Cytologic Evaluation of p16 Staining in Head and Neck Squamous Cell Carcinoma in CytoLyt® vs Formalin-Fixed Material

Abstract

Background

Management of high-risk human papilloma virus (HR-HPV) related oropharyngeal head & neck squamous cell carcinomas (HNSCC) is distinct from HNSCC linked to smoking/alcohol use. HR-HPV+ HNSCC frequently presents as a cervical lymph node metastasis. As fine needle aspiration (FNA) is often the initial diagnostic procedure, evaluating HR-HPV status in cytology specimens is important. p16 overexpression is a surrogate for HR-HPV, however, evaluation of p16 in FNAs remains controversial.

Methods

From September 2015 to December 2016, cytopathologists performed 25 FNAs of neck lymph nodes suspicious for HR-HPV+ HNSCC. Initial passes produced smears for on-site evaluation and CytoLyt® material. Additional passes were formalin-fixed. A CytoLyt® cell block (CCB) and a formalin-fixed cell block (FFCB) were prepared and p16 immunocytochemistry was performed.

Results

In 24 of 25 cases, the FFCB had diffuse (≥70% of cells) strong nuclear/cytoplasmic p16 staining. In all 24 of these cases, HR-HPV was detected by in-situ hybridization. Corresponding CCB had weak-moderate p16 staining in <70% of the cells (range 5–70%) in 17 cases, 4 had weak-moderate diffuse staining and 4 were acellular. Percentage of p16-positive cells was significantly higher with FFCB than CCB (formalin: 94±2%; CytoLyt®: 38±7%; two-tailed paired Student’s t test p < 0.001 and Fisher’s exact test, p < 0.001).

Conclusion

The fixative used had a drastic impact on p16 staining explaining the staining variability reported in the literature. FFCBs showed a diffuse staining pattern which correlated with HR-HPV status while CCBs show a weaker and inconsistent staining pattern that is more difficult to interpret.

Precis:

In head and neck squamous cell carcinoma HPV status is often evaluated by using p16 staining as a surrogate marker regardless of collection methods and fixation. We found CytoLyt® fixation inhibits p16 immunoreactivity in FNA material while formalin does not.

BACKGROUND

High-risk human papilloma virus (HR-HPV) related oropharyngeal head & neck squamous cell carcinomas (HNSCC) have different demographics and prognosis when compared with non-HR-HPV related HNSCC which is usually linked to smoking and alcohol use. In HR-HPV+ oropharyngeal HNSCC the patients tend to be younger, of higher socioeconomic standing and often with no history of tobacco or alcohol abuse.¹ The primary tumor is commonly located in the oropharynx and the patients often have metastatic disease to the neck lymph nodes at initial presentation. HR-HPV+ oropharyngeal HNSCC are associated with prolonged survival and are more radiosensitive than HR-HPV negative HNSCC due to an intact apoptotic response to radiation.^1,2 As a result, a combination of chemotherapy and radiation is the preferred definitive treatment in lieu of surgical resection in HR-HPV+ oropharyngeal HNSCC. Therefore, the accurate evaluation of the HR-HPV status is critical in the management of these patients. The National Comprehensive Cancer Network (NCCN) and the College of American Pathologists (CAP) recommends p16 and/or HR-HPV testing as part of the work up of all cervical metastatic squamous cell carcinomas with a known oropharyngeal primary not previously tested for HR-HPV, a suspected oropharyngeal primary or with an occult primary.^3,4 p16 is frequently used as the surrogate marker for HR-HPV infection.⁵ Currently, a squamous lesion is considered to be associated with HR-HPV when there is overexpression of the p16 protein, which is defined by greater than 70% strong nuclear and cytoplasmic staining by immunohistochemistry (IHC) in formalin fixed paraffin embedded (FFPE) tissue biopsy specimens.⁶ In fine needle aspiration (FNA) specimens, CAP makes no recommendation against any specific testing methodology for HR-HPV testing. When negative, however, they recommend testing should be performed on tissue if it becomes available.⁴

As HR-HPV+ oropharyngeal HNSCC often presents as a cervical lymph node metastasis from an occult primary, FNA is often the initial diagnostic procedure. Unlike surgical pathologic material, current literature does not support a reproducible universal recommendation for determining what extent of p16 staining correlates with positive HR-HPV status in FNA specimens. Additionally the collection and preparation of cytologic material collected for cell block can drastically vary depending on the laboratory with many laboratories utilizing material collected in alcohol instead of formalin.⁷ Given the different effects alcohol and formalin could potentially have on the p16 protein, we sought to evaluate if the discrepant results observed in the literature are due to different fixation techniques.

