Skip to main content

Advertisement

SpringerLink
Log in

Effect of Formalin Fixation for Near-Infrared Fluorescence Imaging with an Antibody-Dye Conjugate in Head and Neck Cancer Patients

  • Research Article
  • Published:
Molecular Imaging and Biology Aims and scope Submit manuscript

Abstract

Purpose

This study evaluated the effect of formalin fixation for near-infrared (NIR) fluorescence imaging of an antibody-dye complex (panitumumab-IRDye800CW) that was intravenously administered to patients with head and neck squamous cell carcinoma (HNSCC) scheduled to undergo surgery of curative intent.

Procedures

HNSCC patients were infused with 25 or 50 mg of panitumumab-IRDye800CW followed by surgery 1–5 days later. Following resection, primary tumor specimens were imaged in a closed-field fluorescence imaging device, before and after formalin fixation. The fluorescence images of formalin-fixed specimens were compared with images prior to formalin fixation. Regions of interest were drawn on the primary tumor and on the adjacent normal tissue on the fluorescence images. The mean fluorescence intensity (MFI) and tumor-to-background ratios (TBRs) of the fresh and formalin-fixed tissues were compared.

Results

Of the 30 enrolled patients, 20 tissue specimens were eligible for this study. Formalin fixation led to an average of 10 % shrinkage in tumor specimen size (p < 0.0001). Tumor MFI in formalin-fixed specimens was on average 10.9 % lower than that in the fresh specimens (p = 0.0002). However, no statistical difference was found between the TBRs of the fresh specimens and those of the formalin-fixed specimens (p = 0.85).

Conclusions

Despite the 11 % decrease in MFI between fresh and formalin-fixed tissue specimens, the relative difference between tumor and normal tissue as measured in TBR remained unchanged. This data suggests that evaluation of formalin-fixed tissue for assessing the accuracy of fluorescence-guided surgery approaches could provide a valid, yet more flexible, alternative to fresh tissue analysis.

Trial Registration

NCT02415881

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.

Similar content being viewed by others

References

  1. Orosco RK, Tapia VJ, Califano JA, Clary B, Cohen EEW, Kane C et al (2018) Positive surgical margins in the 10 most common solid cancers. Sci Rep 8(1):5686

    Article  Google Scholar 

  2. Eldeeb H, Macmillan C, Elwell C, Hammod A (2012) The effect of the surgical margins on the outcome of patients with head and neck squamous cell carcinoma: single institution experience. Cancer Biol Med 9(1):29–33

    PubMed  PubMed Central  Google Scholar 

  3. Ettl T, El-Gindi A, Hautmann M, Gosau M, Weber F, Rohrmeier C et al (2016) Positive frozen section margins predict local recurrence in R0-resected squamous cell carcinoma of the head and neck. Oral Oncol 55:17–23

    Article  Google Scholar 

  4. Rosenthal EL, Warram JM, de Boer E, Chung TK, Korb ML, Brandwein-Gensler M, Strong TV, Schmalbach CE, Morlandt AB, Agarwal G, Hartman YE, Carroll WR, Richman JS, Clemons LK, Nabell LM, Zinn KR (2015) Safety and tumor specificity of cetuximab-IRDye800 for surgical navigation in head and neck cancer. Clin Cancer Res 21(16):3658–3666

    Article  CAS  Google Scholar 

  5. Zhang RR, Schroeder AB, Grudzinski JJ, Rosenthal EL, Warram JM, Pinchuk AN, Eliceiri KW, Kuo JS, Weichert JP (2017) Beyond the margins: real-time detection of cancer using targeted fluorophores. Nat Rev Clin Oncol 14(6):347–364

    Article  CAS  Google Scholar 

  6. Gao RW, Teraphongphom NT, van den Berg NS, Martin BA, Oberhelman NJ, Divi V et al (2018) Determination of tumor margins with surgical specimen mapping using near-infrared fluorescence. Cancer Res 78(17):5144–5154

    Article  CAS  Google Scholar 

  7. Lu G, van den Berg NS, Martin BA, Nishio N, Hart ZP, van Keulen S, Fakurnejad S, Chirita SU, Raymundo RC, Yi G, Zhou Q, Fisher GA, Rosenthal EL, Poultsides GA (2020) Tumour-specific fluorescence-guided surgery for pancreatic cancer using panitumumab-IRDye800CW: a phase 1 single-centre, open-label, single-arm, dose-escalation study. Lancet Gastroenterol Hepatol 5:753–764

