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Immunohistochemical Expression of p16 in Melanocytic Lesions: An Updated Review and Meta-analysis.

Melanoma is the most fatal cutaneous neoplasm. Surgical resection is curative early in the disease, but chemotherapy, biotherapy, and immunotherapy remain ineffective for more advanced melanoma, with mean survival from first detection of metastases being only 6 to 9 months. (1) Accurate pathologic identification of the primary melanoma is critical for optimal clinical management. Unfortunately, diagnosis by standard histologic criteria can be very difficult, and distinction of melanoma from an atypical nevus may be impossible in some cases.

Misdiagnosis of melanoma, mainly underdiagnosis, constitutes 13% of total pathology-related medical malpractice lawsuits, the second largest group of malpractice claims. (2) Recurrent problems include melanoma misdiagnosed as a melanocytic nevus (without disclosure of diagnostic doubt), chronically inflamed nevus, Spitz nevus, and dysplastic/ atypical nevus. (2-4) These lesions are often difficult to diagnose, and interpretation may be subjective, leading to a range of diagnoses from different pathologists, including expert dermatopathologists. In a study (5) wherein 11 expert pathologists reviewed 37 classic melanocytic lesions, there was total agreement in only 30% of cases. The potentially high rates of misdiagnosis and the possibility that they may contribute to an unfavorable outcome are of great concern to dermatologists and pathologists. The problem is that distinction between benign and malignant melanocytic lesions is often quite difficult for general pathologists, and even for skilled dermatopathologists. (6)

Additionally, a large study surveying 207 dermatopathologists interpreting melanocytic lesions in 10 US states revealed that the majority of dermatopathologists' interpretive behavior was altered because of concerns about malpractice. One of the alterations in behavior included ordering additional tests. (7) Immunohistochemistry is likely the most common additional test used by pathologists to assist in the distinction between benign and malignant melanocytic lesions. Unfortunately, there is no single marker or panel of markers that can achieve this desired end with absolute certainty in all cases. Immunohistochemistry is used by general pathologists and dermatopathologists as an adjunctive diagnostic tool primarily because of accessibility, turnaround time, and familiarity of interpretation. The emerging field of molecular diagnostics is promising; however, it is currently impractical for daily use because it may require a molecular pathologist to interpret the findings, the test is typically expensive, and turnaround time is longer. Therefore, acquisition of molecular tests for diagnostic purposes is mostly reserved for the most difficult cases.

A few immunohistochemical markers have been proposed and used to assist the pathologist with melanocytic lesions. (8-13) Currently, a panel comprising HMB45, Ki-67, and p16 is commonly used to seek assistance in distinguishing benign from malignant lesions. (8) Although the panel may be helpful in some cases, it variably lacks the confidence to provide definitive answers sought by the pathologist. Each marker has its own advantages and disadvantages, dependent on the case; for example, Ki-67 is difficult to interpret in settings with extensive lymphocytic inflammation, although dual labeling with a cytoplasmic marker, such as MART-1, with contrasting chromogen has been proposed to alleviate this problem. (14,15) Additionally, HMB45 may not be as useful in blue nevus-related lesions and malignant blue nevus cases. HMB45 and Ki-67, nevertheless, have been used for some time, and there is substantial literature supportive of their use with difficult melanocytic lesions. However, p16 has been less evaluated and reviewed for its efficacy and practicality for adjunctive diagnostic use in melanocytic lesions. Therefore, herein we review p16 immunohistochemistry as a marker for diagnostic use, particularly for the distinction of nevi from melanoma.

BACKGROUND

Cyclin-dependent kinase inhibitors (CDKIs) are essential proteins involved in withdrawing from cell cycle progression. Lack of CDKIs allows activation of cyclin-dependent kinases, which may then phosphorylate retinoblastoma protein (Rb). Unregulated hypophosphorylated Rb initially sequesters E2F transcription factors, thereby acting as a tumor suppressor. The phosphorylation of Rb releases the bound E2F. Free E2F then acts to progress cell cycle progression by the transcription of genes required for mitotic S phase entry and DNA synthesis. (16-18)

There are 2 families of CDKIs. (17) The CIP/KIP family of kinase inhibitors includes p21, p27, and p57. The INK4 family of CDKIs include [p15.sup.INK4b], [p16.sup.INK4a] (referred to herein as p16), [p18.sup.INK4c], and [p19.sup.INK4d]. The most studied among these are p21 and p16. (16) It is thought that induction of cellular senescence is established and/or maintained by either or both of the p53/p21 and p16/pRb pathways. (19-22) The upregulation of p16 has been shown to respond to hypermitogenic/oncogenic signals such as overexpression of RAS, MAP kinases, or Myc. Multiple other stimuli for upregulation of p16 have also been proposed, such as response to DNA damage. (16-19)

Among the CDKIs, there has been great interest in studying the relation of p16 with melanoma. This may partially be due to the linkage of chromosome 9p21, which includes the p16 gene, to familial melanoma. Although germline mutations of p16 were strongly correlated with familial melanoma, there was no definitive proof that p16 mutations or deletions were absolute requirements for melanoma, because not all melanomas have p16 mutations. Interestingly, few nondiseased controls were also detected to have p16 mutations. Atypical/dysplastic nevi were also analyzed and had a lesser rate of p16 mutations compared with melanomas. (23)

The role of p16 in cellular senescence, particularly in melanocytes, is also of great importance, because senescence is considered to be a critical barrier for tumorigenesis or progression of melanoma. (24) In vitro cell culture studies further support this view. Analysis of human melanocytes revealed that cells depleted of p16 displayed enhanced proliferation and an extended replicative lifespan in the presence of replication-associated DNA damage. (25) Additionally, similar findings were seen with mouse melanocyte culture studies. Mouse Ink4a-[Arf.sup.-/-] melanocytes were shown to readily become immortal upon culture, whereas wild-type Ink4a-[Arf.sup.+/+] cells senesced within 4 to 5 weeks of culture. Restoration of p16 by retroviral vectors containing normal p16 complementary DNAs into Ink4a-[Arf.sup.-/-] melanocytes stopped growth of the cells. (26)

