benign breast tissue in patients with triple-negative high-grade breast carcinomas: breast p53 signature?

    P53 Alteration in Morphologically Normal/Benign Breast Tissue in Patients With Triple Negative High-grade Breast Carcinomas: Breast p53 Signature? Xi Wang MD, Moritz Stolla MD, Brian Z. Ring PhD, Qi Yang BS, Todd S. Laughlin BS, Paul G. Rothberg PhD, Kristin Skinner MD, David G.Hicks MD PII: DOI: Reference:

S0046-8177(16)30092-2 doi: 10.1016/j.humpath.2016.05.011 YHUPA 3909

To appear in:

Human Pathology

Received date: Revised date: Accepted date:

22 March 2016 3 May 2016 12 May 2016

Please cite this article as: Wang Xi, Stolla Moritz, Ring Brian Z., Yang Qi, Laughlin Todd S., Rothberg Paul G., Skinner Kristin, G.Hicks David, P53 Alteration in Morphologically Normal/Benign Breast Tissue in Patients With Triple Negative Highgrade Breast Carcinomas: Breast p53 Signature?, Human Pathology (2016), doi: 10.1016/j.humpath.2016.05.011

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ACCEPTED MANUSCRIPT P53 Alteration in Morphologically Normal/Benign Breast Tissue in Patients With Triple Negative Highgrade Breast Carcinomas: Breast p53 Signature?

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Wang, Xi MD1; Stolla, Moritz MD1; Ring, Brian Z. PhD2; Yang, Qi BS1; Laughlin, Todd S. BS1; Rothberg, Paul G. PhD1; Skinner, Kristin MD3; Hicks, David G. MD1

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1. Dept. of Pathology; 3. Dept. of Surgery, University of Rochester Medical Center, 601 Elmwood Ave. Rochester. NY 14642, U.S.A.

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2. College of Life Science, Institute of Personalized and Genomic Medicine, Huazhong University of Science and Technology, Wuhan, China

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Corresponding Author: Xi Wang, Department of Pathology and Laboratory Medicine, University of Rochester, 601 Elmwood Ave., Rochester, NY 14642. Phone: 585-273-1802; Fax 585-275-3637; email:

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[email protected]

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Funding: No outside funding

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Conflict of interest: The authors declare that they have no conflict of interest.

Running head: p53 in Normal/Benign Breast Tissue: Breast p53 Signature?

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Key wards: p53, benign breast tissue, triple negative high-grade breast carcinoma

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ACCEPTED MANUSCRIPT Abstract: P53 alterations have been identified in approximately 23% of breast carcinomas, particularly in hormone receptors negative high-grade carcinomas. It is considered to be an early event in breast carcinogenesis.

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Nevertheless, the putative precursor lesion of high-grade breast carcinoma remains elusive. Breast

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excision specimens from 93 triple negative high-grade invasive ductal carcinomas, 48 ER+/PR+/Her2non-high-grade invasive ductal carcinomas, and 50 mammoplasty breasts were selected. At least 2 tissue

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blocks with tumor and adjacent benign tissue were sectioned and subjected to immunohistochemistry (IHC) staining for p53. TP53 gene sequencing was performed on select tumors. Further IHC staining for ER and Ki67 was performed on consecutive sections of tissue with p53-positive normal/benign cells.

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Fifty-one of the 93 (55%) high-grade carcinomas were positive for p53 alteration, while only 3 of the 48 (6.25%) non-high-grade carcinomas were p53 altered. Focal p53 positivity in adjacent normal/benign

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breast tissue was identified in 19 cases, with 18 of them also had p53 alteration in their carcinomas. Only one case had focal p53 staining in normal/benign tissue but the tumor was negative for p53 alteration. No p53 staining positivity was identified in the mammoplasty specimens. The p53-stained normal/benign

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cells were ER negative and did not show an increase in the Ki67 labeling index. These findings indicate that the p53 staining positivity in normal/benign breast tissue is not a random event. It could be

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considered as the “p53 signature” in breast and serve as an indicator for future potential risk of p53-

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positive high-grade breast carcinoma.

