Clinical Focus

  • Cytopathology
  • Head & Neck Pathology
  • Fine Needle Aspiration Biopsy
  • Anatomic and Clinical Pathology

Academic Appointments

  • Clinical Associate Professor, Pathology

Administrative Appointments

  • Director of Cytopathology Service, Stanford University School of Medicine, Department of Pathology (2020 - Present)
  • Associate Director of Cytopathology Service, Stanford University School of Medicine, Department of Pathology (2018 - 2020)
  • Director of Head & Neck Pathology Service, Stanford University School of Medicine, Department of Pathology (2018 - Present)

Professional Education

  • Board Certification: American Board of Pathology, Anatomic and Clinical Pathology (2014)
  • Board Certification: American Board of Pathology, Cytopathology (2015)
  • Fellowship: Massachusetts General Hospital, Department of Pathology (2015) MA
  • Residency: Johns Hopkins School of Medicine, Department of Pathology (2014) MD
  • Medical Education: Vanderbilt University School of Medicine (2010) TN

Graduate and Fellowship Programs

  • Cytopathology (Fellowship Program)

All Publications

  • Morphological diversity of cancer cells predicts prognosis across tumor types. Journal of the National Cancer Institute Sali, R., Jiang, Y., Attaranzadeh, A., Holmes, B., Li, R. 2023


    Intratumor heterogeneity drives disease progression and treatment resistance, which can lead to poor patient outcomes. Here, we present a computational approach for quantification of cancer cell diversity in routine hematoxylin and eosin (H&E)-stained histopathology images.We analyzed publicly available digitized whole slide H&E images for a total of 2000 patients. Four tumor types were included: lung, head and neck, colon and rectal cancers, representing major histology subtypes (adenocarcinomas and squamous cell carcinomas). We performed single-cell analysis on H&E images and trained a deep convolutional autoencoder to automatically learn feature representations of individual cancer nuclei. We then computed features of intra-nuclear variability and inter-nuclear diversity to quantify tumor heterogeneity. Finally, we used these features to build a machine learning model to predict patient prognosis.A total of 68 million cancer cells were segmented and analyzed for nuclear image features. We discovered multiple morphological subtypes of cancer cells (range: 15-20) that co-exist within the same tumor, each with distinct phenotypic characteristics. Moreover, we showed that a higher morphological diversity is associated with chromosome instability and genomic aneuploidy. A machine learning model based on morphological diversity demonstrated independent prognostic values across tumor types (hazard ratio range: 1.62-3.23, P < 0.035) in validation cohorts and further improved prognostication when combined with clinical risk factors.Our study provides a practical approach for quantifying intratumor heterogeneity based on routine histopathology images. The cancer cell diversity score can be used to refine risk stratification and inform personalized treatment strategies.

    View details for DOI 10.1093/jnci/djad243

    View details for PubMedID 37982756

  • Parosteal Lipoma Overlying an Osteochondroma of the Hyoid Bone: A Case Report and Literature Review. The Laryngoscope Green, A., Wei, E. X., Chang-Graham, A., Holmes, B., Smith, A., Sung, C. K. 2023


    Parosteal lipomas and osteochondromas of the head and neck are uncommon benign tumors, constituting a small fraction of lipoma and bone tumor cases. We present a unique case of a 66-year-old male with a parosteal lipoma overlying an osteochondroma in the anterior midline neck, causing dysphagia. Surgical excision confirmed the diagnosis, and a literature review revealed similar cases predominantly adjacent to the mandible or calvaria. This case emphasizes the need to have parosteal lipoma and osteochondroma on the differential diagnosis for patients presenting with a firm mass of the central neck, especially with a history of trauma. Laryngoscope, 2023.

