Surgical Resection Of Residual Breast Cancer: Stem Cells that Neoadjuvant Chemotherapy Leave Behind SuEllen Pommier, Ph.D., Cynthia Jackson, M.S., Jennifer.

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Surgical Resection Of Residual Breast Cancer: Stem Cells that Neoadjuvant Chemotherapy Leave Behind SuEllen Pommier, Ph.D., Cynthia Jackson, M.S., Jennifer Peckham, M.S., Patrick Muller, B.S., Mary Condron, M.D., Nora Jameson, M.S. and Rodney F. Pommier, M.D.

Breast Cancer Heterogeneity Breast cancers with similar pathology do not all respond to treatment equally well. There is a substantial recurrence rate observed in both ER- and ER+ breast cancers, irrespective of the use of chemotherapy or hormonal treatment. Lancet 2005; 365: 1687-1717 Lancet 2005; 365: 1687-1717

There are Two Ways to Treat Breast Cancer Chemo Naive NAC Surgery Neoadjuvant Chemotherapy Surgery Adjuvant Chemotherapy Residual Disease? Assess response

Statistics on Residual Disease (RD) RD is still present in 70-90% of patients treated with chemotherapy. RD correlates with poorer survival. Earl et al. BMC Med. 2015, 13:234 Earl H. et al. BMC Med. 2015, 13:234

Breast Cancer Stem Cell Research What cells survive chemotherapy? Thought that cancer stem and progenitor cells may initiate and maintain the tumor proliferation. They harbor oncogenic mutations. May be responsible for high recurrence rate. Hypothesis: BCSC will be present in RD and harbor oncogenic mutations. This is best accomplished through the investigation of fresh surgical specimens whenever possible. Cancer Res 2006; 66:9339-9344 1a. Specifically we are examining the role of Cancer Stem cells in these processes. 2. We hope that by our investigation of CSC we will also Cancer Res 2006; 66:9339-44 Cancer Res 2008; 68: 3243-50 J Natl Cancer Inst 2008; 100:672-79 J Natl Cancer Inst 2006; 98:1777-85

Objective: To determine correlations between: BCSC frequencies Study Design Objective: To determine correlations between: BCSC frequencies BCSC mutations and residual disease in tumors treated with neoadjuvant chemotherapy (NAC). -To determine correlations between the frequencies of breast cancer stem cells, their mutations, and their response to chemo, 3 cohorts were enrolled in our study: 38 cancer patients who had not been treated with chemotherapy 11 patients who had been treated w/ chemo 20 healthy breast reduction patients (validated by Pathology benign tissue) the BCSCs were isolated as described previously and the DNA was sequenced using the Ion Torrent hot spot oncogene panel.

Tissue Samples: Fresh Surgical Specimens obtained from 2 sets of patients Chemo Naive NAC Neoadjuvant Chemotherapy Surgery Surgery Adjuvant Chemotherapy To test this hypothesis we compared tumors obtained from two sets of patients

Patient Demographics, Tumor Characteristics NAIVE NAC Patients n = 37 n = 11 SIG. specimens N=195 N = 55 Age (yrs) 57 50 p = NS range 28-90 35-64 Tumors n =39 n= 11 Stage (%) p =NS I 16 (41) 2 (18) II 18 (46) 7 (64) III 4 (10.5) 1 (9) IV 1 (2.5) Hormone R ER+PR+/-H2+ 3 (7.5) 1 (9) ER+PR+/-H2- 30 (77) 7 (64) TNBR 2 (5) 2 (18) ER-PR-H2+ 4 (10.5)

Treatment Regimens of NAC No. of patients Treatments 9 doxorubicin/cyclophosphamide, followed by paclitaxel 1 Same as above, plus trastuzumab Docetaxel; docetaxel/trastuzumab/pertuzumab, trastuzumab/pertuzumab only; denosumab Chemotherapy was uniform

