(Related to Figure 3). Relationships between TCA cycle labeling and clinical factors.

(Related to Figure 3). TCA cycle metabolite abundance does not correlate with overall survival.

(Related to Figure 2). Transcriptomic data for NSCLC and adjacent lung tissue fragments.

(Related to Figure 3). Overall and recurrence-free survival summary.

Related to Figures 4 and 6). PDX Isotopologue Data.

(Related to Figure 1). Patient Isotopologue Data.

(Related to Figures 5 and 6): Treatment with IACS-010759 reduces distant metastasis.

Background Nucleophosmin 1 (NPM1) mutations define a major molecular subtype of acute myeloid leukemia (AML). Although RT-PCR and next-generation sequencing (NGS) remain the gold standard for diagnosis and minimal/measurable residual disease (MRD) assessment, NPM1 mutant-specific immunohistochemistry (NPM1m IHC) using monoclonal antibodies presents a rapid and cost-effective alternative. Correlation between NPM1m IHC patterns and specific NPM1 mutation subtypes remain underexplored. Objectives Evaluate the diagnostic utility of NPM1m IHC in AML with NPM1 mutation and to assess its correlation with mutation subtypes, morphologic features, cytogenetic profiles, co-mutations, and clinical outcomes. Methods A retrospective cohort of 36 AML cases with confirmed NPM1 mutations (2018-2024) were analyzed for clinicopathologic variables, cytogenetics, PCR/NGS data, and NPM1m IHC results with subgroup analysis between Type A (n=28) and non-Type A (n=8) mutations. Results NPM1m IHC positivity was observed in all insertional NPM1 mutation subtypes with two distinct staining patterns: homogeneous and two-toned, with two-toned pattern more frequent in Type A mutations. Non-Type A mutations were more often associated with complex karyotypes and showed a trend toward less frequent FLT3 co-mutations. Monocytic differentiation and cup-like nuclear morphology were assessed in both subgroups. No significant difference in survival or remission rates was observed between groups, although Type A mutations exhibited higher rates of dysplasia (p=0.03). Conclusion NPM1m IHC is a sensitive and timely surrogate marker for insertional NPM1 mutations, correlating well with molecular results and useful in both immediate diagnosis and MRD evaluation. This study reveals subtype-specific morphologic and cytogenetic associations and highlights the need for further investigation into non-insertional NPM1 mutations and their detection challenges.

(Related to Figure 2). Epithelial and myeloid cell contributions to gene expression and 13C labeling features.

(Related to Figure 1). TCA cycle labeling and metabolite abundance in tumors and lungs from NSCLC patents.

(Related to Figure 1). Clinical and pathological data from patients recruited to this study.

(Related to Figure 4). Development of patient-derived xenografts from malignant tumors in the lung.

PDXs derived from primary NSCLC retain histological, molecular, and metabolic characteristics. A and B, Summary of histological and molecular characterization of donor tumors (A) and PDX models (B). C, H&E staining of NSCLC PDXs. D, Engraftment success of primary NSCLCs and lung metastases considering the metabolic phenotype of the patient’s tumor. “High” and “low” TCA cycle enrichment was defined in Fig. 3B and C. E, Patient samples (lung, n = 64; primary NSCLC, n = 66) are plotted from Fig. 1C. Mice bearing NSCLC PDXs (mx57, n = 8; mx73, n = 6; mx95, n = 3; mx148, n = 3) were infused with [U-13C]glucose. Average TCA cycle enrichment was calculated and compared with primary NSCLC and adjacent lung tissue. Differences in enrichment were determined by a Kruskal–Wallis test with Dunn’s test. Data are expressed as average and SD. ADC, adenocarcinoma; NS, not statistically significant; Pleo, pleomorphic; SQCC, squamous cell carcinoma. Scale bar, 200 μm. ****, P<0.0001. NS, not significant.

Ceruminous adenomas are benign neoplasms that arise from ceruminous glands in the external auditory canal. While these tumors are currently regarded as a single entity, they are divided into three histologically diverse subtypes: ceruminous syringocystadenoma papilliferum, ceruminous pleomorphic adenoma, and ceruminous adenoma not otherwise specified (NOS). Given the similarities of two of these subtypes to other tumors that occur at multiple anatomic sites, it is currently unclear whether ceruminous adenomas are truly a unified group. In this study, we performed targeted molecular profiling of 11 cases of ceruminous adenoma to clarify their classification. We identified BRAF V600E mutations (via PCR and/or immunohistochemistry) in five ceruminous syringocystadenomas papilliferum. We also identified HMGA2::WIF1 fusions (via RNA sequencing) in five ceruminous adenomas NOS and one ceruminous pleomorphic adenoma. Tumors with HMGA2::WIF1 fusion did not display the canalicular adenoma-like morphology seen in salivary gland pleomorphic adenomas with this fusion. Overall, these findings suggest that the three subtypes of ceruminous adenoma represent two biologically distinct groups. Recurrent BRAF V600E mutations in ceruminous syringocystadenoma papilliferum are parallel to those in cutaneous syringocystadenoma papilliferum. Histologic and molecular concordance suggests that ceruminous syringocystadenoma papilliferum should be part of the broader syringocystadenoma papilliferum category rather than a subtype of ceruminous adenoma. Conversely, HMGA2::WIF1 fusions in ceruminous adenoma NOS and ceruminous pleomorphic adenoma suggest that a stromal component may not be an essential point of distinction between these groups. These residual true ceruminous adenomas all likely represent a specialized form of mixed tumor unique to the external ear.

