**5. The refutation episode**

Two years following the publication of the above-mentioned article and several months after the presentation of a poster at the Hodgkin Lymphoma Meeting in Kiel, a scientist, from Germany, contacted me indirectly, informing me that he wanted to investigate our findings. He proposed to find out whether he could reproduce our data in his lab. He also requested to make any fresh-frozen CHL tissues available to him, mainly those which showed an MV-antigen positivity.

In their labs, the German scientists had collected 44 frozen CHL samples, of which, only 18 were selected for their high quality RNA, as determined by the Agilent technology, as well as for classic H/RS cell morphology. Using UV-laser beam single cell microdissection, some 100 H/RS cells were pooled for each experiment. Total RNA was extracted and RT-PCR was performed with primers from three MV genes, which were different from those we had used in Israel [40].

Five CHL-derived cell lines were found to be negative for the three MV transcripts. Moreover, the 18 selected German cases did not show, either, evidence of any of the three viral transcripts. The GAPDH gene, used as a housekeeper gene for these experiments, displayed a large amount of RNA. At this point, the German group had received the 22 snap-frozen CHL samples from our laboratory. The cases had been chosen, as requested for their positive MV antigens, as shown on FFPE tissues. However, the selection highlighted only 7 of these cases, chosen for the high RNA quality and the classic H/RS cell features [40].


*NP, nucleoprotein; HA, hemagglutinin; MV, measles virus; RT-PCR, reverse transcriptase-polymerase chain reaction; ISH, in situ hybridization; and IHC, immunohistochemistry.*

#### **Table 4.**

*Comparison of results obtained by three assays.*

All 25 RNA samples disclosed negative results with the three MV transcripts, as studied in Essen labs. Back to Israel, 5 of 12 German histological unstained slides, subjected to IHC, were found to express MV antigens in the H/RS cells. A similar discrepancy in favor of IHC was also obtained in our series (**Table 4**).

The German scientists invited one of my colleagues and myself to co-author their manuscript. At every stage, we were reluctant to accept the invitation, due to the concern for a permanent damage to our hypothesis. Moreover, we did not curb our criticism, which covered several aspects of the manuscript. Prominent among these was the tight selection of the cases (both those from Germany and Israel). The methods used in Essen differed markedly from ours, notably the single cell microdissection, a different strategy for the RT-PCR and the choice of primers. Following our criticism, minor changes were introduced, but the German group did not go all the way: "….due to their worry that the manuscript will lose its sharp edge and might not be accepted by a highly rated journal."

The Scott collaborators of the Essen scientists reported above selected a group of patients, many of whom having suffered from measles, mainly as children. We consider this selection as counterproductive, since these patients should have been protected from the development of CHL. The very fact that they were not refutes the prevalent thesis that exposure of a child, aged 1–4 years, to MV infection protects the subject from CHL at a young-adult age [41]. The Scottish group may have had their own agenda, which might have unsettled the question raised by us. In addition, the antibody used by the Scott group is problematic: the clone 49–41 was purchased by us from Argene-Biosoft, Varilhes, France (# 11-045). It was not an anti-nucleoprotein antibody, as stated, and was positive in our lab for 37 of our 72 CHL cases (data not shown). Moreover, not unlike the German scientists, the Scotts used the GAPDH gene as a housekeeping gene, and we have already mentioned why it was not appropriate.

I have to admit, that we not only considered ourselves as the minor partner, but that we were such indeed! We had been recurrently reassured by the German scientists and by then had our two names included in the manuscript as co-authors. However, when we read the finalized doublet articles (in addition, the article by Wilson et al., weighted heavily on the balance) [41], published in the International Journal of Cancer, we realized that we had made a huge mistake.

The days which followed the double publication were crucial, since we were not sure, as co-authors, whether we were entitled to carry out an "insider" censure. When we read the final paper, we grasped the experience in terms of a possible deception. In addition to the impediments mentioned above, we also noted that the housekeeping gene, the GAPDH, might not be an adequate choice at all, since we had shown that CHL is most probably of low MV-RNA abundance. In contrast, the Essen group had described in relation with GAPDH a large amount of RNA [40]. Moreover, of the seven Israeli cases, selected for the experiment in Germany, five had been shown by us to present a faint MV-RNA positivity only (two) or were negative (three).

