directly infiltrating the tumoural glands (intra-epithelial) and those infiltrating only the neoplastic stroma around the glands (intratumoural). Quantification showed high numbers of intra-epithelial CD3+, CD4+, CD8+, CD20+ and CD68+ cells in MSI compared with MSS cancers (P<0.001, P=0.01, P<0.001, P<0.001 and P=0.006, respectively). Higher microvessel density (MVD) was observed in MSI tumours compared with their MSS counterpart. CONCLUSIONS: Mixed morphology, reflecting tumour heterogeneity, is an important feature of MSI tumours and may have both diagnostic and therapeutic impact. The inflammatory reaction also presented with significant differences in MSI vs MSS colorectal tumours. MSI cancers showed mainly infiltration by cytotoxic T-cells in both the tumour and the close border around the tumour, as well as increased intra-epithelial infiltration in contrast to MSS tumours. The type of immune cell and the compartment it resides in (intratumoural or intra-epithelial) depend both on MSI status and morphology. Finally, MSI tumours showed a higher angiogenic capacity represented by an increased MVD, hinting for possible therapeutic consequences.

Single-Centre 20-Year Experience with Surgical Treatment of Thymic Tumours.

BACKGROUND: Large single-centre institutional series on thymic tumours are rare. Complete resection remains the mainstay of successful treatment. Characteristics and survival were reviewed in all patients treated between 19932013. METHODS: Hospital databases revealed 134 patients with pathologically-proven thymic tumour. Follow-up (median 63 months) was through patient notes and telephone contact with general practitioner. RESULTS: Patients were classified in Masaoka-Koga stages: I: 50 (37%); Ila: 14 (10%); lib: 14 (10%); III: 27 (20%); IVa: 19 (14%); IVb: 4 (3%); unknown: 6 (5%). According to WHO classification, pathological subtypes were A: 19 (14%); AB: 25 (19%); B1: 21 (16%); B2: 31 (23%); B3: 15 (11%); thymic carcinoma: 23 (17%). Parathymic syndromes were diagnosed in 45 patients: myasthenia gravis (84%); pure red-cell aplasia (4%); hypogammaglobulinemia (2%); and others. 124 patients (93%) underwent surgery with complete resection in 104 (84%). Surgical approach was: sternotomy: 79; thoracotomy: 35; cervicotomy: 2; other/unknown: 8. In 73 patients (59%) no biopsy was taken prior to surgical resection, 25 were treated with induction chemotherapy, 36 received adjuvant radiotherapy. Hospital mortality was 0.81%. 35 patients died during follow-up (13 of tumour or treatment-related causes). Overall and recurrence-free survival at 5, 10, and 15 years were 86%; 64%; 47% and 67%; 49%; and 31%, respectively and were significantly (p < 0.01) different according to Masaoka-Koga stage. There was a significant association between WHO classification and Masaoka-Koga stages I-IIa-IIb versus III-IVa-IVb (p < 0.01). CONCLUSIONS: Operability and complete resectability of thymic tumours in our experience is high resulting in prolonged overall and recurrence-free survival. Masaoka-Koga stage is an important predictor for survival and shows a significant association with WHO classification.

Single-centre 20-year experience with surgical treatment of thymic tumours.

BACKGROUND: Large single-centre institutional series on thymic tumours are rare. Complete resection remains the mainstay of successful treatment. Characteristics and survival were reviewed in all patients treated between 1993-2013. METHODS: Hospital databases revealed 134 patients with pathologically-proven thymic tumour. Follow-up (median 63 months) was through patient notes and telephone contact with general practitioner. RESULTS: Patients were classified in Masaoka-Koga stages: I: 50 (37%); IIa: 14 (10%); IIb: 14 (10%); III: 27 (20%); IVa: 19 (14%); IVb: 4 (3%); unknown: 6 (5%). According to WHO classification, pathological subtypes were A: 19 (14%); AB: 25 (19%); B1: 21 (16%); B2: 31 (23%); B3: 15 (11%); thymic carcinoma: 23 (17%). Parathymic syndromes were diagnosed in 45 patients : myasthenia gravis (84%); pure red-cell aplasia (4%); hypogammaglobulinemia (2%); and others. 124 patients (93%) underwent surgery with complete resection in 104 (84%). Surgical approach was: sternotomy: 79; thoracotomy: 35; cervicotomy: 2; other/unknown: 8. In 73 patients (59%) no biopsy was taken prior to surgical resection, 25 were treated with induction chemotherapy, 36 received adjuvant radiotherapy. Hospital mortality was 0.81%. 35 patients died during follow-up (13 of tumour or treatment-related causes). Overall and recurrence-free survival at 5, 10, and 15 years were 86%; 64%; 47% and 67%; 49%; and 31%, respectively and were significantly (p < 0.01) different according to Masaoka-Koga stage. There was a significant association between WHO classification and Masaoka-Koga stages I-IIa-IIb versus III-IVa-IVb (p < 0.01). CONCLUSIONS: Operability and complete resectability of thymic tumours in our experience is high resulting in prolonged overall and recurrence-free survival. Masaoka-Koga stage is an important predictor for survival and shows a significant association with WHO classification.

