Smoking status in acute myeloid leukemia is associated with worse overall survival and independent of prior nonhematopoietic malignancies, cytogenetic abnormalities, and WHO category.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy with several patient- and disease-associated variables known to impact prognosis. Tobacco smoking is an environmental factor associated with a greater incidence of AML, but there have been limited studies that evaluated smoking toward overall survival. We retrospectively searched for AML cases and collected clinical and diagnostic data for each case. We also used an independent next-generation sequencing (NGS) data set to assess for a distinct mutational signature associated with smoking. When stratified by smoking status, there was a greater number of males, patients aged ≥60 years, and patients with ≥2 comorbidities within the smoking category (P < .05). Survival analysis demonstrated decreased survival probability in the smokers, male smokers, smokers with 1 other comorbidity, and smokers without a prior history of nonhematopoietic malignancy (P < .05) as compared to nonsmokers. Smoking was associated with a decrease in survival within the World Health Organization categories of AML, not otherwise specified (AML NOS; P = .035) and AML with recurrent genetic abnormalities (AML RGA; P = .002). Multivariate analysis showed that patients who were smokers had a greater hazard ratio than nonsmokers after adjusting for the other covariates. Our findings demonstrated that smoking was independently associated with decreased overall survival after adjusting for other potentially confounding factors. In addition, our results suggest that a mutational signature can be recognized using NGS data in a subset of AML patients who smoke.

Copy Number Loss at Chromosome 14q11.2 Correlates With the Proportion of T Cells in Biopsies and Helps Identify T-Cell Neoplasms.

CONTEXT.­: Evidence of T-cell clonality is often critical in supporting the diagnosis of a T-cell lymphoma. OBJECTIVES.­: To retrospectively explore the significance of copy number losses at the 14q11.2 T-cell receptor α locus in relation to the presence of a T-cell neoplasm and proportion of T cells by targeted next-generation sequencing. DESIGN.­: Targeted next-generation sequencing data from 139 tissue biopsies, including T-cell lymphomas, B-cell lymphomas, classic Hodgkin lymphomas, nonhematopoietic malignancies, and normal samples, were reviewed for copy number losses involving the T-cell receptor α gene segments at chr14q11.2. RESULTS.­: We found that biallelic or homozygous deletion of 14q11.2 was found in most (28 of 33, 84.8%) T-cell lymphomas. The magnitude of 14q11.2 loss showed a statistically significant correlation with the proportion of T cells in lymphoma tissue samples. Copy number losses could also be detected in other lymphomas with high numbers of T cells (8 of 32, 25% of B-cell lymphomas, 4 of 4 classical Hodgkin lymphomas), though biallelic/homozygous deletion of 14q11.2 was not significantly observed outside of T-cell lymphomas. Most nonhematopoietic neoplasms and normal tissues (59 of 64, 92.2%) showed no significant copy number losses involving the T-cell receptor α locus at chr14q11.2. CONCLUSIONS.­: Analysis of copy number losses at the T-cell receptor α locus chr14q11.2 with targeted next-generation sequencing can potentially be used to estimate the proportion of T cells and detect T-cell neoplasms.

Indolent T-lymphoblastic proliferation: A systematic review of the literature analyzing the epidemiologic, clinical, and pathologic features of 45 cases.

An indolent T-lymphoblastic proliferation (iT-LBP) is a rare benign disorder characterized by an abnormal expansion of immature T-cells, which morphologically can mimic malignancy. Since the first case was described in 1999, dozens more have been reported in the literature. However, the epidemiologic, clinical, pathologic, and biologic features of this disease have not been well described. Here, we retrospectively reviewed all known cases reported in the literature to better understand this entity. A PubMed search up to January 2022 highlighted 25 papers describing cases/case series of iT-LBP, one of which was a case presentation in a slide workshop. Except for 9 of the cases in one of the papers, where it was evident that the number of CD3+/TdT+ cells were too few to conform with a diagnosis of iT-LBP, all papers and all the cases reported were included in the study amounting to a total of 45 cases. Clinicopathologic characteristics were analyzed using descriptive statistics and frequencies. Our analysis highlighted the previously known association with Castleman disease and Castleman-like features and underlined its association with dendritic cell proliferations in general, as well as uncovering high frequency of concurrence with hepatocellular carcinoma and autoimmune diseases, most notably myasthenia gravis, paraneoplastic pemphigus and paraneoplastic autoimmune multiorgan syndrome. Furthermore, the co-expression of CD4 and CD8 and high prevalence of extranodal disease and recurrences were other less well described features that were revealed.