**147**

*A Role for the Measles Virus in Oncology DOI: http://dx.doi.org/10.5772/intechopen.84342*

p53 by targeting it for degradation in the proteasome.

on our experience, as described above [42, 43].

higher mortality were evident [44].

**6. New evidence**

An independent putative evidence for a role of the MV in carcinogenesis is that part of the MV proteins that interacts with and stabilizes the Pirh2 protein, an ubiquitin E3 ligase, by preventing its ubiquitination. Pirh2 indeed negatively regulates

We set out to delve into endometrial carcinoma (EC), looking for the footprints of the MV in this cancer. Our cohort of EC included 49 patients, 36 of whom had a known follow-up. Using IHC and several anti-MV antibodies, we disclosed that 26 of the 36 (72%) did express MV antigens in tumor cells, mainly in type I EC (**Figure 6**). In addition, 16/21 of the patients displayed positivity for MV-RNA by ISH. When type II EC was allied with MV, then deeper myometrial invasion and a

Our next endeavor concerned the possible role of MV in lung cancer. Our interest was first raised by the probable relevance of Pirh2 in lung cancer. Indeed, Pirh2, an E3 ubiquitin ligase, was found to be expressed in lung cancer, in which it inhibits p53 [45]. It was later found that the P-protein of the MV inhibited the function of Pirh2 (see description in [48]). This indirect association between lung cancer, Pirh2 and p53, and MV led us to the following study. We identified 65 non-small cell lung cancers, for which FFPE tissues were available over 10 years. We limited our study to the IHC analysis with several anti-MV antibodies (**Figure 7**). Of the cases included, 54/65 were positive for MV antigens. These 54 positive cases were associated with older patient age, with positive Pirh2 expression, as well as with an improved survival [46]. The following study concerned breast cancer and the MV-IHC expression. We identified 131 breast cancer cases, with preserved FFPE tissues. Of these cases, 64% were positive for the MV antigens (**Figure 8**). Positive MV expression was associated with ER (*p* = 0.018); with Ki-67 < 40% (*p* = 0.029); with low or intermediate grade (*p* = 0.037); with age < 50 (*p* = 0.039); and with p53 overexpression (*p* = 0.049) (**Table 5**). It seems,

therefore, that the MV may play a role in the development of this cancer [47].

We have speculated whether the MV phosphoprotein, through its relation with the Pirh2, ubiquitin ligase, by itself negatively related with the p53 expression, may have more relevance, *a priori,* than other MV antigens to the pathogenesis of the

Altogether, our criticism of the article, in which we took officially a part, is in principle that the German scientists carried out an investigation, which was by their declared intention, a duplication of our own research [3]. However, their study was anything, except a duplicate of ours! It stands out independently, with major flaws, notably a very strong selection of cases, the use of questionable primers of a housekeeping gene which is not appropriate for the expected amount of retrieved MV-RNA. It is of note that when confronted with our criticism regarding the manuscript, the German group seemed to agree, but did not modify the manuscript: "…for fear of weakening our data". Moreover, they insisted on mentioning the relevance of the findings to the CHL pathogenesis, which was not appropriate at this stage nor was it agreed upon [40]! At this point, we insist that the impact of the European group refutation, on a possible role of MV in CHL, was extremely ruinous to our position and thesis. Moreover, its repercussions are felt to this day: for the reasons mentioned above, not a single investigator has accepted to engage in this project anymore, together with us or independently. By so doing, the two research groups have categorically "buried" our hypothesis. At the same time, we felt a release from the constraints which might have led us freeze in our position and we allowed ourselves again to indulge in the research concerning the measles virus and cancer. Therefore, we first wrote a review

#### *A Role for the Measles Virus in Oncology DOI: http://dx.doi.org/10.5772/intechopen.84342*

*Viruses and Viral Infections in Developing Countries*

*ISH, in situ hybridization; and IHC, immunohistochemistry.*

*Comparison of results obtained by three assays.*

**Table 4.**

not be accepted by a highly rated journal."

