POU2F3 in SCLC: Clinicopathologic and Genomic Analysis With a Focus on Its Diagnostic Utility in Neuroendocrine-Low SCLC

Abstract

Introduction: POU2F3 is a recent marker of a small cell lung carcinoma (SCLC) subtype related to chemosensory tuft cells (SCLC-P). The characteristics of SCLC-P have not been fully defined, and the data on POU2F3 expression in other lung tumors are scarce.

Methods: We screened 254 SCLC for POU2F3 expression and comprehensively analyzed histopathologic, genomic, and clinical characteristics of POU2F3-positive tumors. We also explored POU2F3 expression in other major lung cancer types (n = 433) and a targeted set of potential diagnostic mimics of SCLC (n = 123).

Results: POU2F3 was expressed in 30 of 254 (12%) SCLC and was strongly associated with low expression of standard neuroendocrine markers (synaptophysin, chromogranin A, CD56, INSM1). Notably, POU2F3 was expressed in 75% of SCLC with entirely negative or minimal neuroendocrine marker expression (15/20) and was helpful in supporting the diagnosis of SCLC in such cases. Broad targeted next-generation sequencing revealed that SCLC-P (n = 12) exhibited enrichment in several alterations, including PTEN inactivation, MYC amplifications, and 20q13 amplifications, but similar rates of RB1 and TP53 alterations as other SCLC (n = 155). Beyond SCLC, POU2F3 expression was exclusively limited to large cell neuroendocrine carcinoma (12%) and basaloid squamous cell carcinoma (22%).

Conclusions: This is the largest cohort of SCLC-P clinical samples to date, where we describe the diagnostic utility of POU2F3 in a challenging subset of SCLC with low or absent expression of standard neuroendocrine markers. The distinct genomic alterations in SCLC-P may offer a novel avenue for therapeutic targeting. The role of POU2F3 in a narrow subset of other lung cancer types warrants further study.

Keywords: Neuroendocrine-low; POU2F3; SCLC-P; Small cell lung carcinoma.

Figure 1.. Immunohistochemical characteristics of SCLC-P.

(A) Dot plots depicting expression of various markers in POU2F3-positive (n=30) versus POU2F3-negative SCLC (n=142 for all markers, except n=127 for Ki67 and n=122 for TTF-1). Mean and standard deviation for each comparison are graphically depicted by grey and red lines, respectively (see Supplementary Table 3 for details). (B) Bar graph depicting the proportion of POU2F3-positive cases among SCLC groups defined by extent of NE marker labeling, as expressed by the NE-score (average H-score for all four conventional NE markers: synaptophysin, chromogranin A, CD56, and INSM1). *75% refers to proportion of POU2F3-positive cases among all NE-extremely low/negative SCLC (H-score 0–50). Abbreviations: CHRA chromogranin A, NE neuroendocrine, SYN synaptophysin

Figure 2.. Illustration of preferential POU2F3 expression in SCLC with low or absent expression of standard NE markers.

Case in the left panel illustrates a NE-high SCLC (NE-score = 229) showing strong diffuse expression of all conventional NE markers and TTF-1, and negative POU2F3. Other cases are examples of NE-low (Case IDs 23 and 25) or entirely negative (Case ID 29) SCLC with robust expression of POU2F3. Abbreviations: CHRA chromogranin A, NE neuroendocrine, SYN synaptophysin

Figure 3.. Illustration of a case in which POU2F3 supported the diagnosis of SCLC (Case ID 28).

High-grade tumor showing histologic features of SCLC on H&E (high N:C ratio, nuclear molding, and finely granular chromatin) and high Ki-67 proliferative index (70%). However, all NE markers (with the exception of rare cells labeling weakly with INSM1) and TTF-1 are negative. Various other markers were initially evaluated to exclude an alternative diagnosis (including p40 to exclude squamous cell carcinoma), and were negative. Subsequently performed POU2F3 provides direct support for the diagnosis of SCLC. Abbreviations: CHRA chromogranin A, H&E hematoxylin and eosin, IHC immunohistochemistry, N:C nuclear-to-cytoplasmic, NE neuroendocrine, SYN synaptophysin

Figure 4.. Clinicopathologic and genomic characteristic of SCLC-P.

(A) Table comparing clinical and histologic features of SCLC-P (POU2F3-positive) vs other SCLC (POU2F3-negative). (B) Univariate Kaplan-Meyer analysis comparing overall survival in patients with SCLC-P vs other SCLC. (C) Summary of molecular characteristics of SCLC-P. The most prevalent (≥33%) genomic alterations in SCLC-P are shown, as well as genes with divergent distribution in SCLC-P compared to other SCLC. †Rb IHC was performed on all SCLC cases, and the rate of shown RB1 alterations incorporates genomic and IHC findings. ^#Amplifications of the 20q13 locus involved several genes, including NCOA3 in all 5 cases, and AURKA and GNAS in 3 of 5 cases (Case ID 29, 22 and 16). (D) MYC expression by IHC in SCLC-P vs other SCLC. Abbreviations: ADC adenocarcinoma, IHC immunohistochemistry, LCNEC large cell neuroendocrine carcinoma, PY pack years, SCC squamous cell carcinoma

Figure 5.. POU2F3 expression in other major lung cancer types and histologic mimics of SCLC.

(A) Summary of the frequency of POU2F3 expression in other major lung cancer types. ^*Mean (range) of POU2F3 H-score in basaloid SCC: 153 (15–275). ^**Mean (range) of POU2F3 H-score in LCNEC: 204 (85–300). ^#Lymphomas included 20 MALT lymphomas and 9 primary mediastinal B-cell lymphomas. Round cell sarcomas included 10 Ewing/Ewing family sarcomas, 7 CIC-rearranged sarcomas, 1 NCOA2-rearranged sarcoma, 1 BCOR-CCNB3 sarcoma, and 1 desmoplastic round cell tumor. (B) Illustration of representative cases of basaloid SCC (left panel) and LCNEC (right panel) with POU2F3 expression. Confirming the diagnosis of basaloid SCC is diffuse expression of p40 and the lack of NE markers. LCNEC is distinguished from SCLC based on standard morphologic criteria. Abbreviations: LCNEC large cell neuroendocrine carcinoma, NOS, not otherwise specified, SCC squamous cell carcinoma, SMARCA4-UT SMARCA4-deficient undifferentiated tumor

Figure 6.. POU2F3 expression in normal airways.

Triple synaptophysin (brown chromogen), p40 (green chromogen), and POU2F3 (red chromogen) IHC in representative bronchiolar cross-sections shows nuclear POU2F3 expression in rare cells that are distinct from the flask-shaped synaptophysin-positive resident pulmonary neuroendocrine cells, with POU2F3-positive nuclei overlying a discretely separate continuous p40-positive basal cell layer and located at or above the level of ciliated cell nuclei. Abbreviations: IHC immunohistochemistry, SYN synaptophysin