Case Report: Castleman Disease With an Associated Stromal Spindle Cell Proliferation, PDGFRB Mutation and p53 Expression: Clonal Origins of a Rare Disease.

Castleman disease (CD) is a rare lymphoproliferative disorder with distinct clinical subtypes. However, our understanding of the underlying pathogenesis of particular subtypes of CD remains unclear. While the characteristic morphologic changes within UCD, including occasional cases of overgrowth of spindled stromal and follicular dendritic cells have been described, the nature and origin of these spindle cells remain elusive. Few reports have suggested that underlying stromal cells in UCD are clonally neoplastic and may be of fibroblastic reticular cell (FRC) or follicular dendritic cell (FDC) origins given their close clonal relationship. Although certain histomorphologic features may aid diagnosis, there are no specific biomarkers that can differentiate a reactive process mimicking UCD from true UCD. Hence, we describe an index case with morphology consistent with the hyaline vascular subtype of UCD with concomitant atypical smooth muscle actin (SMA)-positive stromal spindle cell proliferation containing a recurrent PDGFRB N666S mutation and upregulation of p53 expression. Further analysis of 21 additional cases of UCD identified increased p53 expression by digital image analysis and SMA positive stromal cells predominantly within the paracortical and intrafollicular areas further strengthening the hypothesis of the stromal cellular derivation and origins of UCD.

A Review of Genetic Abnormalities in Unicentric and Multicentric Castleman Disease.

Castleman disease (CD) is a rare lymphoproliferative disorder known to represent at least four distinct clinicopathologic subtypes. Large advancements in our clinical and histopathologic description of these diverse diseases have been made, resulting in subtyping based on number of enlarged lymph nodes (unicentric versus multicentric), according to viral infection by human herpes virus 8 (HHV-8) and human immunodeficiency virus (HIV), and with relation to clonal plasma cells (POEMS). In recent years, significant molecular and genetic abnormalities associated with CD have been described. However, we continue to lack a foundational understanding of the biological mechanisms driving this disease process. Here, we review all cases of CD with molecular abnormalities described in the literature to date, and correlate cytogenetic, molecular, and genetic abnormalities with disease subtypes and phenotypes. Our review notes complex karyotypes in subsets of cases, specific mutations in PDGFRB N666S in 10% of unicentric CD (UCD) and NCOA4 L261F in 23% of idiopathic multicentric CD (iMCD) cases. Genes affecting chromatin organization and abnormalities in methylation are seen more commonly in iMCD while abnormalities within the mitogen-activated protein kinase (MAPK) and interleukin signaling pathways are more frequent in UCD. Interestingly, there is a paucity of genetic studies evaluating HHV-8 positive multicentric CD (HHV-8+ MCD) and POEMS-associated CD. Our comprehensive review of genetic and molecular abnormalities in CD identifies subtype-specific and novel pathways which may allow for more targeted treatment options and unique biologic therapies.

Mutations in JAK/STAT and NOTCH1 Genes Are Enriched in Post-Transplant Lymphoproliferative Disorders.

Post-transplant lymphoproliferative disorders (PTLD) are diseases occurring in immunocompromised patients after hematopoietic stem cell transplantation (HCT) or solid organ transplantation (SOT). Although PTLD occurs rarely, it may be associated with poor outcomes. In most cases, PTLD is driven by Epstein-Barr virus (EBV) infection. Few studies have investigated the mutational landscape and gene expression profile of PTLD. In our study, we performed targeted deep sequencing and RNA-sequencing (RNA-Seq) on 16 cases of florid follicular hyperplasia (FFH) type PTLD and 15 cases of other PTLD types that include: ten monomorphic (M-PTLD), three polymorphic (P-PTLD), and two classic Hodgkin lymphoma type PTLDs (CHL-PTLD). Our study identified recurrent mutations in JAK3 in five of 15 PTLD cases and one of 16 FFH-PTLD cases, as well as 16 other genes that were mutated in M-PTLD, P-PTLD, CHL-PTLD and FFH-PTLD. Digital image analysis demonstrated significant differences in single cell area, major axis, and diameter when comparing cases of M-PTLD and P-PTLD to FFH-PTLD. No morphometric relationship was identified with regards to a specific genetic mutation. Our findings suggest that immune regulatory pathways play an essential role in PTLD, with the JAK/STAT pathway affected in many PTLDs.

