Multi-scale spatial modeling of immune cell distributions enables survival prediction in primary central nervous system lymphoma.

To understand the clinical significance of the tumor microenvironment (TME), it is essential to study the interactions between malignant and non-malignant cells in clinical specimens. Here, we established a computational framework for a multiplex imaging system to comprehensively characterize spatial contexts of the TME at multiple scales, including close and long-distance spatial interactions between cell type pairs. We applied this framework to a total of 1,393 multiplex imaging data newly generated from 88 primary central nervous system lymphomas with complete follow-up data and identified significant prognostic subgroups mainly shaped by the spatial context. A supervised analysis confirmed a significant contribution of spatial context in predicting patient survival. In particular, we found an opposite prognostic value of macrophage infiltration depending on its proximity to specific cell types. Altogether, we provide a comprehensive framework to analyze spatial cellular interaction that can be broadly applied to other technologies and tumor contexts.

Large B-cell Lymphomas of Immune-Privileged Sites Relapse via Parallel Clonal Evolution from a Common Progenitor B Cell.

Large B-cell lymphoma of immune-privileged sites (LBCL-IP) arise in immune sanctuaries including the testis and central nervous system (CNS). After initially reaching complete response, relapses occur in almost 50% of patients, typically at other immune-privileged sites. Resolution of the clonal relationships and evolutionary patterns of LBCL-IP is required to understand the unique clinical behavior. We collected a unique set of 33 primary-relapse LBCL-IP sample pairs and performed next-generation sequencing for copy number, mutation, translocation, and immunoglobulin clonality analysis. All LBCL-IP sample pairs were clonally related, and both tumors developed from a common progenitor cell (CPC) with MYD88 and TBL1XR1 mutations and/or BCL6 translocations in 30/33 cases, indicating that these are early genetic events. This was succeeded by intermediate genetic events including shared, as well as unique alterations in targets of aberrant somatic hypermutation (aSHM), CD79B mutations, and 9p21.3/CDKN2A loss. Genetic alterations in genes involved in immune escape (HLA, CD274/PDCD1LG2) were predominantly unique in primary and relapse samples and thus considered late genetic events. Together, this study indicates that primary and relapsed LBCL-IP follow an early parallel evolutionary pattern where the CPC contains genetic alterations that support prolonged survival/proliferation and retention in a memory B-cell state, followed by germinal center reentry, aSHM and immune escape. SIGNIFICANCE: Genomic analyses reveal that primary and relapse LBCL-IP originate from a common progenitor cell with a small set of genetic alterations, followed by extensive parallel diversification, elucidating the clonal evolution of LBCL-IP.

ALL-tRNAseq enables robust tRNA profiling in tissue samples.

Transfer RNAs (tRNAs) are small adaptor RNAs essential for mRNA translation. Alterations in the cellular tRNA population can directly affect mRNA decoding rates and translational efficiency during cancer development and progression. To evaluate changes in the composition of the tRNA pool, multiple sequencing approaches have been developed to overcome reverse transcription blocks caused by the stable structures of these molecules and their numerous base modifications. However, it remains unclear whether current sequencing protocols faithfully capture tRNAs existing in cells or tissues. This is specifically challenging for clinical tissue samples that often present variable RNA qualities. For this reason, we developed ALL-tRNAseq, which combines the highly processive MarathonRT and RNA demethylation for the robust assessment of tRNA expression, together with a randomized adapter ligation strategy prior to reverse transcription to assess tRNA fragmentation levels in both cell lines and tissues. Incorporation of tRNA fragments not only informed on sample integrity but also significantly improved tRNA profiling of tissue samples. Our data showed that our profiling strategy effectively improves classification of oncogenic signatures in glioblastoma and diffuse large B-cell lymphoma tissues, particularly for samples presenting higher levels of RNA fragmentation, further highlighting the utility of ALL-tRNAseq for translational research.

Pathology review identifies frequent misdiagnoses in recurrent classic Hodgkin lymphoma in a nationwide cohort: implications for clinical and epidemiological studies.