    View details for DOI 10.1002/lary.31192

    View details for PubMedID 37965970

  • Targeted proteomic quantitation of NRF2 signaling and predictive biomarkers in HNSCC. Molecular & cellular proteomics : MCP Wamsley, N. T., Wilkerson, E. M., Guan, L., LaPak, K. M., Schrank, T. P., Holmes, B. J., Sprung, R. W., Gilmore, P. E., Gerndt, S. P., Jackson, R. S., Paniello, R. C., Pipkorn, P., Puram, S. V., Rich, J. T., Townsend, R. R., Zevallos, J. P., Zolkind, P., Le, Q. T., Goldfarb, D., Ben Major, M. 2023: 100647


    The NFE2L2(NRF2) oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry (MS)-based targeted proteomics assay based on internal standard triggered parallel reaction monitoring (IS-PRM) to quantify 69 NRF2 pathway components and targets, as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved an existing IS-PRM acquisition algorithm, called SureQuantTM, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded (FFPE) HNSCCs, NRF2 signaling intensity positively correlated with NRF2 activating mutations and with SOX2 protein expression. Protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus (HPV) infection status. CDKN2A (p16) protein expression positively correlated with the HPV oncogenic E7 protein, and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or FFPE archived tissues in under 90 minutes.

    View details for DOI 10.1016/j.mcpro.2023.100647

    View details for PubMedID 37716475

  • Galectin-1 mediates chronic STING activation in tumors to promote metastasis through MDSC recruitment. Cancer research Nambiar, D. K., Viswanathan, V., Cao, H., Zhang, W., Guan, L., Chamoli, M., Holmes, B., Kong, C., Hildebrand, R., Koong, A. J., von Eyben, R., Plevritis, S., Li, L., Giaccia, A., Engleman, E., Le, Q. T. 2023


    The immune system plays a crucial role in the regulation of metastasis. Tumor cells systemically change immune functions to facilitate metastatic progression. Through this study, we deciphered how tumoral Galectin-1 (Gal1) expression shapes the systemic immune environment to promote metastasis in head and neck cancer (HNC). In multiple preclinical models of HNC and lung cancer in immunogenic mice, Gal1 fostered the establishment of a pre-metastatic niche through polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), which altered the local microenvironment to support metastatic spread. RNA sequencing of MDSCs from pre-metastatic lungs in these models demonstrated the role of PMN-MDSCs in collagen and extracellular matrix remodeling in the pre-metastatic compartment. Gal1 promoted MDSC accumulation in the pre-metastatic niche through the NF-κB signaling axis, triggering enhanced CXCL2-mediated MDSC migration. Mechanistically, Gal1 sustained NF-κB activation in tumor cells by enhancing STING protein stability, leading to prolonged inflammation-driven MDSC expansion. These findings suggest an unexpected pro-tumoral role of STING activation in metastatic progression and establish Gal1 as an endogenous positive regulator of STING in advanced-stage cancers.

    View details for DOI 10.1158/0008-5472.CAN-23-0046

    View details for PubMedID 37409887

  • Association between locoregional failure and NFE2L2/KEAP1/CUL3 pathway mutations in NRG/RTOG 9512: A randomized trial of hyperfractionation vs. conventional fractionation in T2N0 glottic squamous cell carcinoma (SCC). Guan, L., Torres-Saavedra, P. A., Zhao, X., Major, M. B., Holmes, B. J., Nguyen, N., Kumaravelu, P., Hodge, T., Diehn, M., Zevallos, J., Emami, B., Sagar, S. M., Morrison, W. H., Schultz, C. J., Caudell, J. J., Jones, C. U., Yom, S. S., Harris, J., Le, Q., Hayes, D. N. LIPPINCOTT WILLIAMS & WILKINS. 2023
  • NFE2L2 mutations enhance radioresistance in head and neck cancer by modulating intratumoral myeloid cells. Cancer research Guan, L., Nambiar, D. K., Cao, H., Viswanathan, V., Kwok, S., Hui, A. B., Hou, Y., Hildebrand, R., von Eyben, R., Holmes, B. J., Zhao, J., Kong, C. S., Wamsley, N., Zhang, W., Major, M. B., Seol, S. W., Sunwoo, J. B., Hayes, D. N., Diehn, M., Le, Q. T. 2023