A meta-analysis of SARS-CoV-2 patients identifies the combinatorial significance of D-dimer, C-reactive protein, lymphocyte, and neutrophil values as a predictor of disease severity.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), known to be the causative agent of COVID-19, has led to a worldwide pandemic. At presentation, individual clinical laboratory blood values, such as lymphocyte counts or C-reactive protein (CRP) levels, may be abnormal and associated with disease severity. However, combinatorial interpretation of these laboratory blood values, in the context of COVID-19, remains a challenge. METHODS: To assess the significance of multiple laboratory blood values in patients with SARS-CoV-2 and develop a COVID-19 predictive equation, we conducted a literature search using PubMed to seek articles that included defined laboratory data points along with clinical disease progression. We identified 9846 papers, selecting primary studies with at least 20 patients for univariate analysis to identify clinical variables predicting nonsevere and severe COVID-19 cases. Multiple regression analysis was performed on a training set of patient studies to generate severity predictor equations, and subsequently tested on a validation cohort of 151 patients who had a median duration of observation of 14 days. RESULTS: Two COVID-19 predictive equations were generated: one using four variables (CRP, D-dimer levels, lymphocyte count, and neutrophil count), and another using three variables (CRP, lymphocyte count, and neutrophil count). In adult and pediatric populations, the predictive equations exhibited high specificity, sensitivity, positive predictive values, and negative predictive values. CONCLUSION: Using the generated equations, the outcomes of COVID-19 patients can be predicted using commonly obtained clinical laboratory data. These predictive equations may inform future studies evaluating the long-term follow-up of COVID-19 patients.

A comprehensive analysis of RHOA mutation positive and negative angioimmunoblastic T-cell lymphomas by targeted deep sequencing, expression profiling and single cell digital image analysis.

Angioimmunoblastic T­cell lymphoma (AITL) is a uniquely aggressive mature T­cell neoplasm. In recent years, recurrent genetic mutations in ras homolog family member A (RHOA), tet methylcytosine dioxygenase 2 (TET2), DNA methyltransferase 3 alpha (DNMT3A) and isocitrate dehydrogenase [NADP(+)] 2 (IDH2) have been identified as associated with AITL. However, a deep molecular study assessing both DNA mutations and RNA expression profile combined with digital image analysis is lacking. The present study aimed to evaluate the significance of molecular and morphologic features by high resolution digital image analysis in several cases of AITL. To do so, a total of 18 separate tissues from 10 patients with AITL were collected and analyzed. The results identified recurrent mutations in RHOA, TET2, DNMT3A, and IDH2, and demonstrated increased DNA mutations in coding, promoter and CCCTC binding factor (CTCF) binding sites in RHOA mutated AITLs vs. RHOA non­mutated cases, as well as increased overall survival in RHOA mutated patients. In addition, single cell computational digital image analysis morphologically characterized RHOA mutated AITL cells as distinct from cells from RHOA mutation negative patients. Computational analysis of single cell morphological parameters revealed that RHOA mutated cells have decreased eccentricity (more circular) compared with RHOA non­mutated AITL cells. In conclusion, the results from the present study expand our understanding of AITL and demonstrate that there are specific cell biological and morphological manifestations of RHOA mutations in cases of AITL.

LIM domain only 2 (LMO2) expression distinguishes T-lymphoblastic leukemia/lymphoma from indolent T-lymphoblastic proliferations.