METHODS

At Memorial Sloan Kettering Cancer Center (MSK), from September 2015 to December 2016, cytopathologists performed 25 consecutive FNAs of cervical neck lymph nodes on patients who either had a previous diagnosis of oropharyngeal HNSCC with unknown p16 status or were determined to have HNSCC at the time of the procedure by the performing cytopathologist. The 25 patients were comprised of 22 males and 3 females with an average age of 60 years old (range 47 – 76). (Table 1) The FNA was typically performed under ultrasound guidance using a 1 inch, 25-gauge needle attached to a 10cc syringe. The initial passes were performed for Diff-Quik stained and ethanol fixed smears while the needles were rinsed in CytoLyt®. The performing cytopathologist would perform rapid on-site evaluation (ROSE) of the Diff-Quik stained smears to determine adequacy and obtain a preliminary result. Once a diagnosis of metastatic HNSCC was rendered, and an area of the lymph node with sufficient viability was identified, the cytopathologist would perform one to two more passes targeting this area. These passes were then allowed to clot before being transferred into formalin for fixation. From the collected material, a CytoLyt® cell block (CCB) and a formalin-fixed cell block (FFCB) were made as recently described.⁸

Table1.

Patient Demographics

Patient #	Age at Dx	Gender	Smoking Hx (pack years)	Alcohol use
1	57	M	Never	Social
2	51	M	2.5	Social
3	58	F	36	9 yrs abstinent
4	69	M	7	Social
5	47	M	0.5	Heavy
6	62	M	Never	Social
7	57	M	Never	Social
8	65	M	Never	Never
9	65	M	Never	Social
10	49	M	Never	Social
11	57	M	~30 cigars/yr	Social
12	67	M	12	Heavy
13	72	M	Never	Social
14	58	M	40	Social
15	76	M	10	Social
16	53	F	Never	Occasional
17	53	M	Never	Social
18	61	M	Never	Social
19	66	F	15	Daily
20	73	M	Never	Daily
21	55	M	15	Social
22	65	M	10	Social
23	62	M	Never	Daily
24	49	M	Never	Heavy
25	62	M	Never	Occasional

Abbreviations: Dx, diagnosis; Hx, history

Both the CCB and FFCB were cut prospectively and heat-induced epitope retrieval (HIER) was used to prepare the unstained slides. Immunocytochemistry (ICC) staining was then performed on each using a mouse anti-human monoclonal p16 antibody (Clone E6H4; antibody concentrate 1 ug/ml; Ventana Medical Systems Inc, Tucson, AZ). This process was followed regardless of whether tumor had basaloid morphology typical of HR-HPV+ oropharyngeal HNSCC or was overtly keratinizing. Once this was complete, each specimen was evaluated for percentage of the tumor cells staining as well as the strength of both cytoplasmic and nuclear staining. Percentage of positive tumor staining and staining intensity was carefully estimated as is routinely done in our clinical practice by the same cytopathologist to ensure consistency.

Chart review of all patients was performed retrospectively to determine the patient’s smoking and alcohol use, how they were treated and clinical outcomes. Direct evaluation of HR-HPV using in situ hybridization (Clone 16, 18, 26, 31, 33, 35, 39, 41, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82, E6/E7 mRNA; RTU; Advanced Cell Diagnostics, Newark, California) targeting the transcripts of E6 and E7 mRNA was performed in all cases retrospectively.

Statistical analyses were performed using the SPSS software 22.0 (IBM Corporation, New York, NY, U.S.). For each fixation condition, the percentage of positive tumor cells was compared using two-tailed paired Student’s t test. Also, p16 immunopositivity was determined using the same criteria applied to surgical specimen, being ≥70% of tumor cells showing nuclear and cytoplasmic staining as defined by the CAP guideline ⁴. The rate of p16 immunopositivity was computed and compared using Fisher’s exact test. P values less than 0.05 were considered to be statistically significant.

RESULTS

The FFCBs displayed a consistent staining pattern for p16 by ICC, with 24 of the 25 specimens being diffusely positive (moderate to strong cytoplasmic and nuclear staining in >70% of cells). (Figure 1) Retrospectively, we performed HR-HPV in situ hybridization on all 24 cases that showed diffuse p16 staining. In all cases high risk HR-HPV was detected. (Figure 2) HR-HPV in situ hybridization performed on the one case that showed only focal p16 staining was non-contributory due to insufficient tumor remaining on deeper levels. The CCB showed a more variable staining pattern, but in all cases p16 stained a lower percentage of cells and with less intensity than the formalin-fixed counterpart. (Figure 3) Only 4 cases showed weak to moderate p16 staining in ≥70% of cells while 6 cases showed weak to moderate staining in 10% or less of tumor cells. 11 cases stained between 10 and 69% of the cells and in four cases the CCB was inadequate for evaluation due to scant cellularity. (Table 2) (Figure 4) We attempted to stain 2 of the CCB cases with the p16 antibody excluding HIER, with no recovery of antigenicity. Only 11 cases had surgical material available for p16 staining, all showed concordant results with the formalin fixed material.

