Mutational impact of APOBEC3A and APOBEC3B in a human cell line and comparisons to breast cancer.

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in signature C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which protein(s) are responsible. Here we report the development of a selectable system to quantify genome mutation and demonstrate its utility by comparing the mutagenic activities of three leading candidates-APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and similar TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from independent clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs 70% of the time and APOBEC3B 50% of the time (Y = C/T; W = A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes contribute in a combinatorial manner to the overall mutation landscape. Although the vast majority of APOBEC3A- and APOBEC3B-induced single base substitution mutations occur outside of predicted chromosomal DNA hairpin structures, whole genome sequence analyses and supporting biochemical studies also indicate that both enzymes are capable of deaminating the single-stranded loop regions of DNA hairpins at elevated rates. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.

Primary mucosal melanomas of the head and neck are characterised by overexpression of the DNA mutating enzyme APOBEC3B.

AIMS: Primary head/neck mucosal melanomas (MMs) are rare and exhibit aggressive biologic behaviour and elevated mutational loads. The molecular mechanisms responsible for high genomic instability observed in head/neck MMs remain elusive. The DNA cytosine deaminase APOBEC3B (A3B) constitutes a major endogenous source of mutation in human cancer. A3B-related mutations are identified through C-to-T/-G base substitutions in 5'-TCA/T motifs. Herein, we present immunohistochemical and genomic data supportive of a role for A3B in head/neck MMs. METHODS AND RESULTS: A3B protein levels were assessed in oral (n = 13) and sinonasal (n = 13) melanomas, and oral melanocytic nevi (n = 13) by immunohistochemistry using a custom rabbit α-A3B mAb (5210-87-13). Heterogeneous, selective-to-diffuse, nuclear only, A3B immunopositivity was observed in 12 of 13 (92.3%) oral melanomas (H-score range = 9-72, median = 40) and 8 of 13 (62%) sinonasal melanomas (H-score range = 1-110, median = 24). Two cases negative for A3B showed prominent cytoplasmic staining consistent with A3G. A3B protein levels were significantly higher in oral and sinonasal MMs than intraoral melanocytic nevi (P < 0.0001 and P = 0.0022, respectively), which were A3B-negative (H-score range = 1-8, median = 4). A3B levels, however, did not differ significantly between oral and sinonasal tumours (P > 0.99). NGS performed in 10 sinonasal MMs revealed missense NRAS mutations in 50% of the studied cases and one each KIT and HRAS mutations. Publicly available whole-genome sequencing (WGS) data disclosed that the number of C-to-T mutations and APOBEC3 enrichment score were markedly elevated in head/neck MMs (n = 2). CONCLUSION: The above data strongly indicate a possible role for the mutagenic enzyme A3B in head/neck melanomagenesis, but not benign melanocytic neoplasms.

DNA Deamination Is Required for Human APOBEC3A-Driven Hepatocellular Carcinoma In Vivo.

Although the APOBEC3 family of single-stranded DNA cytosine deaminases is well-known for its antiviral factors, these enzymes are rapidly gaining attention as prominent sources of mutation in cancer. APOBEC3's signature single-base substitutions, C-to-T and C-to-G in TCA and TCT motifs, are evident in over 70% of human malignancies and dominate the mutational landscape of numerous individual tumors. Recent murine studies have established cause-and-effect relationships, with both human APOBEC3A and APOBEC3B proving capable of promoting tumor formation in vivo. Here, we investigate the molecular mechanism of APOBEC3A-driven tumor development using the murine Fah liver complementation and regeneration system. First, we show that APOBEC3A alone is capable of driving tumor development (without Tp53 knockdown as utilized in prior studies). Second, we show that the catalytic glutamic acid residue of APOBEC3A (E72) is required for tumor formation. Third, we show that an APOBEC3A separation-of-function mutant with compromised DNA deamination activity and wildtype RNA-editing activity is defective in promoting tumor formation. Collectively, these results demonstrate that APOBEC3A is a "master driver" that fuels tumor formation through a DNA deamination-dependent mechanism.

Endogenous APOBEC3B overexpression characterizes HPV-positive and HPV-negative oral epithelial dysplasias and head and neck cancers.