All 25 RNA samples disclosed negative results with the three MV transcripts, as studied in Essen labs. Back to Israel, 5 of 12 German histological unstained slides, subjected to IHC, were found to express MV antigens in the H/RS cells. A similar

RT-PCR 1/16 (6.25) 7/21 (33.3) ISH 7/14 (50) 4/7 (57.1) IHC 25/29 (86.2) 20/29 (68.9) *NP, nucleoprotein; HA, hemagglutinin; MV, measles virus; RT-PCR, reverse transcriptase-polymerase chain reaction;* 

**NP-MV HA-MV Positive/n (%) Positive/n (%)**

The German scientists invited one of my colleagues and myself to co-author their manuscript. At every stage, we were reluctant to accept the invitation, due to the concern for a permanent damage to our hypothesis. Moreover, we did not curb our criticism, which covered several aspects of the manuscript. Prominent among these was the tight selection of the cases (both those from Germany and Israel). The methods used in Essen differed markedly from ours, notably the single cell microdissection, a different strategy for the RT-PCR and the choice of primers. Following our criticism, minor changes were introduced, but the German group did not go all the way: "….due to their worry that the manuscript will lose its sharp edge and might

The Scott collaborators of the Essen scientists reported above selected a group of patients, many of whom having suffered from measles, mainly as children. We consider this selection as counterproductive, since these patients should have been protected from the development of CHL. The very fact that they were not refutes the prevalent thesis that exposure of a child, aged 1–4 years, to MV infection protects the subject from CHL at a young-adult age [41]. The Scottish group may have had their own agenda, which might have unsettled the question raised by us. In addition, the antibody used by the Scott group is problematic: the clone 49–41 was purchased by us from Argene-Biosoft, Varilhes, France (# 11-045). It was not an anti-nucleoprotein antibody, as stated, and was positive in our lab for 37 of our 72 CHL cases (data not shown). Moreover, not unlike the German scientists, the Scotts used the GAPDH gene as a housekeeping gene, and we have already mentioned why it was not appropriate. I have to admit, that we not only considered ourselves as the minor partner, but that we were such indeed! We had been recurrently reassured by the German scientists and by then had our two names included in the manuscript as co-authors. However, when we read the finalized doublet articles (in addition, the article by Wilson et al., weighted heavily on the balance) [41], published in the International

discrepancy in favor of IHC was also obtained in our series (**Table 4**).

Journal of Cancer, we realized that we had made a huge mistake.

The days which followed the double publication were crucial, since we were not sure, as co-authors, whether we were entitled to carry out an "insider" censure. When we read the final paper, we grasped the experience in terms of a possible deception. In addition to the impediments mentioned above, we also noted that the housekeeping gene, the GAPDH, might not be an adequate choice at all, since we had shown that CHL is most probably of low MV-RNA abundance. In contrast, the Essen group had described in relation with GAPDH a large amount of RNA [40]. Moreover, of the seven Israeli cases, selected for the experiment in Germany, five had been shown by

us to present a faint MV-RNA positivity only (two) or were negative (three).

**146**

An independent putative evidence for a role of the MV in carcinogenesis is that part of the MV proteins that interacts with and stabilizes the Pirh2 protein, an ubiquitin E3 ligase, by preventing its ubiquitination. Pirh2 indeed negatively regulates p53 by targeting it for degradation in the proteasome.

Altogether, our criticism of the article, in which we took officially a part, is in principle that the German scientists carried out an investigation, which was by their declared intention, a duplication of our own research [3]. However, their study was anything, except a duplicate of ours! It stands out independently, with major flaws, notably a very strong selection of cases, the use of questionable primers of a housekeeping gene which is not appropriate for the expected amount of retrieved MV-RNA. It is of note that when confronted with our criticism regarding the manuscript, the German group seemed to agree, but did not modify the manuscript: "…for fear of weakening our data". Moreover, they insisted on mentioning the relevance of the findings to the CHL pathogenesis, which was not appropriate at this stage nor was it agreed upon [40]!