Within melanocytes, the loss of p16 (by mutation, deletion, or silencing) is thought to be a possible contributing factor for tumorigenesis, consistent with our current understanding of the molecular biology of tumor suppressor genes/products. This is in contrast to malignant human papillomavirus-driven tumors (which include mostly anogenital, gynecologic, and oropharyngeal tumors) that show increased p16 expression by immunohistochemistry. This is thought to be due to the integration and expression of E6 and E7 viral oncoproteins. E7 inactivates pRb, preventing the sequestration of E2F transcription factor. The increase in E2F then leads to upregulation of p16 by a feedback mechanism. E6, on the other hand, acts by degrading p53, preventing apoptosis. (27,28)

Observation of germline mutations within melanoma cases and also through experimental basic science has led to the implication of p16 in the tumorigenesis of melanoma. Many independent groups have therefore studied the possible incorporation of p16 immunohistochemistry for the purpose of distinguishing benign from malignant melanocytic lesions, particularly in sporadic melanoma cases. A review and meta-analysis of studies analyzing nevi, atypical melanocytic lesions, and melanoma is presented.

p16 IMMUNOHISTOCHEMISTRY AND ITS USE IN MELANOCYTIC LESIONS

Multiple studies (29-71) evaluating p16 expression by immunohistochemistry of melanocytic lesions were reviewed, analyzed, and tabulated. Essential information (number of samples, antibody used, collection dates, overall p16 immunohistochemistry results, and general method of interpretation) from each study are listed in the Table. Most studies showed a high proportion of benign cutaneous nevi to stain positively for p16 immunohistochemistry. The reported percentage of benign nevus cases staining positive ranged from 61% to 100%. * Most cases, however, reported p16 positivity close to 100% for benign nevi. For primary cutaneous invasive melanomas, ([dagger]) the percentage of cases with positive p16 staining reported by multiple studies ranged from 12% to 93%. Most studies (10) reported percentages within the 40s to 60s. There were 4 studies that reported less than 40% and 2 studies that reported greater than 80%. Metastatic melanoma cases, ([double dagger]) on the other hand, ranged from 0% to 71% of cases staining for p16 immunohistochemistry. Because each group had differing methodologies and reporting styles, not all studies were included, and the aforementioned range of percentages was derived from studies that defined p16 immunohistochemistry simply as either positive or negative. Still, the definition of what was interpreted as positive versus negative varied from study to study. Excluded were studies ([section]) that used a scale or grading-type system in reporting their p16 immunohistochemistry results, because they did not clearly distinguish a positive versus negative result but rather a gradient of staining patterns or relative expression. It is evident that the range of reported percentages is wide and appears insignificant. In general, however, there does appear to be a decreasing trend of p16 staining going from benign to advanced melanoma (especially metastatic lesions). This may also be highlighted by one of the studies (Ghiorzo et al (45)) that showed 1 subset of primary invasive melanoma with smaller than 0.8-mm Breslow thickness to have 51% relative p16 protein expression, and another subset larger than 0.8 mm to have 21% relative p16 protein expression. Relative expression within this unique study was based on staining intensity derived from using benign nevi as reference for comparison. Unfortunately, however, the wide variation in the numerous studies appears to undermine the possible value of p16 immunohistochemistry in its application for melanocytic lesions. It is uncertain, though, whether this wide variation could be due to subjective interpretation, unstandardized laboratory techniques, source of the antibody, or other factors. There is too much variability to consider; however, it is possible that digital imaging, as used by Ghiorzo et al, (45) may assist in the reduction of intraobserver variability, although variations could also exist among imaging hardware and software.

Figure 1, a through h, illustrates the variability of p16 immunohistochemistry staining patterns experienced with melanocytic lesions. For baseline comparison, a nevus with corresponding p16 immunohistochemistry characteristically shows strong and diffuse staining pattern of nevocytes (Figure 1, b). A more variable staining pattern is illustrated in primary invasive cutaneous melanoma, in which there is mostly cytoplasmic staining of the invasive cells (Figure 1, d), versus negative staining (Figure 1, f), versus cases in which there is heterogenous positive and negative staining of the invasive cells (Figure 1, h).

Multiple studies have also incorporated specialized types of melanocytic lesions, such as Spitz lesions, (58-61,64,66,68) desmoplastic melanomas, (58,67,71) blue nevus-related lesions, (69,71) and mucosal melanocytic lesions (41,49,62,63); 2 studies (43,70) focused on nodal metastases, and a recent study (S. S. Koh, unpublished data, 2018) compared nevi of pregnancy and nevoid melanomas. The wide range of variable results is further highlighted when analyzing studies from Spitz nevi. The percentage of Spitz nevi cases positive for p16 immunohistochemistry reported by multiple groups (30,34,50,61,64) ranged from 0% to 100%. Thus, the entire range of staining characteristics disfavors the use of p16 immunohistochemistry for spitzoid lesions. Mason et al (64) showed no difference in p16 staining in Spitz nevi and spitzoid melanomas, concluding that p16 was not a useful marker to distinguish the two. Additionally, George et al (60) had similar conclusions for atypical Spitz nevi, having shown that their p16 immunoreactivity is intermediate between those of Spitz nevi and melanoma, although they claimed that p16 was useful for discriminating Spitz nevus from melanoma. In contrast, Hilliard et al (58) (desmoplastic Spitz nevi versus desmoplastic melanoma) and Al Dhaybi et al (61) (Spitz nevi versus spitzoid melanoma) reported profound differences in expression in benign versus malignant cases and proposed p16 immunohistochemistry expression as an aid for differentiation. Although it is inconclusive for diagnostic purposes in spitzoid lesions, Horst et al (66) and Yazdan et al (68) proposed p16 immunohistochemistry as a possible screening tool for 9p21 cytogenetic abnormalities. Similar to spitzoid lesions, multiple studies analyzing nevi and primary invasive melanomas have also had mixed conclusions: some studies (52,62) support p16 immunohistochemistry for diagnostic purposes (differentiating nevi from primary invasive melanoma), and some studies (29,65) do not show convincing evidence. Interestingly, in the 2 studies (43,70) analyzing nodal metastases, both supported the use of p16 immunohistochemistry as a diagnostic utility to distinguish nodal nevi from metastatic melanoma.