The DNA cytosine deaminase APOBEC3B (A3B) is a newly recognized endogenous source of mutations in a range of human tumors, including head/neck cancer. A3B inflicts C-to-T and C-to-G base substitutions in 5'-TCA/T trinucleotide motifs, contributes to accelerated rates of tumor development, and affects clinical outcomes in a variety of cancer types. High-risk human papillomavirus (HPV) infection causes A3B overexpression, and HPV-positive cervical and head/neck cancers are among tumor types with the highest degree of APOBEC signature mutations. A3B overexpression in HPV-positive tumor types is caused by the viral E6/E7 oncoproteins and may be an early off-to-on switch in tumorigenesis. In comparison, less is known about the molecular mechanisms responsible for A3B overexpression in HPV-negative head/neck cancers. Here, we utilize an immunohistochemical approach to determine whether A3B is turned from off-to-on or if it undergoes a more gradual transition to overexpression in HPV-negative head/neck cancers. As positive controls, almost all HPV-positive oral epithelial dysplasias and oropharyngeal cancers showed high levels of nuclear A3B staining regardless of diagnosis. As negative controls, A3B levels were low in phenotypically normal epithelium adjacent to cancer and oral epithelial hyperplasias. Interestingly, HPV-negative and low-grade oral epithelial dysplasias showed intermediate A3B levels, while high-grade oral dysplasias showed high A3B levels similar to oral squamous cell carcinomas. A3B levels were highest in grade 2 and grade 3 oral squamous cell carcinomas. In addition, a strong positive association was found between nuclear A3B and Ki67 scores suggesting a linkage to the cell cycle. Overall, these results support a model in which gradual activation of A3B expression occurs during HPV-negative tumor development and suggest that A3B overexpression may provide a marker for advanced grade oral dysplasia and cancer.

Intraoral Cutaneous Hamartomas-Clinicopathologic and Immunohistochemical Characteristics of 3 Cases.

Intraoral cutaneous hamartomas (ICHs) are uncommon mucosal lesions characterized microscopically by a combination of cutaneous structures, including various stages of follicular and sebaceous elements. Due to their rarity, the clinicopathologic and immunohistochemical attributes of ICHs have not been thoroughly delineated. Three cases of ICH were identified from our records, and formalin-fixed paraffin-embedded sections were immunohistochemically stained with antibodies against androgen receptor, estrogen receptor, and progesterone receptor, p63, factor XIIIα, and CD34. All 3 ICHs involved the buccal mucosa with an M:F ratio = 2:1 and mean age = 42.3 years (age range: 27-61 years). ICHs presented as thickened, painless, white and yellow plaques or nodules of long duration, measuring 0.6-1.5 cm. No history of skin graft in the area of the lesions was reported. Histopathologically, the lesions showed aggregates of rudimentary folliculosebaceous structures. Although well-defined piloerector muscles were present in all cases of ICH, bona fide hair follicles and isolated hair shafts were identified only in 1 case. The overlying oral epithelium exhibited epidermis-like morphological features, while inflammation was generally absent. Immunohistochemically, strong and diffuse nuclear staining for androgen receptor and factor XIIIα was observed in the sebaceous glands, and estrogen receptor and p63 reactivity were confined exclusively to the peripheral basal cells, while progesterone receptor staining was negative in ICHs. CD34 diffusely decorated the lesional stroma. In conclusion, ICH is a rare lesion composed of cutaneous elements in an abnormal location. A predilection for the buccal mucosa is reported in the current study.

A subset of ectomesenchymal chondromyxoid tumours of the tongue show EWSR1 rearrangements and are genetically linked to soft tissue myoepithelial neoplasms: a study of 11 cases.

AIMS: Ectomesenchymal chondromyxoid tumour (ECT) is a rare, benign intraoral neoplasm showing a predilection for the anterior dorsum of the tongue. The World Health Organization includes ECT in the pathological spectrum of soft tissue myoepithelioma. EWS RNA-binding protein 1 gene (EWSR1) rearrangement is found in 45% of cutaneous, soft tissue and bone myoepithelial neoplasms, and pleomorphic adenoma gene 1 (PLAG1) aberrations are found in 37% of EWSR1-negative soft tissue myoepitheliomas. The aim of this study was to evaluate the presence of EWSR1 and PLAG1 rearrangements in ECTs. METHODS AND RESULTS: Eleven formalin-fixed, paraffin-embedded ECTs were evaluated with fluorescence in-situ hybridization probes for EWSR1 (22q12) and PLAG1 (8q12). Among the 11 ECTs tested, three (27.3%) showed EWSR1 rearrangement in >15% of tumour cells, whereas eight (72.7%) cases did not show EWSR1 rearrangement. Eight of nine (89%) ECTs showed gain of EWSR1, probably representing gain of all or part of chromosome 22, in a varying proportion of neoplastic cells ranging between 1.4% and 27.9%. PLAG1 rearrangement was not detected in the successfully hybridized tissue sections (7/11). No correlation was observed between the molecular and histopathological findings, such as morphology of the neoplastic cells, the presence of atypia, and matrical type. CONCLUSIONS: We identified EWSR1 rearrangement in >25% of ECTs. These results suggest that some ECTs are at least genetically related to myoepithelioma of the soft parts. Finally, PLAG1 aberrations do not appear to be critical in the pathogenesis of ECT of the tongue.