Patients treated for classic Hodgkin lymphoma (CHL) have a reported 13-fold increased risk of developing subsequent non-Hodgkin lymphoma (NHL). In light of the growing awareness of CHL mimickers, this study re-assesses this risk based on an in-depth pathology review of a nationwide cohort of patients diagnosed with CHL in the Netherlands (2006-2013) and explores the spectrum of CHL mimickers. Among 2,669 patients with biopsy-proven CHL, 54 were registered with secondary NHL. On review, CHL was confirmed in 25/54 patients. In six of these, the subsequent lymphoma was a primary mediastinal B-cell lymphoma/mediastinal gray zone lymphoma, biologically related to CHL and 19/25 were apparently unrelated B-cell NHL. In 29/54 patients, CHL was reclassified as NHL, including T-cell lymphomas with secondary Hodgkin-like B-blasts (n=15), Epstein Barr virus-positive diffuse large B-cell lymphoma (n=8), CD30+ T-cell lymphoma (n=3) and indolent B-cell proliferations (n=3). Higher age, disseminated disease at presentation, extensive B-cell marker expression and association with Epstein-Barr virus were identified as markers to alert for CHL mimickers. Based on these data, the risk of developing NHL after CHL treatment was re-calculated to 3.6-fold (standardized incidence ratio 3.61; confidence interval: 2.29-5.42). In addition, this study highlights the clinicopathological pitfalls leading to misinterpretation of CHL and consequences for the care of individual patients, interpretation of trials and epidemiological assessments.

Genomic and microenvironmental landscape of stage I follicular lymphoma, compared with stage III/IV.

Although the genomic and immune microenvironmental landscape of follicular lymphoma (FL) has been extensively investigated, little is known about the potential biological differences between stage I and stage III/IV disease. Using next-generation sequencing and immunohistochemistry, 82 FL nodal stage I cases were analyzed and compared with 139 FL stage III/IV nodal cases. Many similarities in mutations, chromosomal copy number aberrations, and microenvironmental cell populations were detected. However, there were also significant differences in microenvironmental and genomic features. CD8+ T cells (P = .02) and STAT6 mutations (false discovery rate [FDR] <0.001) were more frequent in stage I FL. In contrast, programmed cell death protein 1-positive T cells, CD68+/CD163+ macrophages (P < .001), BCL2 translocation (BCL2trl+) (P < .0001), and KMT2D (FDR = 0.003) and CREBBP (FDR = 0.04) mutations were found more frequently in stage III/IV FL. Using clustering, we identified 3 clusters within stage I, and 2 clusters within stage III/IV. The BLC2trl+ stage I cluster was comparable to the BCL2trl+ cluster in stage III/IV. The two BCL2trl- stage I clusters were unique for stage I. One was enriched for CREBBP (95%) and STAT6 (64%) mutations, without BLC6 translocation (BCL6trl), whereas the BCL2trl- stage III/IV cluster contained BCL6trl (64%) with fewer CREBBP (45%) and STAT6 (9%) mutations. The other BCL2trl- stage I cluster was relatively heterogeneous with more copy number aberrations and linker histone mutations. This exploratory study shows that stage I FL is genetically heterogeneous with different underlying oncogenic pathways. Stage I FL BCL2trl- is likely STAT6 driven, whereas BCL2trl- stage III/IV appears to be more BCL6trl driven.

Robust detection of translocations in lymphoma FFPE samples using targeted locus capture-based sequencing.

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.

Epcoritamab induces potent anti-tumor activity against malignant B-cells from patients with DLBCL, FL and MCL, irrespective of prior CD20 monoclonal antibody treatment.

Epcoritamab (DuoBody-CD3xCD20, GEN3013) is a novel bispecific IgG1 antibody redirecting T-cells toward CD20+ tumor cells. Here, we assessed the preclinical efficacy of epcoritamab against primary tumor cells present in the lymph node biopsies from newly diagnosed (ND) and relapsed/refractory (RR) B-NHL patients. In the presence of T-cells from a healthy donor, epcoritamab demonstrated potent activity against primary tumor cells, irrespective of prior treatments, including CD20 mAbs. Median lysis of 65, 74, and 84% were achieved in diffuse large B-cell lymphoma (n = 16), follicular lymphoma (n = 15), and mantle cell lymphoma (n = 8), respectively. Furthermore, in this allogeneic setting, we discovered that the capacity of B-cell tumors to activate T-cells was heterogeneous and showed an inverse association with their surface expression levels of the immune checkpoint molecule Herpesvirus Entry Mediator (HVEM). In the autologous setting, when lymph node (LN)-residing T-cells were the only source of effector cells, the epcoritamab-dependent cytotoxicity strongly correlated with local effector cell-to-target cell ratios. Further analyses revealed that LN-residing-derived or peripheral blood-derived T-cells of B-NHL patients, as well as heathy donor T-cells equally mediated epcoritamab-dependent cytotoxicity. These results show the promise of epcoritamab for treatment of newly-diagnosed or relapsed/refractory B-NHL patients, including those who became refractory to previous CD20-directed therapies.