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ACCEPTED MANUSCRIPT Introduction Breast cancer is a heterogeneous group of diseases. Molecular profiling has sub-classified breast cancer

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into 5 different types: luminal A, luminal B, Her2 expression, triple-negative non-basal, and triple-

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negative basal-like [1, 2]. This inter-tumor heterogeneity, which has clinical and therapeutic implications, is believed to reflect the „„cells of origin‟‟ of cancer, as well as their genetic mutational profiles. The

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terminal ductal lobular unit (TDLU) is the primary site where breast carcinogenesis initiates. A linear multistep progression process to low grade invasive carcinoma, starting from flat epithelial atypia (FEA),

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through atypical ductal hyperplasia (ADH) and low-grade ductal carcinoma in situ (DCIS) is supported by

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their geographic association, similar immunophenotype, and shared genetic alterations[3]. The gene expression profile indicates that the relatively differentiated luminal cells in the differentiation hierarchy of TDLU might be the “cell of origin” of this group of lesions [4], the so-called “family of low-nuclear-

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grade breast neoplasia” [3]; however, the carcinogenesis of high-grade breast carcinoma is believed to be

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a separate “parallel” process, arising from less-differentiated luminal progenitor/stem cells. However, the

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breast neoplasia”.

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morphological precursor lesion of high-grade carcinomas remains elusive compared with “low-grade

The Cancer Genome Atlas Network has systematically studied the common features between ovarian serous carcinoma and basal-like high-grade breast carcinoma and has found many molecular commonalities between them, including p53, RB1 and BRCA1 loss and MYC amplification, which strongly suggests shared molecular-driving events in the carcinogenesis of ovarian serous carcinoma and high-grade basal-like breast carcinoma [5]. Currently, it has been hypothesized that tumor cells seeding from the serous tubal intraepithelial carcinoma (STIC) may serve as the origin of high-grade ovarian serous carcinoma. Identical TP53 mutations have been reported in STICs, as well as in histologically normal tubal epithelium; this phenomenon has been termed the “p53 signature” and is considered the precursor of high-grade ovarian serous carcinoma. 3

ACCEPTED MANUSCRIPT P53 is the second most common molecule (following PI3K) altered in breast carcinomas, with a mutation rate of approximately 23%. P53 is mostly attributable to high-grade breast carcinomas, particularly in

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medullary carcinomas, while its alteration is uncommon in low-grade breast carcinomas [6]. P53

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alteration is an early event in breast carcinogenesis, having been identified as early as in morphologically benign-appearing breast tissue [7-11]. Studies have also shown that p53 alteration in core biopsies of

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benign/normal breast tissue is associated with an increased risk of progression to breast carcinoma [12,

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13].

We studied p53 alteration in normal/benign breast tissue in patients with triple negative high-grade and

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ER+/PR+/Her2- non-high-grade breast cancers, as well as in mammoplasty specimens using immunohistochemical staining. Next, we correlated p53 staining with cellular morphology, ER and Ki67

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Materials and Methods:

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a “p53 signature” in breast tissue.

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status to identify the earliest stage at which p53 alteration initiates to determine whether we could identify

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Case selection: The study was approved by the RSRB at the University of Rochester. The pathology files from 2005 to 2012 were reviewed, and 93 cases with high-grade (modified Bloom-Richardson grade 3) invasive ductal carcinoma, 48 cases with non-high-grade invasive ductal carcinoma (modified BloomRichardson grade 1-2), and 50 cases with benign breast from mammoplasty were selected. The high-grade carcinomas were all ER, PR and Her-2 triple negative (54 basal-like subtype, 2 non-basal subtype, 37 without EGFR/CK5 information). The non-high-grade carcinomas were all ER and/or PR positive, and Her2 negative (35 cases with Ki67 labeling index less than 15% - Luminal A subtype, 13 cases Ki67 equal or more than 20% - luminal B subtype). At least two tissue blocks with tumor and/or normal/benign tissue were selected from each case. Tumor-infiltrating lymphocytes (TILs) were evaluated by referencing the guideline by the International TILs Working Group 2014 [14]. 4

ACCEPTED MANUSCRIPT Immunohistochemistry (IHC): Formalin-fixed paraffin-embedded tissue blocks were sectioned consecutively using a 4-µm thickness. Antigen retrieval was conducted using DAKO EnVisionTM FLEX

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Target Retrieval Solution with a high pH (9.0) for p53 and ER and a low pH for Ki67 (6.0).

