Targeted DNA sequencing in diagnosis of malignant phyllodes tumors with emphasis on tumors with keratin and p63 expression

Abstract

The differential diagnosis of malignant spindle cell neoplasms in the breast most frequently rests between malignant phyllodes tumor (MPT) and metaplastic carcinoma (MBC). Diagnosis of MPT can be challenging due to diffuse stromal overgrowth, keratin (CK) and/or p63 immunopositivity, and absent CD34 expression, which can mimic MBC, especially in core biopsies. Distinction of MPT from MBC has clinical implications, with differences in surgical approach, chemotherapy, and radiation. In this study, we evaluated MPT (78 tumors, 64 patients) for stromal CK, p63, and CD34 expression and profiled a subset (n=31) by targeted next-generation DNA sequencing (NGS), with comparison to MBC (n=44). Most MPT (71%) were CK+ and/or p63+, including 32% CK+ (25/77 focal) and 65% p63+ (32/66 focal, 10/66 patchy, 1/66 diffuse). Thirty-percent of MPT expressed both CK and p63 (20/66), compared to 95% of MBC (40/42, p<0.001). CK and/or p63 were positive in CD34+ and CD34- MPT. Recurrent genetic aberrations in MPT involved TERT, TP53, MED12, CDKN2A, chromatin modifiers, growth factor receptors/ligands, and PI-3K and MAPK pathway genes. Only MED12 (39%, 12/31) and SETD2 (13%, 4/31) were exclusively mutated in MPT and not MBC (p<0.001 and p=0.044, respectively), whereas PIK3R1 mutations were only found in MBC (35%, 13/35, p<0.001). Comparative literature review additionally identified ARID1B, EGFR, FLNA, NRAS, PDGFRB, RAD50, and RARA alterations enriched or exclusively in MPT versus MBC. MED12 was mutated in MPT with diffuse stromal overgrowth (53%, 9/17), CD34- MPT (41%, 7/17), and CK+ and/or p63+ MPT (39%, 9/23), including 36% of CD34- MPT with CK and/or p63 expression. Overall, MED12 mutation and/or CD34 expression were observed in 68% (21/31) MPT, including 61% (14/23) of CK+ and/or p63+ tumors. Our results emphasize the prevalence of CK and p63 expression in MPT and demonstrate diagnostic utility of NGS, especially in MPT with confounding factors that can mimic MBC.