Spatial intratumoural heterogeneity in the expression of GIT1 is associated with poor prognostic outcome in oestrogen receptor positive breast cancer patients with synchronous lymph node metastases

Patient cohort

Primary site and lymph node metastasis tissue samples were obtained from the Pathology Department of Donostia University Hospital from 140 breast patients who were diagnosed between 2000 and 2006, with follow-up data until 2014 with a median follow-up of 8 years and 8 months. Written consent was obtained from patients for the inclusion of their samples in this study and samples were collected in accordance with the Declaration of Helsinki, and approved by local ethics committees (Comite Etico de Investigación Clínica de Euskadi (CEIC-E)). Cases were selected and re-reviewed by two experienced breast pathologists independently. Clinical data was only available for 104 of these patients (Dataset 1). All patients were women with an age range between 27 and 86 years old (median age 58 years old). All primary site tumours were ER+ and 91% of them PR+. Eighty-six percent of patients presented with invasive carcinoma of no special type (NST) and 11% with invasive lobular carcinoma (ILC). Histological grades varied from grade I (21%), grade II (55%) to grade III (15%), according to the Elston-Ellis modification of Scarff-Bloom-Richardson grading system. All patients were surgically treated either by tumorectomy or by mastectomy with axillary lymphadenectomy. All patients underwent hormone therapy with the majority of them receiving radiotherapy (90%) and adjuvant chemotherapy (75%). Table 1 summarizes the clinical and histological characteristics of patients as we previously described⁷.

Tissue microarray construction and immunohistochemistry (IHC)

Representative areas of high tumour load (>70%) were selected after H&E staining and two 1.5mm punch biopsies taken from both primary tumour and lymph node and arrayed non-adjacently to reduce staining bias using a Manual Tissue Arrayer MTA-1 (Beecher Instruments, USA).

Immunohistochemical (IHC) staining was carried out on 5µm slices manually using the Immunohistochemistry Accessory Kit of Bethyl Laboratories (Montgomery, USA). Slides were deparaffinized in xylene and blocked in peroxidase for 30 min. Antigen retrieval was carried out in Epitope Retrieval Buffer (Bethyl Laboratories, Montgomery, USA). Slides were blocked in BSA for 30 min and then incubated for 1h at room temperature with anti-GIT1 rabbit polyclonal antibody (1:100) (IHC-00527 (lot #001), Bethyl Laboratories, Montgomery, USA). Even though this is the same antibody used by the study of Chan et al.⁶, and raised against a GIT1-specific peptide immunogen, we cannot rule out cross-reactivity against other non-GIT1 epitopes. After 1h incubation with secondary anti-Rabbit IHC Antibody and DAB substrate. The slides counterstained with hematoxylin.

GIT1 expression was scored in a blinded fashion by an experienced breast pathologist according to tumour cell staining intensity and categorical scores assigned as follows; 0= negative (0%); 1=1–10%; 2= 11–50%; 3= >50% (Dataset 2). Scores between non-adjacent cores were combined and categorised according to following criteria; GIT1 negative (combined score <1); moderate GIT1 expression (combined score 1–2); and high GIT1 expression (score >2). Examples of the different staining categories are shown in Figure 1 and Supplementary Figure S2. A selection of cases (n=8) were examined both as whole biopsy sections and TMAs, all scoring was concordant. Examples of whole section IHC staining with GIT1 are shown in Supplementary Figure S1 and Supplementary Figure S3.

Figure 1. Example of GIT1 expression patterns found in ER+ breast cancer.

Images were enhanced from the original (Supplementary Figure S2). Representative intensity staining of GIT1 expression (primary tumour) depicting A. negative (score=0); B. weak (score=1); C. moderate (score=2); and D. strong (score=3). Magnification ×400.

Public dataset analysis

GIT1 gene expression levels were analyzed using publicly available databases. For this analyses we interrogated the TCGA dataset which included 525 mixed breast cancer tumours and 22 normal breast samples (Dataset 3)⁸, 2000 mixed breast tumours (Dataset 4)⁹, 570 metastatic breast tumours (Dataset 5)¹⁰, 252 lymph-node negative breast cancer patients (Dataset 6)¹¹, 67 triple negative breast cancer patients (Dataset 7)¹², and 19 primary breast cancer and 19 brain metastasis from HER2 positive breast cancer patients (Dataset 8)¹³.