At this point, we insist that the impact of the European group refutation, on a possible role of MV in CHL, was extremely ruinous to our position and thesis. Moreover, its repercussions are felt to this day: for the reasons mentioned above, not a single investigator has accepted to engage in this project anymore, together with us or independently. By so doing, the two research groups have categorically "buried" our hypothesis. At the same time, we felt a release from the constraints which might have led us freeze in our position and we allowed ourselves again to indulge in the research concerning the measles virus and cancer. Therefore, we first wrote a review on our experience, as described above [42, 43].

## **6. New evidence**

We set out to delve into endometrial carcinoma (EC), looking for the footprints of the MV in this cancer. Our cohort of EC included 49 patients, 36 of whom had a known follow-up. Using IHC and several anti-MV antibodies, we disclosed that 26 of the 36 (72%) did express MV antigens in tumor cells, mainly in type I EC (**Figure 6**). In addition, 16/21 of the patients displayed positivity for MV-RNA by ISH. When type II EC was allied with MV, then deeper myometrial invasion and a higher mortality were evident [44].

Our next endeavor concerned the possible role of MV in lung cancer. Our interest was first raised by the probable relevance of Pirh2 in lung cancer. Indeed, Pirh2, an E3 ubiquitin ligase, was found to be expressed in lung cancer, in which it inhibits p53 [45]. It was later found that the P-protein of the MV inhibited the function of Pirh2 (see description in [48]). This indirect association between lung cancer, Pirh2 and p53, and MV led us to the following study. We identified 65 non-small cell lung cancers, for which FFPE tissues were available over 10 years. We limited our study to the IHC analysis with several anti-MV antibodies (**Figure 7**). Of the cases included, 54/65 were positive for MV antigens. These 54 positive cases were associated with older patient age, with positive Pirh2 expression, as well as with an improved survival [46].

The following study concerned breast cancer and the MV-IHC expression. We identified 131 breast cancer cases, with preserved FFPE tissues. Of these cases, 64% were positive for the MV antigens (**Figure 8**). Positive MV expression was associated with ER (*p* = 0.018); with Ki-67 < 40% (*p* = 0.029); with low or intermediate grade (*p* = 0.037); with age < 50 (*p* = 0.039); and with p53 overexpression (*p* = 0.049) (**Table 5**). It seems, therefore, that the MV may play a role in the development of this cancer [47].

We have speculated whether the MV phosphoprotein, through its relation with the Pirh2, ubiquitin ligase, by itself negatively related with the p53 expression, may have more relevance, *a priori,* than other MV antigens to the pathogenesis of the

#### **Figure 6.**

*Endometrial carcinoma—MV expression in tumor cells and in normal endometrium. a. Cytoplasmic HA-MV antigen is positive. b. Positive immunostain for NP-MV antigen. c. ISH positivity for NP-MV in tumoral glands. d. ISH positivity of surface and glandular normal epithelium of the case shown in c.*

MV-associated cancer [48]. We therefore purchased a robust anti-MV-phosphoprotein antibody (9H4): sc-101,356, Santa Cruz Biotechnology, and used it by Western blot, first, to determine whether we could find a variance between the different types of lung cancers and in addition to determine whether the normal lung tissues around the tumor did express the MV. But, the distribution of lung cancer types in our cohort was clearly in favor of adenocarcinoma, which expressed the P-MV in 11 patients (84.6%). This was also the rate of its expression in the normal lung tissue

**Figure 7.** *Lung cancer—positive for MV. a. Adenocarcinoma. b. Squamous cell carcinoma (IHC with DAB × 240).*

**149**

**Table 5.**

**Figure 8.**

*(immunoperoxidase with DAB × 180).*

**Demographics**

**Pathology**

**Immunohistochemistry**

*Breast cancer is positive for measles virus antigens. a and c. Negative controls. b and d. Positive immunostains* 

Age <50 years >50 years Ethnicity Jewish Bedouin-Arab Birth place Israel/Europe/America Asia/Africa