Chromosome 20 loss is characteristic of breast implant-associated anaplastic large cell lymphoma.

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a very rare type of T-cell lymphoma that is uniquely caused by a single environmental stimulus. Here, we present a comprehensive genetic analysis of a relatively large series of BIA-ALCL (n = 29), for which genome-wide chromosomal copy number aberrations (CNAs) and mutational profiles for a subset (n = 7) were determined. For comparison, CNAs for anaplastic lymphoma kinase (ALK)- nodal anaplastic large cell lymphomas (ALCLs; n = 24) were obtained. CNAs were detected in 94% of BIA-ALCLs, with losses at chromosome 20q13.13 in 66% of the samples. Loss of 20q13.13 is characteristic of BIA-ALCL compared with other classes of ALCL, such as primary cutaneous ALCL and systemic type ALK+ and ALK- ALCL. Mutational patterns confirm that the interleukin-6-JAK1-STAT3 pathway is deregulated. Although this is commonly observed across various types of T-cell lymphomas, the extent of deregulation is significantly higher in BIA-ALCL, as indicated by phosphorylated STAT3 immunohistochemistry. The characteristic loss of chromosome 20 in BIA-ALCL provides further justification to recognize BIA-ALCL as a separate disease entity. Moreover, CNA analysis may serve as a parameter for future diagnostic assays for women with breast implants to distinguish seroma caused by BIA-ALCL from other causes of seroma accumulation, such as infection or trauma.

Treatment of patients with MYC rearrangement positive large B-cell lymphoma with R-CHOP plus lenalidomide: results of a multicenter HOVON phase II trial.

Patients with MYC-rearrangement positive large B-cell lymphoma (MYC+ LBCL) have an inferior prognosis following standard first-line therapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) as compared to patients without MYC rearrangement. Although intensive chemotherapy regimens yield higher remission rates, toxicity remains a concern. Lenalidomide is an oral immunomodulatory drug which downregulates MYC and its target genes thereby providing support using lenalidomide as additional therapeutic option for MYC+ LBCL. A phase II trial was conducted evaluating the efficacy of lenalidomide (15 mg day 1-14) in combination with R-CHOP (R2CHOP) in newly diagnosed MYC+ LBCL patients identified through a nationwide MYC-FISH screening program. The primary endpoint was complete metabolic response (CMR) on centrally reviewed 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)-computer tomography (CT)-scan at end-of-treatment. Secondary endpoints were overall survival (OS), disease-free survival (DFS) and event-free survival (EFS). Eighty-two patients with stage II-IV MYC+ LBCL were treated with 6 cycles of R2CHOP. At EOT, 67% (confidence interval (CI) 58-75%) of the patients reached CMR. With a median follow-up of 25.4 months, 2-year estimates (95% CI) for OS, DFS, EFS were 73% (62-82%), 75% (63-84%) and 63% (52-73%) respectively. In this prospective trial for newly diagnosed MYC+ LBCL patients, we found that administering R2CHOP was safe, and yields comparable CMR and survival rates as in studies applying more intensive chemotherapy regimens. Hence, these findings offer new prospects for MYC+ LBCL patients and warrant comparison in prospective randomized clinical trials. This trial was registered at www.clinicaltrialsregister.eu (#2014-002654-39).

Breast Implant Prevalence in the Dutch Female Population Assessed by Chest Radiographs.

BACKGROUND: Breast implant-related health problems are a subject of fierce debate. Reliable population-based estimates of implant prevalence rates are not available, however, due to a lack of historical registries and incomplete sales data, precluding absolute risk assessments. OBJECTIVES: This study aimed to describe the methodology of a novel procedure to determine Dutch breast implant prevalence based on the evaluation of routine chest radiographs. METHODS: The validity of the new method was first examined in a separate study. Eight reviewers examined a series of 180 chest radiographs with (n = 60) or without (n = 120) a breast implant confirmed by a computed tomography or magnetic resonance imaging scan. After a consensus meeting with best-performing expert reviewers, we reviewed 3000 chest radiographs of women aged 20 to 70 years in 2 large regional hospitals in the Netherlands in 2015. To calculate the national breast implant prevalence, regional prevalence variations were corrected utilizing the National Breast Cancer Screening Program. RESULTS: Eight reviewers scored with a median sensitivity of 71.7% (range, 41.7%-85.0%) and a median specificity of 94.6% (range, 73.4%-97.5%). After a consensus meeting and a reevaluation by best-performing expert reviewers, sensitivity was 79.9% and specificity was 99.2%. The estimated national prevalence of breast implants among women between 20 and 70 years was 3.0%, ranging from 1.7% at 21 to 30 years to 3.9% between 51 and 60 years. CONCLUSIONS: The novel method in this study was validated with a high sensitivity and specificity, resulting in accurate prevalence estimates and providing the opportunity to conduct absolute risk assessment studies on the health consequences of breast implants.

