Tumor-Infiltrating Lymphocytes in Patients Receiving Trastuzumab/Pertuzumab-Based Chemotherapy


Michail Ignatiadis; Gert Van den Eynden; Salgado Roberto; Marco Fornili; Yacine Bareche; Christine Desmedt; Françoise Rothé; Marion Maetens; David Venet; Esther Holgado; Virginia McNally; Astrid Kiermaier; Heidi M. Savage; Timothy R. Wilson; Javier Cortes; Andreas Schneeweiss; Karen Willard-Gallo; Elia Biganzoli; Christos Sotiriou


J Natl Cancer Inst. 2019;111(1):69-77. 

In This Article

Supplementary Materials

TRYPHAENA Study Design

Briefly, 225 patients with centrally confirmed HER2-positive early breast cancer were randomized to receive three different preoperative regimens: either 3 cycles of FEC (Fluorouracil, Epirubicine, Cyclophosphamide) followed by 3 cycles of docetaxel in combination with trastuzumab and pertuzumab that was initiated together with the 1st cycle of FEC or the same chemotherapy regimen in combination with trastuzumab and pertuzumab starting with the first cycle of docetaxel or 6 cycles of an anthracycline-free regimen of docetaxel and carboplatin in combination with trastuzumab and pertuzumab(1). This trial provided evidence that the combination of trastuzumab and pertuzumab with standard chemotherapy is safe in terms of cardiac toxicity (primary objective) and resulted in high pCR rates(1).

The independent ethics committees that approved the TRYPHAENA protocols, and amendments when appropriate, were: CE università Cattolica del S. Cuore -Policl. Gem, Italy; Comitato Etico A.O. San Gerardo di Monza, Italy; EK Heidelberg, Germany; Comisia Nationala de Etica, Romania; Ethic Committee Institute for Oncology and Radiology of Serbia; Agency for medicinal products and medical devices, Bosnia and Herzegovina; Ottawa Hospital Research Ethics Boards; Canada; UBC BCCA Research Ethics Board (BCCA REB), Canada; Comitê de Ética em Pesquisa da PUCRS, Brazil; Comite de Etica em Pesquisa do Centro de Referencia da Saude da Mulher, Brazil; Northern X Ethics Committee, New Zealand; Pharma-Ethics Independent Research Ethics committee, South Africa; Kyungpook

National Uni Hospital, Republic of Korea; TVGH Institutional Review Board, Taiwan; Comité Etico Hospital Vall de Hebron, Spain; Comité de Etico Hospital Universitario Puerta de Hierro, Spain; Western Institutional Review Board, United States; Hospital Angeles Metropolitano, Mexico; Southampton & South West Hampshire LREC (B), UK; Kantonale Ethikkommission Aarau, Switzerland; Korea University Guro Hospital, Republic of Korea; Kantonale Ethikkommission Zürich (KEK), Switzerland; CEIC - Comissão de Ética para Investigação Clínica, Portugal; Regionala Etikprövningsnämnden i Stockholm, Sweden; McGill University Health Centre – Research Ethics Board, Canada; University of Pretoria Research Ethics committee, Faculty of Health Sciences, South Africa; Comité Etico de Euskadi, Spain; Comité Ético de Investigación Clinica Hospital Universitario Reina Sofía, Spain; Central Ethics Committee Agency for medicines and medical devices, Croatia; IRB, China Medical University Hospital, Taiwan; Centro Estatal De Cancerologia Dr Miguel Dorantes Mesa, Mexico; Ministry of Health and Social Welfare, Croatia.

TILs Evaluation

The geometric mean of TIL percentages from the two pathologists was computed after adding 0.5 to values equal to 0 consistently with previous methodological developments on TILs evaluation(2). Agreement between the 2 pathologists was evaluated using the concordance correlation coefficient (CCC) as a summary measure of reproducibility(3). For binary variables with no repetitions, CCC is equivalent to Cohen's kappa.

Sample Processing and RNA Extraction for Gene Expression Profiling

Macrodissection was performed in order to enrich for tumor cell content. If it was possible to obtain a tumor area of at least 5 mm2 containing at least 70% viable tumor cells only those area(s) were macrodissected. If collection of the required amount of tumor cells (less than 70% viable tumor cell content) was not feasible, macrodissection was performed on a minimum of 5mm2 tumor area containing at least 50% viable tumor cells. If this was not feasible or only less than 5mm2 tumor area are available, the total amount of tumor area is dissected.

A proprietary method developed in house was applied to extract RNA using kit reagents that are equivalent to the now commercially available High Pure FFPET RNA Isolation Kit manufactured by Roche Diagnostics.

Single Genes/Signature Scores Calculations and PAM50 Subtype Definition

The signature scores were calculated as a weighted sum of the log-expressions of their genes, with gene-specific weights equal to +1 or -1 depending on the direction of their association with the gene expression immune phenotype. The selected immune genes and three immune gene signature scores were scaled so that the 2.5% and 97.5% quantiles equaled −1 and +1, respectively.

Gene expression levels were made comparable to those of HER2-positive samples from the C9741 NanoString cohort(4) by using the cross-studies normalization of the R package genefu(5) (R package version 2.4.2). A merged data set was obtained by adding the renormalized TRYPHAENA samples to all C9741 samples. PAM50 subtypes were determined using genefu on the merged data set.

Statistical Analysis for Gene Expression Data Generated Using the NanoString® nCounter Assay

Correlation analysis:

Spearman correlation analysis was performed between the baseline TILs, the selected immune genes, the immune gene signatures and the ERBB2 and ESR1 mRNA genes.

Spearman correlation analysis was also performed between the baseline TILs level and the whole 800 gene panel. Genes with spearman coefficient rho higher than 0.30 or below -0.30 and p-values corrected for multiple testing (FDR) of less than .05 were considered statistically significant for the gene ontology (GO) analysis. GO enrichment was performed using the R package GO.db (version 3.4.1) and limma (version 3.32.4).

Association with pCR

The ERBB2, ESR1 and the immune single genes/gene signatures, as well as the HER2 enriched subtype defined with PAM50 were assessed for their association with pCR status using logistic regression. The regression models were evaluated with and without adjustment for baseline clinicopathologic variables including age (<50y vs ≥50y), histology grade (III vs I/II), ER status (positive vs negative), clinical stage (III vs II), chemotherapy (anthracyclines vs non-anthracyclines) and Baseline TILs (10 units increase).

Association with EFS

The prognostic value of ERBB2, ESR1 and the immune single genes/gene signatures, as well as the HER2 enriched subtype defined with PAM50, was assessed using univariate Cox proportional hazard models. The prognostic value of clinicopathological characteristics (age (<50y vs ≥50y), histology grade (III vs I/II), ER status (positive vs negative), clinical stage (III vs II), chemotherapy (anthracyclines vs non-anthracyclines)), PAM50 subtype (HER2-enriched vs non-HER2-enriched), pCR and baseline TILs were evaluated in a multivariate cox proportional hazard model. The proportional hazards assumption was checked by examining scaled Schoenfeld residuals (6).

P-values were corrected for multi-testing with the Benjamini-Hochberg procedure (FDR).


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