Epstein-Barr Virus and immune status imprint the immunogenomics of non-Hodgkin lymphomas occurring in immune-suppressed environments.

Non-Hodgkin lymphomas (NHL) commonly occur in immune-deficient (ID) patients, both HIV-infected and transplanted, and are often EBV-driven with cerebral localization, raising the question of tumor immunogenicity, a critical issue for treatment responses. We investigated the immunogenomics of 68 lymphoproliferative disorders from 51 ID (34 posttransplant, 17 HIV+) and 17 immunocompetent patients. Overall, 72% were Large B Cells Lymphoma (LBCL) and 25% were primary central-nervous-system lymphoma (PCNSL) while 40% were EBV-positive. Tumor whole-exome and RNA sequencing, along with a bioinformatics pipeline allowed analysis of tumor mutational burden (TMB), tumor landscape and microenvironment (TME) and prediction of tumor neoepitopes. Both TMB (2.2 vs 3.4/Mb, p=0.001) and neoepitopes numbers (40 vs 200, p=0.00019) were lower in EBVpositive than in EBV-negative NHL, regardless of the immune status. In contrast both EBV and the immune status influenced the tumor mutational profile, with HNRNPF and STAT3 mutations exclusively observed in EBV-positive and ID NHL, respectively. Peripheral blood T-cell responses against tumor neoepitopes were detected in all EBV-negative cases but in only half EBV-positive ones, including responses against IgH-derived MHC-class-II restricted neoepitopes. The TME analysis showed higher CD8 T cell infiltrates in EBVpositive vs EBV-negative NHL, together with a more tolerogenic profile composed of Tregs, type-M2 macrophages and an increased expression of negative immune-regulators. Our results highlight that the immunogenomics of NHL in patients with immunodeficiency primarily relies on the tumor EBV status, while T cell recognition of tumor- and IgH-specific neoepitopes is conserved in EBV-negative patients, offering potential opportunities for future T cell-based immune therapies.

Long-term outcome and prognosis of mixed histiocytosis (Erdheim-Chester disease and Langerhans Cell Histiocytosis).

Background: Erdheim-Chester disease (ECD) is a rare histiocytosis that may overlap with Langerhans Cell Histiocytosis (LCH). This "mixed" entity is poorly characterized. We here investigated the clinical phenotype, outcome, and prognostic factors of a large cohort of patients with mixed ECD-LCH. Methods: This retrospective study was performed at two referral centers in France and Italy (Pitié-Salpêtrière Hospital, Paris; Meyer Children's Hospital, Florence). We included children and adults with ECD diagnosed in 2000-2022 who had biopsy-proven LCH, available data on clinical presentation, treatment and outcome, and a minimum follow-up of one year. Outcomes included differences in clinical presentation and survival between mixed ECD-LCH and isolated ECD; we also investigated response to treatments and predictors of survival in the mixed cohort. Survival was analyzed using the Kaplan-Maier method and differences in survival with the long-rank test. Cox regression models were used to evaluate the potential impact of age and gender on survival and to identify predictors of non-response and survival. Findings: Out of a cohort of 502 ECD patients, 69 (14%) had mixed ECD-LCH. Compared to isolated ECD, mixed ECD-LCH occurred more frequently in females (51 vs. 26%, p < 0.001) and in patients with multisystem disease (≥4 sites). Mixed ECD-LCH more frequently involved long bones (91 vs. 79%, p = 0.014), central nervous system (51 vs. 34%, p = 0.007), facial/orbit (52 vs. 38%, p = 0.031), lungs (43 vs. 28%, p = 0.009), hypothalamic/pituitary axis (51 vs. 26%, p < 0.001), skin (61 vs. 29%, p < 0.001), and lymph nodes (15 vs. 7%, p = 0.028); the BRAFV600E mutation was also more frequent in mixed ECD-LCH (81 vs. 59%, p < 0.001). Targeted treatments (BRAF and/or MEK inhibitors) induced response more frequently than conventional therapies (interferon-α, chemotherapy), either as first-line (77 vs. 29%, p < 0.001) or as any line (75 vs. 24%, p < 0.001). After a median follow-up of 71 months, 24 patients (35%) died. Survival probability was comparable between ECD alone and mixed ECD-LCH (log-rank p = 0.948). At multivariable analysis, age at diagnosis (HR 1.052, 95% CI 1.008-1.096), associated hematologic conditions (HR 3.030, 95% CI 1.040-8.827), and treatment failure (HR 9.736, 95% CI 2.919-32.481) were associated with an increased risk of death, while lytic bone lesions with a lower risk (HR 0.116, 95% CI 0.031-0.432). Interpretation: Mixed ECD-LCH is a multisystem disease driven by the BRAFV600E mutation and targeted treatments are effective. Age at diagnosis, bone lesion patterns, associated hematologic conditions, and treatment failure are the main predictors of death in mixed ECD-LCH. Funding: None.

