Inter‐ and intra‐tumor heterogeneity of SMAD4 loss in head and neck squamous cell carcinomas

Abstract

Reports regarding the frequency of SMAD4 loss in human head and neck squamous cell carcinoma (HNSCC) vary significantly. We have shown that SMAD4 deletion contributes to HNSCC initiation and progression. Therefore, accurately detecting genetic SMAD4 loss is critical to determine prognosis and therapeutic interventions in personalized medicine. We developed a SMAD4 fluorescence in situ hybridization (FISH) assay to identify chromosomal SMAD4 loss at the single cell level of primary HNSCC specimens and patient derived xenograft (PDX) tumors derived from HNSCCs. SMAD4 heterozygous loss was detected in 35% of primary HNSCCs and 41.3% of PDX tumors. Additionally, 4.3% of PDX tumors had SMAD4 homozygous loss. These frequencies of SMAD4 loss were similar to those in The Cancer Genome Atlas (TCGA). However, we identified significant heterogeneities of SMAD4 loss (partial or complete) among cells within each tumor. We also found that aneuploidy (monosomy and polysomy) contributed greatly to how to define chromosomal SMAD4 deletion. Furthermore, in cultured PDX tumors, SMAD4 mutant cells outcompeted SMAD4 wildtype cells, resulting in establishing homogenous SMAD4 mutant HNSCC cell lines with partial or complete genomic SMAD4 loss, suggesting a survival advantage of SMAD4 mutant cells. Taken together, our study reveals inter- and intra-tumor heterogeneities of SMAD4 chromosomal loss in HNSCCs. Further, SMAD4 FISH assay provides a platform for future clinical diagnosis of SMAD4 chromosomal loss that potentially serves as a molecular marker for prognosis and therapeutic intervention in cancer patients.

The fidelity of cancer cells in PDX models: Characteristics, mechanism and clinical significance

“…Because of proven discrepancies in drug sensitivity in later passage PDXs, subsequent studies have modified the experimental design to use early passage PDX models . A recent study in head and neck squamous cell carcinoma showed genomic SMAD family member 4 (SMAD4) alterations in PDX . Surprisingly, SMAD4 loss has been shown to negatively correlate with cetuximab sensitivity, which is not in agreement with the conclusions of previous studies .…”

“…also showed the acquisition of CNAs in PDX tumors compared to parental tumors, including chromosome 18q deletion and chromosome 20 amplification . A study of head and neck squamous cell carcinomas detected genetic SMAD4 loss in PDX models and a derived cell line …”

Lessons learned from SMAD4 loss in squamous cell carcinomas

“…Furthermore, multiple reports show that single copy loss of SMAD4 occurs in 30% to 50% of HNSCCs; however, other reports suggest reduced SMAD4 immunostaining in <30% of HNSCC cases that may be due to control tissues or poor antibody specificity. In addition, intra‐tumor heterogeneity of genomic SMAD4 loss, as well as aneuploidy of chromosome 18, may also contribute to variations in reported SMAD4 loss . While the genomic loss of SMAD4 is evident in ~50% of SCCs, its detection by immunostaining or RNA expression analyses is not standardized and ideal expression standards for SMAD4 are lacking.…”

“…Overall, 56% of primary head and neck SCCs (HNSCCs) have SMAD4 genomic alterations. 11,12 SMAD4 loss occurs in 35% to 68% of HNSCCs [13][14][15] and in up to 70% of skin SCCs 16 ; whereas SMAD4 point mutations are rare (<5%) in these types of SCCs. 17 Higher rates of genomic loss (56%) compared with point mutations (<5%) are consistent with rates of genetic abnormalities in human cancers.…”

“…In addition, intra-tumor heterogeneity of genomic SMAD4 loss, as well as aneuploidy of chromosome 18, may also contribute to variations in reported SMAD4 loss. 13 While the genomic loss of SMAD4 is evident in~50% of SCCs, its detection by immunostaining or RNA expression analyses is not standardized and ideal expression standards for SMAD4 are lacking. With a central role in tumor development and potential therapeutic response marker as discussed below, a welldefined standard for "SMAD4 loss" is a critical and logical need in future studies.…”