From the molecular level, some authors (29,54) have suggested that p16 loss is not necessary for tumor initiation, perhaps because it is retained mostly in melanoma in situ and primary invasive melanoma. Others (31,34,63) have, in contrast, supported the possibility that p16 may be contributory as a primary event. Some (35) have proposed that the gradual loss of p16 correlates with the progression of melanoma, but is not an initiating event. Most studies did, however, show gradual loss of p16 with melanoma progression (see Table). Furthermore, 2 studies (31,39) correlated loss of p16 with increased Ki-67, supporting its role in the loss of regulating and inhibiting cell cycle entry.

Although some studies incorporated atypical nevi in their studies, most did not analyze p16 immunohistochemistry staining characteristics based on grade of atypia. Chang and Cassarino (69) evaluated atypical cellular blue nevi, including cases with mild, moderate, and severe atypia, versus melanoma (including cases of malignant cellular blue nevi). They found that mildly and moderately atypical cellular blue nevi could be distinguished from severely atypical cellular blue nevi and melanomas when p16 was used within a panel of markers. Unfortunately, however, severely atypical cases showed loss of p16 similar to melanomas, and were therefore not distinguishable from melanoma based on p16 staining. It may be reasonable to raise the possibility that the severely atypical cases could have been underdiagnosed. However, the authors reported low Ki-67 staining pattern for the severely atypical cases that were similar to mild and moderately atypical cellular blue nevi and distinctly different from the high Ki-67 present in melanoma.

Apart from its use in diagnostics, p16 immunohistochemistry expression has also been explored as a potential prognostic marker. Similar to its use for diagnostics, p16 was found to have mixed results as to its role in prognostication. Some studies proposed lack of p16 to be associated with recurrent disease, (32) predicting decreased patient survival, (39,44) and as a predictor of lymph node metastasis. (48) Sanki et al, (51) in contrast, proposed that p16 immunohistochemistry expression did not reliably predict recurrence or survival. Additionally, for sinonasal melanomas, loss of p16 did not correlate with prognosis. (49)

Analysis of multiple studies shows a wide range of results, as previously discussed. The variation could be due to how the observer is interpreting the results. One of the main differences seen among studies is whether positive staining was considered to be nuclear and/or cytoplasmic. Reanalysis was performed by separating studies depending on whether nuclear alone or nuclear and cytoplasmic staining was considered positive for p16 staining (Table). For benign nevi, 89% to 100% of cases positive for p16 was the range reported (31,34,50,54) if only nuclear staining was considered positive, compared with 61% to 100% for those studies (29,31,43,52,61) that used both nuclear and cytoplasmic staining for positivity. For primary invasive melanomas, studies (31,33,34,50,54) considering only nuclear staining had a range of 50% to 68% compared with studies (29,30,37,42,51,52) considering both nuclear and cytoplasmic staining showing a range of 12% to 91%. For metastatic melanomas, studies (33,34,37,56) considering only nuclear staining had a range of 0% to 64% compared with studies (29,43,51) considering both nuclear and cytoplasmic staining showing a range of 2% to 56%. By analyzing the studies as 2 groups (one group interpreting only nuclear staining, and a second group interpreting both nuclear and cytoplasmic staining), it appears that the group considering only nuclear staining characteristics had a shorter range, or less variability, from study to study. These results may suggest that the use of only nuclear staining for interpreting p16 immunohistochemistry for melanocytic lesions may be more optimal. However, it is unclear at the moment whether only nuclear staining is biologically relevant and whether cytoplasmic staining should be considered.

Figure 2 illustrates the staining patterns encountered with p16 immunohistochemistry in melanocytic lesions. Strong staining is usually encountered with benign nevi. Here we see an example of a nevus showing strong cytoplasmic and nuclear staining in many cells (Figure 2, a). In contrast, an example of primary invasive melanoma with the majority of cells staining is present, but the staining pattern is mostly cytoplasmic, with only a few cells showing nuclear staining (Figure 2, b). In this example, if only nuclear staining is considered within this illustrated field, then positivity may be interpreted as being around 5%. However, if cytoplasmic staining is considered, then overall positivity may be interpreted as being 50% to 60%. Depending on the methodology one uses, results may be very different, as illustrated in this example. As experienced by many pathologists, variability of staining is inevitable, as seen in another example (Figure 2, c) of a primary invasive melanoma showing many cells with weak cytoplasmic staining and no nuclear staining (yellow arrow), cytoplasmic staining without nuclear staining (red arrow), and an adjacent cell showing strong nuclear and cytoplasmic staining (green arrow). The point at which to call a positive-staining cell is quite arbitrary, and unfortunately is left to the discretion of the observer.