AIMS: An indolent T-lymphoblastic proliferation (iT-LBP) is a benign, reactive expansion of immature terminal deoxynucleotidyl transferase (TdT)-positive T cells found in extrathymic tissues. iT-LBP can be challenging to distinguish from malignant processes, specifically T-lymphoblastic lymphoma (T-LBL), given the overlapping clinical and histological features. Recently, it has been shown that LIM domain only 2 (LMO2) is overexpressed in T-LBL but not in reactive immature TdT+ T cells in the thymus. On the basis of these findings, the aim of this study was to investigate the expression of LMO2 by using immunohistochemistry and its role in differentiating iT-LBPs from T-LBLs. METHODS AND RESULTS: We retrospectively identified cases of iT-LBP and T-LBL from the pathology archives of four institutions. Seven iT-LBP cases (including five new cases that have not been reported in the literature) and 13 T-LBL cases were analysed. Clinical, morphological, immunophenotypic and molecular data were analysed. Immunohistochemical staining with LMO2 was performed on all iT-LBP and T-LBL cases. A review of five new iT-LBP cases showed similar morphological, immunophenotypic and molecular features to those of previously reported cases. All iT-LBP cases were negative for LMO2 (0/7), whereas 92% of T-LBL cases (12/13) expressed LMO2; the sensitivity was 92% (confidence interval 64-100%) and the specificity was 100% (confidence interval 59-100%). CONCLUSION: We confirm previously published findings that iT-LBP cases show highly overlapping morphological and immunophenotypic features with T-LBL. Importantly, LMO2 expression is a sensitive and specific marker with which to rule out iT-LBP.

Significance of bacterial and viral genotypes as a risk factor in driving cancer (Review).

Microbes have been known to drive human cancers for over half a century. However, despite the association of bacterial and viral infections with a high risk of cancer, most infections do not result in the development of cancer. Additionally, certain bacteria and viruses, considered to drive oncogenesis, are commonly prevalent in the global population. The current study performed a comprehensive meta-analysis of primary literature data to identify particular aspects of microbial genotypes as crucial factors that dictate the cancer risks associated with infection. The results indicated the importance of incorporating microbial genotype information with human genotypes into clinical assays for the more efficient diagnosis and prognosis of patients with cancer. The current review focuses on the importance of microbial genotypes and specific genes and genetic differences that are important to human oncogenesis.

TBL1XR1 Mutations in Primary Marginal Zone Lymphomas of Ocular Adnexa are Associated with Unique Morphometric Phenotypes.

PURPOSE: Extranodal marginal zone B-cell lymphoma (EMZL) of mucosa-associated lymphoid tissue (MALT) that affects the ocular adnexa, also known as ocular adnexal MALT lymphomas (OAML), are low-grade lymphomas that mostly affect elderly individuals. This study was conducted to explore the genetic and microbial drivers of OMAL, and unique morphometric phenotypes associated with these mutations and infections. MATERIALS AND METHODS: In this study, we performed targeted deep sequencing of 8 OAML cases to identify its potential genetic and microbial drivers. We additionally performed computational digital image analysis of cases to determine if morphologic features corresponded to genetic mutations and disease biology. RESULTS: We identified TBL1XR1 as recurrently mutated in OAML (4/8), and mutations in several other oncogenes, tumor suppressors, transcription regulators, and chromatin remodeling genes. Morphologically, OAML cases with mutations in TBL1XR1 showed lymphoma cells with significantly lower circularity and solidity by computational digital image analysis (p-value <0.0001). Additionally, cases of OAML with mutations in TBL1XR1 showed equivalent or increased vascular density compared to cases without mutations in TBL1XR1. Finally, we did not find any infectious microbial organisms associated with OAML. CONCLUSIONS: Our study showed recurrent mutations in TBL1XR1 are associated with unique morphometric phenotypes in OMAL cases. Additionally, mutations in genes associated with the methylation status of histone 3, nuclear factor (NF)-κB pathway, and NOTCH pathway were enriched in OMAL cases. Our findings have biologic and clinical implications as mutations in TBL1XR1 and other genes have the potential to be used as markers for the diagnosis of OAML, and also demonstrate a specific biologic phenotypic manifestation of TBL1XR1 mutations.

The life and death of the germinal center.