    Article  Google Scholar 

  8. Lamberts LE, Koch M, de Jong JS, Adams ALL, Glatz J, Kranendonk MEG, Terwisscha van Scheltinga AGT, Jansen L, de Vries J, Lub-de Hooge MN, Schröder CP, Jorritsma-Smit A, Linssen MD, de Boer E, van der Vegt B, Nagengast WB, Elias SG, Oliveira S, Witkamp AJ, Mali WPTM, van der Wall E, van Diest PJ, de Vries EGE, Ntziachristos V, van Dam GM (2017) Tumor-specific uptake of fluorescent bevacizumab-IRDye800CW microdosing in patients with primary breast cancer: a phase I feasibility study. Clin Cancer Res 23(11):2730–2741

    Article  CAS  Google Scholar 

  9. Koller M, Qiu S-Q, Linssen MD, Jansen L, Kelder W, de Vries J et al (2018) Implementation and benchmarking of a novel analytical framework to clinically evaluate tumor-specific fluorescent tracers. Nat Commun 9(1):3739

    Article  Google Scholar 

  10. van Keulen S, Nishio N, Birkeland A, Fakurnejad S, Martin B, Forouzanfar T et al (2019) The sentinel margin: intraoperative ex vivo specimen mapping using relative fluorescence intensity. Clin Cancer Res 25(15):4656–4662

    Article  Google Scholar 

  11. Fakurnejad S, Krishnan G, van Keulen S, Nishio N, Birkeland AC, Baik FM et al (2019) Intraoperative molecular imaging for ex vivo assessment of peripheral margins in oral squamous cell carcinoma. Front Oncol 9:1476

    Article  Google Scholar 

  12. de Boer E, Warram JM, Tucker MD, Hartman YE, Moore LS, de Jong JS, Chung TK, Korb ML, Zinn KR, van Dam GM, Rosenthal EL, Brandwein-Gensler MS (2015) In vivo fluorescence immunohistochemistry: localization of fluorescently labeled cetuximab in squamous cell carcinomas. Sci Rep 5:10169

    Article  Google Scholar 

  13. Lu G, Fakurnejad S, Martin BA, van den Berg NS, van Keulen S, Nishio N, Zhu AJ, Chirita SU, Zhou Q, Gao RW, Kong CS, Fischbein N, Penta M, Colevas AD, Rosenthal EL (2020) Predicting therapeutic antibody delivery into human head and neck cancers. Clin Cancer Res 26(11):2582–2594

    Article  CAS  Google Scholar 

  14. Nishio N, van den Berg NS, van Keulen S, Martin BA, Fakurnejad S, Teraphongphom N et al (2019) Optical molecular imaging can differentiate metastatic from benign lymph nodes in head and neck cancer. Nat Commun 10(1):5044

    Article  Google Scholar 

  15. van Keulen S, van den Berg NS, Nishio N, Birkeland A, Zhou Q, Lu G, Wang HW, Middendorf L, Forouzanfar T, Martin BA, Colevas AD, Rosenthal EL (2019) Rapid, non-invasive fluorescence margin assessment: optical specimen mapping in oral squamous cell carcinoma. Oral Oncol 88:58–65

    Article  Google Scholar 

  16. Nishio N, van den Berg NS, van Keulen S, Martin BA, Fakurnejad S, Zhou Q, Lu G, Chirita SU, Kaplan MJ, Divi V, Colevas AD, Rosenthal EL (2020) Optimal dosing strategy for fluorescence-guided surgery with panitumumab-IRDye800CW in head and neck cancer. Mol Imaging Biol 22(1):156–164

    Article  CAS  Google Scholar 

  17. van Keulen S, Nishio N, Fakurnejad S, Birkeland A, Martin BA, Lu G, Zhou Q, Chirita SU, Forouzanfar T, Colevas AD, van den Berg NS, Rosenthal EL (2019) The clinical application of fluorescence-guided surgery in head and neck cancer. J Nucl Med 60(6):758–763

    Article  Google Scholar 

  18. van Dam GM, Themelis G, Crane LMA, Harlaar NJ, Pleijhuis RG, Kelder W, Sarantopoulos A, de Jong JS, Arts HJ, van der Zee A, Bart J, Low PS, Ntziachristos V (2011) Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results. Nat Med 17(10):1315–1319

    Article  Google Scholar 

  19. Boogerd LSF, Hoogstins CES, Schaap DP, Kusters M, Handgraaf HJM, van der Valk MJM, Hilling DE, Holman FA, Peeters KCMJ, Mieog JSD, van de Velde CJH, Farina-Sarasqueta A, van Lijnschoten I, Framery B, Pèlegrin A, Gutowski M, Nienhuijs SW, de Hingh IHJT, Nieuwenhuijzen GAP, Rutten HJT, Cailler F, Burggraaf J, Vahrmeijer AL (2018) Safety and effectiveness of SGM-101, a fluorescent antibody targeting carcinoembryonic antigen, for intraoperative detection of colorectal cancer: a dose-escalation pilot study. Lancet Gastroenterol Hepatol. 3(3):181–191