CONCLUDING REMARKS

The use of p16 for diagnostic purposes in melanocytic lesions appears limited. For the differentiation of metastatic lesions, such as in nodal metastasis versus nodal nevi (although based only on 2 reports), there appears to be some evidence supporting its diagnostic utility. However, for the purposes of distinguishing primary cutaneous melanoma from benign lesions, there is currently a lack of substantial evidence to support its use, especially when it is used alone. When it is used in a panel of other melanocytic markers, however, the possibility for diagnostic utility is likely increased. (8) Perhaps other potential markers similar to p16 might provide better use for distinguishing benign from malignant melanocytic lesions in the future, such as with the recent report (72) of p15 immunohistochemistry in melanocytic lesions. Otherwise, currently, because of the variability of results encountered by analyzing multiple studies, p16 appears to have many limitations, especially for differentiating benign from malignant primary lesions. Nevertheless, if interpretation methods and techniques for its use were better defined, perhaps its role for melanocytic lesions might become more acceptable. Because studies considering only nuclear immunohistochemical p16 staining as positive appeared to show more consistent results, limiting the interpretation to nuclear p16 staining may prove to be more accurate, and may improve interobserver variability, thus potentially making it more useful in the routine distinction of benign versus malignant melanocytic lesions.

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Stephen S. Koh, MD, PhD; David S. Cassarino, MD, PhD

Accepted for publication December 13, 2017.

From the Department of Pathology and Dermatopathology, Kaiser Permanente Anaheim Medical Center, Anaheim, California.

The authors have no relevant financial interest in the products or companies described in this article.

Presented at the 16th Spring Seminar of the Korean Pathologists Association of North America (KOPANA); March 3, 2017; San Antonio, Texas.

Corresponding author: Stephen S. Koh, MD, PhD, Department of Pathology and Dermatopathology, Kaiser Permanente Anaheim Medical Center, 3430 E La Palma Ave, Anaheim, CA 92806 (email: stephen.s.koh@kp.org).

* References 29-31, 34, 35, 43, 44, 46, 50, 52-54, 61.

([dagger]) References 29-31, 33-35, 37, 42, 44, 50-54.

([double dagger]) References 29, 33-35, 37, 43-44, 51, 53, 56.

([section]) References 32, 36, 38-40, 45, 47-48, 55, 57, 60, 62-63, 65-67, 69.

Caption: Figure 1. a and b, Nevus with corresponding p 16 staining. Higher magnification of p16 staining provided as inset on lower right corner of b illustrates a clearer view of strong and diffuse nuclear and cytoplasmic staining of dermal nevus cells, c and d, Primary invasive melanoma with corresponding p16 staining. Higher magnification of p16 staining provided as inset on lower right corner of d illustrates a clearer view of partial and mostly cytoplasmic staining of dermal invasive cells, e and f, Primary invasive melanoma with corresponding negative p 16 staining, g and h, Primary invasive melanoma with corresponding heterogenous p16 staining (hematoxylin-eosin, original magnifications x 10 objective [a, c, e, and gl; p16 immunohistochemistry, original magnifications x 10 objective [b, d, f, and h] and X60 objective [b and d, insets]).

Caption: Figure 2. a, Nevus with many cells showing both cytoplasmic and nuclear staining. b, Primary invasive melanoma with mostly cytoplasmic staining. c, Primary invasive melanoma showing weak cytoplasmic staining with negative nuclear staining (yellow arrow), cytoplasmic staining with negative nuclear staining (red arrow), and strong cytoplasmic and strong nuclear staining (green arrow) (p16 immunohistochemistry, original magnification X60 objective).
Results and Highlighted Findings From Each Reviewed Study

                      Study
Source, y             Period             Samples

Reed et al,       NA               20 BN
(29) 1995
                                   18 AN

                                   14 MIS

                                   37 PIM

                                   14 MM

Wang et al,       NA               28 BN (6 Spitz nevi)
(30) 1996
                                   19 PIM

Talve et al,      1973-1982        10 BN
(31) 1997                          79 PIM

Straume and       1981-1989        91 PIM, NM
Akslen, (32)
1997

Piccinin et       NA               8 PIM
al, (33) 1997                      8 MM

Sparrow et        5 yrs or less    35 BN (5 Spitz nevi)
al, (34) 1998
                                   12 MIS
                                   38 PIM
                                   25 MM

Keller-           NA               51 BN + AN
Melchior et
al, (35) 1998                      7 MIS

                                   17 PIM

                                   33 MM

Funk et           NA               24 BN
al, (36)
1998                               2 lentigo simplex

                                   4 MIS, LM

                                   26 PIM, SSM

                                   28 PIM, NM

                                   3 PIM, LMM

                                   5 MM

Morita et         NA               14 PIM, ALM +
al, (37) 1998                        SSM + NM
                                   14 MM,
                                     corresponding
                                     cases

Radhi,            NA               20 PIM with
(38) 1999                            residual dermal
                                     nevi

Straume et        1981-1997        190 PIM
al, (39) 2000
                                   58 MM

Vuhahula et       January 1997-    28 PIM, 50% ALM
al, (40) 2000       December
                    1998

Tanaka et         NA               13 PIM, oral
al, (41) 2001

Pavey et          July 1993-       108 PIM, SSM +
al, (42) 2002       June 1994        NM

Mihic-Probst et   NA               1 5 BN, dermal
al, (43) 2003                      1 8 nodal nevi
                                   26 MM, SLN

                                   10 MM, NSLN

                                   13 MM, organs

Alonso et         1995-2000        9 BN
al, (44) 2004                      26 PIM, radial
                                   66 PIM, vertical
                                   68 MM

Ghiorzo et        NA               33 BN
al, (45) 2004
                                   12 MIS

                                   31 PIM <0.8 mm

                                   9 PIM >0.8 mm

                                   11 MM

Herron et         1994-2001        30 BN
al, (46) 2004

Gray-Schopfer     NA               11 BN
et al,
(47) 2006                          9 AN