Fluorescence in-situ hybridization identifies Mastermind-like 2 (MAML2) rearrangement in odontogenic cysts with mucous prosoplasia: a pilot study.

AIMS: The pathogenesis of intraosseous mucoepidermoid carcinoma (IMEC) remains unknown. Coexistence with odontogenic cysts (ODC) has been reported in 32-48% of IMEC. Furthermore, prosoplastic mucous cells are often seen in the epithelial lining of ODCs. MECT1-MAML2 fusion transcripts have been identified in >66% of salivary gland MEC cases. The aim of this study was to investigate the presence of MAML2 rearrangement in ODCs featuring mucous prosoplasia. METHODS AND RESULTS: Ten cases of ODC with a mucous cell component and three cases of IMEC were evaluated using fluorescence in-situ hybridization. All cases occurred in the mandible. The ODCs exhibited a M:F ratio of 4:1 (mean age 49.2 years), while all IMECs occurred in women (mean age 68.3 years). All three IMECs demonstrated MAML2 rearrangement, in 26-61% of tumour cells. Successful hybridization was observed in nine of 10 cases of ODC. In two of these nine, there was MAML2 rearrangement in 12% and 24% of the lining epithelial cells, while three of the nine showed rearrangement in 7-8% of cells; the remaining four cases were negative. CONCLUSIONS: We identified MAML2 rearrangements in five of nine ODCs lined by mucus-secreting cells. This suggests that at least a subset of ODCs with mucous prosoplasia are characterized by molecular events considered diagnostic for intraosseous and extraosseous MEC.

Localized juvenile spongiotic gingival hyperplasia featuring unusual p16INK4A labeling and negative human papillomavirus status by polymerase chain reaction.

BACKGROUND: Localized juvenile spongiotic gingival hyperplasia (LJSGH) is a distinct type of gingival hyperplastic lesion with specific clinicopathologic features. Evaluation of the morphological characteristics of LJSGH indicates the potential role of human papillomavirus (HPV) infection as an underlying etiopathogenetic mechanism. METHODS: All cases of LJSGH from 2008 to present were retrieved. Clinical and demographic data were collected. HPV status was investigated by p16INK4A immunohistochemistry and HPV-Polymerase chain reaction (PCR). RESULTS: Twenty-one cases of LJSGH were identified, 14 (66.7%) affecting males and seven (33.3%) females (M:F = 2:1, age range: 8-36, mean: 13 years). All lesions were well-demarcated, exophytic, erythematous, and hemorrhagic with granular or slightly papillary surface. Preponderance for the maxillary gingiva (19, 90.5%) was observed. Two (9.5%) patients presented with recurrence 20 and 21 months after excision (mean follow-up: 18.7 months). Histopathologically, all LJSGH lesions featured epithelial hyperplasia with intense neutrophilic exocytosis and spongiosis. All cases demonstrated positivity for p16INK4A with the majority of specimens (47.6%) intensely decorated in >50% of the overlying epithelium with focal immunostaining observed in 47.6% and diffuse in 52.4%. Thirteen cases (61.9%) were negative for HPV DNA by PCR, while two (9.5%) were suspicious for the presence of low levels of HPV DNA but definitive genotyping was not possible. One case (4.8%) displayed positivity for HPV-31. The remaining five cases failed the PCR reaction. CONCLUSIONS: Human papillomavirus does not participate in the pathogenesis of LJSGH. P16INK4A expression in the absence of detectable HPV DNA can likely be attributed to the intense inflammation associated with LJSGH.

Fungal Otitis Externa (Otomycosis) Associated with Aspergillus Flavus: A Case Image.

