Performance of fluorescence in situ hybridization in biliary brushings with equivocal cytology: an institutional experience.

INTRODUCTION: Biliary brushing (BB) cytology has a sensitivity of 15%-65% and specificity approaching 100% for detecting malignancy. Fluorescence in-situ hybridization (FISH) using the UroVysion probe set has been advocated to enhance the detection of malignancies with reported sensitivity of 43%-84%. We sought to evaluate the performance of FISH in BB with equivocal cytology at our institution. MATERIALS AND METHODS: Patients with atypical and suspicious BB with concurrent diagnostic FISH performed at our institution from 2014 to 2021 were identified through a query of our pathology database. FISH (using UroVysion probe set containing centromere enumeration probes to chromosomes 3, 7, and 17) was positive if at least 5 cells demonstrated polysomy. Electronic medical records were reviewed for pathology results and outcomes. Patients were classified malignant if they had positive pathology or documented clinical impression of malignancy and benign if they had negative pathology and/or documented benign clinical course for at least 12 months. RESULTS: We identified 254 equivocal BB (238 atypical/16 suspicious) with concurrent FISH results from 191 patients (105 benign, 86 malignant). 12% (22/191) of patients were FISH positive. Twenty-four percent (21/86) of patients with malignancy had positive FISH but were nonspecific for pancreaticobiliary/ampullary adenocarcinomas. Almost all positive FISH were associated with malignancy (21/22; 95%). There was 1 positive FISH in a patient with primary sclerosing cholangitis who had a benign outcome. CONCLUSIONS: The small number of positive FISH results in BB with equivocal cytology raises the question of the optimal criteria for malignancy. Using only polysomy could result in lower sensitivity.

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma.

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

Specific Polo-Like Kinase 1 Expression in Nodular Lymphocyte-Predominant Hodgkin Lymphoma Suggests an Intact Immune Surveillance Program.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare and relatively indolent B-cell lymphoma. Characteristically, the [lymphocyte-predominant (LP)] tumor cells are embedded in a microenvironment enriched in lymphocytes. More aggressive variants of mature B-cell and peripheral T-cell lymphomas exhibit nuclear expression of the polo-like kinase 1 (PLK1) protein, stabilizing MYC (alias c-myc) and associated with worse clinical outcomes. This study demonstrated expression of PLK1 in the LP cells in 100% of NLPHL cases (n = 76). In contrast, <5% of classic Hodgkin lymphoma cases (n = 70) showed PLK1 expression within the tumor cells. Loss-of-function approaches demonstrated that the expression of PLK1 promoted cell proliferation and increased MYC stability in NLPHL cell lines. Correlation with clinical parameters revealed that the increased expression of PLK1 was associated with advanced-stage disease in patients with NLPHL. A multiplex immunofluorescence panel coupled with artificial intelligence algorithms was used to correlate the composition of the tumor microenvironment with the proliferative stage of LP cells. The results showed that LP cells with PLK1 (high) expression were associated with increased numbers of cytotoxic and T-regulatory T cells. Overall, the findings demonstrate that PLK1 signaling increases NLPHL proliferation and constitutes a potential vulnerability that can be targeted with PLK1 inhibitors. An active immune surveillance program in NLPHL may be a critical mechanism limiting PLK1-dependent tumor growth.

Molecular Analysis of Liquid Vitreous Biopsy Reveals Occult Lymphoma Following Cytology-Negative Biopsies of the Brain and Vitreous.

PURPOSE: Primary central nervous system lymphoma (PCNSL) is a rare but deadly malignancy that principally affects adults in the fifth and sixth decades of life. Despite diagnostic advances in analyses of cerebral spinal fluid and neuroimaging, definitive diagnosis of PCNSL requires primary brain tissue biopsy. While small neurosurgical biopsy volumes are pursued to minimize removal of normal brain tissue, the spatial margins to precisely biopsy pathologic tissue are narrow and can result in missed diagnoses. Furthermore, prior steroid treatment can significantly reduce tumor burden increasing the likelihood of a non-diagnostic biopsy. METHODS: A retrospective case report from a tertiary referral center using a combination of neuroradiological studies, sterotactic tissue biopsy, and molecular testing for genome mutations. RESULTS: A 72-year-old woman with strong suspicion for PCNSL clinically and radiologically, but cerebral spinal fluid and primary brain tissue biopsy were negative for tumor. However, vitreous liquid biopsy molecular testing for a MYD88 mutation as well as B-cell clonality (IGH/IGK rearrangement) were positive, indicating the presence of secondary vitreoretinal lymphoma from PCNSL. Only after autopsy of her brain was histopathological and immunohistochemical evidence of PCNSL confirmed. CONCLUSION: This case illustrates the unique contribution of liquid biopsy neuropathology-oriented molecular testing in a challenging case with high clinical suspicion of PCNSL in which gold-standard diagnostic testing failed to yield a diagnosis.

