Bone marrow (BM) cellularity assessment is a crucial step in the evaluation of BM trephine biopsies for hematologic and nonhematologic disorders. Clinical assessment is based on a semiquantitative ...visual estimation of the hematopoietic and adipocytic components by hematopathologists, which does not provide quantitative information on other stromal compartments. In this study, we developed and validated MarrowQuant 2.0, an efficient, user-friendly digital hematopathology workflow integrated within QuPath software, which serves as BM quantifier for 5 mutually exclusive compartments (bone, hematopoietic, adipocytic, and interstitial/microvasculature areas and other) and derives the cellularity of human BM trephine biopsies. Instance segmentation of individual adipocytes is realized through the adaptation of the machine-learning-based algorithm StarDist. We calculated BM compartments and adipocyte size distributions of hematoxylin and eosin images obtained from 250 bone specimens, from control subjects and patients with acute myeloid leukemia or myelodysplastic syndrome, at diagnosis and follow-up, and measured the agreement of cellularity estimates by MarrowQuant 2.0 against visual scores from 4 hematopathologists. The algorithm was capable of robust BM compartment segmentation with an average mask accuracy of 86%, maximal for bone (99%), hematopoietic (92%), and adipocyte (98%) areas. MarrowQuant 2.0 cellularity score and hematopathologist estimations were highly correlated (R2 = 0.92-0.98, intraclass correlation coefficient ICC = 0.98; interobserver ICC = 0.96). BM compartment segmentation quantitatively confirmed the reciprocity of the hematopoietic and adipocytic compartments. MarrowQuant 2.0 performance was additionally tested for cellularity assessment of specimens prospectively collected from clinical routine diagnosis. After special consideration for the choice of the cellularity equation in specimens with expanded stroma, performance was similar in this setting (R2 = 0.86, n = 42). Thus, we conclude that these validation experiments establish MarrowQuant 2.0 as a reliable tool for BM cellularity assessment. We expect this workflow will serve as a clinical research tool to explore novel biomarkers related to BM stromal components and may contribute to further validation of future digitalized diagnostic hematopathology workstreams.
Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is a rare aggressive T-cell lymphoma most reported in Asia. We performed a comprehensive clinical, pathological and genomic study of 71 ...European MEITL patients (36 males, 35 females, median age 67 years). The majority presented with gastrointestinal involvement and had emergency surgery, and 40% had stage IV disease. The tumors were morphologically classified into two groups: typical (58%) and atypical (i.e., non-monomorphic or with necrosis, angiotropism or starry-sky pattern) (42%), sharing a homogeneous immunophenotypic profile (CD3+ 98% CD4- 94% CD5- 97% CD7+ 97% CD8+ 90% CD56+ 86% CD103+ 80% cytotoxic marker+ 98%) with more frequent expression of TCRgd (50%) than TCRab (32%). MYC expression (30% of cases) partly reflecting MYC gene locus alterations, correlated with non-monomorphic cytology. Almost all cases (97%) harbored deleterious mutation(s) and/or deletion of the SETD2 gene and 90% had defective H3K36 trimethylation. Other frequently mutated genes were STAT5B (57%), JAK3 (50%), TP53 (35%), JAK1 (12.5%), BCOR and ATM (11%). Both TP53 mutations and MYC expression correlated with atypical morphology. The median overall survival (OS) of 63 patients (43/63 only received chemotherapy after initial surgery) was 7.8 months. Multivariate analysis found a strong negative impact on outcome of MYC expression, TP53 mutation, STAT5B mutation and poor performance status while aberrant B-cell marker expression (20% of cases) correlated with better survival. In conclusion, MEITL is an aggressive disease with resistance to conventional therapy, predominantly characterized by driver gene alterations deregulating histone methylation and JAK/STAT signaling and encompasses genetic and morphologic variants associated with very high clinical risk.
Aims
Follicular helper T‐cell (TFH) lymphoma of the angioimmunoblastic‐type (AITL), one of the most prevalent T‐cell lymphomas, typically encompasses proliferation of high endothelial venules and ...Epstein–Barr virus‐positive immunoblasts, but neither infection with HHV8 nor association with Kaposi's sarcoma (KS) have been described. The aims of this study are to characterise the association between AITL and HHV8 infection or KS.
Methods and results
Three male patients aged 49–76 years, HIV‐negative, with concurrent nodal involvement by AITL and KS, were identified from our files and carefully studied. Two patients originated from countries where endemic KS occurs, including one with cutaneous KS. The lymphomas featured abundant vessels, expanded follicular dendritic cells and neoplastic TFH cells PD1+ (three of three), ICOS+ (three of three), CXCL13+ (three of three), CD10+ (two of three), BCL6 (two of three) but lacked EBV+ immunoblasts. The foci of KS consisted of subcapsular proliferations of ERG+, CD31+ and/or CD34+, HHV8+ spindle cells. High‐throughput sequencing showed AITL‐associated mutations in TET2 (three of three), RHOA (G17V) (three of three) and IDH2 (R172) (two of three), which were absent in the microdissected KS component in two cases. Relapses in two patients consisted of AITL, without evidence of KS. No evidence of HHV8 infection was found in a control group of 23 AITL cases.
Conclusion
Concurrent nodal involvement by AITL and KS is rare and identification of both neoplastic components may pose diagnostic challenges. The question of whether the association between AITL and KS may be fortuitous or could reflect the underlying immune dysfunction in AITL remains open.
An association of angioimmunoblastic T‐cell lymphoma and Kaposi sarcoma is reported in lymph node biopsies of three HIV‐negative patients. This raises the question of whether this association could reflect the functional properties of TFH cells and underlying immune dysfunction in AITL.