                                   14 PIM, RGP

                                   14 PIM, VGP

Mihic-Probst et   NA               34 BN
al, (48) 2006
                                   64 PIM

Franchi et        NA               37 PIM, sinonasal
al, (49) 2006

Stefanaki et      NA               20 BN
al, (50) 2007
                                   10 Spitz nevi

                                   16 PIM

Sanki et al,      NA               19 BN + AN
(51) 2007
                                   39 PIM

                                   34 MM

Demirkan et       NA               28 BN
al, (52) 2007                      24 PIM, NM +
                                     LMM + ALM +
                                     SSM

Sini et al,       NA               20 BN
(53) 2008                          22 AN
                                   14 PIM, T1-T2
                                   18 PIM, T3-T4

                                   8 MM

Stefanaki et      NA               41 BN, congenital
al, (54) 2008
                                   20 BN, acquired

                                   16 PIM

Richmond-         January 1998-    109 PIM, LMM +
Sinclair et         December       SSM
al, (55) 2008       1999

De Sa et          1994-2000        125 MIS + PIM
al, (56) 2009                      29 MM

Karim et          1997-1999        8 BN, dermal
al, (57) 2009
                                   7 BN, compound

                                   15 MIS

                                   25 PIM, thin

                                   29 PIM, thick

                                   28 MM

Hilliard et       NA               15 desmoplastic
al, (58)2009                         Spitz
                                   11 desmoplastic
                                     melanoma

Hsieh et al,      NA               32 ALM, 3 MIS +
(59) 2009                            29 PIM

George et al,     1983-2000        27 Spitz nevi
(60) 2010
                                   19 atypical Spitz

                                   42 PIM, 6 with
                                     spitzoid features

Al Dhaybi et      1998-2010        12 BN
al, (61) 2011                      18 Spitz nevi
                                   6 spitzoid
                                     melanoma

Zoroquiain et     1993-2009        19 BN
al, (62) 2012                      15 PAM w/o atypia
                                   2 PAM w atypia
                                   9 PIM

de Andrade et     NA               38 BN, intramucosal
al, (63) 2012
                                   13 PIM

Mason et al,      NA               18 BN, Spitz nevi
(64) 2012                          19 PIM, spitzoid

Tran et al,       1993-2010        19 BN
(65) 2012
                                   18 MM

                                   20 BN, tissue array

                                   21 PIM, tissue array

Horst et al,      July 2000-       22 Spitz nevi,
(66) 2013           June 2010        patient age >50 y
                                   11 Spitz nevi,
                                     patient age <50 y

Blokhin et al,    NA               5 BN, desmoplastic
(67) 2013                            22 PIM,
                                     desmoplastic

Yazdan et al,     NA               12 atypical Spitz,
(68) 2014                            heterozygous
                                     9p21 deletion
                                   13 atypical Spitz,
                                     homozygous 9p21
                                     deletion
                                   16 PIM, with
                                     homozygous 9p21
                                     deletion

Chang and         NA               6 CBN,
Cassarino, (69)                      conventional and
2014                                 mildly atypical
                                   4 CBN, moderately
                                     atypical
                                   5 CBN, severely
                                     atypical
                                   12 melanomas, MIS
                                     + superficial
                                     spreading + in
                                     transit + spitzoid
                                     + nevoid
                                     5 CBN, malignant

Piana et al,      January 2000-    22 nodal nevi with
(70) 2015           January 2014     5 coexistent
                                     metastatic deposit

Donigan et        2007-2015        9 common blue
al, (71) 2017                        nevi
                                   8 cellular blue nevi
                                   12 PIM, spitzoid +
                                     desmoplastic +
                                     nevoid + nodular
                                   3 PIM, blue
                                     nevuslike

                                 p16
                        I mmunohistochemistry
Source, y                      Results                Interpretation

Reed et al,       100% of cases homogeneously         N and C
(29) 1995           positive
                  100% of cases homogeneously
                    positive
                  100% of cases homogeneously
                    positive
                  48% of cases homogeneously
                    positive, 43% variable
                    proportion positive, 9%
                    negative
                  28% of cases homogenously
                    positive, 28% variable
                    proportion positive, 44%
                    negative

Wang et al,       17 cases (61%) positive, all 6     N or C
(30) 1996           Spitz reported as negative
                  3 cases (16%) positive

Talve et al,      10 cases (100%) positive            N
(31) 1997         46 cases (58%) positive

Straume and       8 cases (9%) negative or very       N and C
Akslen, (32)        weak, 4 cases (4%) very strong,
1997                and all remaining cases had
                    intermediate staining

Piccinin et       5/8 cases positive                  N
al, (33) 1997     0/8 cases positive

Sparrow et        89% of cases positive, 1/5 Spitz    N, <5% N
al, (34) 1998       reported negative                 staining
                  92% of cases positive               considered
                  50% of cases positive               negative
                  64% of cases positive

Keller-           44 cases (86%) with uniform         Unspecified
Melchior et         labeling
al, (35) 1998     5 cases (72%) with uniform
                    labeling
                  10 cases (59%) with uniform
                    labeling
                  16 cases (49%) with uniform
                    labeling

Funk et           6 (25%) 0%-10% positive, 6          N + C "mixed
al, (36)            (25%) 10%-50% positive, 12        pattern"
1998                (50%) >50% positive
                  1 (50%) 0%-l 0% positive, 1
                    (50%) 10%-50% positive, 0
                    (0%) >50% positive
                  2 (50%) 0%-l 0% positive, 1
                    (25%) 10%-50% positive, 1
                    (25%) >50% positive
                  1 8 (69%) 0%-l 0% positive, 5
                    (19%) 10%-50% positive, 3
                    (12%) >50% positive
                  17 (61%) 0%-10% positive, 6
                    (21%) 10%-50% positive, 5
                    (18%) >50% positive
                  3 (100%) 0%-10% positive, 0
                    (0%) 10%-50% positive, 0 (0%)
                    >50% positive
                  5 (100%) 0%-10% positive, 0
                    (0%) 10%-50% positive, 0 (0%)
                    >50% positive