Fluorescence-Activated Cell Sorting (FACS) Chemo Naive Post Chemo (NAC) CD49f 13-35 M, 14-32 T, 14-01 TB CD24

Selection and Quantification of Breast Cells Remove CD45+ and CD31+ cells Collect breast stem/progenitor cells Stem/progenitor Cell Markers: CD49f CD24 Measure % breast stem/progenitor markers CD44 EpCAM CSC can be distinguished from non-tumorigenic stem cells by a variance in the cell surface profile. Pan Cytokeratin: 4,5,6,8,13, 18 Hemopoietic and endothial cells Proc Natl Acad Sci 2003; 100:3983-88

BCSCs mutations identified GENETICS BCSCs mutations identified Ion Torrent Hot Spot oncogene panels. ABL1 EZH2 JAK2 PTEN AKT1 FBXW7 IDH2 PTPN11 ALK FGFR1 KDR RB1 APC FGFR2 KIT RET ATM KRAS SMAD4 BRAF FLT3 MET SMARCB1 CDKN2A GNAQ NOTCH1 STK11 CTNNB1 HNF1A NPM1 TP53 EGFR HRAS NRAS VHL ERBB2 IDH1 PDGFRA ERBB4 PIK3CA We used the HotSpot panel to detect mutations in our Breast Cancer and CSC samples. Hotspot regions, including ~2,800 COSMIC mutations of 50 oncogenes and tumor suppressor genes. 300,000- 500,000 reads/specimen; 1800 reads / amplicon; 95% 100x coverage; 50% 500x coverage

Cellular Changes in Post Chemotherapy Residual Disease Results Cellular Changes in Post Chemotherapy Residual Disease We also examined post chem tumor samples to determine what sort of effect Chem has on

Post NAC Vascular Cell Changes: Whole tumor Stem Cells. 2014 Jan;32(1):145-56. doi: 10.1002/stem.1556. CD44 expressed on cancer-associated fibroblasts is a functional molecule supporting the stemness and drug resistance of malignant cancer cells in the tumor microenvironment. Kinugasa Y1, Matsui T, Takakura N.

Pathologic Responses to NAC (N=11) 9/11 patients 2/11 patients Mostly viable tumor Densely cellular Brisk mitotic rates Patchy response No treatment effect Small foci of tumor Minimal residual

Naïve vs NAC Stromal Cell Changes (%) lin-CD49f-CD24- increase p=0.04

CD49f-CD24- Cell Populations % Ave. Staining

Naïve vs NAC Cell Changes (%)

Stem/progenitor Cell Changes (%)

Stem/progenitor Cell Changes in CD49f and CD24 Expression (%) Differ in frequency and CD44

EpCam %: Naïve vs NAC Stem/progenitor Cells CD49f+24+ CD49f+24- CD49f-24+ CD49f-24-

CD44 %: Naïve vs NAC Stem/progenitor Cells CD44 inc in 2 of 4 populations Dependent of CD49f marker. EpCam not significantly different

Chemo Naïve Vs NAC Stem/Prog Differences

Chemo Naïve Vs NAC Stem/Prog Differences

Clinical Significance? Lin-CD49f-CD24+ 2.43 fold increase Relative increase of 59% Genetic component

BCSCs predominant mutations GENETICS BCSCs predominant mutations ABL1 EZH2 JAK2 PTEN AKT1 FBXW7 IDH2 PTPN11 ALK FGFR1 KDR RB1 APC FGFR2 KIT RET ATM KRAS SMAD4 BRAF FLT3 MET SMARCB1 CDKN2A GNAQ NOTCH1 STK11 CTNNB1 HNF1A NPM1 TP53 EGFR HRAS NRAS VHL ERBB2 IDH1 PDGFRA ERBB4 PIK3CA We used the HotSpot panel to detect mutations in our Breast Cancer and CSC samples. Hotspot regions, including ~2,800 COSMIC mutations of 50 oncogenes and tumor suppressor genes. 300,000- 500,000 reads/specimen; 1800 reads / amplicon; 95% 100x coverage; 50% 500x coverage