The formation, development and dissolution of germinal centers is a major part of immune system function. It is important to differentiate neoplastic processes from follicular hyperplasia and regressive follicular changes. Better understanding of germinal center development and dissolution also provides diagnostic clues to the underlying pathologic process. It is also important in identifying the immune basis of different pathologic entities as well as in immunotherapy decision making and follow up. In this study, we characterize the immunoarchitecture of lymphoid follicles with a focus on germinal center in one representative case, each of commonly encountered benign and malignant lymph node disorders, with morphologic and immunohistochemical alterations of germinal centers. The cases include reactive follicular hyperplasia (FH), florid follicular hyperplasia (FFH), follicular lymphoma (FL), angioimmunoblastic T-cell lymphoma (AITL), hyaline-vascular Castleman disease (HVCD), progressive transformation of germinal centers, nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), lymphocyte-rich classic Hodgkin lymphoma (LR-CHL), human immunodeficiency virus (HIV)-associated follicular dissolution and chronic lymphocytic leukemia (CLL) with proliferation centers (PC). A panel of antibodies were used namely CD3, CD20, CD10, BCL2, BCL6, CD21, CD23, CD35, FOXP1, GCET1, HGAL/GCET2, LMO2, MUM1, IgD, Ki67, PD1 and PD-L1. We found that these entities show distinct immunoarchitectural patterns of germinal center formation, development and regression, particularly, the distribution of mantle zone B-cells, follicular helper T cells (Tfh) and FDC meshworks, confirming the influence of antigenic stimulation and status of immune system in these changes. This also confirms the interrelationship of underlying immunologic mechanisms in these disease processes.

Indolent In Situ B-Cell Neoplasms With MYC Rearrangements Show Somatic Mutations in MYC and TNFRSF14 by Next-generation Sequencing.

Systemic high-grade B-cell lymphomas (HGBCLs) with MYC gene rearrangements are clinically aggressive. In situ lesions with indolent behavior have not been described to date. We have identified 2 cases of in situ B-cell neoplasms with MYC rearrangements (IS-BCN, MYC) occurring, and focally confined to ≤4 lymphoid follicles in otherwise healthy individuals and without clinical progression despite minimal intervention (surgical only). Morphologically similar to systemic HGBCLs, the low power view of these lesions showed a starry sky pattern with numerous mitotic figures. High power imaging demonstrated these cells to be medium-large in size with irregular nuclear contours, immature chromatin, and prominent nucleoli. Immunophenotypically these cells were light chain restricted, positive for CD20, CD10, c-Myc, and dim or negative for BCL2 with a Ki67 proliferative index of >95%. By fluorescence in situ hybridization studies, we detected MYC translocations in these cells but no rearrangements in BCL2 or BCL6. Microdissection of neoplastic cells in these patients followed by targeted next-generation sequencing identified a mutation in MYC, D2N, and an indel in TNFRSF14. Mutations in ID3 or TCF3 were not identified. Although rare, these lesions should be separated from HGBCLs involving follicles but with systemic spread which has been previously described. Unlike systemic lymphomas with MYC gene rearrangements, these in situ B-cell neoplasms with MYC rearrangements did not require systemic therapy and no progression has been seen in either patient beyond 1 year (29 and 16 mo). Our work offers pathologic and biologic insight into the early process of B-cell neoplasia.

Molecular genetic testing methodologies in hematopoietic diseases: current and future methods.

INTRODUCTION: Rapid technological advancements in clinical molecular genetics have increased our diagnostic and prognostic capabilities in health care. Understanding these assays, as well as how they may change over time, is critical for pathologists, clinicians, and translational researchers alike. METHODS: This review provides a practical summary and basic reference for current molecular genetic technologies, as well as new testing methodologies that are in use, gaining momentum, or anticipated to contribute more broadly in the future. RESULTS: Here, we discuss DNA and RNA based methodologies including classic assays such as the polymerase chain reaction (PCR), Sanger sequencing, and microarrays, to more cutting-edge next-generation sequencing (NGS) based assays and emerging molecular technologies such as cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA), and NGS-based detection of infectious disease organisms. CONCLUSION: This review serves as a basic foundation for knowledge in current and emerging clinical molecular genetic technologies.