Establishment of an efficient pathologic diagnostic platform using core needle biopsy for salivary gland carcinoma

doi:10.21203/rs.3.rs-4703440/v1

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Establishment of an efficient pathologic diagnostic platform using core needle biopsy for salivary gland carcinoma

https://doi.org/10.21203/rs.3.rs-4703440/v1

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Purpose:

A major problem in establishing treatment strategies for salivary gland cancer is the difficulty of preoperative histologic typing. In recent years, genetic evaluation has become essential for salivary gland tumor diagnosis. The core needle biopsy (CNB) allows a small piece of the center of the tumor to be taken intact and analyzed in detail. The purpose of this study was to clarify the efficacy and the limitations of CNB in the preoperative diagnosis, and to establish a generalizable preoperative histologic typing platform.

Methods: Paired preoperative 20-gauge core needle biopsy (CNB) and surgical specimens from 41 patients with malignant salivary gland tumors were reviewed. Morphological evaluation, immunohistochemical evaluation, and break-apart fluorescence in situ hybridization (FISH) assay were performed as histologic typing methods for CNB. The quality of biopsy specimens, diagnostic accuracy, and immunostaining concordance rates between biopsy and surgical specimens were analyzed.

Results: For CNB, 95% (39/41) of the specimens were of high quality, allowing adequate morphologic, immunohistologic, and genomic analysis. Two patients had unanalyzable specimens due to cystic fluid or tumor firmness. Overall, 75% (31/41) had correct preoperative histologic typing. Compared to other histologic types, carcinoma ex pleomorphic adenoma (CXPA) and salivary duct carcinoma arising from CXPA had a significantly lower concordance rate for histologic typing (50% vs. 89.6%, p=0.016) and poorer HER2 immunostaining concordance rates between CNB and surgical specimens (60% vs. 0%, p=0.001). There were no recurrences due to tumor seeding after CNB.

Conclusions: Detailed analysis of CNB specimens allows for highly accurate determination of salivary gland carcinoma histologic type with molecular diagnosis. However, careful histologic typing is necessary in pathologically heterogeneous tumors.

core needle biopsy

preoperative pathologic diagnosis

histologic type

Heterogeneity

SOX10

Because of the diversity in salivary gland carcinoma histopathologic types, pathologic diagnosis is notoriously difficult ⁽¹⁾. Moreover, in recent years, as the molecular biology of salivary gland carcinoma has become elucidated, genomic information such as fusion genes and gene amplification have been used for pathologic diagnosis in addition to morphologic and immunohistologic information ^{(2, 3)}.

Core needle biopsy (CNB) allows a small piece of the center of the tumor to be taken intact and all of its components to be analyzed in detail ⁽⁴⁾. In addition, immunohistologic status and genomic information such as fusion genes and gene amplification can be extracted, which is expected to dramatically improve diagnostic performance ^{(2, 5, 6)}.

The histologic heterogeneity of salivary gland carcinoma is due to the complexity of salivary gland structures ⁽²⁾. Histogenesis-based diagnostic markers are useful for systematic and effective histologic typing. Our previous reports showed that SRY-related HMG-box10 (SOX10) expression can clearly define two distinct subtypes of salivary gland tumors based on their anatomical site of origin ⁽⁷⁾. To make needle biopsies for the diagnosis of salivary gland cancer more widespread, establishing an efficient pathologic diagnostic platform that uses several immunohistologic markers based on the origin and characteristics of the tumor is urgently needed. It is also necessary to identify problems and limitations in pathological diagnosis using needle biopsy specimens.

Study design and setting

In this retrospective study, medical records, biopsy specimens, and surgical specimens of 139 patients with parotid gland and submandibular gland tumor who underwent primary surgical treatment at the National Cancer Center Hospital of Japman between 2007 and 2020 were reviewed. Of these, we excluded 51 patients with benign tumor, and of the remaining 88 patients with malignant tumor, 41 patients who underwent surgical treatment after preoperative CNB were considered eligible for the analysis. We have been performing preoperative CNB as a first choice on all patients with suspected malignancy since 2017 to improve the accuracy of preoperative diagnosis.

Patient characteristics are shown in Table 1. Median follow-up was 30 (range: 2–169) months. The eighth edition of the tumor–node–metastasis (TNM) classification from the International Union Against Cancer and the American Joint Committee on Cancer was used for clinical staging. Prognostic information was available for all patients. Ethics approval for retrospective biomarker analysis was obtained from our ethics committee (approval number, 2010-77).