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Immunohistochemical staining for p53 (DAKO; clone DO-7, dilution 1:100) was applied to all of the tissue blocks selected. Further ER (DAKO, Kit SK310) and Ki67 (DAKO) staining was applied to the

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consecutive sections of the normal/benign breast tissue that were positive for p53. Staining for p53 in tumor cells was read as positive when >50% of cells showed strong nuclear staining [15]. In

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benign/normal TDLUs, more than ten consecutive luminal cells with strong nuclear staining were read as

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p53 positive, referencing the criteria of the “p53 signature” in the fallopian tube, while scattered singlecell nuclear staining was read as negative[16].

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TP53 gene sequencing: Twelve tumors, including 5 with inconclusive p53 IHC staining and 5 with a discordant staining result (p53 focally positive in normal/benign breast tissues but negative in the tumor),

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were analyzed using Sanger sequencing to detect mutations in exons 4-10 and the adjacent intronic regions of the TP53 gene. Sections were cut from the paraffin blocks of each sample, and genomic DNA

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was purified using the QIAamp system (Qiagen Inc., Valencia, CA) according to the manufacturer‟s

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protocol. Polymerase chain reaction (PCR) was performed in a total volume of 12 L with primers at a final concentration of 1 M each, 50 M of each dNTP, 1 unit of HotStar Taq DNA polymerase, 1.2 L of the 10X buffer provided by the enzyme manufacturer (Qiagen, Inc.) and approximately 50 ng of template DNA. The PCR primers were synthesized with M13 tail sequences appended to the 5‟-end to facilitate sequencing. The gene-specific parts of the primers were located in the adjoining introns and are described in table 1. First, the reactions were kept for 15 minutes at 95˚C, and then they were cycled 35 times at 95˚C for 10 seconds, 63˚C for 15 seconds and 72˚C for 60 seconds, followed by 5 minutes at 72˚C. The primers were purchased from Integrated DNA Technologies (Coralville, IA). The amplicons were treated with ExoSap (Amersham Biosciences, Piscataway, NJ) to remove the primers and dNTPs, and then they were sequenced using the M13 tails as sequencing primers and Applied Biosystems (ABI, 5

ACCEPTED MANUSCRIPT Foster City, CA) BigDye Terminator v.3.1 chemistry. The sequencing reactions were purified using the CleanSeq system (Agencourt Bioscience, Beverly, MA) and then resolved by capillary electrophoresis on

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the ABI 3500XL Genetic Analyzer. Mutations were confirmed by repeat analysis starting with the PCR

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step.

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Statistics: Fisher‟s exact test was used to evaluate the association between TP53 and TIL with grade, and

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logistic regression was used to assess the multivariable associations. All p values were two-sided, and a p

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value less than 0.05 was considered to indicate statistical significance.

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Results:

Of the 93 high-grade carcinomas, 46 were positive for p53 expression by IHC staining (Fig. 1a-1d), and 5

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were negative for p53 expression but had chain-terminating mutations in the TP53 gene (3 nonsense point-mutations, one splice site mutation and one deletion causing a frameshift). The overall p53

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alteration rate was 55%. Of the 48 non-high-grade carcinomas, 1 had p53-IHC positive staining, and 2

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were IHC inconclusive but were verified to be positive for TP53 alteration by sequencing with an alteration rate of 6.25% (Table 2, p value <0.0001).

DCIS was identified in 38 of the 93 (41%) cases with high-grade invasive carcinomas and in 34 of the 48 (71%) cases with non-high-grade invasive carcinomas. The p53 status of the DCIS in all cases matched with their invasive counterpart.

Eighteen of the 93 (19.35%) cases with high-grade carcinomas showed focal p53 positivity in the adjacent benign-appearing breast tissue (Fig. 2a, 2b). Twelve of the 18 cases had tumors with p53-positive IHC 6

ACCEPTED MANUSCRIPT staining, and 5 cases had tumors for which p53 IHC staining was negative but sequencing on the tumor proved to be TP53 chain-terminating mutations. Only one case had a tumor negative for TP53 alteration

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by IHC and sequencing, but adjacent benign/normal tissue showed focal p53 positivity by IHC. In the 48

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cases of non-high-grade carcinomas, only one case had p53-positive staining in adjacent benign/normal breast tissue, while its tumor counterpart was also positive for p53 immunostaining. P53 staining positive

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benign breast tissue was identified more frequently in specimens with high grade carcinomas, compared to the specimens with non-high grade carcinomas (Table 3, p=0.007), and was associated with carcinomas

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that were p53 altered, compared to the carcinomas that were p53 alteration negative (Table 4, p<0.001).