Morita et         9/14 cases positive                 N
al, (37) 1998
                  7/14 cases positive

Radhi,            14 cases (70%) strong N and 6       N or C; see
(38) 1999           (30%) weak N positivity in        comment
                    residual nevi
                  1 8 cases (90%) weak N and no
                    reported strong N positivity in
                    malignant melanocytes

Straume et        Nuclear scores: 28% = 0, 1 7% =     N or C
al, (39) 2000       1, 21% = 2, 4% = 3, 14% =
                    4, 13% = 6, 3% = 9
                  Cytoplasmic scores: 15% = 0, 6%
                    = 1,12% = 2, 34% = 3, 2% =
                    4, 25% = 6, 6% = 9
                  Nuclear scores: 74% = 0, 3% =
                    1, 7%-2, 0%-3, 12%-4,
                  Cytoplasmic scores: 36% = 0,
                    22% = 1,14% = 2, 3% = 3,
                    12% = 4, 9% = 6, 3% = 9

Vuhahula et       18% = 0, 32% = 1,18% = 2,           N
al, (40) 2000       18% = 4, 11 % = [greater
                    than or equal to] 6

Tanaka et         7/13 cases positive (4/8 stage      N > C; see
al, (41) 2001       II, 2/ 3 stage III, 1/2 stage     comment
                    IV)

Pavey et          52 (48%) of tumors with some        N or C
al, (42) 2002       degree of immunoreactivity, as
                    defined by a lesion having
                    more than a rare cell with
                    staining.

Mihic-Probst et   All cases N or C positive           N or C
al, (43) 2003     All cases N or C positive
                  All cases N negative, 6 cases C
                    staining
                  All cases N negative, 3 cases C
                    staining
                  12 cases N negative, 4 cases C
                    staining

Alonso et         9 cases (100%) positive             Unspecified
al, (44) 2004     23 cases (88%) positive
                  59 cases (89%) positive
                  48 cases (71%) positive

Ghiorzo et        100% relative p16 protein           N and C
al, (45) 2004       expression
                  43% relative p16 protein
                    expression
                  51% relative p16 protein
                    expression
                  21% relative p16 protein
                    expression
                  18% relative p16 protein
                    expression

Herron et         100% positive                       Unspecified
al, (46) 2004

Gray-Schopfer     10/11 cases ++ positive, 1/11 +     N and C
et al,              positive
(47) 2006         5/9 cases ++ positive, 4/9 +
                    positive
                  7/14 cases ++ positive, 7/14 +
                    positive
                  3/14 cases ++ positive, 11/14 +
                    positive

Mihic-Probst et   67% [+ or -] 22% of cells           N or C
al, (48) 2006       positive (N + C), 9% [+ or -]
                  16% of cells positive (C only)
                  16% [+ or -] 24% of cells
                    positive (N + C), 34% [+ or -]
                  34% of cells positive (C only)

Franchi et        27 cases (72.9%) showed loss of     N and C
al, (49) 2006       p16 expression

Stefanaki et      20/20 cases positive, moderate      N
al, (50) 2007       to strong staining in >60% of
                    nuclei
                  10/10 cases positive, strong N
                    expression in 40%-70% of
                    nuclei
                  11/16 cases positive,
                    heterogeneous N weak
                    expression from <5% to 40%
                    of nuclei

Sanki et al,      73.7% (N) and 94.7% (C) of cases    N or C
(51) 2007           positive
                  28.2% (N) and 38.5% (C) of cases
                    positive
                  14.7% (N) and 17.6% (C) of cases

Demirkan et       79% of cases positive               N and C
al, (52) 2007     12% of cases positive

Sini et al,       0 cases (0%) showing loss of p16    Unspecified
(53) 2008         0 cases (0%) showing loss of p16
                  1 case (7%) showing loss of p16
                  3 cases (1 7%) showing loss of
                    p16
                  5 cases (62%) showing loss of
                    p16

Stefanaki et      41/41 cases positive,               N
al, (54) 2008       heterogeneous intense >70%
                    nuclei staining
                  20/20 cases positive, diffuse
                    moderate to strong >60% N
                    staining
                  11/20 cases positive,
                    heterogeneous weak expression
                    of <5%-40% nuclei

Richmond-         0% (64 cases), <1% (5 cases),       N and/or C
Sinclair et         1 %-10% (12 cases), 10%-30%
al, (55) 2008       (10 cases), 30%-50% (2 cases),
                    >50% (16 cases)

De Sa et          4% of cases positive                N, <5% N
al, (56) 2009     0% of cases positive                staining
                                                      considered
                                                      negative

Karim et          6.1 (mean), 7 (median), 3-7         N
al, (57) 2009       (range)
                  5.4 (mean), 5 (median), 2-6
                    (range)
                  3.6 (mean); 4 (median); 0-6
                    (range)
                  3.9 (mean), 4 (median), 0-7
                    (range)
                  2.2 (mean), 2 (median), 0-7
                    (range)
                  1.2 (mean), 0 (median), 0-7
                    (range)

Hilliard et       15 cases (100%) positive            N and C
al, (58)2009
                  2 cases (1 8.2%) positive

Hsieh et al,      25 cases (78%) positive, 1/3 MIS    N
(59) 2009           cases positive

George et al,     68% (N) and 80% (C) mean            N or C
(60) 2010           positive dermal tumor cells
                  52% (N) and 65% (C) mean
                    positive dermal tumor cells
                  40% (N) and 57% (C) mean
                    positive dermal tumor cells