Ultrasound (US)-guided CNB and FNAC

For CNB, we used a semi-automatic biopsy device (FINE CORE^® Needle, 20-gauge, length 55 mm, Dr. Japan Co., Ltd. Tokyo, Japan), which was developed under our advice. This device was developed specifically for performing biopsies of head and neck tumors. It was designed to have a short handle and be compact, lightweight, and easy to maneuver. All biopsies were performed under US guidance by head and neck surgeons (K.K., S.Y., G.O., M.F., or M.Y.). Using US, the surgeon confirmed tumor vascularity and ensured that the biopsy needle penetrated completely into the tumor (Fig. 1). In solid-cystic lesions, a biopsy of the solid part was performed. To prevent shrinkage, biopsy specimens were attached to the edges of filter paper cut into 2 x 1 cm pieces and fixed with 10% formalin. To prevent residual tumor from puncture seeding, the surgical skin incision was designed to include the biopsy scar (Fig. 2). Regardless of tumor localization, the puncture point was set on the expected skin incision line.

Pathologic diagnosis

As a preoperative histologic typing platform for CNB specimens of salivary gland carcinoma, in addition to the conventional morphologic evaluation, immunohistochemical evaluation of SOX10, Epithelial membrane antigen (EMA)/Mucin 1 (MUC1), c-kit, p63, Androgen receptor (AR), and Human epidermal growth factor type 2 (HER2); fluorescent in situ hybridization (FISH) analysis of MYB, MAML2,and ETV6; and dual color in situ hybridization (DISH) analysis of HER2 were performed. SOX10, EMA/MUC1, c-kit, p63, AR, and HER2 were ordered reflexively on all biopsy specimens. Immunostaining for other biomarkers, FISH, and DISH were ordered based on initial pathologic findings within 4 days, with considerations of cost effectiveness. All preoperative diagnoses were performed by 2 of our pathologists (T.M. and Y.Y.) within 8 days of specimen submission.

For surgical specimens, the entire tumor was sliced into many sections and thoroughly sampled for detailed evaluation of tumor components and resection margins. Immunostaining for SOX10, EMA/MUC1, c-kit, p63, AR, and HER2 was re-evaluated in surgical specimens and compared as paired samples.

Histology and immunohistochemistry (IHC)

Histologic examination was performed on 4-µm–thick sections cut from formalin-fixed paraffin-embedded (FFPE) tissue and stained with hematoxylin and eosin. A solid pattern of 30% or more was used to define the cutoff for solid type adenoid cystic carcinoma (AdCC) ⁽²⁾. Immunohistochemical staining was performed on FFPE specimens. Antigen retrieval was performed using Target Retrieval Solution pH 9 (Agilent Technologies, Santa Clara, California, USA). All primary antibodies used are summarized in Supplementary Table 1. Nuclear staining of > 10% tumor cells was considered positive for SOX10, p63, and AR, respectively ⁽⁷⁾. Cytoplasmic staining with circumferential membranous accentuation in > 10% tumor cells was considered positive for EMA/MUC1 ⁽⁸⁾. Cytoplasmic or membranous diffuse staining of > 25% tumor cells was considered positive for c-kit ⁽⁹⁾.

HER2 status

HER2 status was assessed based on IHC and DISH. Staining intensity was classified as 0 (no immunoreactivity), 1+ (incomplete membrane staining in > 10% of tumor cells), 2+ (weak to moderate complete membrane staining in > 10% of tumor cells), or 3+ (strong immunoreactivity in > 10% of tumor cells) based on the guidelines for HER2 assessment in breast cancer ^{(10, 11)}. The DISH assay for HER2 was performed on a Ventana BenchMark series instrument (Ventana/Roche, Oro Valley, Arizona, USA). HER2 (labeled with spectrum black) and chromosome 17 (CHR17) reference gene signals (labeled with spectrum red) were enumerated with conventional bright-field microscopy after hematoxylin counterstaining. HER2:CHR17 ratio ≥ 2.0 was defined as HER2 DISH +. IHC 3 + or IHC 2+/DISH + was considered to indicate HER2 amplification.

FISH

FISH was performed on 4-µm–thick FFPE tissue sections from relevant areas for analysis identified by a pathologist (T.M.) and prepared using a standard protocol for probe application. Probes are listed in Supplementary Table 2. FISH images were captured using the Metafer Slide Scanning Platform (MetaSystems, Altlussheim, Germany); 200 non-overlapping tumor nuclei with at least one of each signal were examined. Tumors with > 20% nuclei with fused or split rearrangement signals, were deemed rearrangement positive.

