Residual Tumor Characteristics Following Neoadjuvant Chemotherapy in Breast Cancer

Research Article

Austin J Cancer Clin Res 2015;2(7): 1059.

Residual Tumor Characteristics Following Neoadjuvant Chemotherapy in Breast Cancer

Mani CS¹*, Chander V², Kurian A³, Subramani A³, Mallikarjun VS4, Mahalingam S5 and Ramakrishnan V6

¹Cancer Research and Relief Trust, India

²Department of Pathology, Saveetha Medical College, India

³Department of Pathology, Apollo Specialty Hospitals, India

4Department of Pathology, SRM Medical College, India

5Department of Biotechnology, IIT-Madras, India

6Madras Medical College, Indi

*Corresponding author: Mani CS, Consultant Surgical Oncologist, Cancer Research and Relief Trust, 39, TTk 1st cross street, Alwarpet, Chennai 600018, India

Received: June 16, 2015; Accepted: August 09, 2015; Published: August 12, 2015


Neoadjuvant chemotherapy for breast cancer leaves behind a residual tumor that is substantially different in its characteristics from the original tumor. The histological changes and receptor changes have been systematically evaluated in large studies. However, the nucleolar morphology of the residual cancer has not been well characterized. This study evaluated the nucleolar morphology in the residual cancer using haematoxylin eosin for evaluation of nucleolar number and size in relation to mitotic index, mAgNOR assessment, and an immunohistochemistry panel consisting of Ki-67, p53, CCND1, E-Cad, ER, PR, Her2Neu, CD3, CD8 and CD68. Another sample of patients were studied by exome analysis of a 344 candidate cancer gene panel. The results were collated to infer the nature of the residual tumor. There was an increase in nucleolar number and size following neoadjuvant chemotherapy. The mitotic rate, and positive staining for Ki-67 and CCND1 were reduced in the residual tumor. 344 candidate cancer gene panel exome analyses showed residual tumor with fewer mutations and the more common known driver mutation in breast cancer such as CREBP, ERBB2, PARP, TOP2A, P53 etc. COL1A1 was the only gene mutation demonstrated in the residual tumor that was not found in the untreated primary surgical group.

Keywords: Breast cancer; Nucleoli; mAgNOR; Exome analysis; Neoadjuvant chemotherapy; Residual tumor


mAgNOR: Mean AgNOR; NAC: Neoadjuvant Chemotherapy; NSABP: B18 National Surgical Adjuvant Breast and Bowel Project; QC: Quality Control; ER: Estrogen Receptor; PR: Progesterone Receptor; FISH: Fluorescent in Situ Hybridization; RD: Residual Disease; IHC: Immunohistochemistry


Response to neoadjuvant chemotherapy (NAC) in breast cancer has a wide spectrum. It ranges from complete pathological response to no response, or progression of disease. Chemotherapy results in histologic changes that have been well studied but its effect on nucleolar morphology has received little attention.

It appears that tumor cells remaining after NAC contain a cancer cell population that is intrinsically resistant to chemotherapy. They represent clones that are capable of further growth and cell division and ultimately result in distant metastases. They are unlikely to respond to further chemotherapy especially to the agents directed against the pathways that were previously targeted. It is postulated that these residual cells could be a resistant clone or acquire features of cancer stem cells.

Neoadjuvant chemotherapy is the standard of care in locally advanced breast cancer [1]. It is also being increasingly used in early stage breast cancers that have poor prognosis such as triple negative breast cancer and the Her2 Neu positive cancers [2,3]. Neo adjuvant chemotherapy offers the advantage of early institution of systemic treatment in order to sterilize potential micro metastases before local therapy such as surgery or radiation is performed. It provides an idea of in vivo chemo sensitivity of the tumor. It also improves the chances of breast conservation surgery in a substantial number of patients. The NSABP- B18 trial that addressed the issue of neoadjuvant treatment is the largest randomized controlled trial comparing neoadjuvant chemotherapy to adjuvant chemotherapy and its 9 years follow up study showed no difference in the overall and disease specific survival of patients between the 2 arms [4].

Neoadjuvant treatment is therefore started after obtaining a core needle biopsy that determines the histology, grade and the immunohistochemical markers predictive of treatment options such as hormonal receptors and Her2Neu expression. The tissue obtained in a core needle biopsy is limited but adequate to obtain the necessary information. Concerns about tumor heterogeneity and lack of adequate tissue to evaluate novel prognostic markers remain with such an approach. NAC alters the tumor in remarkable ways [5]. It fragments the tumor into islands of necrosis and fibrosis with residual viable tumor in between. These residual tumor cells may differ substantially from the original tumors in many respects. The histology changes to more of a lobular type, histiocytoid cellular changes are noted, and expression of hormonal receptors are altered significantly, while Her2Neu receptor expression remains constant. Molecular profiling and whole genome sequencing of the residual tumors have been studied to gain insight into chemo resistance and the outcome of these patients. But less is known on how the nucleolus responds to neoadjuvant treatment and contributes to the architectural and molecular changes of the residual tumor.

The purpose of the current study was to systematically evaluate the changes in nucleolar morphology due to neoadjuvant chemotherapy after NAC. We also wanted to understand whether nucleolar morphology and function in the residual cancer cells after chemotherapy contribute to either the resistance or stemness.


Consecutive breast cancer patients who consented to this study at the Cancer Research and Relief Trust between 2013 and 2014 were recruited to this study after obtaining approval from the ethics committee and institutional review board. Concept study of 11 patients with infiltrating ductal carcinoma who had received NAC with anthracycline or taxol containing regimens were taken to study the nucleolar morphology before and after chemotherapy on routine H&E staining (Sample A).

An additional cohort of 46 patients, 7of whom had been treated with NAC was taken up for mAgNOR scoring and detailed studies with an immunohistochemistry (IHC) panel containing CCND1, p53, Bcl2, Ki-67, E-Cad, ER, PR, Her2Neu CD3, CD8 and CD68 (Sample B).

The staining protocol consisted of numerous steps in which reagents were incubated for pre-determined times at specific temperatures. The paraffin embedded tissues that were used for the original hematoxylin and eosin stained sections were used for IHC. Sections were cut at 3 micron thickness. A standard IHC technique was performed using VENTANA BENCHMARK XT.

Tissues obtained from a third set of 11 patients with residual tumor who had received NAC were analyzed by exome sequencing with a 344 gene panel (Sample C). The exome analysis procedure consisted of DNA/RNA extraction, QC, DNA library preparation, enrichment capture of comprehensive candidate cancer genes, cluster amplification, and running of sample on Illumina HiSeq platform, generating quality report of raw data and advanced data analysis.

Genomic DNA was isolated to meet quality specifications. The genomic DNA was sheared and used to perform enrichment using the probes in the cancer gene panel. Cancer gene panel targets about 344 genes with an approximate target region of 7MB. Captured fragments were adapted to produce libraries that were sequenced on Illumina HiSeq 2000 to generate paired end 2 x 100bp sequence reads to produce 100 x coverage. The generated sequence data were analyzed after quality control for variant calling and annotation. Bioanalyzer plots used at Bioanalyzer plots were used at every step to assess library size and qPCR was used for measuring the quantity of the library before sequencing.

The results were computed and analyzed using SPSS2.


The number of nucleoli per cell was counted in tissue obtained from patients in Sample A. The mean number of nucleoli per cell was distinctly higher in the post treatment residual viable cells withmacroenlargement of the nucleoli (Figures 1, 2, 3).