Risk Stratification of Oral Potentially Malignant Disorders in Fanconi Anemia Patients Using Autofluorescence Imaging and Cytology-On-A Chip Assay

Abstract

Fanconi anemia (FA) is a hereditary genomic instability disorder with a predisposition to leukemia and oral squamous cell carcinomas (OSCCs). Hematopoietic stem cell transplantation (HSCT) facilitates cure of bone marrow failure and leukemia and thus extends life expectancy in FA patients; however, survival of hematologic malignancies increases the risk of OSCC in these patients. We developed a “cytology-on-a-chip” (COC)–based brush biopsy assay for monitoring patients with oral potentially malignant disorders (OPMDs). Using this COC assay, we measured and correlated the cellular morphometry and Minichromosome Maintenance Complex Component 2 (MCM2) expression levels in brush biopsy samples of FA patients’ OPMD with clinical risk indicators such as loss of autofluorescence (LOF), HSCT status, and mutational profiles identified by next-generation sequencing. Statistically significant differences were found in several cytology measurements based on high-risk indicators such as LOF-positive and HSCT-positive status, including greater variation in cell area and chromatin distribution, higher MCM2 expression levels, and greater numbers of white blood cells and cells with enlarged nuclei. Higher OPMD risk scores were associated with differences in the frequency of nuclear aberrations and differed based on LOF and HSCT statuses. We identified mutation of FAT1 gene in five and NOTCH-2 and TP53 genes in two cases of FA patients’ OPMD. The high-risk OPMD of a non-FA patient harbored FAT1, CASP8, and TP63 mutations. Use of COC assay in combination with visualization of LOF holds promise for the early diagnosis of high-risk OPMD. These minimally invasive diagnostic tools are valuable for long-term surveillance of OSCC in FA patients and avoidance of unwarranted scalpel biopsies.

Point‐of‐care oral cytology tool for the screening and assessment of potentially malignant oral lesions

“…The current study has provided a key step toward the development of new tools that could pave the way for new capabilities in the area of “precision lesion diagnostics.” Helping to push forward this theme, we have demonstrated the usefulness of temporal changes in the numerical index in a pilot study of patients with Fanconi anemia . These efforts demonstrated strong potential for patient‐specific temporal changes in the lesion numerical index to track early signs of disease for this high‐risk population.…”

“…Helping to push forward this theme, we have demonstrated the usefulness of temporal changes in the numerical index in a pilot study of patients with Fanconi anemia. 13 These efforts demonstrated strong potential for patient-specific temporal changes in the lesion numerical index to track early signs of disease for this high-risk population. Plans currently are in place to: 1) evaluate the POCOCT's precision lesion diagnostic capabilities through a prospective longitudinal study of malignant transformation and cancer recurrence; and 2) move the POCOCT into a clinical trial to assess its diagnostic performance versus routine care in primary care clinics.…”

“…11,12 These efforts included the development of an "enhanced gold-standard" adjudication process 12 that was used to correlate brush cytology measurements with 6 levels of histopathological diagnosis, ranging from benign, to OED, to OSCC. The same approach demonstrated strong promise for OSCC surveillance in patients with Fanconi anemia 13 and for the development of a cytology-based numerical risk index for cancer progression. 14 Overall, these past efforts have demonstrated that microfluidic-based cell capture systems with integrated imaging and embedded diagnostic algorithms can yield diagnostic accuracies that rival and exceed the capabilities of previously developed adjunct devices.…”

Development of a cytology-based multivariate analytical risk index for oral cancer

“…Of these six patients, MARIO scores increased significantly (+>40%) for two patients, suggesting progression; decreased (->40%) for two patients, suggesting non-progression or regression; and remained unchanged for the remaining two patients (+/-5%), indicating non-progression or stable disease. [22] One of the first two patients demonstrated a 27-fold increase in their MARIO, self-reported a recent pharyngeal cancer diagnosis within the 2-year period, and was notified to seek additional guidance from the and neck surgeon. This small pilot, proof-of concept study demonstrated that the MARIO score has potential to predict accurately the malignancy risk of OPMD in two patients as confirmed by follow-up scalpel biopsy, though significant further evaluation is needed to validate the MARIO for surveillance applications.…”

Two enemies, one fight: An update of oral cancer in patients with Fanconi anemia

“…Noninvasive methods used in patients with FA include the loss of heterozygosity (LOH) screening of brushed oral cells 124 and a programmable bio-nanochip brush biopsy test. 125,126 Although the outcomes of these methods seem promising, they still have limitations. For example, Smetsers et al found that LOH was significantly associated with HNSCC in patients with FA.…”