Statistical analysis

Differences between any two groups were analyzed using Fisher’s exact test. R and its command input platform, ezR, were used for analysis ⁽¹²⁾. A value of P < 0.05 was considered to be statistically significant.

Details of preoperative histologic typing by CNB

In the 41 patients who underwent CNB, 95% (39 patients) had a high-quality specimen that enabled adequate morphologic, immunohistologic, and genomic analysis (Fig. 3). In two patients with high-grade mucoepidermoid carcinoma and squamous cell carcinoma, respectively, analyzable specimens were not available due to cystic fluid or tumor firmness. The results of the histologic typing analysis are shown in Fig. 4.

In total, 75% (31/41) had a correct preoperative histologic typing. It was possible to successfully determine the histologic type for even solid type AdCC. However, carcinoma ex pleomorphic adenoma (CXPA) and salivary duct carcinoma arising from CXPA were significantly less able to determine the histologic type than other histologic types (50% vs 89%, p = 0.0164) (Table 2).

Concordance of immunostaining in paired CNB and surgical specimens

The immunostaining findings in paired CNB and surgical specimens of all 41 patients are shown in Fig. 4. Concordance between CNB and surgical specimen results for SOX10, EMA/MUC1, c-kit, p63, AR, and HER2 immunostaining were 94% (37/39), 97% (38/39), 94% (37/39), 94% (37/39), 94% (37/39), and 82% (32/39), respectively. CXPA and salivary duct carcinoma arising from CXPA had significantly more HER2 IHC discordance than other histologic types (70% vs 0%, p = 7.80E-06) (Table 2). Tumors with discordant HER2 IHC status had heterogenous HER2 expression (Fig. 3).

Safety and adverse events of CNB

There were no cases of hospitalization or emergency consultation due to hematoma or facial nerve palsy after CNB. The clinicopathologic information of all 41 patients are shown in Fig. 4. In total, there was one case of local recurrence, however, no recurrence due to tumor seeding by needle puncture was observed.

In salivary gland carcinoma, it is critical to accurately assess preoperative biopsy specimens morphologically, immunohistologically, and genomically to determine histologic type. We performed a retrospective analysis of 41 patients with salivary gland carcinoma treated at a single institution with a uniform treatment strategy and diagnostic method. We found the following: 1) detailed analysis of CNB specimens can determine the histologic type with high accuracy; and 2) it is challenging to determine the histologic type of heterogeneous tumors such as CXPA from small CNB specimens.

Most studies of histologic typing with CNB have focused on benign tumors; few studies have evaluated malignant tumors. In general, malignant tumors are more difficult to classify histologically than benign tumors. Walsh et al. reported a 69% histologic typing rate for 29 malignant lesions based on needle biopsy ⁽¹³⁾. Novoa et al. performed CNB on 22 malignant salivary gland tumors and successfully histologically classified 17 (77%) of them ⁽¹⁴⁾. In this study, we established an efficient preoperative histologic typing platform for salivary gland carcinoma based on CNB. This platform consists of six immunohistologic markers: SOX10, EMA/MUC1, c-kit, p63, AR, and HER2. First, SOX10 clearly differentiated between tumors originating from the acini or intercalated ducts and tumors originating from the striated or excretory ducts ⁽⁷⁾. Second, c-kit and p63 allow for the evaluation of luminal and abluminal cells of biphasic glandular structures, respectively. Third, AR and HER2 are available to evaluate the expression of hormone receptors. Fourth, EMA/MUC1 enables evaluation of glandular differentiation, which is useful for validation of SOX10 status, due to the mutually exclusive relationship between EMA/MUC1 and SOX10. In addition, molecular biology evaluations of gene fusions and amplifications can be performed with FISH and DISH. Due to reliable tumor sampling using US, detailed immunohistologic analysis, and incorporation of genetic information from FISH and DISH, we successfully achieved a favorable histologic typing rate of 75%. This result is consistent with previous reports. It is remarkable that immunohistological and genomic evaluations were possible with a 20-gauge needle.

Sample quality and intratumoral heterogeneity affect histologic typing based on CNB. There were two specimens of inadequate quality in this study: a cystic tumor with a predominantly cystic fluid component and a very clinically firm tumor. Histologically, firm tumor had abundant fibrotic tissue with sclerosis. It is difficult to obtain sufficient tumor samples from such cystic or firm tumors.