Rituximab-PECC induction followed by 90 Y-ibritumomab tiuxetan consolidation in relapsed or refractory DLBCL patients who are ineligible for or have failed ASCT: results from a phase II HOVON study.

Patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) after, or ineligible for, autologous stem cell transplantation (ASCT) have a dismal prognosis. This phase II study evaluated treatment with R-PECC (rituximab, prednisolone, etoposide, chlorambucil, lomustine), every 28 days for 4 cycles in 62 patients, followed by radio-immunotherapy consolidation with 90 Y-ibritumomab tiuxetan in responsive patients. Primary endpoints were failure-free survival (FFS) and incidence of grade ≥3 adverse events from start of 90 Y-ibritumomab tiuxetan. The overall response rate after R-PECC was 50%. Twenty-nine of 31 responsive patients proceeded to 90 Y-ibritumomab tiuxetan. Five out of 15 partial remission patients converted to complete remission after 90 Y-ibritumomab tiuxetan. One-year FFS and overall survival (OS) from start of 90 Y-ibritumomab tiuxetan was 52% (95% confidence interval [CI], 33-68%) and 62% (95% CI, 42-77%), respectively. One-year FFS and OS from start of R-PECC was 28% (95% CI, 17-39%) and 49% (95% CI, 36-61%), respectively. Toxicities of R-PECC and 90 Y-ibritumomab tiuxetan were mainly haematological. In conclusion, for relapsed DLBCL patients the largely oral R-PECC regimen achieves promising response rates, combined with an acceptable safety profile. Consolidation with 90 Y-ibritumomab tiuxetan resulted in long-term response durations in approximately one third of the patients that received it.

Interobserver variation in CD30 immunohistochemistry interpretation; consequences for patient selection for targeted treatment.

AIMS: CD30 immunohistochemistry (IHC) in malignant lymphoma is used for selection of patients in clinical trials using brentuximab vedotin, an antibody drug-conjugate targeting the CD30 molecule. For reliable implementation in daily practice and meaningful selection of patients for clinical trials, information on technical variation and interobserver reproducibility of CD30 immunohistochemistry (IHC) staining is required. METHODS AND RESULTS: We conducted a three-round reproducibility assessment of CD30 scoring for categorised frequency and intensity, including a technical validation, a 'live polling' pre- and post-instruction scoring round and a web-based round including individual scoring with additional IHC information to mimic daily diagnostic practice. Agreement in all three scoring rounds was poor to fair (κ = 0.12-0.35 for CD30-positive tumour cell percentage and κ = 0.16-0.41 for staining intensity), even when allowing for one category of freedom in percentage of tumour cell positivity (κ = 0.30-0.61). The first round with CD30 staining performed in five independent laboratories showed objective differences in staining intensity. In the second round, approximately half the pathologists changed their opinion on CD30 frequency after a discussion on potential pitfalls, highlighting hesitancy in decision-making. Using fictional cut-off points for percentage of tumour cell positivity, agreement was still suboptimal (κ = 0.35-0.60). CONCLUSIONS: Lack of agreement in cases with heterogeneous expression is shown to influence patient eligibility for treatment with brentuximab vedotin, both in clinical practice and within the context of clinical trials, and limits the potential predictive value of the relative frequency of CD30-positive neoplastic cells for clinical response.

Benign and malignant tumors in Rubinstein-Taybi syndrome.