Altered X-chromosome inactivation predisposes to autoimmunity.

In mammals, males and females show marked differences in immune responses. Males are globally more sensitive to infectious diseases, while females are more susceptible to systemic autoimmunity. X-chromosome inactivation (XCI), the epigenetic mechanism ensuring the silencing of one X in females, may participate in these sex biases. We perturbed the expression of the trigger of XCI, the noncoding RNA Xist, in female mice. This resulted in reactivation of genes on the inactive X, including members of the Toll-like receptor 7 (TLR7) signaling pathway, in monocyte/macrophages and dendritic and B cells. Consequently, female mice spontaneously developed inflammatory signs typical of lupus, including anti-nucleic acid autoantibodies, increased frequencies of age-associated and germinal center B cells, and expansion of monocyte/macrophages and dendritic cells. Mechanistically, TLR7 signaling is dysregulated in macrophages, leading to sustained expression of target genes upon stimulation. These findings provide a direct link between maintenance of XCI and female-biased autoimmune manifestations and highlight altered XCI as a cause of autoimmunity.

Prognosis of a Heterogeneous TRG Pathological Response to Neoadjuvant Chemotherapy in Patients who Undergo Resection for Colorectal Liver Metastases.

BACKGROUND: Optimal management of colorectal liver metastasis (CRLM) is based on a combination of chemotherapy and surgical resection. The tumor regression grade (TRG) score is a histological scoring system to evaluate response to chemotherapy. The prognosis of a heterogeneous response in cases of multiple metastases has not been evaluated according to the TRG score. PATIENTS AND METHODS: All patients who underwent liver resection for multiple CRLM after neoadjuvant chemotherapy in two tertiary centers from January 2015 to April 2019 were retrospectively included. Oncological characteristics and outcome between TRG 1-2-3 (good response group), TRG 4-5 (poor response group) and heterogeneous TRG (good and poor TRG among different lesions within the same patient) groups were compared. RESULTS: Among the 327 patients included, 134 (41.0%) had good response (TRG 1-2-3), 120 (36.7%) had poor response (TRG 4-5), and 73 (22.3%) had heterogeneous response. The type and number of cycles of chemotherapy, k-Ras mutational status, and tumor number or size did not differ between the three groups. Use of irinotecan-based and anti-VEGF neoadjuvant therapy was associated with better TRG response [irinotecan-based: hazard ratio (OR) = 1.744; p = 0.045; anti-VEGF neoadjuvant therapy: 2.054; p = 0.005). Overall survival (OS) was higher in the 1-2-3 TRG group than in the heterogeneous TRG group (2-year OS = 81.3% vs. 60.3%, respectively; p = 0.003) and the 4-5 TRG group (2-year OS = 81.3% vs. 55.0%, respectively; p = 0.012) and similar between the heterogeneous and 4-5 TRG groups. CONCLUSIONS: The proportion of heterogeneous pathological response according to TRG is 22.3%, and the prognosis is comparable to that of poor pathological response.

Deep learning-based phenotyping reclassifies combined hepatocellular-cholangiocarcinoma.

Primary liver cancer arises either from hepatocytic or biliary lineage cells, giving rise to hepatocellular carcinoma (HCC) or intrahepatic cholangiocarcinoma (ICCA). Combined hepatocellular- cholangiocarcinomas (cHCC-CCA) exhibit equivocal or mixed features of both, causing diagnostic uncertainty and difficulty in determining proper management. Here, we perform a comprehensive deep learning-based phenotyping of multiple cohorts of patients. We show that deep learning can reproduce the diagnosis of HCC vs. CCA with a high performance. We analyze a series of 405 cHCC-CCA patients and demonstrate that the model can reclassify the tumors as HCC or ICCA, and that the predictions are consistent with clinical outcomes, genetic alterations and in situ spatial gene expression profiling. This type of approach could improve treatment decisions and ultimately clinical outcome for patients with rare and biphenotypic cancers such as cHCC-CCA.

Kidney dysfunction due to AA amyloidosis in a morbidly obese female.