Figure 1:

A) H&E of squamous cell carcinoma. B) Diffuse p16 staining on FFCB

Figure 2:

A) H&E of squamous cell carcinoma. B) Diffuse p16 staining on FFCB. C) Positive for HR-HPV by in situ hybridization

Figure 3:

Three representative cases of squamous cell carcinoma (A,D,G) with side by side comparison of p16 staining on the CCB (B,E,H) and the FFCB (C,F,I) respectively

Table 2:

Evaluation of p16 staining and HPV

	% p16 Staining in		High risk HPV
Patient	CCB	FFCB	By mRNAish
1	5 w	95 s	+
2	50 w	99 s	+
3	NC	5 m	NC
4	100 w	100 s	+
5	25 m	75 s	+
6	NC	95 s	+
7	25 m	90 m-s	+
8	5 w	90 m-s	+
9	10 w	95 s	+
10	30 m	70 m	+
11	10 w	90 s	+
12	50 w-m	99 s	+
13	5 m	95 m-s	+
14	50 m	99 s	+
15	NC	90 s	+
16	5 w	90 m-s	+
17	NC	99 s	+
18	30 w	95 s	+
19	70 s	95 s	+
20	40 w-s	100 m-s	+
21	90 m	100 s	+
22	90 m	100 s	+
23	15 w-m	99 s	+
24	25 w-m	100 s	+
25	60 m	99 s	+

Abbreviations: CCB, CytoLyt® cell block; FFB, formalin-fixed cell block; mRNAish, mRNA in situ hybridization; s, strong; m, medium; w, weak; NC, non-contributory

Figure 4:

Comparison of p16 staining in tumor cells in CytoLyt® and Formalin

The percentage of p16-positive tumor cells was significantly higher in specimens with FFCB compared with those CCB (Mean±standard errors of mean, formalin: 94±2%; CytoLyt®: 38±7%; two-tailed paired Student’s t test, p < 0.001).

Using a cutoff value of ≥70% of tumor cells with p16 staining to determine p16 immunopositivity as suggested by the CAP guideline ⁴, the rate of p16 positivity was significantly different between CytoLyt®-fixed samples (positive rate: 19%, 4 of 21 samples) and formalin-fixed samples (positive rate: 100%, 24 of 24 samples; Fisher’s exact test, p < 0.001).

Seventeen of the 25 patients were treated with a combination of chemotherapy and radiation, 4 were treated with surgical resection of disease followed by chemotherapy and radiation, 1 with surgical resection and post-op radiation only, 1 by surgical resection alone and 2 were not treated at our hospital. Of the 23 patients who received treatment at MSK, 2 developed lung metastasis 9 months after completing initial treatment and one locally recurred; with 2 having died of disease, 20 have no evidence of disease (Range: 25 – 38 months from completion of treatment). Of the 17 patients treated with only chemotherapy and radiation, 16 are NED after completing treatment and 1 patient recurred with lung metastasis after 9 months. (Table 3) The overall response to therapy, especially the response to chemotherapy and radiation, is consistent with HR-HPV+ HNSCC.

Table 3:

Treatment and Clinical Outcomes of Patients

Patient	Initial Treatment	Status	Disease free Interval(Months)
1	Surgical resection + Cisplatin + Rx (30 Gy)	NED	41
2	Cisplatin + Rx (60 Gy)	NED	38
3	Cisplatin + Rx	NED	38
4	Cisplatin + Rx (70 Gy)	NED	36
5	Cisplatin + Rx (70 Gy)	Recurred with lung metastasis after 9 months
6	Cisplatin + Rx (60 Gy)	NED	35
7	Surgical resection + Cisplatin + Rx (30 Gy)	Recurred locally, DOD
8	Cetuximab + Rx(70 Gy)	NED	34
9	Surgical resection + Cisplatin + Rx	Recurred with lung metastasis after 9 months, DOD
10	Cisplatin + Rx (60 Gy)	NED	29
11	Surgical resection + Cisplatin + Rx (30 Gy)	NED	29
12	Cisplatin + Rx (70 Gy)	NED	29
13	Cisplatin + Rx (70 Gy)	NED	27
14	Cisplatin + Rx (70 Gy)	NED	28
15	none	DOD
16	Cisplatin + Rx (70 Gy)	NED	28
17	Surgical resection + post op RT	NED	26
18	Cisplatin + Rx (70 Gy)	NED	27
19	Cisplatin switched to CarboTaxol + Rx (70 Gy)	NED	26
20	Cisplatin + Rx (70 Gy)	NED	26
21	Cisplatin + Rx	NED	26
22	Cisplatin + Rx	NED	25
23	Sur Res	NED	27
24	Cisplatin + Rx (70 Gy)	NED	25
25	none	Lost to follow up