ETV6 Deficiency Unlocks ERG-Dependent Microsatellite Enhancers to Drive Aberrant Gene Activation in B-Lymphoblastic Leukemia.

Distal enhancers play critical roles in sustaining oncogenic gene-expression programs. We identify aberrant enhancer-like activation of GGAA tandem repeats as a characteristic feature of B-cell acute lymphoblastic leukemia (B-ALL) with genetic defects of the ETV6 transcriptional repressor, including ETV6-RUNX1+ and ETV6-null B-ALL. We show that GGAA repeat enhancers are direct activators of previously identified ETV6-RUNX1+/- like B-ALL "signature" genes, including the likely leukemogenic driver EPOR. When restored to ETV6-deficient B-ALL cells, ETV6 directly binds to GGAA repeat enhancers, represses their acetylation, downregulates adjacent genes, and inhibits B-ALL growth. In ETV6-deficient B-ALL cells, we find that the ETS transcription factor ERG directly binds to GGAA microsatellite enhancers and is required for sustained activation of repeat enhancer-activated genes. Together, our findings reveal an epigenetic gatekeeper function of the ETV6 tumor suppressor gene and establish microsatellite enhancers as a key mechanism underlying the unique gene-expression program of ETV6-RUNX1+/- like B-ALL. SIGNIFICANCE: We find a unifying mechanism underlying a leukemia subtype-defining gene-expression signature that relies on repetitive elements with poor conservation between humans and rodents. The ability of ETV6 to antagonize promiscuous, nonphysiologic ERG activity may shed light on other roles of these key regulators in hematolymphoid development and human disease. See related commentary by Mercher, p. 2. This article is highlighted in the In This Issue feature, p. 1.

One-Step Multiplexed Droplet Digital Polymerase Chain Reaction for Quantification of p190 BCR-ABL1 Fusion Transcript in B-Lymphoblastic Leukemia.

CONTEXT.­: Quantification and detection of the t(9;22) (BCR-ABL1) translocation in chronic myelogenous leukemia and B-lymphoblastic leukemia are important for directing treatment protocols and monitoring disease relapse. However, quantification using traditional reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is dependent on a calibration curve and is prone to laboratory-to-laboratory variation. Droplet digital polymerase chain reaction (ddPCR) is a novel method that allows for highly sensitive absolute quantification of transcript copy number. As such, ddPCR is a good candidate for disease monitoring, an assay requiring reproducible measurements with high specificity and sensitivity. OBJECTIVE.­: To compare results of ddPCR and RT-qPCR BCR-ABL1 fusion transcript measurements of patient samples and determine if either method is superior. DESIGN.­: We optimized and standardized a 1-step multiplexed ddPCR assay to detect BCR-ABL1 p190 and ABL1 e10 transcripts. The ddPCR optimization included varying cycle number and primer concentration with standardization of droplet generation and droplet number and analyses to improve data sensitivity. Following optimization, ddPCR measurements were performed on clinical samples and compared with traditional RT-qPCR results. RESULTS.­: Droplet digital polymerase chain reaction was able to detect the BCR-ABL1 p190 transcript to 0.001% (1:10-5) with a calculated limit of detection and limit of quantitation of 4.1 and 5.3 transcripts, respectively. When tested on patient samples, ddPCR was able to identify 20% more positives than a laboratory-developed 2-step RT-qPCR assay. CONCLUSIONS.­: Droplet digital polymerase chain reaction demonstrated increased detection of BCR-ABL1 compared with RT-qPCR. Improved detection of BCR-ABL1 p190 and the potential for improved standardization across multiple laboratories makes ddPCR a suitable method for disease monitoring in patients with acute B-lymphoblastic leukemia.