Another factor that interferes with histologic typing is intratumoral heterogeneity ⁽¹⁵⁾. In this study, CXPA, which is histologically highly heterogenous, had a low concordance rate for HER2 immunostaining and histologic type between CNB and surgical specimens. The impact of intratumor heterogeneity on concordance of biomarker profiles between CNB and surgical specimens has been well examined in the field of breast cancer ^(15–17). Concordance between CNB and surgical specimen for estrogen receptor, progesterone receptor, HER2, and Ki67 immunostaining was reported to be 94.1%, 88.1%, 84.5%, and 88.1%, respectively ^{(16, 17)}. Repeated HER2 testing with immunohistochemical staining or FISH of surgical specimens is considered reasonable, especially when the surgical specimens has tumor characteristics and morphology that differ from those found in CNB specimens ⁽¹⁸⁾. In salivary gland carcinoma, HER2 assessment is also considered appropriate for both CNB and surgical specimens in highly histologically heterogeneous tumors such as CXPA ^(15–17).

There are concerns that CNB poses a higher risk of complications than FNAC because it is more invasive. Hematoma is uncommon with both: 0.03% for cytology and 0.6% for CNB. Facial nerve palsy occurred in 0.075% of patients who underwent CNB, but was reported to be temporary due to local anesthesia ^{(5, 19)}. CNB might pose a greater risk of tumor seeding due to the larger gauge of the needle used^{(20, 21)}. Previous reports showed that tumor seeding occurred in 0.01–0.00012% of FNACs and 0.1–0.001% of CNBs ⁽²¹⁾. An increased risk of tumor displacement has been reported with the use of 18-gauge or larger needles ^{(5, 21)}. In this study, we used a 20-gauge needle to minimize the risk of tumor seeding. We did not see tumor seeding after CNB in our limited number of cases. However, long-term safety for low-grade carcinoma was not fully evaluated. We made every effort to reduce dissemination, including excision of the biopsy scar at the time of surgery or multiple biopsies from the same skin puncture wound.

The main limitation of this study is selection bias due to its retrospective nature. Moreover, due to the relatively rarity of salivary gland carcinoma, a large cohort study was not possible. However, the exclusion criteria and treatment strategy were consistent and pathologic information on biopsy or surgical specimens was well annotated with clinical information. All patients with salivary gland carcinoma treated during the study period at a single institution were examined without exception. Notwithstanding, further research is warranted to verify the findings of this study.

In conclusion, detailed analysis of 20-gauge needle biopsy specimens allows for highly accurate determination of salivary gland carcinoma histologic type. However, careful histologic typing is necessary in pathologically heterogeneous tumors.

Funding

This study was founded by Japan Society for the Promotion of Science (JSPS) KAKENHI grants to KK (22K09662) and TM (23K06476).

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was approved by the ethical review board of the National Cancer Center Hospital (approval number, 2010-77). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Consent to Participate

Informed consent was obtained from all individual participants included in the study.

Consent for publication

Consent for publication was obtained for every individual person’s data included in the study.

Availability of data and materials

The datasets used and/or analyzed during the current study are available for the corresponding author on reasonable request.

Code Availability

Not Applicable.

Authors’ contribution

Conceived and designed the work: K.K. T.M. Provision of study material or patients: K.K., S.Y., G.O., Y.M., A.S., K.E., T.S., Y.H., F.M. Pathological analysis and FISH probe preparation: T.M., E.R., A.Y., Y.Y. Data analysis and interpretation: K.K., T.M, M.K. Statistical analysis: K.K. Manuscript writing: K.K. T.M. Revised the manuscript: T.M., S.Y., G.O., Y.S., M.K. Approved final version: T.M. All authors red and approved the final manuscript.

Acknowledgements

Not applicable.

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No competing interests reported.

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You are reading this latest preprint version

Establishment of an efficient pathologic diagnostic platform using core needle biopsy for salivary gland carcinoma

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Study design and setting

Ultrasound (US)-guided CNB and FNAC

Pathologic diagnosis

Histology and immunohistochemistry (IHC)

HER2 status

FISH

Statistical analysis

Results

Details of preoperative histologic typing by CNB

Concordance of immunostaining in paired CNB and surgical specimens

Safety and adverse events of CNB

Discussion

Conclusion

Declarations

Funding

Conflict of Interest

Ethical approval

Consent to Participate

Consent for publication

Availability of data and materials

Code Availability

Authors’ contribution

Acknowledgements

References

Additional Declarations

Supplementary Files

Status:

Version 1