BACKGROUND: Esophageal carcinoma (EC) and gastric cardiac adenocarcinoma (GCA) have high incidence rates in the Chaoshan region of South China. Multifocal esophageal and cardiac cancer (MECC) is commonly observed in this region in clinical practice. However, the genomic characteristics of MECC remains unclear.
METHODS: In this study, a total of 2123 clinical samples of EC and GCA were analyzed to determine the frequency of multifocal tumors, as well as their occurrence sites and pathological types. Cox proportional hazards regression was used to model the relationship between age, sex, and tumor state concerning survival in our analysis of the cohort of 541 patients with available follow-up data. We performed whole-genome sequencing on 20 tumor foci and 10 normal samples from 10 MECC patients to infer clonal structure on 6 MECC patients to explore genome characteristics.
RESULTS: The MECC rate of EC and GCA was 5.65% (121 of 2123). Age and sex were potential factors that may influence the risk of MECC (p < 0.001). Furthermore, MECC patients showed worse survival compared with single tumor patients. We found that 12 foci from 6 patients were multicentric origin model (MC), which exhibited significant heterogeneity of variations in paired foci and had an increased number of germline mutations in immune genes compared to metastatic model. In MC cases, different lesions in the same patient were driven by distinct mutation and copy number variation (CNV) events. Although TP53 and other driver mutation genes have a high frequency in the samples, their mutation sites show significant heterogeneity in paired tumor specimens. On the other hand, CNV genes exhibited higher concordance in paired samples, especially in the amplification of oncogenes and the deletion of tumor suppressor genes.
CONCLUSIONS: The extent of inter-tumor heterogeneity suggests both monoclonal and polyclonal origins of MECC, which could provide insight into the genome diversity of MECC and guide clinical implementation.

Follicular helper T-cell (TFH) lymphoma harbors recurrent mutations of RHOAG17V, IDH2R172, TET2, and DNMT3A. TET2 and DNMT3A mutations are the most frequently affected genes in clonal hematopoiesis (CH). The aim of our study was to investigate the frequency of CH in bone marrow biopsies (BMB) of TFH/angioimmunoblastic T-cell lymphoma (TFH-AITL) patients and its association with myeloid neoplasms. A total of 29 BMB from 22 patients with a diagnosis of TFH-AITL were analyzed by next-generation sequencing (NGS) with a custom panel. Morphologically, 5 BMB revealed that TFH-AITL infiltrates of >5% of bone marrow (BM) cellularity confirmed in 4 cases by NGS-based T-cell clonality. IDH2R172 was demonstrated only in 1 (3%) of 29, and RHOAG17V in 2 (7%) of 29 samples. TET2 and DNMT3A were identified in 24 (83%) of 29 and 17 (59%) of 29 BMB, respectively. In the parallel lymph node the frequencies of mutations were 27% (IDH2R172), 64% (RHOAG17V), 86% (TET2), and 50% (DNMT3A). TET2 and/or DNMT3A mutations identical in lymph node and BMB were present in 18 (82%) of 22 patients, regardless of BM infiltration. In 3 cases the CH mutations were detected 13, 41, and 145 months before TFH-AITL diagnosis. Cases with TET2/DNMT3A mutations and BM variant allele frequencies >40% (7/18, 39%) showed lower blood counts. However, only low platelet count was statistically significant (P = .024). Myeloid neoplasms and/or myelodysplastic syndrome-related mutations were identified in 4 cases (4/22; 18%); all with high TET2 variant allele frequencies (>40%; P = .0114). In conclusion, CH is present in 82% of TFH-AITL and can be demonstrated up to 145 months before TFH-AITL diagnosis. NGS T-cell clonality analysis is an excellent tool to confirm TFH-AITL BM infiltration. Concurrent myeloid neoplasms were identified in 18% of the cases and were associated with TET2 mutations with high allelic burden (>40%). We demonstrated that myeloid neoplasms might occur simultaneously or precede the diagnosis of TFH lymphoma.