A 48-year-old man presented with a chief complaint of intermittent right ear otorrhea of several-month duration, occasional otalgia and progressive unilateral hearing impairment. He also reported frequent episodes of headache and pressure in the sinuses and maxilla. Previous systemic treatment with antibiotics failed to alleviate the symptoms. A head/neck CT showed completely normal mastoid, middle ear and external auditory canal regions without any evidence of opacification or bone erosion. Otoscopic examination of the right ear disclosed aggregates of dried, brown, fibrillar material and debris occluding the external auditory canal and obstructing the otherwise intact tympanic membrane. Dilation of the external auditory canal or thickening of the tympanic membrane were not appreciated. The canal was debrided and the fibrillar material was placed in formalin. Histopathologic examination revealed numerous branching, septated fungal hyphae organized in densely-packed clusters. In other areas, the fungal hyphae abutted or were attached to lamellated collections of orthokeratin. As highlighted by GMS staining, the fungi were morphologically compatible with Aspergillus species. The clinicopathologic findings supported a diagnosis of fungal otitis externa, while the numerous anucleate squamous cells were compatible with colonization of an underlying, probably developing, cholesteatoma. Culture of material isolated from the external auditory canal confirmed the presence of Aspergillus flavus. In this illustrative case, we present the main clinical and microscopic characteristics of Aspergillus-related otomycosis developing in the setting of a tautochronous cholesteatoma.

SMARCB1-Deficient Skull Base Chondrosarcoma with 12p Duplication Presenting as Somatic-Type Malignancy Arising from Metastatic Seminoma.

Somatic-type malignancy (STM) can occur infrequently within a primary or metastatic testicular germ cell tumor (TGCT) and is associated with dismal prognosis and survival. STM with chondrosarcomatous features is exceedingly rare and head and neck involvement has not been previously documented. A 39-year-old white man presented with nasal obstruction and epistaxis. Imaging disclosed a 6.9-cm expansile tumor involving the nasal cavity and skull base with intraorbital and intracranial extension. The histopathologic properties of the tumor were compatible with chondrosarcoma, grade II-III. Immunohistochemically, malignant cells were strongly and diffusely positive for S100 and epithelial markers, and showed loss of SMARCB1 expression. IDH1/2 mutations were not detected. Following whole-body PET scan, a 7.0-cm left testicular mass was discovered and diagnosed as seminoma with syncytiotrophoblastic cells, stage pT3NXM1b. Extensive retroperitoneal, mediastinal, and supraclavicular lymphadenopathy was also noticed. Histopathologic examination of the left supraclavicular lymph node revealed metastatic seminoma. By FISH, most metastatic nodal seminoma cells harbored 1 to 4 copies of isochromosome 12p, while the chondrosarcoma featured duplication of 12p. Presence of a malignant TGCT with disseminated supradiaphragmatic lymphadenopathy, the unique immunophenotypic properties of the skull-based chondrosarcoma and lack of IDH1/2 aberrations with gain of 12p strongly support the diagnosis of STM chondrosarcoma arising from metastatic TGCT. The patient did not respond to chemotherapy and succumbed three months after diagnosis. Although exceedingly uncommon, metastasis to the head and neck may occur in patients with TGCT. This case of STM chondrosarcoma demonstrated divergent immunophenotypic and molecular characteristics compared to "typical" examples of head and neck chondrosarcoma. High index of suspicion is advised regarding the diagnosis of lesions that present with otherwise typical histomorphology but unexpected immunohistochemical or molecular features.

Comparison between p16 INK4A immunohistochemistry and human papillomavirus polymerase chain reaction assay in oral papillary squamous cell carcinoma.

PURPOSE: Oral papillary squamous cell carcinoma (OPSCC) is a histologic variant of SCC with a growth pattern suggesting human papillomavirus (HPV) infection. The aim of this study was to investigate the presence of HPV genotypes in OPSCC. MATERIALS AND METHODS: All cases with a histologic diagnosis of OPSCC from 1993 through 2008 were retrieved and confirmed. Immunohistochemical evaluation for the surrogate marker p16(INK4A) and HPV polymerase chain reaction were performed in tissue and DNA derived from formalin-fixed paraffin-embedded tissue. RESULTS: Forty-four patients with confirmed OPSCC (mean age, 71.96 yr; female-to-male ratio, 1.75:1) comprised the study population. The most common site of involvement was the gingiva followed by the palate and buccal mucosa. Forty cases exhibited an invasive component, 1 was noninvasive, and in 3 cases invasion could not be confirmed owing to suboptimal thickness of the biopsy. Paraffin tissue blocks were available in 41 cases. Twenty-three cases (56.1%) exhibited positive p16(INK4A) staining, which was primarily weak to moderate with a generally focal pattern. Polymerase chain reaction assays were negative for HPV DNA in all cases. CONCLUSIONS: In this study, there was a clinical predilection of OPSCC in older women, with most cases occurring in the masticatory mucosa. Weak to moderate and focal p16(INK4A) staining was appreciated in contrast to reported staining properties in genital and oropharyngeal PSCC. Failure of the polymerase chain reaction assay to exhibit transcriptionally active HPV genotypes suggests that HPV is not associated with OPSCC tumorigenesis.