Tumor diameter <10 mm >10 mm Axillary LNs None Involvement DCIS component Yes No Grade Low/intermediate G1–2 High G3

ER Positive Negative PR Positive Negative HER2 Positive Negative Ki67 Positive Negative p53 Positive Negative NP-MV Positive Negative HA-MV Positive Negative

*Distribution of demographics, clinics, pathology, and MV expression in breast cancer patients.*

**Variable Variable distribution**

*A Role for the Measles Virus in Oncology DOI: http://dx.doi.org/10.5772/intechopen.84342* *A Role for the Measles Virus in Oncology DOI: http://dx.doi.org/10.5772/intechopen.84342*

#### **Figure 8.**

*Viruses and Viral Infections in Developing Countries*

MV-associated cancer [48]. We therefore purchased a robust anti-MV-phosphoprotein antibody (9H4): sc-101,356, Santa Cruz Biotechnology, and used it by Western blot, first, to determine whether we could find a variance between the different types of lung cancers and in addition to determine whether the normal lung tissues around the tumor did express the MV. But, the distribution of lung cancer types in our cohort was clearly in favor of adenocarcinoma, which expressed the P-MV in 11 patients (84.6%). This was also the rate of its expression in the normal lung tissue

*Endometrial carcinoma—MV expression in tumor cells and in normal endometrium. a. Cytoplasmic HA-MV antigen is positive. b. Positive immunostain for NP-MV antigen. c. ISH positivity for NP-MV in tumoral* 

*glands. d. ISH positivity of surface and glandular normal epithelium of the case shown in c.*

*Lung cancer—positive for MV. a. Adenocarcinoma. b. Squamous cell carcinoma (IHC with DAB × 240).*

**148**

**Figure 7.**

**Figure 6.**

*Breast cancer is positive for measles virus antigens. a and c. Negative controls. b and d. Positive immunostains (immunoperoxidase with DAB × 180).*


**Table 5.**

*Distribution of demographics, clinics, pathology, and MV expression in breast cancer patients.*


#### **Table 6.**

*Western blotting using a phosphoprotein-MV antibody.*

#### **Figure 9.**

*Western blotting with an antiphosphoprotein-MV antibody on fresh frozen tissues of non-small cell lung cancer. Most cases show positive expression both in tumor tissue (T) and in nonadjacent nonneoplastic lung tissue (H). In a few cases, the MV expression is found in the tumor only.*


#### **Table 7.**

*Tissue microarray using a phosphoprotein-MV antibody.*

(**Table 6**; **Figure 9**). The second step was to look for the expression of the P-MV in a tissue microarray (one tissue microarray slide). We compared 10 lung cancers, 13 colon cancers, 14 stomach cancers, and 15 breast cancers. Breast cancer expressed the MV antigen in 75% in the nucleus and 50% in the cytoplasm. Stomach cancers expressed the antigen in 73.7% in the nucleus and 21% in the cytoplasm. Colon cancers expressed the MV in 68.4% in the nucleus (**Table 7**). The tissue microarray study had demonstrated a nuclear expression of MV antigens, of which we have not been aware before. This may be due on the one hand to the tiny size of the tissue section. On the other hand, it may be specific to the present anti-P-MV antibody. In any case, various immunostains have shown a marked shuttling of antigens between cytoplasm and nucleus in various tissue sections and with varied antigens [49] (**Table 7**).

Our experience with melanoma and benign melanocytic nevi is shown in **Table 8**. The table shows the expression of several MV antigens and of Pirh2. The expression of NP-MV is the strongest and resembles that of Pirh2. A similar trend, but with a more limited expression, is found in benign melanocytic nevi. It is of note that the expression of the P-protein in melanomas is the mirror image of that of the NP-MV antigen. This was a preliminary exercise and no follow-up is yet

**151**

at least in CHL [51].

desmoid tumor.