Primary mediastinal large B-cell lymphoma (PMBL) is a mature large B-cell lymphoma of putative thymic B-cell origin involving the mediastinum with younger age distribution and better prognosis than ...diffuse large B-cell lymphoma (DLBCL), not otherwise specified. Recently, based on gene expression profile analysis and morphologic findings, cases of PMBL without mediastinal involvement have been reported. In this study, we analyzed 3 cases of nodal DLBCL with morphologic features of PMBL presenting in submandibular or supraclavicular lymph nodes, in middle-aged to elderly patients, 2 of them without clinical or radiologic evidence of mediastinal involvement. The 3 patients presented with stage I/II disease and had excellent response to R-CHOP/R-EPOCH therapy. The 3 cases showed MAL expression and were positive for CD23 and/or CD30. All 3 cases expressed cyclin D1 with copy number gains of CCND1 gene but without rearrangement. There was no rearrangement of CIITA or PDL1/PDL2. Reverse transcriptase-multiplex ligation-dependent probe amplification, a mRNA-based gene expression profile analysis revealed high probability of PMBL (87.6%, 98.7%, and 99%) in these 3 cases. Targeted next-generation sequencing analysis showed SOCS1 mutations in the 3 cases, and TNFAIP3 and XPO1 mutations in one, further supporting the diagnosis of PMBL. In conclusion, we report 3 cases of nodal PMBL, 2 of them without mediastinal mass, and expression of cyclin D1 due to copy number gains of CCND1 gene, a diagnostic pitfall with mantle cell lymphoma and DLBCL, not otherwise specified.
Kaposi sarcoma is the most common human herpesvirus 8 (HHV-8)–related disease described after solid organ transplantation. Multicentric Castleman disease and hemophagocytic syndrome are other ...potential HHV-8–induced entities but are less frequently reported. We describe the case of a liver transplant recipient who presented with an acute febrile illness 1 year after transplantation with a rapidly fatal outcome. Autopsy revealed 3 distinct HHV-8–related entitiesKaposi sarcoma, HHV-8-associated multicentric Castleman disease with microlymphomas and a severe hemophagocytic syndrome. Retrospective serologic tests suggested that HHV-8 was likely transmitted by the seropositive donor at the time of transplantation. To our knowledge, this is the first case of copresentation of 3 clinical presentations of HHV-8–mediated human disease in the post-transplant setting. Considering the absence of systematic screening of organ donors/recipients for HHV-8 infection, HHV-8–related illness should be suspected in transplant recipients who present with acute febrile illness, systemic symptoms, lymphadenopathies, and/or multiorgan failure to rapidly document the diagnosis and provide timely an adequate treatment.
Chromogenic immunohistochemistry (IHC) is omnipresent in cancer diagnosis, but has also been criticized for its technical limit in quantifying the level of protein expression on tissue sections, thus ...potentially masking clinically relevant data. Shifting from qualitative to quantitative, immunofluorescence (IF) has recently gained attention, yet the question of how precisely IF can quantify antigen expression remains unanswered, regarding in particular its technical limitations and applicability to multiple markers. Here we introduce microfluidic precision IF, which accurately quantifies the target expression level in a continuous scale based on microfluidic IF staining of standard tissue sections and low-complexity automated image analysis. We show that the level of HER2 protein expression, as continuously quantified using microfluidic precision IF in 25 breast cancer cases, including several cases with equivocal IHC result, can predict the number of HER2 gene copies as assessed by fluorescence in situ hybridization (FISH). Finally, we demonstrate that the working principle of this technology is not restricted to HER2 but can be extended to other biomarkers. We anticipate that our method has the potential of providing automated, fast and high-quality quantitative in situ biomarker data using low-cost immunofluorescence assays, as increasingly required in the era of individually tailored cancer therapy.
Acinar cell carcinomas (ACCs) of the pancreas are a heterogeneous group of neoplasms showing a wide spectrum of morphological features including acinar, solid, glandular, and trabecular architecture. ...In addition, uncommon cytological aspects have recently been described and include oncocytic, spindle, clear, and pleomorphic cell types. This wide histological spectrum represents a challenge in the diagnostic task for pathologists. Molecular mechanisms involved in the onset and progression of ACCs are not completely known, but, in general, they differ from those observed in ductal adenocarcinomas or neuroendocrine neoplasms of the pancreas and frequently include alterations in the APC/β-catenin pathway. In the present paper, we describe a new variant of ACC showing thyroid-like follicular features and
CTNNB1
mutation. This phenotype needs to be included in the spectrum of morphological presentation of ACC.
Fluorescence in situ hybridization (FISH) is one of the recommended techniques for human epidermal growth factor receptor 2 (HER2) status assessment on cancer tissues. Here we develop ...microfluidics-assisted FISH (MA-FISH), in which hybridization of the FISH probes with their target DNA strands is obtained by applying square-wave oscillatory flows of diluted probe solutions in a thin microfluidic chamber of 5 μl volume. By optimizing the experimental parameters, MA-FISH decreases the consumption of the expensive probe solution by a factor 5 with respect to the standard technique, and reduces the hybridization time to 4 h, which is four times faster than in the standard protocol. To validate the method, we blindly conducted HER2 MA-FISH on 51 formalin-fixed paraffin-embedded tissue slides of 17 breast cancer samples, and compared the results with standard HER2 FISH testing. HER2 status classification was determined according to published guidelines, based on average number of HER2 copies per cell and average HER2/CEP17 ratio. Excellent agreement was observed between the two methods, supporting the validity of MA-FISH and further promoting its short hybridization time and reduced reagent consumption.
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