Abbreviations: DOD, dead of disease; Gy, gray; NED, no evidence of disease; Rx, radiation

DISCUSSION

Patients with HR-HPV associated oropharyngeal HNSCC often present with only palpable cervical lymphadenopathy with no history of malignancy. In patients with this presentation the initial clinical differential diagnoses include reactive lymph node, lymphoma, metastatic papillary carcinoma, a branchial cleft cyst, and metastatic HNSCC among others. In this setting, FNA is often performed as it has the ability to determine the cause of the lymphadenopathy. In the setting of metastatic HNSCC, ROSE is important as it provides an opportunity to make the diagnosis at the time of the procedure and properly triage material for ancillary testing such as p16 IHC. Ultrasound guided fine needle aspiration (US-FNA) has many advantages in this setting even though the lymph nodes are palpable. Frequently, these lymph nodes are cystic or necrotic and US-FNA enables the performing cytologist to target areas that appear solid or viable. Most importantly, once an area of sufficiently viable tumor is identified, US-FNA allows for accurate targeting of that area to ensure designated passes for cell block contain viable tumor. Additionally, it is also less invasive than core needle and excisional biopsies and is associated with fewer complications. Establishing the diagnosis of HR-HPV+ HNSCC by US-FNA allows proper clinical management of the patient even when the primary site cannot be identified. In our study, 20% (5 of 25) of the patients underwent surgical exploration of the base of tongue and/or tonsils in attempt to locate the primary site, but no carcinoma was detected in any of the resections or biopsies. Given that the primary tumor is located within the oropharynx in the majority of HR-HPV+ HNSCC, the oral cavity and oropharynx are covered by the radiation field in these cases.³

Though establishing HR-HPV status in HNSCC is critical in patient management, the use of FNA material to make this assessment is not fully accepted. Various papers have reported on the role of p16 immunocytochemistry (ICC) in cytology specimens with conflicting results.^1,2,9–15 Different studies have used different percentages of p16 immunoreactivity to correlate with HR-HPV status, while others have suggested that cytologic material cannot reliably be used. For instance, Xu et al. and Jalaly et al. reported weaker staining for p16 on cytology specimens and observed that staining as little as 10% or 15% of tumor cells, respectively, had a high concordance with HR-HPV related disease.^2,11 Begum et al. reported strong p16 staining on viable tumor cells while observing diminished or absent staining in degraded tumor cells. Grimes et al. observed strong and diffuse staining for p16 in cases with HR-HPV related disease.^9,12 Upon review of these studies, it was noted that the effect of processing on the immunoreactivity, including type of fixative used, had not been standardized. Although immunostains are generally validated using formalin-fixed paraffin embedded tissue, several cytology laboratories use material previously fixed in an alcohol-based medium prior to cell block processing.⁷ The type of fixative can cause decrease antigenicity of certain antibodies, including other nuclear markers such as MIB1 and ER as well as cytoplasmic stains such as S100.^16–18 This decrease in antigenicity might be attributed to the differences in the effect of each fixative on the cells obtained. Formalin fixes specimens through the formation of intra and inter-molecular cross-links, while alcohol removes water from the free carboxyl, hydroxyl, amino, amido and imino groups of the proteins resulting in protein coagulation and tissue shrinkage.¹⁹ When comparing the studies where the fixation material was specified Xu et al. and Jalaly et al. observed low/weak p16 expression in HR-HPV+ HNSCC in alcohol fixed material while Begum et al. reported diffuse staining in formalin-fixed material. These findings support the results in this current study.^2,9,11

CONCLUSION

CytoLyt® has been shown to decrease the degree of immunostaining of certain antibodies, but as far as we are aware, its effect on the p16 immunostain has not been reported.¹³ The reason that suboptimal immunoreactivity with this antibody is particularly important is that, unlike most diagnostic antibodies in pathology, which are not quantitative, the interpretation of p16 depends on quantitative cut-offs. In this study, we found the fixative used had a drastic impact on p16 staining and we believe this explains the staining variability reported in the literature. p16 expression in cytologic material collected in formalin showed a diffuse staining pattern which correlated with HR-HPV status while CCBs show a weaker and inconsistent staining pattern that is more difficult to interpret.