Al Dhaybi et      All positive                        N and C
al, (61) 2011     All positive
                  All negative

Zoroquiain et     7.63 [+ or -] 3.24                  N
al, (62) 2012     11 [+ or -]1.69
                  12 [+ or -] 0
                  3.3 [+ or -] 1.8

de Andrade et     Strongly expressed in more than     N
al, (63) 2012       70% of cells in all cases
                  Only one oral melanoma was
                    positive in 35% of neoplastic
                    cells. Other 12 cases negative.
Mason et al,      15 cases (83%) positive             N or C
(64) 2012         15 cases (79%) positive

Tran et al,       40% median percentage of cells      N
(65) 2012           positive
                  0% median percentage of cells
                    positive
                  45% median percentage of cells
                    positive
                  20% median percentage of cells

Horst et al,      6.1 (mean), 7 (median)              N
(66) 2013
                  6.0 (mean), 6 (median)

Blokhin et al,    All positive                        Unspecified
(67) 2013           6 cases negative, 6 cases with
                    <25% of cells staining, 4 cases
                    with 25%-50% of cells
                    staining, and 6 cases with
                    diffuse positive staining

Yazdan et al,     8 cases (67%) positive              N
(68) 2014
                  0 cases (0%) positive

                  0 cases (0%) positive

Chang and         69.58% [+ or -] 8.23% positive      N and C
Cassarino, (69)     melanocytes
2014
                  66.88% [+ or -] 12.56% positive
                    melanocytes
                  20.2% [+ or -] 11.96% positive
                    melanocytes
                  13.92% [+ or -] 6.32% positive
                    melanocytes

                  6% [+ or -] 3.24% positive
                    melanocytes

Piana et al,      All 22 nodal nevi components        N and C
(70) 2015           positive and all 5 metastatic
                    components negative

Donigan et        0 cases positive                    Unspecified
al, (71) 2017
                  3 cases positive
                  7 cases positive

                  1 case positive

Source, y                P16 Antibody

Reed et al,       Pharmingen Inc, San
(29) 1995           Diego, California; Santa
                    Cruz Biotechnology Inc,
                    Santa Cruz, California,

Wang et al,       Pharmingen
(30) 1996

Talve et al,      Pharmingen
(31) 1997

Straume and       SC-469, Santa Cruz
Akslen, (32)
1997

Piccinin et       151262, Pharmingen;
al, (33) 1997       sc468, Santa Cruz

Sparrow et        Pharmingen; Santa Cruz
al, (34) 1998

Keller-           G175-405 Pharmingen
Melchior et
al, (35) 1998

Funk et           G1 75-405, Pharmingen
al, (36)
1998

Morita et         Pharmingen
al, (37) 1998

Radhi,            Pharmingen Canada
(38) 1999

Straume et        SC-468, Santa Cruz
al, (39) 2000

Vuhahula et       SC-468, Santa Cruz
al, (40) 2000

Tanaka et         G1 75-405, Pharmingen
al, (41) 2001

Pavey et          G1 75-405, Pharmingen
al, (42) 2002

Mihic-Probst et   Neomarkers
al, (43) 2003

Alonso et         F12, Santa Cruz
al, (44) 2004

Ghiorzo et        G1 75-405, Pharmingen;
al, (45) 2004       SC-468 C-20 and SC-467
                    N-20, Santa Cruz

Herron et         Santa Cruz
al, (46) 2004

Gray-Schopfer     Novocastra
et al,
(47) 2006

Mihic-Probst et   Neomarkers
al, (48) 2006

Franchi et        E6H4, Dakocytomation
al, (49) 2006

Stefanaki et      1F4, Santa Cruz
al, (50) 2007

Sanki et al,      Neomarkers
(51) 2007

Demirkan et       6H12, Novocastra,
al, (52) 2007       Norwell, Massachusetts

Sini et al,       16P04JC-2, Lab Vision/
(53) 2008           Neomarkers, Fremont,
                    California

Stefanaki et      1F4, Santa Cruz
al, (54) 2008

Richmond-         G175-405, BD
Sinclair et         Pharmingen, San Diego,
al, (55) 2008       California

De Sa et          SCI 661-F12, Santa Cruz
al, (56) 2009

Karim et          AB-4 16p04, Neomarkers
al, (57) 2009

Hilliard et       Cell Marque, Hot
al, (58)2009        Springs, Arkansas

Hsieh et al,      16P04, Labvision,
(59) 2009           Fremont, California

George et al,     G175-405, Becton
(60) 2010           Dickinson, Franklin
                    Lakes, New Jersey

Al Dhaybi et      Mtm Laboratories,
al, (61) 2011       Heidelberg, Germany

Zoroquiain et     Abcam, Cambridge, United
al, (62) 2012       Kingdom

de Andrade et     SC-1661, Santa Cruz
al, (63) 2012

Mason et al,      E6H4, mtm Laboratories,
(64) 2012           Heidelberg, Germany

Tran et al,       N20 and C20, Santa Cruz
(65) 2012

Horst et al,      E6H4, Ventana, Tucson,
(66) 2013           Arizona

Blokhin et al,    E6H4, Dakocytomation,
(67) 2013           mtm laboratories,
                    Germany

Yazdan et al,     JC8, Santa Cruz
(68) 2014

Chang and         E6H4, Roche, Tucson,
Cassarino, (69)     Arizona
2014

Piana et al,      E6H4, Ventana
(70) 2015

Donigan et        Santa Cruz
al, (71) 2017

Source, y                    Comments

Reed et al,
(29) 1995

Wang et al,
(30) 1996

Talve et al,
(31) 1997

Straume and
Akslen, (32)
1997

Piccinin et
al, (33) 1997

Sparrow et
al, (34) 1998

Keller-           Uniform labeling
Melchior et         considered when at least
al, (35) 1998       two-thirds of
                    melanocytic cells
                    positive.

