**2. Materials and methods**

The basic part of our study (2003–2004) concerned 154 patients with CHL diagnosed between 1975 and 2000 at the Soroka University Medical Centre in Beer-Sheva, Israel, whose tissues were available for research. Only pretreatment biopsies with confirmed histological type, by two hematopathologists were included. Clinical data were retrieved from the patients' charts and when those were not found, the details were searched in the Israel Population Registry by linkage.

#### **2.1 Immunohistochemistry**

Immunophenotyping was performed on formalin-fixed, paraffin-embedded (FFPE) tissues by the avidin-biotin peroxidase complex method and the Vectastain kit by Vector Laboratories (Burlingame, CA, USA) as described. The EBV/LMP1 antibody was purchased from Dako (Glostrup, Denmark); the anti-MV antibodies were either commercial: anti-nucleoprotein (NP)-MV; antihemagglutinin (HA)-MV; anti-matrix (M)-MV; and anti-phosphoprotein (P)-MV were purchased from Chemicon International Inc. (Temecula, CA, USA). Further experimental antibodies were a gift from Birrer et al. [30]: anti-NP, H14; and from Schneider-Schaulies et al. [31]: anti-NP, L39/22; L39/61 or anti-HA antibodies, K83; L77. The antibody specificity had been demonstrated by Western blotting. The staining was considered positive if 10% of H/RS cells or more were positive. For stringency purposes, we requested positivity for two MV antigens, for a case to be considered positive. A murine neuroblastoma cell line permanently infected by MV was used as a positive control; the same cell line, but non-infected was used as a negative control. These cell lines were also the controls used for the RT-PCR and ISH assays. Twenty-five cases of non-Hodgkin lymphoma were also tested by IHC.

#### **2.2 Reverse transcriptase-polymerase chain reaction**

Total RNA was obtained by extraction of snap-frozen lymph node samples from part of our patients, when available. For this goal, EZ-RNA Total RNA Isolation Kit (Biological Industries Co., Beit Ha-Emek, Israel) was employed, using the manufacturer's instructions. To each sample, 40 U of RNA-guard (Promega, Madison, WI, USA) were added. One step RT-PCR was performed with Ready To Go ™ RT-PCR beads (Amersham Pharmacia Biotech Europe GbmH, Freiburg, Germany). A measure of 3 μl of RNA (2-6 μg) was used for each reaction. For the primers used in this study, see Table 1 from Ref. [3]. Nested PCR was carried out, using SUPER-NOVA DNA polymerase (Roche Molecular Biochemicals, Mannheim, Germany).

**141**

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

related with low amounts of RNA.

(*GCTGGAAGCTGACACCTTTC)*, labeled [γ-

**2.3** *In situ* **hybridization on paraffin sections**

**2.4 Comparison between IHC, RT-PCR, and ISH**

**2.5 Clinicopathological correlations**

exact test was used as appropriate.

**3. Preliminary results**

single NP-MV or HA-MV antibody was considered sufficient.

Total RNA was also retrieved from several FFPE tissues from CHL patients, using the Paraffin Block RNA Isolation Kit (Ambion Inc., The RNA Company, Austin, TX. USA) and RT-PCR followed by nested PCR carried out with 10 μl RNA, as described above. As controls, we used seven cases of non-Hodgkin lymphomas, one CHL which was negative for MV by IHC. As housekeeping genes, we used β-actin and protein phosphatase 2C β (pp2Cβ) considered appropriate for experiments

To confirm our RT-PCR findings, the cDNA products of the nested PCR were blotted to GeneScreen (NEN Research Products, Boston, MA, USA) and hybridized to either an NP-MV probe (*CAATCCCTGGAGATTCCTCA)* or to a HA-MV probe