Immunophenotypic switch in cutaneous T-cell lymphoma: A series of three cases and review of the literature.

Primary cutaneous T-cell lymphoma (CTCL) comprises a heterogeneous group of neoplasms with variable clinical behavior. Immunophenotypic switch (IS) is a phenomenon that occurs during lymphoma progression and is defined by an alteration in the immunophenotypic expression of a tumor with retention of its genotypic signature. This has been well-recognized in hematopoietic neoplasms; however, it has been rarely reported in CTCL and its clinical implications are not well understood. We present the clinical, histopathologic, immunophenotypic, and genetic findings of three cases of CTCL that demonstrated IS post treatment with variable outcomes. We add our cases to the small number previously reported to increase awareness of this phenomenon and its diagnostic challenge.

Primary cutaneous follicle center lymphoma with extensive plasmacytic differentiation and t(14;18) in both the lymphoid and plasma cell components.

Primary cutaneous follicle center lymphoma (PCFCL) is the most common cutaneous B-cell lymphoma. The typical immunophenotype includes expression of both CD20 and BCL6, with the majority of cases lacking expression of CD10, BCL2, and the characteristic t(14;18)/IGH-BCL2 rearrangement seen in systemic follicular lymphoma (FL). Plasmacytic differentiation (PD) is an uncommon finding in both systemic and cutaneous FLs and presents a diagnostic challenge when present, leading to the potential for misdiagnosis as marginal zone lymphoma (MZL). Limited reports have described light chain restriction in the plasma cell component of FLs with PD, and rare cases of PCFCL with PD have been described. While the IGH-BCL2 translocation has been identified in a subset of FLs with PD, the presence of the BCL2 translocation in monotypic plasma cells of PCFCL has not been previously described to our knowledge. Here, we report a case of PCFCL with extensive PD in a 77-year-old woman that was favored to represent primary cutaneous MZL on an initial punch biopsy. Excisional biopsy, however, revealed that the atypical lymphocytes expressed CD10, BCL6, and BCL2, while the plasma cell component demonstrated light-chain lambda restriction. FISH studies showed the presence of an IGH-BCL2 translocation in both the lymphocytic and plasmacytic components.

TP53 mutations and CDKN2A mutations/deletions are highly recurrent molecular alterations in the malignant progression of sinonasal papillomas.

Sinonasal papillomas are benign epithelial tumors of the sinonasal tract that are associated with a synchronous or metachronous sinonasal carcinoma in a subset of cases. Our group recently identified mutually exclusive EGFR mutations and human papillomavirus (HPV) infection in inverted sinonasal papillomas and frequent KRAS mutations in oncocytic sinonasal papillomas. We also demonstrated concordant mutational and HPV infection status in sinonasal papilloma-associated sinonasal carcinomas, confirming a clonal relationship between these tumors. Despite our emerging understanding of the oncogenic mechanisms driving formation of sinonasal papillomas, little is currently known about the molecular mechanisms of malignant progression to sinonasal carcinoma. In the present study, we utilized targeted next-generation DNA sequencing to characterize the molecular landscape of a large cohort of sinonasal papilloma-associated sinonasal carcinomas. As expected, EGFR or KRAS mutations were present in the vast majority of tumors. In addition, highly recurrent TP53 mutations, CDKN2A mutations, and/or CDKN2A copy-number losses were detected; overall, nearly all tumors (n = 28/29; 96.6%) harbored at least one TP53 or CDKN2A alteration. TERT copy-number gains also occurred frequently (27.6%); however, no TERT promoter mutations were identified. Other recurrent molecular alterations included NFE2L2 and PIK3CA mutations and SOX2, CCND1, MYC, FGFR1, and EGFR copy-number gains. Importantly, TP53 mutations and CDKN2A alterations were not detected in matched sinonasal papillomas, suggesting that these molecular events are associated with malignant transformation. Compared to aerodigestive tract squamous cell carcinomas from The Cancer Genome Atlas (TCGA) project, sinonasal papilloma-associated sinonasal carcinomas have a distinct molecular phenotype, including more frequent EGFR, KRAS, and CDKN2A mutations, TERT copy-number gains, and low-risk human papillomavirus (HPV) infection. These findings shed light on the molecular mechanisms of malignant progression of sinonasal papillomas and may have important diagnostic and therapeutic implications for patients with advanced sinonasal cancer.