Cytopathology of salivary gland myoepithelial carcinoma: A study of 13 cases and review of the literature.

INTRODUCTION: Myoepithelial carcinoma (MECA) is an infrequently recognized salivary gland (SG) neoplasm that commonly develops within a preexisting pleomorphic adenoma (MECA ex PA). Fine-needle aspiration (FNA) biopsy reports of this neoplasm are largely restricted to small series and single case reports. METHODS: Our cytopathology files were searched for examples of SG MECA/MECA ex PA having confirmatory histopathologic verification. Conventional FNA biopsy smears were performed, and exfoliative specimens processed using standard techniques. RESULTS: Thirteen cases from 9 patients (M:F = 3.5:1; age range: 36 to 95 years, mean age = 60 years) met inclusion criteria. FNA biopsy sites included parotid gland (4), trunk (2), scalp (2), and neck (2). Exfoliative specimens included pleural fluid (1), bronchial brushing (1), and bronchoalveolar lavage (1). Most cases were metastatic deposits (8; 62%), 4 were primary neoplasms, and 1 a local recurrence. FNA diagnoses were MECA ex PA (6; 46%), myoepithelial neoplasm (2), PA (2), basaloid neoplasm (1), atypical myoepithelial cells (1), and myxoma (1). Ancillary testing in 2 cases showed positive staining for myoepithelial markers. Cytologic features were that of a low-grade neoplasm composed principally of epithelioid/polygonal cells exhibiting minimal if any cytologic atypia. Myxoid and chondromyxoid stroma was often the dominant feature in MECA ex PA aspirates. CONCLUSION: In the primary setting, a cytologic diagnosis of MECA/MECA ex PA is extremely challenging if at all possible. Due to overwhelming amounts of stroma, the diagnosis may be challenging in some cases of metastatic MECA ex PA.

Mycobacterial Spindle Cell Pseudotumor of the Nasal Cavity.

BACKGROUND: Mycobacterial spindle cell pseudotumor (MSCP) represents an uncommon tumor-like proliferation associated with nontuberculous mycobacterial infection, i.e., M. avium intracellulare, affecting primarily the lymph nodes of immunocompromised men in their 5th decade. Involvement of the nasal cavity by MSCP is exceedingly rare with only 3 well-documented examples in the literature. METHODS: A 74-year-old, HIV-negative, man presented with a 0.5-cm nodule of the left nasal cavity clinically presenting as a "nasal polyp." His medical history was significant for colonic adenocarcinoma, cutaneous basal cell carcinoma, and chronic lymphocytic leukemia (CLL) transforming to B-cell prolymphocytic leukemia, responsive to chemotherapy. The patient was diagnosed with prostatic adenocarcinoma treated with radiotherapy two months before the nasal lesion was detected. No lymph node enlargement, pulmonary involvement or hepatosplenomegaly were noticed. The nasal nodule was surgically excised and histopathologically examined to rule out metastatic disease or CLL relapse. RESULTS: Microscopically, the lesion comprised a well-circumscribed, monotonous, spindle cell population in a vaguely storiform arrangement mixed with a heavy infiltrate of neutrophils and sparse lymphocytes. The spindle cells featured finely granular rich eosinophilic cytoplasm with rounded, oval to epithelioid, or elongated nuclei with vesicular chromatin and one or two distinct nucleoli. The lesional cells lacked overt cytologic atypia and showed occasional regular mitoses. The surface epithelium was intact or focally ulcerated. By immunohistochemistry, the spindle cell population stained strongly and diffusely for CD68 and was negative for AE1/AE3, SMA, CD34, and PSA. CD3 highlighted scattered lymphocytes. Ziehl-Neelsen stain disclosed numerous intracytoplasmic acid-fast bacilli. A diagnosis of MSCP was rendered. No recurrences were observed during a 24-month follow-up period. CONCLUSION: Although exceptionally rare, MSCP should be considered in the differential diagnosis of nodular lesions of the nasal cavity that are characterized microscopically by marked spindle cell proliferation in a vague storiform pattern, admixed with a lymphocytic or mixed inflammatory infiltrate. A negative medical history for HIV infection and medication-induced immunosuppression should not preclude a diagnosis of MSCP, particularly in extranodal sites. Once the diagnosis is established, prognosis appears to be excellent for nasal MSCP following conservative surgical excision.