    Radiotherapy is one of the primary treatments of head and neck squamous cell carcinoma (HNSCC), which has a high risk of locoregional failure (LRF). Presently, there is no reliable predictive biomarker of radioresistance in HNSCC. Here, we found that mutations in NFE2L2, which encodes Nrf2, are associated with a significantly higher rate of LRF in patients with oral cavity cancer treated with surgery and adjuvant (chemo)radiotherapy but not in those treated with surgery alone. Somatic mutation of NFE2L2 led to Nrf2 activation and radioresistance in HNSCC cells. Tumors harboring mutant Nrf2E79Q were substantially more radioresistant than tumors with wild-type Nrf2 in immunocompetent mice, while the difference was diminished in immunocompromised mice. Nrf2E79Q enhanced radioresistance through increased recruitment of intratumoral polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and reduction of M1-polarized macrophages. Treatment with the glutaminase inhibitor CB-839 overcame the radioresistance induced by Nrf2E79Q or Nrf2E79K. Radiotherapy increased expression of PMN-MDSC-attracting chemokines, including CXCL1, CXLC3 and CSF3, in Nrf2E79Q-expressing tumors via the TLR4, which could be reversed by CB-839. This study provides insights into the impact of NFE2L2 mutations on radioresistance and suggests that CB-839 can increase radiosensitivity by switching intratumoral myeloid cells to an anti-tumor phenotype, supporting clinical testing of CB-839 with radiation in HNSCC with NFE2L2 mutations.

    View details for DOI 10.1158/0008-5472.CAN-22-1903

    View details for PubMedID 36652552

  • CDKN2A/B Loss in High-Grade Transformation of Salivary Gland Carcinoma: High-Grade Malignant Potential in Histologically Low-Grade Component Nakaguro, M., Faquin, W., Snuderl, M., Baloch, Z., Cantley, R., Compton, M., Ely, K., Holmes, B., Hu, R., Kerr, D., Montone, K., Nishino, M., Pantanowitz, L., Rossi, E., Nagao, T., Sadow, P. SPRINGERNATURE. 2022: 894-895
  • Detection of cryptogenic malignancies from metagenomic whole genome sequencing of body fluids. Genome medicine Gu, W., Talevich, E., Hsu, E., Qi, Z., Urisman, A., Federman, S., Gopez, A., Arevalo, S., Gottschall, M., Liao, L., Tung, J., Chen, L., Lim, H., Ho, C., Kasowski, M., Oak, J., Holmes, B. J., Yeh, I., Yu, J., Wang, L., Miller, S., DeRisi, J. L., Prakash, S., Simko, J., Chiu, C. Y. 2021; 13 (1): 98


    BACKGROUND: Metagenomic next-generation sequencing (mNGS) of body fluids is an emerging approach to identify occult pathogens in undiagnosed patients. We hypothesized that metagenomic testing can be simultaneously used to detect malignant neoplasms in addition to infectious pathogens.METHODS: From two independent studies (n = 205), we used human data generated from a metagenomic sequencing pipeline to simultaneously screen for malignancies by copy number variation (CNV) detection. In the first case-control study, we analyzed body fluid samples (n = 124) from patients with a clinical diagnosis of either malignancy (positive cases, n = 65) or infection (negative controls, n = 59). In a second verification cohort, we analyzed a series of consecutive cases (n = 81) sent to cytology for malignancy workup that included malignant positives (n = 32), negatives (n = 18), or cases with an unclear gold standard (n = 31).RESULTS: The overall CNV test sensitivity across all studies was 87% (55 of 63) in patients with malignancies confirmed by conventional cytology and/or flow cytometry testing and 68% (23 of 34) in patients who were ultimately diagnosed with cancer but negative by conventional testing. Specificity was 100% (95% CI 95-100%) with no false positives detected in 77 negative controls. In one example, a patient hospitalized with an unknown pulmonary illness had non-diagnostic lung biopsies, while CNVs implicating a malignancy were detectable from bronchoalveolar fluid.CONCLUSIONS: Metagenomic sequencing of body fluids can be used to identify undetected malignant neoplasms through copy number variation detection. This study illustrates the potential clinical utility of a single metagenomic test to uncover the cause of undiagnosed acute illnesses due to cancer or infection using the same specimen.