Gene expression profiling reveals clear differences between EBV-positive and EBV-negative posttransplant lymphoproliferative disorders.

Posttransplant patients are at risk of developing a potentially life-threatening posttransplantation lymphoproliferative disorder (PTLD), most often of diffuse large B cell lymphoma (DLBCL) morphology and associated with Epstein-Barr Virus (EBV) infection. The aim of this study was to characterize the clinicopathological and molecular-genetic characteristics of posttransplant DLBCL and to elucidate whether EBV(+) and EBV(-) posttransplant DLBCL are biologically different. We performed gene expression profiling studies on 48 DLBCL of which 33 arose posttransplantation (PT-DLBCL; 72% EBV+) and 15 in immunocompetent hosts (IC-DLBCL; none EBV+). Unsupervised hierarchical analysis showed clustering of samples related to EBV-status rather than immune status. Except for decreased T cell signaling these cases were inseparable from EBV(-) IC-DLBCL. In contrast, a viral response signature clearly segregated EBV(+) PT-DLBCL from EBV(-) PT-DLBCL and IC-DLBCL cases that were intermixed. The broad EBV latency profile (LMP1+/EBNA2+) was expressed in 59% of EBV(+) PT-DLBCL and associated with a more elaborate inflammatory response compared to intermediate latency (LMP1+/EBNA2-). Inference analysis revealed a role for innate and tolerogenic immune responses (including VSIG4 and IDO1) in EBV(+) PT-DLBCL. In conclusion we can state that the EBV signature is the most determining factor in the pathogenesis of EBV(+) PT-DLBCL.

Molecular pathogenesis of B-cell posttransplant lymphoproliferative disorder: what do we know so far?

Posttransplant lymphoproliferative disorder (PTLD) is a potentially fatal disease that arises in 2%-10% of solid organ and hematopoietic stem cell transplants and is most frequently of B-cell origin. This very heterogeneous disorder ranges from benign lymphoproliferations to malignant lymphomas, and despite the clear association with Epstein-Barr Virus (EBV) infection, its etiology is still obscure. Although a number of risk factors have been identified (EBV serostatus, graft type, and immunosuppressive regimen), it is currently not possible to predict which transplant patient will eventually develop PTLD. Genetic studies have linked translocations (involving C-MYC, IGH, BCL-2), various copy number variations, DNA mutations (PIM1, PAX5, C-MYC, RhoH/TTF), and polymorphisms in both the host (IFN-gamma, IL-10, TGF-beta, HLA) and the EBV genome to B-cell PTLD development. Furthermore, the tumor microenvironment seems to play an important role in the course of disease representing a local niche that can allow antitumor immune responses even in an immunocompromised host. Taken together, B-cell PTLD pathogenesis is very complex due to the interplay of many different (patient-dependent) factors and requires thorough molecular analysis for the development of novel tailored therapies. This review aims at giving a global overview of the currently known parameters that contribute to the development of B-cell PTLD.

Pitfall in the diagnosis of endometrial cancer: case report of an endometrioid adenocarcinoma arising from uterine adenomyosis.

BACKGROUND: The development of cancer from adenomyotic foci is a rare occurrence. The diagnosis is frequently delayed because of the absence of tumor in the eutopic endometrium. CASE REPORT: We present a case of a 64-year-old postmenopausal woman with irregular vaginal bleeding and dull abdominal pain. Hysteroscopy was negative and hormonal treatment was continued. Nine months later, persisting symptoms necessitated endometrial biopsy revealing an atrophic endometrium. Hydrosonography suggested an endometrial polyp of 14 x 7 mm with a surrounding regular thin endometrium and a diffusely inhomogeneous ultrasonographic pattern throughout the myometrium. Hysteroscopic excision of the endometrial polyp was performed. Biopsies obtained during operative hysteroscopy showed a well differentiated endometrioid endometrial carcinoma. A laparoscopically assisted vaginal hysterectomy with bilateral salpingo-oophorectomy, pelvic lymphadenectomy and peritoneal cytology was performed. Pathologic examination revealed an atrophic endometrium and a Stage IB (FIGO 2009) well differentiated endometrioid endometrial carcinoma with prominent squamous differentiation originating from nodular adenomyosis. This ectopic localization of the endometrioid carcinoma added to a diagnostic delay of 12 months. CONCLUSION: Endometrial cancer arising from uterine adenomyosis may be difficult to diagnose. Awareness of this entity and careful ultrasonography are likely to reduce diagnostic delay.