Signet-ring cell sinus histiocytosis: report of the clinicopathologic characteristics of 4 cases with emphasis on the differential diagnosis of signet-ring cell lesions.

Signet-ring cell sinus histiocytosis (SRCSH) represents a distinctly rare reactive phenomenon predominantly affecting axillary and pelvic lymph nodes (LNs) of individuals with breast or prostatic adenocarcinoma. Reports of SRCSH in the literature are sparse with only 12 previous examples, thus underscoring the rarity of this process. Here, we report 4 additional SRCSH cases affecting 2 women and 2 men (M/F = 1:1; age range: 50-71 years; mean age = 61 years). In the 2 men, pelvic LNs were excised during radical cystoprostatectomy for genitourinary cancer, whereas in one woman, SRCSH was incidentally discovered in axillary LNs during mastectomy for breast adenocarcinoma. The other female patient presented with a history of aortic valve replacement and enlarged supraclavicular LNs. Microscopically, all involved LNs exhibited marked distention with filling of the subcapsular and medullary sinuses by sheets of signet-ring histiocytes containing a singular large, cytoplasmic vacuole and a crescentic nucleus. Overt cytologic atypia, pleomorphism, and mitoses were absent. Erythrophagocytosis and occasional fibrosis were appreciated. None of the LNs with SRCSH showed evidence of metastatic tumor. Immunohistochemically, signet-ring sinus histiocytes were invariably positive for CD68 and CD163 but were negative for pancytokeratins. The histopathologic characteristics of SRCSH, albeit bland, in conjunction with the patient's medical history, may be misinterpreted as metastatic adenocarcinoma with signet-ring cell configuration. Immunohistochemical confirmation of the histiocytic lineage of the lesional cells in SRCSH usually suffices for rendering an accurate diagnosis. The underlying pathogenetic mechanism and possible biologic significance of SRCSH remain currently unknown.

Intracellular calprotectin (S100A8/A9) facilitates DNA damage responses and promotes apoptosis in head and neck squamous cell carcinoma.

OBJECTIVES: In head and neck squamous cell carcinoma (HNSCC), poor prognosis and low survival rates are associated with downregulated calprotectin. Calprotectin (S100A8/A9) inhibits cancer cell migration and invasion and facilitates G2/M cell cycle arrest. We investigated whether S100A8/A9 regulates DNA damage responses (DDR) and apoptosis in HNSCC after chemoradiation. MATERIALS AND METHODS: Human HNSCC cases in TCGA were analyzed for relationships between S100A8/A9 and expression of apoptosis-related genes. Next, S100A8/A9-expressing and non-expressing carcinoma lines (two different lineages) were exposed to genotoxic agents and assessed for 53BP1 and γH2AX expression and percent of viable/dead cells. Finally, S100A8/A9-wild-type and S100A8/A9null C57BL/6j mice were treated with 4-NQO to induce oral dysplastic and carcinomatous lesions, which were compared for levels of 53BP1. RESULTS: In S100A8/A9-high HNSCC tumors, apoptosis-related caspase family member genes were upregulated, whereas genes limiting apoptosis were significantly downregulated based on TCGA analyses. After X-irradiation or camptothecin treatment, S100A8/A9-expressing carcinoma cells (i.e., TR146 and KB-S100A8/A9) showed significantly higher 53BP1 and γH2AX expression, DNA fragmentation, proportions of dead cells, and greater sensitivity to cisplatin than wild-type KB or TR146-S100A8/A9-KD cells. Interestingly, KB-S100A8/A9Δ113-114 cells showed similar 53BP1 and γH2AX levels to S100A8/A9-negative KB and KB-EGFP cells. After 4-NQO treatment, 53BP1 expression in oral lesions was significantly greater in calprotectin+/+ than S100A8/A9null mice. CONCLUSIONS: In HNSCC cells, intracellular calprotectin is strongly suggested to potentiate DDR and promote apoptosis in response to genotoxic agents. Hence, patients with S100A8/A9-high HNSCC may encounter more favorable outcomes because more tumor cells enter apoptosis with increased sensitivity to chemoradiation therapy.

