P63 Expression in Primary Mediastinal Lymphomas

doi:10.21203/rs.3.rs-5036152/v1

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P63 Expression in Primary Mediastinal Lymphomas

https://doi.org/10.21203/rs.3.rs-5036152/v1

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p63 protein is frequently used as an immunohistochemical marker in the pathologic diagnosis of non-hematological solid tumors. In malignant lymphoproliferative disorders, Primary mediastinal large B-cell lymphoma (PMLBCL) and classic Hodgkin lymphoma (CHL) represent two major types of mature B-cell lymphoma of the mediastinum. They have morphological and phenotypic overlap features, making this differential diagnosis challenging.

This study aims to evaluate the diagnostic utility of p63 immunohistochemistry (IHC) in distinguishing these entities.

In this single-center retrospective cohort study, we analyzed p63 expression using a tissue microarray approach in 44 cases of PMLBCL, 15 CHL cases and 7 Grey Zone Lymphoma (GZL). Diagnoses were made by experienced pathologists familiar with the nuances of lymphoma diagnosis, alongside clinical, morphological and immunohistochemical information. P63 expression was correlated with clinicopathological features.

P63 nuclear expression was found in 26/44 (59%) cases of PMLCBL, followed by only 4/7 cases of GZL and 1/15 cases CHL.

For PMLBCL, p63 demonstrated a sensitivity of 59%, specificity of 77%, a positive predictive value (PPV) of 83%, and a negative predictive value (NPV) of 49.7%.

Survival analysis revealed no significant differences in metastasis-free survival based on p63 expression across PMLBCL, CHL, and GZL.

These findings demonstrates that p63 has potential diagnostic value in differentiating PMLBCL from CHL and may be used as a potential tool in diagnosis of thoracic lymphoma. However, It has no prognostic value.

p63

Primary mediastinal large B cell lymphoma

Hodgkin's lymphoma

Immunohistochemistry

Diagnosis

prognosis

Lymphoma represents a major health issue worldwide. It is a common malignancy which incidence is increasing hastily.

Primary mediastinal large B cell lymphoma (PMLBCL) and classic Hodgkin lymphoma (CHL) constitute the most common causes of bulky anterior mediastinal masses in young patients, and the two major lymphoma of mediastinal B-cell origin. While, PMLBCL is an aggressive non-Hodgkin’s lymphoma subtype, well defined in the World Health Organization (WHO) classification of lymphoid (1)and thymic (2) tumors, CHL is characterized by a better prognosis, emphasizing the importance of accurate identification at diagnosis. However, differential diagnosis between these 2 entities can be challenging in daily practice.

PMLBCL and CHL have overlap features in terms of histomorphology, immunohistochemistry profiles and gene expression patterns(3,4) ,which sometimes make the differential diagnosis complicated.

In accordance with these findings, the recent WHO classification described the entity of ‘gray-zone’ lymphoma presenting transitional features between PMLBCL and mediastinal CHL, hence their name Gray Zone Lymphoma (GZL), rendering the precise diagnosis of these mediastinal lymphoproliferations more challenging. Under the circumstances, investigating distinguishing protein expression could help to establish the right diagnosis.

In this study, we aim to investigate the expression status and prognostic significance of P63 in primarly mediastinal lymphoma.

the p63 gene, which maps to chromosome 3q27–28, bears strong homology to the tumor suppressor gene p53. p63 protein exhibits a consistent expression pattern in normal tissues such as urothelium, squamous epithelia, the basal glandular cells of prostate and breast, as well as basal bronchial cells of the lung, thymus, and also in a subset of lymphocytes in the germinal center of morphologically normal lymph nodes(5,6). However, in malignancy, p63 is rarely mutated, and studies of primary human tumors and cell lines suggest that p63 may promote tumor development.

It is found in squamous cell carcinomas of the esophagus, head and neck, and lungs (7). Although numerous studies have investigated the role of p63 in lymphomagenesis, its role remains uncertain, and further studies are needed focusing on the p63 phenotype in hematopoietic tumors(5–9).