    View details for DOI 10.1186/s13073-021-00912-z

    View details for PubMedID 34074327

  • Fine needle aspiration of salivary gland carcinomas with high-grade transformation: A multi-institutional study of 22 cases and review of the literature. Cancer cytopathology Nakaguro, M., Faquin, W. C., Baloch, Z. W., Cantley, R. L., Compton, M. L., Ely, K. A., Holmes, B. J., Hu, R., Kerr, D. A., Montone, K. T., Nishino, M., Pantanowitz, L., Rossi, E. D., Sadow, P. M. 2020


    BACKGROUND: High-grade transformation (HGT) is a rare process whereby conventional low- to intermediate-grade salivary gland carcinomas (SGC) transform into high-grade, poorly or undifferentiated malignancies with focal or complete loss of their conventional histomorphologic features. Because tumors with HGT are associated with a worse prognosis than their conventional counterparts, preoperative recognition of HGT may be of benefit for optimal patient management. Using a multi-institutional approach, we describe the largest fine needle aspiration (FNA) cohort of salivary gland carcinomas with HGT.METHODS: The archives of 9 large academic medical centers were searched, and 22 cases of SGC with HGT were identified by surgical excision accompanied by preoperative FNA. Clinical and cytomorphologic features were retrospectively reviewed.RESULTS: The male-to-female ratio was 14:8, and the mean patient age was 60.2 years. The average tumor size was 3.6 cm, and 19 cases were from the parotid gland. Acinic cell carcinoma with HGT was the most common tumor subtype, comprising 12 cases with HGT, followed by adenoid cystic carcinoma, secretory carcinoma, and other subtypes. Eighteen cases were classified as malignant; however, a specific diagnosis of HGT was not made. Sixteen cases contained a high-grade cytologic component, and 7 cases had a mixture of both conventional and high-grade components retrospectively.CONCLUSIONS: SGC with HGT should be considered in the differential diagnosis of a salivary gland aspirate exhibiting high-grade cytomorphologic features. The presence of distinct tumor populations, conventional and high-grade, should prompt consideration of HGT, especially when the conventional component is acinic cell carcinoma or adenoid cystic carcinoma.

    View details for DOI 10.1002/cncy.22388

    View details for PubMedID 33211402

  • Salivary Gland Cytopathology and Histopathology: Practical Insights and Updates From the World Health Organization Classification AJSP-REVIEWS AND REPORTS Holmes, B. J. 2020; 25 (5): 199–200
  • Pilot study of loss of the p53/p63 target gene PERP at the surgical margin as a potential predictor of local relapse in head and neck squamous cell carcinoma. Head & neck Holmes, B. J., von Eyben, R., Attardi, L. D., Kong, C. S., Le, Q. T., Nathan, C. O. 2020


    PERP (p53 apoptosis effector related to PMP22) localizes to desmosomes and suppresses squamous cell carcinoma development. Loss of PERP leads to worse local control in head and neck squamous cell carcinoma (HNSCC), likely by destabilizing desmosomes. We evaluated PERP loss at HNSCC surgical margins as a predictor of local relapse.Combining discovery (n = 17) and validation (n = 31) cohorts, we examined membranous PERP protein expression by immunohistochemistry in surgical mucosal margins with competing risk analysis of the relationship between local relapse and PERP expression.Of the 44 analyzable patients, the 2-year cumulative incidence of local relapse was 44.4% for the PERP-negative group and 16.4% for the PERP-positive group (P = .01). A trend toward worse progression-free survival (P = .09) and overall survival (P = .06) was observed with loss of PERP.PERP loss at surgical margins is associated with higher risk of local recurrence in HNSCC, warranting further evaluation in a larger prospective study.

    View details for DOI 10.1002/hed.26358

    View details for PubMedID 33034918

  • Model for Assessing Cytopathology Workload, Efficiency, and Wellness Li, Y., Cederlof, K., Cowan, T., De Leon, F., Mendoza, K., de Jesus, A., Kong, C., Holmes, B. NATURE PUBLISHING GROUP. 2020: 1871–72
  • Macrocystic (Mammary Analogue) Secretory Carcinoma: An Unusual Variant and a Pitfall in the Differential Diagnosis of Cystic Lesions in the Head and Neck. The American journal of surgical pathology Hernandez-Prera, J. C., Holmes, B. J., Valentino, A., Harshan, M., Bacchi, C. E., Petersson, F., Liu, K. K., Najfeld, V., Wenig, B. M. 2019