Histologic transformation (HT) is common following targeted therapy in adenocarcinoma. However, whether the transformed tumor is a new component or a combined neuroendocrine carcinoma (C-NEC) remains controversial. We aimed to explore the relationship between pulmonary C-NEC and HT. Macro-dissection was performed on different components of surgically resected C-NEC samples. Molecular alterations and clonal evolution were analyzed using whole exome sequencing (WES). The gene statuses for TP53 and RB1 were determined using immunohistochemistry (IHC) and WES to analyze the relationship between C-NEC and reported HT. Sixteen combined small-cell lung cancer patients and five combined large-cell neuroendocrine carcinoma patients were enrolled. The frequency of p53 and Rb inactivation, assessed using IHC in NEC and non-NEC components, was 76.2/76.2% and 66.7/61.9%, respectively. The expression consistency between the components was 81.0 and 85.7% for p53 and Rb, respectively. The frequencies of TP53, RB1, and EGFR mutations, assessed using WES in NEC and non-NEC components, were 81.0/81.0%, 28.6/28.6%, and 42.9/42.9%, respectively. The concordance rates for TP53, RB1, and EGFR were 90.5, 71.4, and 90.5%, respectively. The consistency rate between IHC and WES was 81.0 and 61.9% for TP53 and RB1, respectively. The different components had a common clonal origin for the 21 C-NECs in the clonal analysis, consistent with previous studies on HT. Our study shows that IHC is more sensitive for Rb detection and C-NEC, and the reported HT may be due to differences in evaluations between pathologist and clinicians. Assessing the p53/Rb and EGFR status for such cases would help in recognizing potential transformation cases or uncovering potential combined components.

UNASSIGNED: An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease. Although its pathogenesis is still poorly understood, recent evidence suggests that AAA displays autoimmune disease characteristics. Particularly, T cells responding to AAA-related antigens in the aortic wall may contribute to an initial immune response. Single-cell RNA (scRNA) T cell receptor (TCR) and B cell receptor (BCR) sequencing is a powerful tool for investigating clonality. However, difficulties such as limited numbers of isolated cells must be considered during implementation and data analysis, making biological interpretation challenging. Here, we perform a representative single-cell immune repertoire analysis in experimental murine AAA and show a reliable bioinformatic processing pipeline highlighting opportunities and limitations of this approach.
UNASSIGNED: We performed scRNA TCR and BCR sequencing of isolated lymphocytes from the infrarenal aorta of male C57BL/6J mice 3, 7, 14, and 28 days after AAA induction via elastase perfusion of the aorta. Sham-operated mice at days 3 and 28 and non-operated mice served as controls.
UNASSIGNED: Comparison of complementarity-determining region (CDR3) length distribution of 179 B cells and 796 T cells revealed neither differences between AAA and control nor between the disease stages. We found no clonal expansion of B cells in AAA. For T cells, we identified several clones in 11 of 16 AAA samples and one of eight control samples. Immune receptor repertoire comparison indicated that only a few clones were shared between the individual AAA samples. The most frequently used V-genes in the TCR beta chain in AAA were TRBV3, TRBV19, and the splicing variant TRBV12-2 + TRBV13-2.
UNASSIGNED: We found no clonal expansion of B cells but evidence for clonal expansion of T cells in elastase-induced AAA in mice. Our findings imply that a more precise characterization of TCR and BCR distribution requires a more extensive number of lymphocytes to prevent undersampling and potentially detect rare clones. Thus, further experiments are necessary to confirm our findings. In summary, this paper examines TCR and BCR sequencing results, identifies limitations and pitfalls, and offers guidance for future studies.

Approximately one-third of patients with diffuse large B-cell lymphoma (DLBCL) relapse and often require salvage chemotherapy followed by autologous stem cell transplantation. In most cases, the clonal relationship between the first diagnosis and subsequent relapse is not assessed, thereby potentially missing the identification of second primary lymphoma. In this study, the clonal relationship of 59 paired DLBCL diagnoses and recurrences was established by next-generation sequencing-based detection of immunoglobulin gene rearrangements. Among 50 patients with interpretable results, 43 patients (86%) developed clonally related relapsed disease. This was observed in 100% of early recurrences (<2 years), 80% of the recurrences with an interval between 2 and 5 years, and 73% of late recurrences (≥5 years). On the other hand, 7 (14%) out of 50 patients displayed different dominant clonotypes in primary DLBCL and clinical recurrences, confirming the occurrence of second primary DLBCL; 37% of DLBCL recurrences that occurred ≥4 years after diagnosis were shown to be second primary lymphomas. The clonally unrelated cases were Epstein-Barr virus positive in 43% of the cases, whereas this was only 5% in the relapsed DLBCL cases. In conclusion, next-generation sequencing-based clonality testing in late recurrences should be considered in routine diagnostics to distinguish relapse from second primary lymphoma, as this latter group of patients with DLBCL may benefit from less-intensive treatment strategies.