2.1 Material

Initially, 69 cases of mediastinal lymphoma of B cell origin were reviewed GZL were retrieved from the pathology archives in the pathology department in Abderrahmen Mami Hospital in Tunisia. Biopsy materials were obtained by transthoracic needle biopsy in all cases. Three cases were removed from the study due to shortfall of samples either by quality or quantity-based standards. Finally, 66 cases were selected categorized as follow: PMLBCL (N = 44), CHL (N = 15), GZL (N = 7).

All cases were located at the mediastinum. Two of the authors reviewed and validated the cases according to WHO criteria prior to inclusion. These cases were selected between the period from 2012 to 2021.

Primary mediastinal large B-cell lymphoma (PMLBCL) is defined as a mature aggressive large B-cell lymphoma of putative thymic B-cell origin arising at the mediastinum, with distinctive clinical, immunophenotypic and genotypic features. Cases that arise outside the mediastinum are probably very uncommon and cannot be confidently recognized without gene expression profiling studies [1,2].

CHL are lymphoid neoplasms composed of large dysplastic mononuclear and multinucleated cells surrounded by a variable mixture of mature non-neoplastic inflammatory cells. Abundant band-like and/or more diffuse collagen fibrosis may be present. The neoplastic cells are often ringed by T cells in a rosette-like pattern [1,2].

GZL is a B cell lymphoma, unclassifiable, with features intermediate between diffuse large B cell lymphoma and classic Hodgkin lymphoma, it presents a unique challenge due to its complex histologic characteristics with variable microscopic appearance across different areas of the tissue sample. In some regions, the lymphoma may resemble classic Hodgkin lymphoma, while in others, it may mimic the features of primary mediastinal large B cell lymphoma. This heterogeneous presentation complicates diagnosis and requires careful evaluation to distinguish between the overlapping features of these distinct lymphoma types.(1,2)

Tissue microarray blocks were constructed for PMLBCL, CHL and GZL. These sections were used for immunohistochemical analysis. These cases were not previously studied in the context of p63 expression analyses.

The characteristics of the patients and the clinical data, including the disease status, laboratory values, treatment response, and clinical outcome and survival status were collected.

The study was approved by the Research and ethic Committee of the University Hospital of Abselrahmen Mami.

2.2 Methods

The primary antibodies used for routine pathologic examination are: CD20, CD3, CD15 and CD30. In some cases, ki67 and CLA were used.

Additionally, immunohistochemistry for P63(clone 4A4) was performed to determine their usefulness in the differential diagnosis. Tissues and cell lines known to express p63 were used as positive controls.

Immunohistochemical staining was carried out using streptoavidin-biotin immunoperoxidase technique (Dako-cytomation, Glostrup, Denmark). Three to five μm thick sections cut from formalin-fixed, paraffin-embedded blocks, mounted on positive charged slides, were deparaffinized in xylene and rehydrated in graded alcohol. The mounted sections were immersed and boiled in ready to use Dako target retrieval solution (PH 6.0) in a microwave for 20 min, and then washed in phosphate buffer saline (PBS) (pH 7.3). Thereafter, blocking of endogenous peroxidase activity with 6% H2O2 in methanol was carried out.

The slides were then incubated overnight using P63. Expression of p63 was considered positive if the percentage of positive cells was equal or higher than 5% with a strong nuclear staining.

2.3 Statistical analysis

To evaluate the relationship between the positivity of immunohistochemistry marker p63 and the diagnosis of Primary Mediastinal Large B-Cell Lymphoma (PMLBCL) Pearson correlation coefficient was calculated to quantify the strength and direction of the linear relationship. The predictive power of p63 was determined using the R squared value, QQ plot was used to determine validity of our linear regression model.

Additionally, for survival analysis, Kaplan-Meier survival curves were plotted to compare metastasis-free survival between p63+ and p63- groups within each lymphoma subtype (PMLBCL, CHL and GZL) assessing the hazard ratios. The statistical significance of differences between survival curves was calculated for the 3 subgroups using the log-rank (Mantel-Cox) test and the Gehan-Breslow-Wilcoxon test.