Rubinstein-Taybi syndrome (RSTS) is a multiple congenital anomalies syndrome associated with mutations in CREBBP (70%) and EP300 (5-10%). Previous reports have suggested an increased incidence of specific benign and possibly also malignant tumors. We identified all known individuals diagnosed with RSTS in the Netherlands until 2015 (n = 87) and studied the incidence and character of neoplastic tumors in relation to their CREBBP/EP300 alterations. The population-based Dutch RSTS data are compared to similar data of the Dutch general population and to an overview of case reports and series of all RSTS individuals with tumors reported in the literature to date. Using the Nationwide Network and Registry of Histopathology and Cytopathology in the Netherlands (PALGA Foundation), 35 benign and malignant tumors were observed in 26/87 individuals. Meningiomas and pilomatricomas were the most frequent benign tumors and their incidence was significantly elevated in comparison to the general Dutch population. Five malignant tumors were observed in four persons with RSTS (medulloblastoma; diffuse large-cell B-cell lymphoma; breast cancer; non-small cell lung carcinoma; colon carcinoma). No clear genotype-phenotype correlation became evident. The Dutch population-based data and reported case studies underscore the increased incidence of meningiomas and pilomatricomas in individuals with RSTS. There is no supporting evidence for an increased risk for malignant tumors in individuals with RSTS, however, due to the small numbers this risk may not be fully dismissed.

Breast Implants and the Risk of Anaplastic Large-Cell Lymphoma in the Breast.

IMPORTANCE: Breast implants are among the most commonly used medical devices. Since 2008, the number of women with breast implants diagnosed with anaplastic large-cell lymphoma in the breast (breast-ALCL) has increased, and several reports have suggested an association between breast implants and risk of breast-ALCL. However, relative and absolute risks of breast-ALCL in women with implants are still unknown, precluding evidence-based counseling about implants. OBJECTIVE: To determine relative and absolute risks of breast-ALCL in women with breast implants. DESIGN, SETTING, AND PARTICIPANTS: Through the population-based nationwide Dutch pathology registry we identified all patients diagnosed with primary non-Hodgkin lymphoma in the breast between 1990 and 2016 and retrieved clinical data, including breast implant status, from the treating physicians. We estimated the odds ratio (OR) of ALCL associated with breast implants in a case-control design, comparing implant prevalence between women with breast-ALCL and women with other types of breast lymphoma. Cumulative risk of breast-ALCL was derived from the age-specific prevalence of breast implants in Dutch women, estimated from an examination of 3000 chest x-rays and time trends from implant sales. MAIN OUTCOMES AND MEASURES: Relative and absolute risks of breast-ALCL in women with breast implants. RESULTS: Among 43 patients with breast-ALCL (median age, 59 years), 32 had ipsilateral breast implants, compared with 1 among 146 women with other primary breast lymphomas (OR, 421.8; 95% CI, 52.6-3385.2). Implants among breast-ALCL cases were more often macrotextured (23 macrotextured of 28 total implants of known type, 82%) than expected (49 193 sold macrotextured implants of total sold 109 449 between 2010 and 2015, 45%) based on sales data (P < .001). The estimated prevalence of breast implants in women aged 20 to 70 years was 3.3%. Cumulative risks of breast-ALCL in women with implants were 29 per million at 50 years and 82 per million at 70 years. The number of women with implants needed to cause 1 breast-ALCL case before age 75 years was 6920. CONCLUSIONS AND RELEVANCE: Breast implants are associated with increased risk of breast-ALCL, but the absolute risk remains small. Our results emphasize the need for increased awareness among the public, medical professionals, and regulatory bodies, promotion of alternative cosmetic procedures, and alertness to signs and symptoms of breast-ALCL in women with implants.

Breast Implant-Associated Anaplastic Large-Cell Lymphoma in a Transgender Woman.

Breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL) is a rare but serious complication in patients with breast implants, Patients are at risk of BIA-ALCL whether they receive breast implants for cosmetic reasons or for reconstructive purposes after surgery for breast cancer or prophylactic mastectomy. During the past decade, an increased number of reports have addressed BIA-ALCL. Herein, we describe BIA-ALCL in a transgender woman. The patient received breast implants as part of her gender transition and was diagnosed with BIA-ALCL 20 years later. The patient underwent several revisional operations in the 20 years after her primary breast surgery to treat unexplained pain with low-grade fever, severe capsular contracture (Baker grade III-IV), and several instances of implant rupture. In July 2016, the patient presented to our office with "late-onset" periprosthetic seroma 5 years after her last revisional breast surgery. She was diagnosed with BIA-ALCL without capsular invasion based on results of cytologic analysis of the periprosthetic seroma and histologic evaluation of the periprosthetic capsule. This diagnosis was verified further by results of immunohistochemical testing, which indicated expression of CD30 and T-cell markers in the periprosthetic seroma only. Our intentions with this case report are to demonstrate that all patients who undergo breast implantation, including transgender women, are at risk of BIA-ALCL and to highlight the importance of cytomorphologic and immunohistochemical screening of seroma fluid in patients with late-onset periprosthetic seroma. LEVEL OF EVIDENCE: 5.