    Mammary analogue secretory carcinoma (MASC) is a relatively recently described salivary gland adenocarcinoma characterized by ETV6-NTRK3 gene fusion and in most cases indolent clinical behavior. The majority of tumors show an admixture of microcystic, solid, and tubular growth patterns but only a few cases with dominant macrocystic growth have been reported. We report 15 cases of macrocystic MASC. There were 11 men and 4 women (17 to 88y age range, average 47y). The patients presented with a painless cystic mass, the majority in the region of the parotid gland (n=13), as well as in submandibular gland (n=1) and the neck (n=1). All tumors were circumscribed measuring 1.0 to 4.0cm in greatest diameter (mean: 1.75cm). Twelve tumors were unilocular, while 3 were multilocular. The cystic spaces were predominantly lined by a single epithelial cell layer with focal areas in which the epithelium was multilayered with papillary and hobnail features. In 3 of the cases there were more solid foci of intracystic tumor characterized by papillary and/or microcystic growth. The neoplastic cells were round to oval with hyperchromatic to vesicular nuclei with centrally located nucleoli and eosinophilic or vacuolated cytoplasm. Tumor cells showed strong positivity for S100 protein and mammaglobin, while DOG1 was uniformly negative. A minority of cases showed focal p63 reactivity predominantly limited to the periphery of the cystic lining. ETV6 gene rearrangement was identified in 9 cases. Macrocystic MASC can simulate benign and malignant salivary gland lesions and needs to be included in the differential diagnosis of cystic lesions in the head and neck. To the best of our knowledge, our report represents the first series of macrocystic MASCs wholly focusing on this unusual variant.

    View details for DOI 10.1097/PAS.0000000000001309

    View details for PubMedID 31464708

  • A Young Woman With a Rapidly GrowingThoracic Tumor. Chest Lai, Y. K., Holmes, B., Guo, H. H. 2019; 155 (5): e145–e148


    CASE PRESENTATION: A 38-year-old woman presented with 2months of dry cough, progressiveshortness of breath, central chest pain, nausea, vomiting, and dizziness. She was previously healthy and was not taking any medications. She denied fever, night sweats, or weight loss. She had a two pack-year smoking history and had quit smoking at 27 years of age. She denied drug use and had no recent travel history. Family history was pertinent for ovarian cancer, breast cancer, and colon cancer.

    View details for DOI 10.1016/j.chest.2018.12.014

    View details for PubMedID 31060712

  • Diagnostic and Grading Challenges in Oncocytic Cell (OC) and Clear Cell (CC) of Mucoepidermoid Carcinoma (MEC) Strosberg, C., Hernandez-Prera, J., Holmes, B., Moreira, F., Wenig, B. NATURE PUBLISHING GROUP. 2019
  • Oncologic management of sinonasal undifferentiated carcinoma. Current opinion in otolaryngology & head and neck surgery Tyler, M. A., Holmes, B., Patel, Z. M. 2018


    PURPOSE OF REVIEW: This article reviews the latest treatment paradigms in sinonasal undifferentiated carcinoma (SNUC).RECENT FINDINGS: The aggressive biology and associated advanced presentation of SNUC make successful treatment a challenge shared across medical specialties. Still, studies reporting outcomes in SNUC indicate that an aggressive treatment strategy consisting of surgery, radiation and chemotherapy offers the best chance of prolonged survival.SUMMARY: Successful treatment of SNUC requires highly specialized care at tertiary cancer treatment facilities. A better understanding of the biology of the disease coupled with increasing outcome reporting will lead to optimized treatment regimens.

    View details for PubMedID 30507692

  • Virus-associated carcinomas of the head & neck: Update from the 2017 WHO classification. Annals of diagnostic pathology Holmes, B. J., Wenig, B. M. 2018; 38: 29–42


    Virus-associated carcinomas of the head and neck represent an unusual confluence of infections spread by viral transmission and cellular dysregulation resulting in carcinogenesis. While much remains to be elucidated about the exact progression from infection to cancer, a basic framework of viral biology can complement the pathologist's understanding of morphology. This context informs the pathologist's everyday practice, including selecting ancillary studies, communicating prognostically relevant findings, and participating in treatment planning. By comparing and contrasting the salient features of human papillomavirus-associated oropharyngeal carcinoma and Epstein-Barr virus-associated nasopharyngeal carcinoma, this review summarizes recent evidence to guide current practice.

    View details for PubMedID 30415111