The results were considered statistically significant if P-value was <0.05. All data were analyzed with Microsoft excel.

3.1 Clinical and pathological findings

Cases were predominantly female (45 Vs 21) and patient’s age ranged from 16 to 77 with a mean age of 35.8 years old.

In the PMLBCL group, there were 14 males and 30 females, and the mean age was 37.9 years (16-77 years). All these cases have mediastinal presentation with or without lymph node involvement in respectively 4 and 16 cases.

The CHL group accounts 15 cases, all of sclero-nodular subtype. Four of these cases were rich in tumor cell, there were 6 males and 9 females, and the mean age was 36.75 years (16-62 years).

Most patients with PMLBCL were treated with rituximab-based chemotherapy such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) (13 of 16, 81.3%) while most CHL patients (12 of 13, 92.3%) were managed by ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine). For GZL, patients were treated as for PMLBCL.

Pathological findings showed significant differences between PMLBCL and CHL. Architecture was mostly diffuse in PMLBCL in 32 cases, nodular in CHL (9 cases), diffuse and nodular in GZL in 4 cases with p<0,001. Necrosis was present in 2 cases of CHL, 14 cases of PMLBCL and 3 cases of GZL. In the three cases of GZL, the fibrosis was present as dense collagenous bands. Most cases of PMLBCL ware characterized by alveolar pattern of the fibrosis (29 cases, 44%) (Figure 1). CHL presented alveolar fibrosis (8 cases) or dense fibrous bands (7cases) (Figure 2). Necrosis and fibrosis were not discriminate parameters to the diagnosis of these lymphoproliferative diseases (respectively, p=0,27, p=0,811).

3.2 Immunohistochemistry study and expression of p63

The large, atypical cells in all cases of PMLBCL were CD20 positive with high percentage of positive cells (70% to 100%) and strong staining intensity.

A subset of 26 PMLBCL cases (59%) showed nuclear positivity with P63 in 30–80% of the neoplastic cells (Figure 1), while 18 (41 %) cases were negative. Only one case of CHL was positive for p63 showing moderate nuclear positivity and heterogenous distribution in 20% of tumor cells (Figure 2).

Four (57.1%) cases of GZL were positive for p63. Expression of p63 was preferentially present in tumor cells of PMLBCL (p < 0.001).

In PMLBCL nuclear expression of p63 demonstrated a 59% sensitivity, 77% specificity, 83% positive predictive value (PPV) and 49.7% negative predictive value (NPV).

The statistical analysis revealed a significant correlation between the immunohistochemical marker p63 and the diagnosis of Primary Mediastinal Large B-Cell Lymphoma (PMLBCL). The Pearson correlation coefficient (r) was 0.3634 (95% CI = 0.1331 - 0.5565), indicating a moderately positive relationship. The R squared value showed that 13.21% of the variance in diagnosis could be explained by p63 expression (p = 0.0027). The QQ plot demonstrated that the residuals followed a normal distribution, confirming that the collected data aligns well with the expected distribution, as illustrated in (Figure 3). These findings highlight the potential utility of p63 as a diagnostic marker for distinguishing PMLBCL from Classical Hodgkin Lymphoma (CHL).

3.3 Survival study

The Kaplan-Meier curve as plotted in figure 3 for general population n=66, illustrating metastasis-free survival over a period of 40 months (Figure 4). The survival rate drops significantly within the first 10 months, then the curve stabilizes, suggesting that patients who survive beyond the first 10 months without metastasis have a more favorable long-term prognosis.

Survival analysis of categorized population by p63 expression (p63+ and p63-) as depicted in (Figure 5) showed that in all three lymphoma subtypes, the log-rank (Mantel-Cox) test did not reveal statistically significant differences in survival between the p63+ and p63- groups. We concluded that p63 expression has no prognostic value.

In this study, we characterized p63 expression in 44 cases of PMLBCL, 15 of CHL, and 7 cases of GZL. P63 expression was positive in 59% of PMLBCL cases, significantly higher than in CHL, where it was only 0.06%. Expression of p63 wasn’t significantly conclusive for GZL, as it was expressed in nearly half cases.