Kidneys are commonly involved in systemic amyloidosis. Systemic AA amyloidosis is known to be associated with states of chronic inflammation such as autoimmune conditions, chronic infections, and malignancies. Obesity is increasingly recognized to be a risk factor for low-grade, chronic inflammation. We report a 48-year-old female with morbid obesity who presented with unexplained persistent mild kidney dysfunction and low-grade proteinuria. Attempt at evaluating the cause of kidney dysfunction included performing kidney biopsy despite technical challenges. Kidney biopsy showed AA amyloidosis with predominant vascular deposition, explaining the absence of nephrotic-range proteinuria. Evaluation for secondary causes of systemic AA amyloidosis was negative. While our patient was treated with sleeve gastrectomy for morbid obesity with reasonable response, it is likely that ongoing chronic inflammation, reflected by her laboratory markers, resulted in AA amyloidosis. Treatment with anakinra, an interleukin-1 antagonist, led to improvement in the laboratory markers in the next 6 months, and her kidney function remained stable. This report highlights an important cause of kidney dysfunction in morbid obesity, an atypical presentation of AA amyloidosis, and emphasizes the value of kidney biopsy in such patients.

Golgi localization of SARS-CoV-2 spike protein and interaction with furin in cerebral COVID-19 microangiopathy: a clue to the central nervous system involvement?

In a neuropathological series of 20 COVID-19 cases, we analyzed six cases (three biopsies and three autopsies) with multiple foci predominantly affecting the white matter as shown by MRI. The cases presented with microhemorrhages evocative of small artery diseases. This COVID-19 associated cerebral microangiopathy (CCM) was characterized by perivascular changes: arterioles were surrounded by vacuolized tissue, clustered macrophages, large axonal swellings and a crown arrangement of aquaporin-4 immunoreactivity. There was evidence of blood-brain-barrier leakage. Fibrinoid necrosis, vascular occlusion, perivascular cuffing and demyelination were absent. While no viral particle or viral RNA was found in the brain, the SARS-CoV-2 spike protein was detected in the Golgi apparatus of brain endothelial cells where it closely associated with furin, a host protease known to play a key role in virus replication. Endothelial cells in culture were not permissive to SARS-CoV-2 replication. The distribution of the spike protein in brain endothelial cells differed from that observed in pneumocytes. In the latter, the diffuse cytoplasmic labeling suggested a complete replication cycle with viral release, notably through the lysosomal pathway. In contrast, in cerebral endothelial cells the excretion cycle was blocked in the Golgi apparatus. Interruption of the excretion cycle could explain the difficulty of SARS-CoV-2 to infect endothelial cells in vitro and to produce viral RNA in the brain. Specific metabolism of the virus in brain endothelial cells could weaken the cell walls and eventually lead to the characteristic lesions of COVID-19 associated cerebral microangiopathy. Furin as a modulator of vascular permeability could provide some clues for the control of late effects of microangiopathy.

Neurolymphomatosis: involvement of peripheral nervous system revealing hematologic malignancy, a report of nine cases.

BACKGROUND AND AIM: Neurolymphomatosis is defined as an infiltration of the peripheral nervous system (PNS) by malignant lymphoma cells. It is a rare entity and diagnosis is complicated especially when PNS involvement is the initial and leading symptom. To improve knowledge of the disorder and shorten the time to diagnosis, we report a series of nine patients without a history of hematologic malignancy, who were diagnosed with neurolymphomatosis after evaluation and workup of peripheral neuropathy. METHODS: The patients were included from the Department of Clinical Neurophysiology at Pitié Salpêtrière and Nancy Hospitals over a period of 15 years. Diagnosis of neurolymphomatosis was confirmed by histopathologic examination for each patient. We characterized their clinical, electrophysiological, biological, imaging, and histopathologic features. RESULTS: The neuropathy was characterized by pain (78%), proximal involvement (44%) or of all four limbs (67%), asymmetrical or with multifocal distribution (78%), abundant fibrillation (78%), a tendency to worsen rapidly, and significant associated weight loss (67%). Neurolymphomatosis was diagnosed principally on nerve biopsy (89%) identifying infiltration of lymphoid cells, atypical cells (78%), a monoclonal population (78%), and supported by fluorodeoxyglucose-positron emission tomography, spine or plexus MRI, cerebrospinal fluid analysis, and blood lymphocyte immunophenotyping. Six patients had systemic disease and three impairment limited to the PNS. In the latter case, progression could be unpredictable and may be diffuse and explosive, sometimes occurring years after a seemingly indolent course. INTERPRETATION: This study provides better knowledge and understanding of neurolymphomatosis when neuropathy is the initial presentation.

OCT2 expression in histiocytoses.