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No p53-positive staining was identified in the 50 mammoplasty specimens.

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Immunohistochemical staining for ER and Ki67 on the consecutive sections of 18 cases with high-grade carcinoma and focal p53 positivity in normal/benign breast tissue showed that those p53-positive -

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normal/benign cells were ER negative (Fig. 2c) and had a low Ki67 labeling index (Fig. 2d), while their

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tumor counterpart was ER negative but with a high Ki67 labeling index up to 90% (1c & 1d).

Twenty-five of the 93 (27%) high-grade tumors had significantly increased lymphocytes in the tumor stroma compared with 1 of 48 (2%) non-high-grade tumors (Table 5, p value=0.002). Seventeen of the 25 (68%) high-grade tumors with TILs had p53 alteration, while 8 (32%) were p53 alteration negative. P53 alteration and tumor grade are independent predictors of TILs via multivariable logistic regression with p values of 0.022 and 0.024, respectively. Nevertheless, in the 19 cases (including high-grade and non-highgrade cases) with focal p53 IHC positivity in benign/normal tissue, only 6 (32%) were associated with mildly increased lymphocyte infiltration.

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ACCEPTED MANUSCRIPT The morphology of these p53-positive normal/benign cells appeared to have slightly enlarged nuclei, with a mildly increased nuclear/cytoplasm ratio and a relatively prominent nucleoli compared with the adjacent

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p53-negative normal/benign luminal cells. However, they are morphologically within the limits of benign

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glands (Fig. 1a).

Discussion:

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Consistent with the previously reported literature, we found a much higher p53 alteration frequency in the high-grade breast carcinomas compared with non-high-grade carcinomas. The associated DCIS showed a

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similar p53-staining phenotype as their invasive carcinoma counterpart in these cases, indicating p53 alteration had occurred prior to the development of invasion in the process of carcinogenesis. Indeed, p53

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alteration is an early event in breast carcinogenesis, having been identified in normal/benign breast tissue by many studies [7-10]. Rohan et al. analyzed p53 status in normal/benign breast tissue by

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immunohistochemistry in patients who subsequently developed breast cancer and found that it was

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associated with an increased risk of progression to breast cancer [12]. They further demonstrated this association by combining TP53 sequencing of normal/benign breast tissue [11, 13]. The limitations of

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these studies are as follows. First, the populations studied were randomly selected without emphasizing the association of p53 alteration with high-grade breast carcinomas. Second, the tissue evaluated was very limited, mostly on core biopsies, precluding finding focal p53 alterations that will usually be the case. Third, there were no clear criteria for p53 positivity in normal/benign breast tissue; any nuclear staining could be considered positive. To overcome these limitations, our study focused on the population with high-grade ER/PR/Her2-negative breast carcinomas. We evaluated more than two whole-tissue sections containing normal/benign tissue in excisional specimens (lumpectomy/mastectomy) and referenced the criteria of the “p53 signature” that had previously been defined in the fallopian tube. We found that patients with high-grade breast carcinomas had a significantly higher frequency of focal p53 positivity in their normal/benign tissue than patients with non-high-grade breast cancer or patients without a history of 8

ACCEPTED MANUSCRIPT breast cancer, and the p53 positive normal/benign tissues were almost exclusively among the cases with p53 altered carcinomas. Interestingly, these p53-positive normal/benign cells were also ER negative,

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which was similar to their high-grade tumor counterpart. These results indicate that the cells harboring

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p53 alterations are very likely the committed precursor cells for p53-positive triple negative high-grade

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carcinomas.

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Nevertheless, these p53-positive cells in normal/benign breast tissue are not yet carcinoma cells. Immunohistochemical staining for Ki67 on consecutive sections showed that these cells remain inactive

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with a low Ki67 labeling index, while Ki67 expression is present in up to 90% in their tumor cell counterpart, which suggests that further genetic/epigenetic changes are needed to trigger unlimited tumor

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growth for complete malignant transformation. There were 5 cases that showed focal p53 positivity (indicating missense mutation) in their normal/benign cells with their tumor counterpart being p53

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negative by IHC but positive for chain-terminating mutations detected by DNA sequencing. These cases

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transformation.