Cutaneous follicle center lymphomas with plasmacytic differentiation.

Follicle center lymphomas, including primary cutaneous follicle center lymphoma (PCFCL), may rarely show plasmacytic differentiation. Such cases can pose a diagnostic challenge and can be mistaken for other lymphomas that more commonly include plasma cells. Here, we report four cases of PCFCL and one case of systemic follicular lymphoma involving the skin with associated monotypic plasma cells, including the clinical, morphologic and immunophenotypic features.

Enabling Precision Oncology Through Precision Diagnostics.

Genomic testing enables clinical management to be tailored to individual cancer patients based on the molecular alterations present within cancer cells. Genomic sequencing results can be applied to detect and classify cancer, predict prognosis, and target therapies. Next-generation sequencing has revolutionized the field of cancer genomics by enabling rapid and cost-effective sequencing of large portions of the genome. With this technology, precision oncology is quickly becoming a realized paradigm for managing the treatment of cancer patients. However, many challenges must be overcome to efficiently implement the transition of next-generation sequencing from research applications to routine clinical practice, including using specimens commonly available in the clinical setting; determining how to process, store, and manage large amounts of sequencing data; determining how to interpret and prioritize molecular findings; and coordinating health professionals from multiple disciplines.

PAX8 expression and TERT promoter mutations in the nested variant of urothelial carcinoma: a clinicopathologic study with immunohistochemical and molecular correlates.

The nested variant of urothelial carcinoma, a frequent mimic of benign lesions on limited specimens, has been associated with high-stage disease including metastases at presentation. While PAX8 immunohistochemistry has been noted to be infrequently present in urothelial carcinoma in general, it has not been studied specifically in a cohort of nested urothelial carcinomas. Furthermore, TERT promoter mutation status is a potentially valuable biomarker for diagnosis of urothelial carcinoma and for noninvasive disease monitoring that has been observed in a majority of urothelial carcinoma and has previously been seen to be prevalent in multiple variant morphologies of urothelial carcinoma, including the nested variant. Twenty-five primary and three metastatic samples of nested urothelial carcinoma, along with 16 benign cases, were identified in a multicenter retrospective record review. PAX8 immunohistochemical stain was performed on all cases. In addition, TERT mutation analysis by allele-specific PCR was performed on 21 of the primary nested urothelial carcinoma cases and all benign cases. Positive PAX8 expression was identified in 52% (13 of 25) primary cases and 67% (2 of 3) metastatic cases of nested urothelial carcinoma; 50% (1 of 2) cases of large nested urothelial carcinoma were positive for PAX8. PAX8 expression was negative in the benign urothelium in all cases. TERT promoter mutation was observed in 83% (15 of 18) nested urothelial carcinoma cases and in 6% (1 of 16) of the benign cases. Recognition of the prevalence of positive PAX8 staining in this clinically relevant variant of urothelial carcinoma is essential to avoiding inaccurate or delayed diagnosis during the diagnostic workup of bladder lesions suspicious for nested variant of urothelial carcinoma. Moreover, the prevalence of TERT promoter mutations in nested urothelial carcinoma is similar to that of conventional urothelial carcinoma, further supporting its use as a biomarker that is stable across morphologic variants of urothelial carcinoma.

Cervical Mesonephric Adenocarcinoma With Novel FGFR2 Mutation.

Mesonephric adenocarcinoma is a rare tumor, accounting for <1% of cervical cancers. Well-differentiated mesonephric adenocarcinoma can be difficult to distinguish from diffuse mesonephric hyperplasia. Herein, we report a case of well-differentiated mesonephric adenocarcinoma with an FGFR2 mutation not previously reported in the literature. Nonselective tyrosine kinase inhibitors or FGFR2 inhibitors may represent options for targeted therapy.

Expression of the checkpoint receptors LAG-3, TIM-3 and VISTA in peripheral T cell lymphomas.