Intraoral salivary lymphoepithelial carcinoma: clinicopathologic and immunophenotypic characterization of 3 cases indicates elevated programmed death-ligand 1 expression.

OBJECTIVE: Intraoral salivary lymphoepithelial carcinoma (ISLEC) is a rare malignancy with programmed death-ligand 1 (PD-L1) expression levels that have been greatly understudied. We examined the clinicopathologic and immunophenotypic characteristics, including PD-L1 levels, of 3 cases of ISLEC. STUDY DESIGN: We searched the archives of 2 oral and maxillofacial pathology laboratories for specimens diagnosed as ISLEC between 1985 and 2022. We collected patient demographic and clinical data. Immunostaining for AE1/AE3, CK7, CD3, CD20, p16, p53, Ki67, and PD-L1 (SP263), as well as Epstein-Barr virus-encoded small RNAs (EBER) in situ hybridization (ISH) were performed. RESULTS: All 3 cases affected males aged 42 to 84 years (median = 61y) and involved the floor of the mouth, soft palate/uvula, and tongue. The lesions showed diffuse infiltration by non-keratinizing sheets and islands of undifferentiated carcinoma cells with associated dense lymphoplasmacytic inflammation. Immunohistochemically, all tumors showed AE1/AE3 positivity, selective p53 staining, and negativity for CK7 and p16. Ki67 highlighted 20%-80% of lesional cells. The inflammatory infiltrate comprised a mixed population of T and B lymphocytes. EBER ISH was positive in one case. All ISLECs displayed membranous, focal-to-diffuse, PD-L1 staining with tumor proportion score > 95% in two and 40-50% in the third case. CONCLUSIONS: The clinicopathologic and immunophenotypic characteristics of the cases we examined highlight the rarity of ISLEC and indicate overall high PD-L1 levels in this type of malignancy, rendering patients with ISLEC potential candidates for targeted α-PD-1/PD-L1 immunotherapy.

Human APOBEC3B promotes tumor heterogeneity in vivo including signature mutations and metastases.

The antiviral DNA cytosine deaminase APOBEC3B has been implicated as a source of mutation in many different cancers. Despite over 10 years of work, a causal relationship has yet to be established between APOBEC3B and any stage of carcinogenesis. Here we report a murine model that expresses tumor-like levels of human APOBEC3B after Cre-mediated recombination. Animals appear to develop normally with full-body expression of APOBEC3B. However, adult males manifest infertility and older animals of both sexes show accelerated rates of tumorigenesis (mostly lymphomas or hepatocellular carcinomas). Interestingly, primary tumors also show overt heterogeneity, and a subset spreads to secondary sites. Both primary and metastatic tumors exhibit increased frequencies of C-to-T mutations in TC dinucleotide motifs consistent with the established biochemical activity of APOBEC3B. Elevated levels of structural variation and insertion-deletion mutations also accumulate in these tumors. Together, these studies provide the first cause-and-effect demonstration that human APOBEC3B is an oncoprotein capable of causing a wide range of genetic changes and driving tumor formation in vivo .

Human APOBEC3B promotes tumor development in vivo including signature mutations and metastases.

The antiviral DNA cytosine deaminase APOBEC3B has been implicated as a source of mutation in many cancers. However, despite years of work, a causal relationship has yet to be established in vivo. Here, we report a murine model that expresses tumor-like levels of human APOBEC3B. Animals expressing full-body APOBEC3B appear to develop normally. However, adult males manifest infertility, and older animals of both sexes show accelerated rates of carcinogenesis, visual and molecular tumor heterogeneity, and metastasis. Both primary and metastatic tumors exhibit increased frequencies of C-to-T mutations in TC dinucleotide motifs consistent with the established biochemical activity of APOBEC3B. Enrichment for APOBEC3B-attributable single base substitution mutations also associates with elevated levels of insertion-deletion mutations and structural variations. APOBEC3B catalytic activity is required for all of these phenotypes. Together, these studies provide a cause-and-effect demonstration that human APOBEC3B is capable of driving both tumor initiation and evolution in vivo.