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indicate that further alteration of the TP53 gene might have occurred during the latter stage of malignant

Tumor-infiltrating lymphocytes (TILs) are a common phenomenon in breast carcinomas and are reportedly associated with clinical prognosis [17]. Consistent with what has been reported in the literature, we found that high-grade invasive carcinomas were more often associated with TILs than with non-high-grade tumors. Our study further indicated that the increased TILs were associated with the p53 status in high-grade tumors. However, p53 IHC positivity in normal/benign tissue is not associated with increased infiltration by lymphocytes, indicating that TILs might be a phenomenon associated with a fullblown malignant process.

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ACCEPTED MANUSCRIPT It will be optimal if high-sensitivity TP53 sequencing could be conducted on IHC-positive normal/benign luminal cells to prove the presence of TP53 missense mutations. However, this remains technically

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difficult at this moment due to the limited numbers of cells of interest. Published studies on sequencing

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TP53 in normal/benign tissue mostly extracted DNA non-specifically without a correlation with cell morphology and IHC staining. Even in the studies by Rohan et al., the epithelium in the region of interest

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was microdissected but not specifically in p53-positive cells. Nevertheless, it is well accepted that the nuclear accumulation of p53 by IHC is highly correlated with a missense mutation [18, 19] and that this

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accumulation could be an independent parameter in terms of evaluating p53 status in cells.

Conclusion:

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P53 could be altered in benign-appearing breast glands in patients with high-grade breast carcinomas. The special morphology and p53 positivity of these benign-appearing cells could be considered as the “p53

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signature” of breast tissue and serve as an indicator for future risk of p53-positive high-grade breast

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carcinoma, similar as p53 signature in fallopian tube for the risk of ovarian serous papillary carcinoma. Further studies are needed to help better define the early stages of the development of these aggressive

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breast carcinomas.

References: [1] Kapp AV, Jeffrey SS, Langerod A, Borresen-Dale AL, Han W, Noh DY, et al. Discovery and validation of breast cancer subtypes. BMC genomics 2006, 7:231. [2] Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proceedings of the National Academy of Sciences of the United States of America 2003, 100:8418-23. [3] Abdel-Fatah TMA, Powe DG, Hodi Z, Reis-Filho JS, Lee AHS, Ellis IO. Morphologic and molecular evolutionary pathways of low nuclear grade invasive breast cancers and their putative precursor lesions: further evidence to support the concept of low nuclear grade breast neoplasia family. Am J Surg Pathol 2008, 32:513-23. [4] Visvader JE, Stingl J. Mammary stem cells and the differentiation hierarchy: current status and perspectives. Genes & development 2014, 28:1143-58. 10

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[5] Cancer Genome Atlas N. Comprehensive molecular portraits of human breast tumours. Nature 2012, 490:61-70. [6] Borresen-Dale AL. TP53 and breast cancer. Human mutation 2003, 21:292-300. [7] Younes M, Lebovitz RM, Bommer KE, Cagle PT, Morton D, Khan S, et al. p53 accumulation in benign breast biopsy specimens. Human pathology 1995, 26:155-8. [8] Schmitt FC, Leal C, Lopes C. p53 protein expression and nuclear DNA content in breast intraductal proliferations. The Journal of pathology 1995, 176:233-41. [9] Millikan R, Hulka B, Thor A, Zhang Y, Edgerton S, Zhang X. p53 mutations in benign breast tissue. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 1995, 13:2293300. [10] Lisboa BW, Vogtlander S, Gilster T, Riethdorf L, Milde-Langosch K, Loning T. Molecular and immunohistochemical analysis of p53 mutations in scrapings and tissue from preinvasive and invasive breast cancer. Virchows Archiv : an international journal of pathology 1997, 431:375-81. [11] Kandel R, Li SQ, Ozcelik H, Rohan T. p53 protein accumulation and mutations in normal and benign breast tissue. International journal of cancer Journal international du cancer 2000, 87:73-8. [12] Rohan TE, Hartwick W, Miller AB, Kandel RA. Immunohistochemical detection of c-erbB-2 and p53 in benign breast disease and breast cancer risk. Journal of the National Cancer Institute 1998, 90:1262-9. [13] Kabat GC, Kandel RA, Glass AG, Jones JG, Olson N, Duggan C, et al. A Cohort Study of p53 Mutations and Protein Accumulation in Benign Breast Tissue and Subsequent Breast Cancer Risk. Journal of oncology 2011, 2011:970804. [14] Salgado R, Denkert C, Demaria S, Sirtaine N, Klauschen F, Pruneri G, et al. The evaluation of tumorinfiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 2015, 26:259-71. [15] Yemelyanova A, Vang R, Kshirsagar M, Lu D, Marks MA, Shih IM, et al. Immunohistochemical staining patterns of p53 can serve as a surrogate marker for TP53 mutations in ovarian carcinoma: an immunohistochemical and nucleotide sequencing analysis. Modern Pathol 2011, 24:1248-53. [16] Crum CP, Drapkin R, Miron A, Ince TA, Muto M, Kindelberger DW, et al. The distal fallopian tube: a new model for pelvic serous carcinogenesis. Current opinion in obstetrics & gynecology 2007, 19:3-9. [17] Ibrahim EM, Al-Foheidi ME, Al-Mansour MM, Kazkaz GA. The prognostic value of tumor-infiltrating lymphocytes in triple-negative breast cancer: a meta-analysis. Breast Cancer Res Treat 2014, 148:46776. [18] Yemelyanova A, Vang R, Kshirsagar M, Lu D, Marks MA, Shih IM, et al. Immunohistochemical staining patterns of p53 can serve as a surrogate marker for TP53 mutations in ovarian carcinoma: an immunohistochemical and nucleotide sequencing analysis. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2011, 24:1248-53. [19] Alsner J, Jensen V, Kyndi M, Offersen BV, Vu P, Borresen-Dale A-L, et al. A comparison between p53 accumulation determined by immunohistochemistry and TP53 mutations as prognostic variables in tumours from breast cancer patients. Acta Oncol 2008, 47:600-7.