Prominent Mutations: Naïve Breast Cancer Tumors, Stem/Progenitor Cells and CAFs PIK3CA TP53 Pre Chemo n= 38 12 (31.6%) 2 (5.3%) Total Tumor tissue 9 (23.4) CD49f+24+ 6 (15.8) 1 (2.6) CD49f+24- 4 (10.5) CD49f-CD24+ 5 (13.2) CD49f-CD24- CAF + SMARCB1 homozygous in Epithelioid sarcoma; ours our heterozygous. In Rabdoid tumor deregulates G1-S transition. Stk11- serine threonine Kinase in cervical cancer PI3K (HRAS) pathway disruption seen in many cancers PDGFRA:tyrosine kinase receptor These growth factors are mitogens for cells of mesenchymal origin. plays a role in organ development, wound healing, and tumor progression and stromal tumors MET: proto-oncogene tyrosine-kinase activity papillary renal carcinoma. Includes samples: 12-110 T 13-04 T 13-10 T Core 13-25 T 13-29 T 13-50 T 14-02 T 14-09 T 14-11 T Core 14-12 T 14-13 T 14-18 T 14-19 T 14-21 T 14-32 T 14-33 T 14-35 T 14-42 T 14-44 T

Prominent Mutations: Following NAC treatment Tumors, Stem/Progenitor Cells and CAFs PIK3CA TP53 NAC n= 11 4 (36.4%) Total Tumor tissue 4 (36.4) 2 (18.2) CD4f+24+ 1(9.1) CD49f+24- 1 (9.1) CD49f-CD24+ 3 (9.1) 2(18.2) CD49f-CD24- CAF +

Contribution of Genetic Mutations Cell Population Relative Increase Cells/tumor Mutation Incidence % CD49f+ CD24- Constant; Inc.CD44 CD49f-CD24+ 59% 31.6 - 36.4 CD49f-CD24- CAF+ 24.5% 5.3 - 36.4 In NAC tumors: BCSC populations remain constant or increase in cell number Mutation incidence higher in post-chemo tumors Mutations are distributed throughout the different BCSC populations and more prevalent 13-35 M, 14-32 T, 14-01 TB

Histopathology found little effect from NAC treatment. Summary RD found in the majority of breast cancer patients treated with neoadjuvant chemotherapy. Histopathology found little effect from NAC treatment. NAC tumors contained viable cells.

There may be a cumulative effect. Summary Increase in the frequency of PIK3CA and TP53 mutations NAC tumors vs Naïve. Increase in cancer stem/progenitors cells in NAC tumors that harbor these mutations. There may be a cumulative effect. J Natl Cancer Inst 2008; 100:672-79

Stem/progenitor cells are present in RD. Conclusion Stem/progenitor cells are present in RD. They are viable, increased in number, and harbor higher mutation frequencies. These findings may explain the higher recurrence and poorer survival rates in patients with RD.

Acknowledgements Surgical Oncology Fellow Rodney Pommier, MD Mary d'Alelio Arpana Naik, MD Statistics John Vetto, MD Knight Cancer Inst. Core Plastic and Reconstructive Surgery Juliana Hansen, MD Pathology David Sauer, MD Funding Megan Troxell, MD Janet E. Bowen Foundation Lab Personnel Patrick Muller, BS Jennifer Peckham, MS Cynthia Jackson, MS Nora Jameson, MS

Surgical Specimen Sample Size BrCa: naïve: 0.05 -1.0 g NAC: 0.01-1.0g Reduction: 0.7- 3.0 g Adequate: 0.2 g

PROTEIN RESULTS Patient variation CSC are ER and PR negative Similar to benign These graphs represent our results . If cells fall Below the gray bar CSC they are ER neg. CSC are ER neg even among ER+ tumors. Some patient variation but over, quite negative. The same is true for the PR. IL-6 and Il_8 mediators of ER activity