Abstract In patients with non–small cell lung cancer (NSCLC), the relationship between tumor metabolism and clinical outcomes is unknown. Here, 13C-labeled nutrients were intraoperatively infused into more than 90 patients with surgically resectable pulmonary lesions, and metabolic properties of resected tumors were correlated with survival. In NSCLCs infused with 13C-glucose, high 13C enrichment in tricarboxylic acid (TCA) cycle intermediates conferred a HR of 3.8 for early death, typically with metastasis. To test whether these features reflect requirements for metastasis, we generated patient-derived xenografts that spontaneously metastasize to multiple organs. Treatment with an electron transport chain (ETC) inhibitor reduced glucose-derived TCA cycle labeling but did not suppress subcutaneous tumor growth. However, ETC blockade reduced the abundance of circulating cancer cells and suppressed xenograft metastatic burden in distant organs. Our data demonstrate that isotope labeling can identify metabolic properties associated with metastasis in patients and that blocking the ETC suppresses metastasis in mice. Significance: Intraoperative 13C-glucose infusions in patients with NSCLC show that tumors with high labeling of TCA cycle intermediates progress rapidly, resulting in metastasis and early death. Blocking this pathway suppresses metastasis of human NSCLC cells in mice.

PDXs generated from primary NSCLCs spontaneously metastasize in NSG mice. A, Flow cytometry analysis of lung tissue from a mouse engrafted with mx73. Cells were stained with mouse lineage markers (CD45, CD31, and TER119) and HLA. B, Bioluminescence of mouse lungs bearing metastases from mx73. C, IHC staining for Ki67-positive cells in lung metastasis from mx148. D, Percentage of HLA-ABC–expressing cells detected in the lungs of mice bearing NSCLC PDXs. Each dot represents one mouse. Data are expressed as average and SD. The numerator and denominator reflect the number of mice with detectable HLA-ABC–expressing cells in the lung and the number of PDX-bearing mice analyzed, respectively. E, CT scans of brain metastases in patients 73 and 148. Yellow arrowheads indicate metastatic lesions. F, Flow cytometry analysis of mouse brains with metastatic cells from PDX mx73 (left) and PDX mx148 (right). G, H&E staining and IHC for CK7 in subcutaneous tumors and brains of mice bearing PDX mx73 (left) and PDX mx148 (right). H, Representative bioluminescence images of brains of mice with subcutaneous mx73 and mx148 PDXs. I, Summary of brain metastases observed in NSCLC PDXs. J, Representative bioluminescence images of liver metastases. K, Summary of liver metastasis in NSCLC PDXs. All data are expressed as average and SD. NT, not tested; SQ, subcutaneous.

Objective To illustrate patterns of cytogenetic abnormalities that promote progression and/or transformation in myelodysplastic syndrome. Methods In this study we evaluated three different data sets to identify recurrent cytogenetic abnormalities (RCAs) to delineate the cytogenetic evolutionary trajectories and their clinical significance. Results Datasets 1 and 2 were 2402 cross sectional samples from Mitelman database of Chromosome Aberrations and Gene Fusions in Cancer; these were used to discover RCAs and to validate them. Dataset 3 was a cohort of 163 institutional patients with serial samples from 35 % of them. This was used to further validate RCAs identified in the cross-sectional data, and their clinical impact. We identified MDS subtype associated RCAs, and some exclusive RCAs (Xp-, 2q-, 17q-, 21q-) that led to disease progression or transformation to leukemia. Evolutionary pathway analysis had shown temporal acquisition of RCAs. Therefore, presence of two or more RCAs suggests cooperative or complementary role in disease progression or transformation. Patients with one or more of these RCAs had poor prognosis and high risk for transformation. Genes frequently altered in MDS are mapped to some of the RCAs and suggest a close correlation between RCAs and molecular alterations in MDS. Karyotypic complexity, clonal evolution, loss of 17p had poor clinical outcomes. Conclusion This study identified a unique combination of RCAs that are components in distinct cytogenetic trajectories. Some of these were primary changes while others were secondary or tertiary changes. Acquiring specific additional aberrations predicts progression or transformation to leukemia.

ABSTRACT:PCR-based fragment analysis of the T-cell receptor (TCR) gene is used extensively in diagnostic labs to assess clonality in T-cell populations in multiple tissue sites. Of the numerous TCR assays that have been reported, studies assessing use on biopsies suspicious for mycosis fungoides specifically are lacking. We compared clonality findings from a previously run 2-tube/2-fluorochrome dye assay to a redesigned 1-tube/1-fluorochrome dye assay on formalin-fixed skin biopsies. Overall, the accuracy of the 2-tube assay was marginally better (75.7% vs. 71.4%), when using clinical history combined with histologic diagnosis as the gold standard. The 2-tube assay had better sensitivity (73.7% vs. 65.8%), while the 1-tube assay had superior specificity (93.8% vs. 87.5%). Clonality results were easier to interpret with the 1-tube assay. In nearly 19% of cases, a change of assays on the same biopsy resulted in a change of clonality interpretation. For laboratories that change TCR-γ clonality assays, follow-up biopsies for mycosis fungoides assessment may result in a change of diagnosis.