AIMS: Peripheral T cell lymphomas represent approximately 10%-15% of non-Hodgkin lymphomas and are characterised by an aggressive clinical courses and poor outcomes. Ligands provided by constituents of the tumour microenvironment engage receptors expressed by malignant T cells, promoting tumour growth and chemotherapy resistance. In addition to stimulatory receptors that promote the growth and survival of malignant T cells, recent studies suggest that homologous inhibitory receptors may have an opposing effect and function as tumour suppressors. For example, recent data suggest that programmed cell death 1 blockade may lead to increased lymphoma growth. Therefore, the identification of alternative checkpoint receptors in T cell lymphoproliferative neoplasms is an important and clinically relevant question. METHODS: The checkpoint receptors T cell immunoglobulin-3 (TIM-3), V-domain Ig-containing suppressor of T cell activation (VISTA) and lymphocyte-activation gene 3 (LAG-3) play fundamental roles in peripheral tolerance, and their ligands are exploited by many solid tumours to evade host immunity. However, their expression in T cell lymphoproliferative neoplasms has not been evaluated. In this study, we evaluated the expression of TIM-3, VISTA and LAG-3 in a cohort of peripheral T cell lymphomas cases by immunohistochemistry and flow cytometric analysis. RESULTS: Our results demonstrate that TIM-3, VISTA and LAG-3 expression is rarely identified within a large cohort of T cell lymphomas and its tumour microenvironment. CONCLUSIONS: Our data suggest that immune-regulatory roles for TIM-3, VISTA and LAG-3 may be predominant in lymphomas subsets different than the ones analysed in the current study. However, a potential role for these checkpoint receptors as tumour suppressors in T cell lymphomas remains to be elucidated.

TERT- beyond the territory: Usage of PCR-based TERT promoter assay in defining urothelial carcinoma in a case of long-standing prostatic adenocarcinoma.

Bladder cancer continues to be a source of disease burden worldwide. In patients with a long-standing history of prostate cancer, distinguishing between new/independent and synchronous poorly differentiated urothelial carcinoma and residual/recurrent high grade/poorly differentiated prostatic adenocarcinoma or prostatic adenocarcinoma with therapy-related changes can be diagnostically and therapeutically challenging. In the present case report, along with morphological features, immunohistochemical (IHC) studies and a novel polymerase chain reaction (PCR) based telomerase reverse transcriptase (TERT) promoter mutation assay were used as essential ancillary tools in reaching a final diagnosis. This is important as spatially, topographically and temporally multi-focal and multi-differentiating tumors can behave differently with different prognostic and therapeutic connotations.

Colony-Stimulating Factor 1 Receptor (CSF1R) Activates AKT/mTOR Signaling and Promotes T-Cell Lymphoma Viability.

PURPOSE: Peripheral T-cell lymphomas are clinically aggressive and usually fatal, as few complete or durable remissions are achieved with currently available therapies. Recent evidence supports a critical role for lymphoma-associated macrophages during T-cell lymphoma progression, but the specific signals involved in the cross-talk between malignant T cells and their microenvironment are poorly understood. Colony-stimulator factor 1 receptor (CSF1R, CD115) is required for the homeostatic survival of tissue-resident macrophages. Interestingly, its aberrant expression has been reported in a subset of tumors. In this article, we evaluated its expression and oncogenic role in T-cell lymphomas. EXPERIMENTAL DESIGN: Loss-of-function studies, including pharmacologic inhibition with a clinically available tyrosine kinase inhibitor, pexidartinib, were performed in multiple in vitro and in vivo models. In addition, proteomic and genomic screenings were performed to discover signaling pathways that are activated downstream of CSF1R signaling. RESULTS: We observed that CSF1R is aberrantly expressed in many T-cell lymphomas, including a significant number of peripheral and cutaneous T-cell lymphomas. Colony-stimulating factor 1 (CSF1), in an autocrine or paracrine-dependent manner, leads to CSF1R autophosphorylation and activation in malignant T cells. Furthermore, CSF1R signaling was associated with significant changes in gene expression and in the phosphoproteome, implicating PI3K/AKT/mTOR in CSF1R-mediated T-cell lymphoma growth. We also demonstrated that inhibition of CSF1R in vivo and in vitro models is associated with decreased T-cell lymphoma growth. CONCLUSIONS: Collectively, these findings implicate CSF1R in T-cell lymphomagenesis and have significant therapeutic implications.

Next-generation sequencing implicates oncogenic roles for p53 and JAK/STAT signaling in microcystic adnexal carcinomas.