**Table 8.**

*Melanoma and measles virus.*

*A Role for the Measles Virus in Oncology DOI: http://dx.doi.org/10.5772/intechopen.84342*

available. The positivity of 23 benign nevi for NP-MV should be investigated to

**Antigen Level Melanoma Benign nevi P value**

Neg. 10 1 0.16

Neg. 36 12 0.37

Neg. 3 1 1.00

Neg. 12 6 0.79

NP-MV Pos. 46 23

P-protein Pos. 19 10

HA-MV Pos. 19 7

Pirh-2 Pos. 44 19

At this stage, we were motivated to determine whether some categories of tumors might be uniformly negative for MV antigens. One limitation is that these tumors were often showed with a restricted sampling. In our original study, we included 25 NHLs by IHC and 7 cases of non-Hodgkin lymphoma by RT-PCR. They were all negative for MV. Only later did we find an interest in ALK-positive anaplastic large cell lymphomas in this regard. In contrast, these lymphomas were predominantly positive for MV. A large scale study of seminomas showed negativity for the MV antigens. Glioblastomas and mesotheliomas were negative for the antigens (Samuel Ariad, personal communication). I later examined small samples of the following tumors which were also negative for the MV antigen: Merkel-cell tumors, hepatocellular carcinoma, malignant mixed mesodermal tumors, and

In 2010, we revised and expanded a prior study on the role of apoptosis in CHL

[50, 51]. This was undertaken in the light of a consensual point of view: it has been stated that apoptosis in CHL is arrested; this is carried out not only with the help of the NF-κB machinery but also through the expression of EBV-LMP1 that inhibits apoptosis [52–55]. The consensus further determines that this apoptosis hindrance is necessary, since tumor cells in CHL are B cells which cannot express the B-cell receptor (in physiologic conditions, such cells have a strong tendency to die in apoptosis). Thus, apoptosis inhibition is a pre-condition for the survival of H/RS cells [56]. However, in our revision, we found inhibition of H/RS cells apoptosis in 55% of the 217 cases only. Our findings also suggested that NF-κB, specifically the p65 protein, as well as EBV/LMP1 expression did not correlate with apoptosis arrest, in contrast with the consensus view [57]. Of note, it has been shown that although the presence of canonical NF-κB has been considered to be implied, it is the noncanonical NF-κB, which is of predominant relevance in CHL [51]. In the revision we have shown that the most significant association of H/RS cell apoptosis was with p53, the negative expression of which was related with a high apoptotic index (*p* = 0.001). We looked for a link between positive MV expression and factors related with apoptosis. Associations with apoptotic index less than median (*p* = 0.005), with MDM2-positive (*p* = 0.028) and with IκBpositive (*p* = 0.0001), were displayed. Viruses have a propensity to regulate the apoptotic mechanisms of the infected cell. We have hereby proposed a pathogenic mechanism, involving the MV and that is effective in the modulation of apoptosis,

exclude evidence of a pre-malignant condition.

*A Role for the Measles Virus in Oncology DOI: http://dx.doi.org/10.5772/intechopen.84342*


#### **Table 8.**

*Viruses and Viral Infections in Developing Countries*

*Western blotting using a phosphoprotein-MV antibody.*

*In a few cases, the MV expression is found in the tumor only.*

*Tissue microarray using a phosphoprotein-MV antibody.*

**Table 6.**

**Figure 9.**

**Table 7.**

(**Table 6**; **Figure 9**). The second step was to look for the expression of the P-MV in a tissue microarray (one tissue microarray slide). We compared 10 lung cancers, 13 colon cancers, 14 stomach cancers, and 15 breast cancers. Breast cancer expressed the MV antigen in 75% in the nucleus and 50% in the cytoplasm. Stomach cancers expressed the antigen in 73.7% in the nucleus and 21% in the cytoplasm. Colon cancers expressed the MV in 68.4% in the nucleus (**Table 7**). The tissue microarray study had demonstrated a nuclear expression of MV antigens, of which we have not been aware before. This may be due on the one hand to the tiny size of the tissue section. On the other hand, it may be specific to the present anti-P-MV antibody. In any case, various immunostains have shown a marked shuttling of antigens between cytoplasm and nucleus in various tissue sections and with varied