    Article  Google Scholar 

  20. Predina JD, Newton AD, Connolly C, Dunbar A, Baldassari M, Deshpande C et al (2018) Identification of a folate receptor-targeted near-infrared molecular contrast agent to localize pulmonary adenocarcinomas. Mol Ther 26(2):390–403

    Article  CAS  Google Scholar 

  21. Frankel A (2012) Formalin fixation in the “-omics” era: a primer for the surgeon-scientist. ANZ J Surg 82(6):395–402

    Article  Google Scholar 

  22. Hewitt SM, Lewis FA, Cao Y, Conrad RC, Cronin M, Danenberg KD, Goralski TJ, Langmore JP, Raja RG, Williams PM, Palma JF, Warrington JA (2008) Tissue handling and specimen preparation in surgical pathology: issues concerning the recovery of nucleic acids from formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med 132(12):1929–1935

    Article  Google Scholar 

  23. Werner M, Chott A, Fabiano A, Battifora H (2000) Effect of formalin tissue fixation and processing on immunohistochemistry. Am J Surg Pathol 24(7):1016–1019

    Article  CAS  Google Scholar 

  24. Chen C-H, Hsu M-Y, Jiang R-S, Wu S-H, Chen F-J, Liu S-A (2012) Shrinkage of head and neck cancer specimens after formalin fixation. J Chin Med Assoc 75(3):109–113

    Article  Google Scholar 

  25. Tran T, Sundaram CP, Bahler CD, Eble JN, Grignon DJ, Monn MF, Simper NB, Cheng L (2015) Correcting the shrinkage effects of formalin fixation and tissue processing for renal tumors: toward standardization of pathological reporting of tumor size. J Cancer 6(8):759–766

    Article  Google Scholar 

  26. Otali D, Stockard CR, Oelschlager DK, Wan W, Manne U, Watts SA, Grizzle WE (2009) Combined effects of formalin fixation and tissue processing on immunorecognition. Biotech Histochem 84(5):223–247

    Article  CAS  Google Scholar 

Download references

Funding

This project was supported partly by the Stanford Comprehensive Cancer Center, the Netherlands Organization for Scientific Research (Rubicon; 019.171LW.022), the National Institutes of Health and the National Cancer Institute (R01CA190306), the Stanford Molecular Imaging Scholars (SMIS) program (T32CA118681), and a scientific research grant of YOKOYAMA Foundation for Clinical Pharmacology (YRY-1702). Institutional equipment loans were received from LI-COR Biosciences, Inc.

Author information

Author notes

  1. Shrey Kapoor and Guolan Lu contributed equally to this work.

Authors and Affiliations

  1. Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine, Stanford, CA, USA

    Shrey Kapoor, Guolan Lu, Nynke S. van den Berg, Giri Krishnan, Jacqueline Pei, Quan Zhou, Fred M. Baik, Eben L. Rosenthal & Naoki Nishio

  2. Department of Clinical Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Brock A. Martin

  3. Department of Otorhinolaryngology, Nagoya University Graduate School of Medicine, 65, Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan

    Naoki Nishio

Authors
  1. Shrey Kapoor
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guolan Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nynke S. van den Berg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Giri Krishnan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jacqueline Pei
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Quan Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Brock A. Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Fred M. Baik
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Eben L. Rosenthal
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Naoki Nishio
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Naoki Nishio.

Ethics declarations

Conflict of Interest

ELR acts as a consultant for LICOR Biosciences, which manufactures IRDye800, and has equipment loans from this company. All other authors declare that they have no conflict of interest.

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committees and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic Supplementary Material

Supplemental Figure 1

Correlations of tumor MFI and TBR in fresh and formalin-fixed tissue. Tumor MFIs and TBR in the formalin-fixed specimens were found to strongly correlate with those in the fresh specimens. (R2 = 0.93 and 0.97, respectively). (PNG 90 kb)

Supplemental Figure 2

Decrease of fluorescence signals in the same piece of tissue over the 10 times. (PNG 202 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kapoor, S., Lu, G., van den Berg, N.S. et al. Effect of Formalin Fixation for Near-Infrared Fluorescence Imaging with an Antibody-Dye Conjugate in Head and Neck Cancer Patients. Mol Imaging Biol 23, 270–276 (2021). https://doi.org/10.1007/s11307-020-01553-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11307-020-01553-1

Key words

Search

Navigation