While p63 showed a significant statistical correlation with PMLBCL diagnosis, it did not demonstrate prognostic value in any mediastinal lymphoma subtype.

Mediastinal CHL and PMLBCL are believed to originate from post-germinal center B-cells lacking surface immunoglobulin. This is supported by common genetic alterations, gene expression profiles(4,10,11) and immuno-oncologic signatures such as PD-L1 or PD-L2 expression(12,13). Although these lymphoma share morphological features, distinguishing CHL from PMLBCL can be challenging, particularly in the histologic "grey zone" between them. Fine reticulated or alveolar fibrosis without nodular architecture or annular sclerosis typically suggests PMLBCL, however, these features are rarely observed due to limited biopsy material. On the other hand, annular sclerosis and an eosinophil-rich background favor CHL.

PMLBCL often presents with large tumor cells featuring atypical nuclei resembling Hodgkin/Reed-Sternberg or lacunar cells, creating diagnostic challenges. Immunohistochemistry is essential, with standard markers including CD20, CD3, CD30, and CD15. Each marker has limitations, and the entire panel can sometimes be insufficient for a definitive diagnosis. For instance, CD30 is expressed in some PMLBCL tumor cells, CD20 may be moderately positive in some CHL cases, and CD15 interpretation can be complicated in case of a granulocyte-rich PMLBCL background, and it can also be negative in CHL (4,14).

In our study, we focused on p63 expression to assess its diagnostic utility in mediastinal lymphoma, as this marker is widely available in most laboratories, Commonly used in basal or myoepithelial differentiation or in lung carcinoma diagnosis, but its role in hematolymphoid tumors is less explored.

Previous studies reported p63 positivity in anaplastic large cell lymphoma(14), PMLBCL(15,16) and gray zone lymphoma(13), but most of the case absent in CHL.

Our findings revealed that p63 expression in the large atypical cells of PMLBCL was slightly lower than expression of CD20, with heterogeneous expression patterns (20 to 80% of tumor cells). This likely reflects sampling differences in small biopsies where focal positivity may not be fully represented.

Half of GZL cases expressed p63 however statistical analysis did not support a diagnostic correlation. These findings align with studies by Kim et al.(16) and Eberle et al. (13), reinforcing the ambiguous classification of GZL.

Our study highlights the utility of p63 in distinguishing PMLBCL from CHL in mediastinal biopsies and recommends its use alongside morphology and other IHC markers, especially in small biopsy materials. However, pathologists should be mindful of the marker's heterogeneous expression in limited samples.

In survival analysis using Kaplan-Meier curves, no significant differences were observed in metastasis-free survival between p63-positive and p63-negative groups across the three lymphoma subtypes. Consequently, p63 expression does not hold any prognostic value in any of the three studied mediastinal lymphomas.

This study is one of the first to specifically evaluate p63 expression in thoracic lymphoma, building on earlier suggestions by Kim et al. (16) of p63 and GATA-3's diagnostic utility. With a larger sample size, our study emphasizes the need for further research, recommending a prospective study with larger, more robust tissue samples to better assess p63 expression in PMLBCL.

In conclusion, heterogenous nuclear expression of p63 in tumor cells was observed in a substantial proportion of PMLBCL suggesting its potential utility as a diagnostic immunohistochemical marker for distinguishing CHL from PMLBCL. However, p63 expression didn’t show any prognostic value regarding any mediastinal lymphoma.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Ethical Approval/ Informed consent

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.