*Western blotting with an antiphosphoprotein-MV antibody on fresh frozen tissues of non-small cell lung cancer. Most cases show positive expression both in tumor tissue (T) and in nonadjacent nonneoplastic lung tissue (H).* 

**Organ Nucleus Cytoplasm Stroma Nucleus Cytoplasm Stroma** Lung 10 (52.6%) 2 (11.1%) 0 9 (47.4%) 16 (88.9%) 18 (100%) Colon 13 (68.4%) 7 (36.8%) 4 (23.5%) 6 (31.6%) 12 (63.2%) 13 (76.5%) Stomach 14 (73.7%) 4 (21%) 0 5 (26.3%) 15 (79%) 19 (100%) Breast 15 (75%) 10 (50%) 2 (10%) 5 (25%) 10 (50%) 18 (90%)

**Positive staining % Negative staining %**

**Pathology Positive (%) Negative (%)** Adenocarcinoma 11 (84.6%) 2 (15.4%) Squamous cell carcinoma 3 (100%) 0 Large cell carcinoma 1 (100%) 0 Bronchioloalveolar carcinoma 1 (100%) 0

Our experience with melanoma and benign melanocytic nevi is shown in **Table 8**. The table shows the expression of several MV antigens and of Pirh2. The expression of NP-MV is the strongest and resembles that of Pirh2. A similar trend, but with a more limited expression, is found in benign melanocytic nevi. It is of note that the expression of the P-protein in melanomas is the mirror image of that of the NP-MV antigen. This was a preliminary exercise and no follow-up is yet

**150**

antigens [49] (**Table 7**).

*Melanoma and measles virus.*

available. The positivity of 23 benign nevi for NP-MV should be investigated to exclude evidence of a pre-malignant condition.

At this stage, we were motivated to determine whether some categories of tumors might be uniformly negative for MV antigens. One limitation is that these tumors were often showed with a restricted sampling. In our original study, we included 25 NHLs by IHC and 7 cases of non-Hodgkin lymphoma by RT-PCR. They were all negative for MV. Only later did we find an interest in ALK-positive anaplastic large cell lymphomas in this regard. In contrast, these lymphomas were predominantly positive for MV. A large scale study of seminomas showed negativity for the MV antigens. Glioblastomas and mesotheliomas were negative for the antigens (Samuel Ariad, personal communication). I later examined small samples of the following tumors which were also negative for the MV antigen: Merkel-cell tumors, hepatocellular carcinoma, malignant mixed mesodermal tumors, and desmoid tumor.

In 2010, we revised and expanded a prior study on the role of apoptosis in CHL [50, 51]. This was undertaken in the light of a consensual point of view: it has been stated that apoptosis in CHL is arrested; this is carried out not only with the help of the NF-κB machinery but also through the expression of EBV-LMP1 that inhibits apoptosis [52–55]. The consensus further determines that this apoptosis hindrance is necessary, since tumor cells in CHL are B cells which cannot express the B-cell receptor (in physiologic conditions, such cells have a strong tendency to die in apoptosis). Thus, apoptosis inhibition is a pre-condition for the survival of H/RS cells [56]. However, in our revision, we found inhibition of H/RS cells apoptosis in 55% of the 217 cases only. Our findings also suggested that NF-κB, specifically the p65 protein, as well as EBV/LMP1 expression did not correlate with apoptosis arrest, in contrast with the consensus view [57]. Of note, it has been shown that although the presence of canonical NF-κB has been considered to be implied, it is the noncanonical NF-κB, which is of predominant relevance in CHL [51]. In the revision we have shown that the most significant association of H/RS cell apoptosis was with p53, the negative expression of which was related with a high apoptotic index (*p* = 0.001). We looked for a link between positive MV expression and factors related with apoptosis. Associations with apoptotic index less than median (*p* = 0.005), with MDM2-positive (*p* = 0.028) and with IκBpositive (*p* = 0.0001), were displayed. Viruses have a propensity to regulate the apoptotic mechanisms of the infected cell. We have hereby proposed a pathogenic mechanism, involving the MV and that is effective in the modulation of apoptosis, at least in CHL [51].