Diagnosis of histiocytosis can be difficult and one of the biggest challenges is to distinguish between reactive and neoplastic histiocytes on histology alone. Recently, OCT2 nuclear expression was reported in Rosai-Dorfman disease (RDD). Our purpose was to expand the testing of OCT2 on a broader variety of sporadic or H syndrome-related histiocytoses. Cases of histiocytoses were retrieved from the files of Ambroise Paré Pathology Department. All slides and molecular analyses were reviewed, and staining was completed with immunohistochemistry for OCT2. A total of 156 samples from different localizations were tested. Among sporadic cases, 52 patients had RDD, and 10 patients had mixed histiocytosis combining RDD with Erdheim Chester disease (ECD, n = 8), Langerhans cell histiocytosis (LCH, n = 2) or juvenile xanthogranuloma (JXG, n = 1). All these patients were positive for OCT2 in RDD characteristic histiocytes. Twenty-three patients had ECD and all but two (91% - 21/23) were negative for OCT2. By contrast, OCT2 was positive in 11/27 (41%) LCH and 6/16 (38%) JXG. Among the 10 samples of H syndrome-associated histiocytosis, 3 had typical RDD histology, 6 had unclassified histiocytosis, and one had mixed RDD-LCH; all were positive for OCT2. On 16 samples of granulomatous lymphadenitis, OCT2 was negative in epithelioid histiocytes. Our study shows that OCT2 has a sensitivity of 100% for RDD cases and mixed histiocytoses with an RDD component. It is negative in 92% of ECD but expressed in at least 38% of LCH, JXG, and C group histiocytoses. Finally, OCT2 is positive in all H syndrome-related histiocytoses, independent of their histology.

Prevalence, patterns and outcomes of cardiac involvement in Erdheim-Chester disease.

AIMS: Cardiac involvement of Erdheim-Chester disease (ECD), a rare L group histiocytosis, has been reported to be associated with poor outcomes, but systematic studies are lacking. The present study aimed to investigate the prevalence, clinical features, imaging features, and prognosis of cardiac involvement in ECD in a large series. METHODS AND RESULTS: All patients with ECD who underwent cardiac magnetic resonance (CMR) imaging between 2003 and 2019 at a French tertiary center were retrospectively included. Primary outcome was all-cause mortality. Secondary outcomes were pericarditis, cardiac tamponade, conduction disorders, device implantation and coronary artery disease (CAD). A total of 200 patients were included [63 (54-71) years, 30% female, 58% BRAFV600E mutated]. Median follow-up was 5.5 years (3.3-9 years). On CMR, right atrioventricular sulcus infiltration was observed in 37% of patients, and pericardial effusion was seen in 24% of patients. In total, 8 patients (4%) had pericarditis (7 acute, 1 constrictive), 10 patients (5%) had cardiac tamponade, 5 patients (2.5%) had ECD-related high-degree conduction disorders, and 45 patients (23%) had CAD. Overall, cardiac involvement was present in 96 patients (48%) and was associated with BRAFV600E mutation [Odds ratio (OR) = 7.4, 95% confidence interval (CI) (3.5-16.8), P < 0.001] and ECD-related clinical events [OR = 5, 95%CI (1.5-21.2), P = 0.004] but not with lower survival in multivariate analysis [adjusted hazard ratio (HR) = 1.4, 95% CI (0.8-2.5), P = 0.2]. CONCLUSION: Cardiac involvement is present in nearly half of ECD patients and is associated with BRAFV600E mutation and complications (pericarditis, cardiac tamponade, and conduction disorders) but not with lower survival.

Clinical phenotypes and long-term outcome of kidney involvement in Erdheim-Chester histiocytosis.

Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis that frequently infiltrates the peri-kidney space ("hairy kidney" appearance), kidney pelvis and proximal ureters, leading to obstructive uropathy. Here, we analyzed the clinical characteristics, imaging findings and long-term kidney outcome of a large multicenter cohort comprising 195 consecutive patients with ECD. Retroperitoneal peri-kidney or peri-ureteral involvement was detected at diagnosis in 147 patients. Of them, 70 had hydronephrosis (bilateral in 47), and 16 with kidney atrophy (unilateral in 14). Kidney vascular peduncle infiltration was found in 60 patients, and kidney artery stenosis in 31. The estimated glomerular filtration rate (eGFR) at diagnosis was significantly lower in patients with than in those without peri-kidney involvement (median 74 vs. 98 mL/min/1.73 m2). Ureteral stenting often failed to achieve kidney function recovery. A total of 181 patients received medical therapies: first-line treatments included interferon-α (61%), BRAF-inhibitors (17%), mTOR-inhibitors (7%), or other drugs (15%). These therapies were efficacious for ECD but rarely induced kidney function improvement (one-year eGFR increase over 25% in under 10% of patients). After a median of 43 months, 19% of patients died and 5% developed kidney failure. Among patients with peri-kidney involvement, 44% developed chronic kidney disease (CKD) 3-5 at five years vs. 5% of those without. Unadjusted predictors of advanced CKD and kidney failure/death were age over 50 years, hypertension, BRAFV600E mutation, and baseline eGFR. At multivariable analysis, cardiovascular comorbidities were associated with advanced CKD, and age over 50 years with kidney failure/death. Thus, kidney involvement is common in ECD and can lead to CKD or kidney failure despite effective medical therapies or urological procedures.