Microcystic adnexal carcinoma is a locally aggressive sweat gland carcinoma characterized by its infiltrative growth and histopathologic overlap with benign adnexal tumors, often posing challenges to both diagnosis and management. Understanding the molecular underpinnings of microcystic adnexal carcinoma may allow for more accurate diagnosis and identify potential targetable oncogenic drivers. We characterized 18 microcystic adnexal carcinomas by targeted, multiplexed PCR-based DNA next-generation sequencing of the coding sequence of over 400 cancer-relevant genes. The majority of cases had relatively few (<8) prioritized somatic mutations, and lacked an ultraviolet (UV) signature. The most recurrent mutation was TP53 inactivation in four (22%) tumors. Frame-preserving insertions affecting the kinase domain of JAK1 were detected in three (17%) cases, and were nonoverlapping with TP53 mutations. Seven (39%) cases demonstrated copy number gain of at least one oncogene. By immunohistochemistry, p53 expression was significantly higher in microcystic adnexal carcinomas with TP53 mutations compared with those without such mutations and syringomas. Similarly, phospho-STAT3 expression was significantly higher in microcystic adnexal carcinomas harboring JAK1 kinase insertions compared with those with wild-type JAK1 and syringomas. In conclusion, microcystic adnexal carcinomas are molecularly heterogeneous tumors, with inactivated p53 or activated JAK/STAT signaling in a subset. Unlike most other nonmelanoma skin cancers involving sun-exposed areas, most microcystic adnexal carcinomas lack evidence of UV damage, and hence likely originate from a relatively photo-protected progenitor population in the dermis. These findings have implications for the biology, diagnosis, and treatment of microcystic adnexal carcinomas, including potential for therapeutic targeting of p53 or the JAK/STAT pathway in advanced tumors.

Sinonasal Papillomas and Carcinomas: A Contemporary Update With Review of an Emerging Molecular Classification.

CONTEXT.­: Sinonasal papillomas and carcinomas are uncommon head and neck neoplasms that comprise a broad clinicopathologic and morphologic spectrum, and thus frequently represent a diagnostic challenge for surgical pathologists. Recent molecular interrogation of these tumors has delineated a number of recurrent alterations that correspond to distinct entities with potential diagnostic and/or therapeutic clinical utility. OBJECTIVE.­: To summarize the salient clinicopathologic, morphologic, and molecular features of sinonasal papillomas and carcinomas. DATA SOURCES.­: Review of pertinent literature regarding sinonasal papillomas and sinonasal carcinomas. CONCLUSIONS.­: Despite their relative rarity in many surgical pathology practices, sinonasal papillomas and carcinomas frequently demonstrate characteristic morphologic features that are important for accurate diagnosis. Given our emerging understanding of the molecular basis for these tumors, judicious use of available ancillary tools-including immunohistochemistry and in situ hybridization-may be helpful in subsets of cases, whereas additional molecular testing may be useful for diagnostically challenging and/or clinically aggressive sinonasal tumors.

The EGFR T790M Mutation Is Acquired through AICDA-Mediated Deamination of 5-Methylcytosine following TKI Treatment in Lung Cancer.

: Almost all patients with EGFR-driven lung cancer who are treated with EGFR tyrosine kinase inhibitors (TKI) develop resistance to treatment. A single base (c.2369C>T) transition mutation, EGFR T790M, is the most frequent resistance event after first-generation exposure to EGFR TKIs. Whether T790M mutation is acquired or is selected from a preexisting clone has been a matter of significant debate. In this study, we show that treatment with EGFR TKIs leads to activation of the NFκB pathway, which in turn induces expression of activation-induced cytidine deaminase (AICDA). In turn, AICDA causes deamination of 5-methylcytosine to thymine at position c.2369 to generate the T790M mutation. Pharmacologic inhibition of the NFκB pathway or knockout of AICDA decreased the frequency or prevented the development of T790M mutation, respectively. In addition, patients treated with first-line EGFR TKI displayed increased expression of AICDA and detection of the T790M mutation upon progression. These results identify the mechanism of T790M acquisition and present an opportunity to target the process to delay or prevent it. SIGNIFICANCE: These findings identify the mechanism behind acquisition of a common resistance mutation to TKI treatment in lung cancer.