Author reply to Comment on: Frequent CTLA4-CD28 gene fusion in diverse types of T-cell lymphoma, by Yoo et al.

Abstract

We read with interest the comments raised by Gong et al.1 on our recently published study, 2 and appreciate these important and critical remarks.We agree that contamination is a potential important problem with polymerase chain reaction (PCR)-based work. To avoid the possibility of contamination, we confirmed our data before completion of our manuscript. We performed the PCR experiment using new experimental reagents at a different laboratory. We also repeated the PCR to detect the fusion using two different set of primers, the new primer set producing longer amplicons than the old primer set. All these efforts produced identical results, confirming that our samples were not contaminated by PCR amplicons.As the direct fusion of one exon of CTLA4 to an exon of CD28 is less likely than fusion breakpoints within the introns, we confirmed direct fusion of the two exons at the genomic DNA level with other experiments before submitting our manuscript. As shown in Figure 1, direct fusion of exon 3 of CTLA4 to exon 4 of CD28 was confirmed by PCR using primers from intron 2 of CTLA4 and the 3' region of CD28 using genomic DNA from formalin-fixed, paraffin-embedded samples. PCR products were validated by Sanger sequencing. DNA from normal blood was used as a control as well as no template controls. These data confirm that the fusion is present at the DNA level and is not the result of contamination.We also tried to perform fluorescent in situ hybridization experiments. However it was difficult to design probes which can distinguish the fusion segment because the CD28 and CTLA4 genes are located very close to each other. As shown in our manuscript 2 in Online Supplementary Figure S5, the result was not helpful for delineating the fusion signal from the normal signal.

Quality improvement of biomarker assessment in breast cancer

“…Overall positivity rates were 87.2% for ER, 71.3% for PR and 9.9% for HER2, which, for ER-and PR, is in line with previous studies [48][49][50] , whereas for HER2 this is somewhat lower than the percentages of 15%-25% that are often referred to [7,14,16,26,[51][52][53] . Although we only included synoptic pathology reports, there is no reason to assume that our synoptic dataset may have been selective, since data from the Dutch Breast Cancer Audit (NBCA), which also holds data from narrative pathology reports, show similar receptor positivity rates [49] . Moreover, over 80% of (pre)malignant breast lesions are currently reported via the synoptic PALGA protocol by Dutch pathologists [54] , which results in an increased overall completeness of reports [55] and it enables easy and error-free data extraction.…”

“…A nationwide multidisciplinary breast cancer audit (NBCA) has already been implemented in breast cancer care in the Netherlands [49] , yet currently there is only one pathology indicator, i.e. whether the PALGA protocol is used for reporting on (pre)malignant breast lesions [61] .…”

“…Furthermore, pathologists and their laboratories may feel best addressed by their own, case-mix adjusted, "mirror" data visualized against other national laboratories. Indeed, in a previous nationwide breast cancer audit, a HER2-outlier hospital critically evaluated their lab process and found that they used a different approach to HER2-positivity [49] .…”

“…Accurate and reproducible biomarker assessment is of key-importance for identification of individual breast cancer patients who may benefit from specific treatment modalities. However, many studies, in which sets of IBC slides were reviewed by multiple pathologists, found substantial differences in receptor-assessment between testing laboratories [18,[46][47][48][49][50][51][52][53][54][55][56] , while reproducibility of histologic grading was no more than moderate [57][58][59][60] . This raises the question whether this also holds true for biomarker assessment in daily clinical practice.…”

Genetic Landscape and Classification of Peripheral T Cell Lymphomas

“…Among structural abnormalities, CTLA4-CD28 fusion genes have been identified in AITL and several other PTCL subtypes, though analysis of different PTCL cohorts has uncovered variation in the frequency of this finding [23, 49, 50]. The resultant fusion protein is thought to transform inhibitory T cell signals into activating signals, and may represent a target for anti-CTLA4 immunotherapy [23].…”