Funk et
al, (36)
1998

Morita et
al, (37) 1998

Radhi,            Author analyzed both C
(38) 1999           and N staining.
                    However, only N
                    immunoreactivity was
                    considered as a sign of
                    p16 positivity.

Straume et        MMs are corresponding
al, (39) 2000       cases.
                  p16 staining was reported
                    through a
                    semiquantitative scoring
                    system. A 3-point
                    percentage of stained
                    cells score (0 = absent,
                    1 = <10%, 2 = 10%-
                    50%, 3 = >50%) was
                    multiplied by a 3-point
                    N intensity score (0 =
                    negative, 1 = weak, 2 =
                    moderate, 3 strong) for
                    highest value of 9.

Vuhahula et       p16 staining was reported
al, (40) 2000       through a
                    semiquantitative scoring
                    system. A 3-point
                    percentage of stained
                    cells score (0 = absent,
                    1 = <10%, 2 = 10%-
                    50%, 3 = >50%) was
                    multiplied by a 3-point
                    N intensity score (0 to 3
                    = no staining to strong
                    staining) for highest
                    value of 9.

Tanaka et         Positive if N was stronger
al, (41) 2001       than C.

Pavey et
al, (42) 2002

Mihic-Probst et
al, (43) 2003

Alonso et
al, (44) 2004

Ghiorzo et        Expression is relative
al, (45) 2004       compared with nevi
                    (100%) analyzed by
                    digital imaging
                    techniques. PIM also
                    stratified by superficial
                    spreading melanoma
                    versus nodular
                    melanoma and by Clark
                    levels.

Herron et
al, (46) 2004

Gray-Schopfer     Immunostaining scored as
et al,              ++ (51 %-100% of cells
(47) 2006           immunostained), + (up
                    to 50%), or 0.

Mihic-Probst et
al, (48) 2006

Franchi et
al, (49) 2006

Stefanaki et
al, (50) 2007

Sanki et al,      Also additional data
(51) 2007           analysis based on type of
                    nevi, thick versus thin
                    melanoma, type of
                    metastasis, and
                    pathologic variables of
                    melanoma.

Demirkan et
al, (52) 2007

Sini et al,
(53) 2008

Stefanaki et
al, (54) 2008

Richmond-         % of cells with
Sinclair et         immunopositivity.
al, (55) 2008

De Sa et          MIS and PIM were grouped
al, (56) 2009       in their global
                    assessment as primary
                    tumor. Separated by
                    tumor thickness; [less than
                    or equal to] 1.0 mm had 7.9%
                    positive p16 staining and
                    >1.0 mm had 0%
                    positive p16 staining.

Karim et          p16 staining was reported
al, (57) 2009       through a
                    semiquantitative scoring
                    system. A 3-point N
                    intensity score (0 = no
                    staining, 1 = weak
                    staining, 2 = moderate
                    staining, 3 = strong
                    staining) was added to a
                    4-point percentage of
                    stained cells score (0 =
                    0%, 1 = 1 %-5%, 2 =
                    6%-10%, 3 = 11 %-
                    30%, 4 = >30%) for a
                    value ranging from 0 to
                     7.

Hilliard et       Intensity and percentage of
al, (58)2009        positive tumor cells also
                    analyzed separately.

Hsieh et al,
(59) 2009

George et al,     Epidermal component also
(60) 2010           evaluated separately.

Al Dhaybi et
al, (61) 2011

Zoroquiain et     Conjunctival lesions.
al, (62) 2012       p16 staining was
                    reported through a
                    semiquantitative scoring
                    system. A 4-point
                    percentage of stained
                    cells score (0 = absent,
                    1 = 1%-10%, 2 = 11%-
                    50%, 3 = 51 %-80%, 4
                    = 81 %-100%) was
                    multiplied by a 3-point
                    N intensity score (0 =
                    negative, 1 = weak, 2 =
                    moderate, 3 strong) for a
                    value ranging from 0 to
                    12.

de Andrade et     Oral lesions.
al, (63) 2012

Mason et al,
(64) 2012

Tran et al,
(65) 2012

Horst et al,      p16 staining was reported
(66) 2013           through a
                    semiquantitative scoring
                    system. A 3-point N
                    intensity score (0 =
                    negative, 1 = weak, 2 =
                    moderate, 3 = strong)
                    was added to a 4-point
                    percentage of stained
                    cells score (0 = 0%, 1 =
                    1 %-25%, 2 = 2 6%-
                    50%, 3 = 51 %-75%, 4
                    = >76%-100%) for a
                    value ranging from 0 to
                    7.

Blokhin et al,
(67) 2013

Yazdan et al,     Complete absence of
(68) 2014           staining and aggregates
                    of tumor cells with
                    complete loss were
                    considered negative.

Chang and         p16 intensity was also
Cassarino, (69)     separately graded.
2014

Piana et al,
(70) 2015

Donigan et
al, (71) 2017

Abbreviations: ALM, acral lentiginous melanoma; AN, atypical/
dysplastic nevi; BN, benign nevi/melanocytic lesion; C, cytoplasmic;
CBN, cellular blue nevus; LM, lentigo maligna; LMM, lentigo maligna
melanoma; MIS, melanoma in situ; MM, metastatic melanoma; N, nuclear;
NA, not available; NM, nodular melanoma; NSLN, nonsentinel lymph node;
PAM, primary acquired melanosis (conjunctival); PIM, primary invasive
melanoma; RGP, radial growth phase; SLN, sentinel lymph node; SSM,
superficial spreading melanoma; VGP, vertical growth phase.
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Author:Koh, Stephen S.; Cassarino, David S.
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Date:Jul 1, 2018
Words:8440
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