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Figure legends:

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Fig. 1: High grade invasive ductal carcinoma (100 X): (a) H&E, (b) p53 strongly positive, (c) ER

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negative, (d) High Ki67 labeling index.

Fig. 2: Adjacent normal/benign breast TDLU (100 X): (a) H&E, (b) Focal p53 positive, (c) ER negative

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with appropriate internal control, (d) Ki67 labeling index is not increased.

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ACCEPTED MANUSCRIPT Table 1: Sequences of oligodeoxyribonucleotides

Sequence (5’3’)*

NG_01713.2 region

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5001-24149

1*

GCTGAGGACCTGGTCMTCTGA

2

GGGATACGGCCAGGCATTGAAG

3

TCACTTGTGCCCTGACTTTCAAC

4

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(53TP-)

Position in

Position in Gene

11235-11255

Intron 3

11612-11591

Intron 4

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Name

Intron 4

CCACTGACAACCACCCTTAACC

12748-12726

Intron 6

5

CTTGCCACAGGTCTCCCCAAG

13202-13222

Intron 6

6

GGGTCAGAGGCAAGCAGAGG

13417-13436

Intron 7

7

CAGGTAGGACCTGATTTCCTTAC

13658-13680

Intron 7

8

GCATTTTGAGTGTTAGACTGGAAAC

14075-14051

Intron 9

9

CCATCTTTTAACTCAGGTACTGTG

16776-16799

Intron 9

10

TATGGCTTTCCAACCTAGGAAGG

17003-16981

Intron 10

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12268-12290

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*Other than the bases A, T, C and G, the 53TP-1 oligo uses the ambiguity code ‘M’ for a mixed A/C to accommodate a common polymorphism (rs17883323)

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ACCEPTED MANUSCRIPT Table 2: P53 alteration (by IHC and/or sequencing) in BC P53 altered (n)

P53 not altered

Total (n)

51

42

93

Non-HG

3

45

48

N/A

N/A

54

87

P<0.0001

191

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Total

50

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Mammoplasty

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tumors

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HG tumors

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(n)

Table 3: P53 alteration (by IHC) in normal/benign breast tissue, comparing HG and non-HG

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groups

P53 negative (n)

Total (n)

18

75

93

Breast with Non-HG tumors

1

47

48

Mammoplasty

0

50

50

19

172

191

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Total

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Breast with HG tumors

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Focal P53 positive (n)

P=0.007

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Table 4: Correlation of P53 status in tumors (including HG and non-HG) and normal/benign breast

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tissue P53 - benign

total

P53 + tumors

19

35

54

P53 - tumors

1

86

total

20

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Table 5: TIL in BC

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Total (n)

68

93

1

47

48

26

115

141

25

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Non-HG tumors

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HG tumors

Without TIL (n)

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With TIL (n)

P=0.002

87

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P<0.001

Total

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P53 +benign

15

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Fig. 1

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Fig. 2

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