Serum vascular endothelial growth factor is associated with cardiovascular involvement and response to therapy in Erdheim-Chester disease.

Erdheim-Chester disease (ECD) is a rare histiocytosis, considered to be an inflammatory myeloid neoplasm. Tropism for specific involvements of the disease remains unexplained. Vascular endothelial growth factor-A (VEGF) is implicated in cancer pathophysiology and mutations of the RAS oncogene have been shown to induce upregulation of VEGF gene expression. We therefore hypothesized that VEGF might play a particular role in ECD pathophysiology. We conducted a retrospective, single-center study to assess serum VEGF (sVEGF) concentrations and determine whether they were associated with the characteristics of ECD patients, and to determine whether VEGF was expressed by histiocytes. We evaluated 247 ECD patients, 53.4% of whom had sVEGF levels above the normal range (>500 pg/mL). Patients with high sVEGF levels more frequently had cardiac and vascular involvement (58.3% vs. 41.4%, P=0.008 and 70.5% vs. 48.3%, P=0.0004, respectively). In treatment-naïve patients (n=135), the association of C-reactive protein >5 mg/L and sVEGF >500 pg/mL was strongly associated with vascular involvement (odds ratio=5.54 [95% confidence interval: 2.39-13.62], P<0.001), and independently associated with cardiac involvement (odds ratio=3.18 [95% confidence interval: 1.34-7.83], P=0.010) after adjustment for the presence of the BRAF V600E mutation. Changes in sVEGF concentration on treatment were associated with a response of cardiac involvement on consecutive cardiac magnetic resonance images. All histological samples analyzed (n=24) displayed histiocytes with intracytoplasmic expression of VEGF, which was moderate to high in more than 90% of cases. Our study suggests a role for VEGF in cardiac and vascular involvement in ECD.

Nestin as a diagnostic and prognostic marker for combined hepatocellular-cholangiocarcinoma.

BACKGROUND & AIMS: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) associated with a poor prognosis. Given the challenges in its identification and its clinical implications, biomarkers are critically needed. We aimed to investigate the diagnostic and prognostic value of the immunohistochemical expression of Nestin, a progenitor cell marker, in a large multicentric series of PLCs. METHODS: We collected 461 cHCC-CCA samples from 32 different clinical centers. Control cases included 368 hepatocellular carcinomas (HCCs) and 221 intrahepatic cholangiocarcinomas (iCCAs). Nestin immunohistochemistry was performed on whole tumor sections. Diagnostic and prognostic performances of Nestin expression were determined using receiver-operating characteristic curves and Cox regression modeling. RESULTS: Nestin was able to distinguish cHCC-CCA from HCC with AUCs of 0.85 and 0.86 on surgical and biopsy samples, respectively. Performance was lower for the distinction of cHCC-CCA from iCCA (AUCs of 0.59 and 0.60). Nestin, however, showed a high prognostic value, allowing identification of the subset of cHCC-CCA ("Nestin High", >30% neoplastic cells with positive staining) associated with the worst clinical outcome (shorter disease-free and overall survival) after surgical resection and liver transplantation, as well as when assessment was performed on biopsies. CONCLUSION: We show in different clinical settings that Nestin has diagnostic value and that it is a useful biomarker to identify the subset of cHCC-CCA associated with the worst clinical outcome. Nestin immunohistochemistry may be used to refine risk stratification and improve treatment allocation for patients with this highly aggressive malignancy. LAY SUMMARY: There are different types of primary liver cancers (i.e. cancers that originate in the liver). Accurately identifying a specific subtype of primary liver cancer (and determining its associated prognosis) is important as it can have a major impact on treatment allocation. Herein, we show that a protein called Nestin could be used to refine risk stratification and improve treatment allocation for patients with combined hepatocellular carcinoma, a rare but highly aggressive subtype of primary liver cancer.