Semi-Quantitative Characterization of Post-Transplant Lymphoproliferative Disorder Morphological Subtypes with [18F]FDG PET/CT.

Background: Post-transplant lymphoproliferative disorder (PTLD) is a complication of organ transplantation classified according to the WHO as nondestructive, polymorphic, monomorphic, and classic Hodgkin Lymphoma subtypes. In this retrospective study, we investigated the potential of semi-quantitative 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) PET/computed tomography (CT)-based parameters to differentiate between the PTLD morphological subtypes. Methods: 96 patients with histopathologically confirmed PTLD and baseline [18F]FDG PET/CT between 2009 and 2019 were included. Extracted semi-quantitative measurements included: Maximum, peak, and mean standardized uptake value (SUVmax, SUVpeak, and SUVmean). Results: Median SUVs were highest for monomorphic PTLD followed by polymorphic and nondestructive subtypes. The median SUVpeak at the biopsy site was significantly higher in monomorphic PTLD (17.8, interquartile range (IQR):16) than in polymorphic subtypes (9.8, IQR:13.4) and nondestructive (4.1, IQR:6.1) (p = 0.04 and p ≤ 0.01, respectively). An SUVpeak ≥ 24.8 was always indicative of a monomorphic PTLD in our dataset. Nevertheless, there was a considerable overlap in SUV across the different morphologies. Conclusion: The median SUVpeak at the biopsy site was significantly higher in monomorphic PTLD than polymorphic and nondestructive subtypes. However, due to significant SUV overlap across the different subtypes, these values may only serve as an indication of PTLD morphology, and SUV-based parameters cannot replace histopathological classification.

A novel patient-derived tumorgraft model with TRAF1-ALK anaplastic large-cell lymphoma translocation.

Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kB (NFkB) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NFkB signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies.

Lymph node biopsies in a general internal medicine department: algorithm or individualized decision-making?

BACKGROUND: Lymphadenopathy (LA) imposes a diagnostic challenge in internal medicine. Exclusion of malignancy is the primary concern. METHODS: A retrospective case series, including 40 adult patients from the general internal medicine department who underwent lymph node biopsy (LNB) at a single university hospital. Demographics, clinical data and histopathological diagnoses were registered. By means of the latest medical record, we obtained a final diagnosis for each patient and subsequently searched for variables correlated with malignancy. Follow-up was at least one year in 95% of cases. RESULTS: The prevalence of malignancy was 58%. Older age (p = 0.02) was significantly correlated with malignancy. The presence of painful lymphadenopathy at clinical examination (p = 0.02) was significantly associated with a benign outcome. No single or combination of baseline variables satisfactorily excluded malignancy. Histopathological analysis correctly predicted malignancy in 93% of cases. In two cases, an initial diagnosis of benign non-specific lymphadenopathy was reversed to non-Hodgkin lymphoma. In one case the pathological diagnosis was inconclusive. CONCLUSION: Rather than following a universal algorithm to determine the need for LNB in patients with LA, we call for individualized decision-making in each case, carefully appreciating all available information. Additionally, one should keep in mind that false-negative results occur due to sampling errors. Therefore, a minimal number of cases should end with a final diagnosis of benign non-specific lymphadenopathy. Intensive, multidisciplinary cooperation with surgeon and pathologist is needed. Moreover, clinical follow-up should be at least one year.

Subclinical GvHD in non-irradiated F1 hybrids: severe lymphoid-tissue GvHD causing prolonged immune dysfunction.

GvHD is an important complication of allogeneic hematopoietic SCT. Parent-in-F1 models are frequently used to study GvHD immunobiology; the characteristics of parent-in-F1 GvHD vary between strain combinations and induction protocols. Here, we observed that a high-dose challenge of non-irradiated B6DBA2F1 and B6SJLF1 recipients with C57BL/6 splenocytes left the majority of recipients clinically healthy, while inducing progressive high-grade donor T-cell chimerism. We investigated this previously undescribed pattern of parent-in-F1 T-cell alloreactivity and studied the effect of serial parental splenocyte infusions on epithelial and lymphohematopoietic tissues. The majority of recipients of 4 weekly splenocyte infusions showed long-term survival with gradual establishment of high-grade donor chimerism and without any signs of epithelial-tissue GvHD. A minority of recipients showed BM failure type of GvHD and, respectively, graft rejection. Moreover, long-term F1 chimeras showed protracted pancytopenia, and in peripheral lymphoid tissues severe lymphopenia and near-complete eradication of APCs and dysfunction in antigen-presenting capacity in remaining APC. Hematopoiesis and lymphoid tissue composition recovered only after multilineage donor chimerism had established. In conclusion, we report on a novel type of parent-in-F1 hybrid GvHD, where a cumulative high dose of C57BL/6 parental splenocytes in non-irradiated F1 mice induces subclinical but severe hematolymphoid-tissue GvHD, causing prolonged immuno-incompetence.