Data availability:

The authors declare that any datasets used can be accessed

Alaggio, R., Amador, C., Anagnostopoulos, I. et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms. Leukemia 36, 1720–1748 (2022).
Tsao MS, Nicholson AG, Maleszewski JJ, Marx A, Travis WD. Introduction to 2021 WHO Classification of Thoracic Tumors. J Thorac Oncol. 2022 Jan,17(1):e1-e4. doi: 10.1016/j.jtho.2021.09.017. PMID: 34930611.
Rosenwald A, Wright G, Leroy K, Yu X, Gaulard P, Gascoyne RD, et al. Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma. J Exp Med. 2003 Sep 15,198(6):851–62.
Savage KJ, Monti S, Kutok JL, Cattoretti G, Neuberg D, De Leval L, et al. The molecular signature of mediastinal large B-cell lymphoma differs from that of other diffuse large B-cell lymphomas and shares features with classical Hodgkin lymphoma. Blood. 2003 Dec 1,102(12):3871–9.
Di Como CJ, Urist MJ, Babayan I, Drobnjak M, Hedvat CV, Teruya-Feldstein J, et al. p63 expression profiles in human normal and tumor tissues. Clin Cancer Res. 2002 Feb,8(2):494–501.
Nylander K, Vojtesek B, Nenutil R, Lindgren B, Roos G, Zhanxiang W, et al. Differential expression of p63 isoforms in normal tissues and neoplastic cells. J Pathol. 2002 Dec,198(4):417–27.
Hibi K, Trink B, Patturajan M, Westra WH, Caballero OL, Hill DE, et al. AIS is an oncogene amplified in squamous cell carcinoma. Proc Natl Acad Sci U S A. 2000 May 9,97(10):5462–7.
Yamaguchi H, Inokuchi K, Sakuma Y, Dan K. Mutation of the p51/p63 gene is associated with blastic crisis in chronic myelogenous leukemia. Leukemia. 2001 Nov,15(11):1729–34.
Hedvat CV, Teruya-Feldstein J, Puig P, Capodieci P, Dudas M, Pica N, et al. Expression of p63 in diffuse large B-cell lymphoma. Appl Immunohistochem Mol Morphol. 2005 Sep,13(3):237–42.
Dunleavy K, Grant C, Eberle FC, Pittaluga S, Jaffe ES, Wilson WH. Gray Zone Lymphoma: Better Treated Like Hodgkin Lymphoma or Mediastinal Large B-Cell Lymphoma? Curr Hematol Malig Rep. 2012 Sep,7(3):241–7.
Calaminici M, Piper K, Lee AM, Norton AJ. CD23 expression in mediastinal large B-cell lymphomas. Histopathology. 2004 Dec,45(6):619–24.
Tanaka Y, Maeshima AM, Nomoto J, Makita S, Fukuhara S, Munakata W, et al. Expression pattern of PD-L1 and PD-L2 in classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, and gray zone lymphoma. Eur J Haematol. 2018 May,100(5):511–7.
Eberle FC, Salaverria I, Steidl C, Summers TA Jr, Pittaluga S, Neriah SB, Rodriguez-Canales J, Xi L, Ylaya K, Liewehr D, Dunleavy K, Wilson WH, Hewitt SM, Raffeld M, Gascoyne RD, Siebert R, Jaffe ES. Gray zone lymphoma: chromosomal aberrations with immunophenotypic and clinical correlations. Mod Pathol. 2011 Dec,24(12):1586-97. doi: 10.1038/modpathol.2011.116. Epub 2011 Aug 5.
Gualco G, Weiss LM, Bacchi CE. Expression of p63 in anaplastic large cell lymphoma but not in classical Hodgkin’s lymphoma. Hum Pathol. 2008 Oct,39(10):1505–10.
Hoeller S, Zihler D, Zlobec I, Obermann EC, Pileri SA, Dirnhofer S, et al. BOB.1, CD79a and cyclin E are the most appropriate markers to discriminate classical Hodgkin’s lymphoma from primary mediastinal large B-cell lymphoma. Histopathology. 2010 Jan,56(2):217–28.
Kim HJ, Kim HK, Park G, Min SK, Cha HJ, Lee H, et al. Comparative pathologic analysis of mediastinal B-cell lymphomas: selective expression of p63 but no GATA3 optimally differentiates primary mediastinal large B-cell lymphoma from classic Hodgkin lymphoma. Diagn Pathol. 2019 Dec 12,14(1):133.

No competing interests reported.

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P63 Expression in Primary Mediastinal Lymphomas

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Abstract

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1. Introduction

2. Materials and Methods

3. Results

4. Discussion

5. Conclusion

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