The prognostic value of skin and blood T-cell receptor clonality in mycosis fungoides is a matter of debate. Our aim was to ascertain the relation between the presence of a monoclonal T-cell population in the blood and in the skin with response to treatment in patients with mycosis fungoides.
Clinical features and follow-up data were retrospectively collected and analyzed in 94 patients with mycosis fungoides seen at a cutaneous lymphoma clinic in a single tertiary center. All patients had results of polymerase chain reaction analysis of T-cell receptor gamma gene rearrangement in lesional skin and in peripheral blood at time of diagnosis. Association of response to treatment with clonality in the tissue and in the blood was assessed.
T-cell monoclonality was detected in the skin in 30 of 94 patients, in the blood in 12 of 94 cases and the same clone was found in both tissues in 6 of 94 patients. The presence of a polyclonal T-cell population in the circulation was associated with complete response (P = .006). Lack of response to treatment (stable disease or progression of disease) was associated with T-cell clonality in skin (P = .009), in blood (P = .002) and in both tissues (P < .001). A multivariate analysis showed that T-cell monoclonality in the skin is independently associated with lack of response of mycosis fungoides to therapy.
Blood and skin should be studied for T-cell clonality as part of the routine initial workup, even in patients with early-stage disease.

Clonality assessment by the detection of immunoglobulin (IG) gene rearrangements is an important method to determine whether two concurrent or subsequent lymphoid malignancies in one patient are clonally related. Here, we report the detailed clonality analysis in a patient with a diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) followed by a histiocytic sarcoma (HS), in which we were able to study clonal evolution by applying next generation sequencing (NGS) to identify IG rearrangements and gene mutations. Using the sequence information of the NGS-based IG clonality analysis, multiple related subclones could be distinguished in the PAX5 P80R-mutated B-ALL. Notably, only one of these subclones evolved into HS after acquiring a RAF1 mutation. This case demonstrates that NGS-based IG clonality assessment and mutation analysis provide clear added value for clonal comparison and thereby improves clinicobiological understanding.

The 2022 Annual Review Issue of The Journal of Pathology, Recent Advances in Pathology, contains 15 invited reviews on research areas of growing importance in pathology. This year, the articles include those that focus on digital pathology, employing modern imaging techniques and software to enable improved diagnostic and research applications to study human diseases. This subject area includes the ability to identify specific genetic alterations through the morphological changes they induce, as well as integrating digital and computational pathology with \'omics technologies. Other reviews in this issue include an updated evaluation of mutational patterns (mutation signatures) in cancer, the applications of lineage tracing in human tissues, and single cell sequencing technologies to uncover tumour evolution and tumour heterogeneity. The tissue microenvironment is covered in reviews specifically dealing with proteolytic control of epidermal differentiation, cancer-associated fibroblasts, field cancerisation, and host factors that determine tumour immunity. All of the reviews contained in this issue are the work of invited experts selected to discuss the considerable recent progress in their respective fields and are freely available online (https://onlinelibrary.wiley.com/journal/10969896). © 2022 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The dynamical process of cell division that underpins homeostasis in the human body cannot be directly observed in vivo, but instead is measurable from the pattern of somatic genetic or epigenetic mutations that accrue in tissues over an individual\'s lifetime. Because somatic mutations are heritable, they serve as natural lineage tracing markers that delineate clonal expansions. Mathematical analysis of the distribution of somatic clone sizes gives a quantitative readout of the rates of cell birth, death, and replacement. In this review we explore the broad range of somatic mutation types that have been used for lineage tracing in human tissues, introduce the mathematical concepts used to infer dynamical information from these clone size data, and discuss the insights of this lineage tracing approach for our understanding of homeostasis and cancer development. We use the human colon as a particularly instructive exemplar tissue. There is a rich history of human somatic cell dynamics surreptitiously written into the cell genomes that is being uncovered by advances in sequencing and careful mathematical analysis lineage of tracing data. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A 62-year-old man visited the Department of Otorhinolaryngology at our hospital with a chief complaint of a pharyngeal mass. He was admitted to our department with a diagnosis of T-cell lymphoma based on a biopsy of a mesopharyngeal tumor. Although clonality analysis was not performed due to the lack of an appropriate sample, we considered the possibility of lymphoma-type (Lugano classification stage II) adult T-cell leukemia-lymphoma (ATL), as the anti-HTLV-1 antibody was positive. During the course of the disease, the peripheral blood smear revealed atypical lymphocytes with cleaved nuclei, and inverse PCR was performed with DNA extracted from those cells; however, the result showed that the pattern of HTLV-1 proviral DNA integration sites was polyclonal. Further, we performed RNA in situ hybridization targeting HTLV-1 bZIP factor (HBZ-ISH) using the formalin-fixed paraffin-embedded (FFPE) tissue samples of the mesopharyngeal tumor, and a high expression of HBZ was found in the tumor cells, leading to the diagnosis of ATL. These findings suggest the effectiveness of the novel diagnostic method using FFPE tissue samples for ATL.