The digoxigenin (DIG)-labeled antisense and sense probes to MV-specific mRNAs were prepared by linearizing the plasmids containing NP-MV including positions 105–586, and those including HA-MV encompassing positions 8147– 8482 in PGEM-T Easy Vectors (Promega), with *Sal*I or *Sac*II. *In vitro* transcription was performed with the DIG-RNA Labeling kit, with SP6 for NP antisense and HA sense probes and T7 for NP sense and HA antisense DIG-labeled probes. The quality of the probes was determined by dot blotting, using a DIG-DNA detection kit (Roche Molecular Biochemicals), according to the manufacturer instructions, with minor modifications. *In situ* hybridization was performed, using Ogata's technique with certain modifications [32]. Paraffin sections were deparaffinized, rehydrated, and submitted to microwave in the presence of 10 mM MgCl2 buffer (pH 6) for 5 min at 750 W. Sections were allowed to cool down for 20 min and then digested with 20 μg/ml proteinase K for 10 min at 37**°**C. They were then fixed in paraformaldehyde in 0.1 M phosphate buffer containing 50% formamide, 10 mM Tris-HCl (pH 7.6), 200 μg/ml tRNA, 1 x Denhardt's solution, 10% dextran sulphate, 600 mM NaCl, 0.25% SDS, and 1 mM EDTA, pH 8.0. Hybridized DIG-labeled probes were detected by the DIG-Nucleic Acid Detection kit (Roche Molecular Biochemicals), followed by blocking with 1.5% of the blocking reagent included in the kit. The color reaction was stopped with 10 mM Tris-HCl (pH 7.6) and 1 mM EDTA. Sections were then fixed in 4% paraformaldehyde in phosphate-buffered saline and stained with hematoxylin. In addition to the neuroblastoma cell lines used as positive and negative controls, CHL cases negative for MV by IHC and NHL served as controls.

This was performed on a sampling of cases. For this purpose, IHC positivity to a

Emphasis was given to MV IHC and to EBV-LMP1 expression, as RT-PCR and ISH disclosed much less results. For contingency table analysis, the χ2 or Fisher's

The lymph node tissues of 82 of 154 CHL patients were positive for at least two MV antigens (**Figure 3**). Neutralization of immunostaining was made possible by

32 P] ATP (Amersham).

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

*Viruses and Viral Infections in Developing Countries*

be due also to the lymph node mode of fixation [29].

**2. Materials and methods**

**2.1 Immunohistochemistry**

At the time our thesis was formulated, the type of MV neural infection known as subacute sclerosing panencephalitis (SSPE) was considered one of the very few persistent MV infections, most being CNS slow viral infections. However, it was difficult to grasp a possible association between MV and cancer, which would not account for the long lapse of time necessary for the malignant transformation. In that context, the absence of a persistent MV infection might be counterproductive. The alternative being that against all odds, MV may be oncogenic, and the MV is not among the consensually recognized oncogenic viruses! An additional clue to MV causing a persistent infection beyond the CNS is the absence of membrane expression of hemagglutinin MV antigens, although this lack of expression might

The basic part of our study (2003–2004) concerned 154 patients with CHL diagnosed between 1975 and 2000 at the Soroka University Medical Centre in Beer-Sheva, Israel, whose tissues were available for research. Only pretreatment biopsies with confirmed histological type, by two hematopathologists were included. Clinical data were retrieved from the patients' charts and when those were not found, the details were searched in the Israel Population Registry by linkage.

Immunophenotyping was performed on formalin-fixed, paraffin-embedded

Total RNA was obtained by extraction of snap-frozen lymph node samples from part of our patients, when available. For this goal, EZ-RNA Total RNA Isolation Kit (Biological Industries Co., Beit Ha-Emek, Israel) was employed, using the manufacturer's instructions. To each sample, 40 U of RNA-guard (Promega, Madison, WI, USA) were added. One step RT-PCR was performed with Ready To Go ™ RT-PCR beads (Amersham Pharmacia Biotech Europe GbmH, Freiburg, Germany). A measure of 3 μl of RNA (2-6 μg) was used for each reaction. For the primers used in this study, see Table 1 from Ref. [3]. Nested PCR was carried out, using SUPER-NOVA

DNA polymerase (Roche Molecular Biochemicals, Mannheim, Germany).

(FFPE) tissues by the avidin-biotin peroxidase complex method and the Vectastain kit by Vector Laboratories (Burlingame, CA, USA) as described. The EBV/LMP1 antibody was purchased from Dako (Glostrup, Denmark); the anti-MV antibodies were either commercial: anti-nucleoprotein (NP)-MV; antihemagglutinin (HA)-MV; anti-matrix (M)-MV; and anti-phosphoprotein (P)-MV were purchased from Chemicon International Inc. (Temecula, CA, USA). Further experimental antibodies were a gift from Birrer et al. [30]: anti-NP, H14; and from Schneider-Schaulies et al. [31]: anti-NP, L39/22; L39/61 or anti-HA antibodies, K83; L77. The antibody specificity had been demonstrated by Western blotting. The staining was considered positive if 10% of H/RS cells or more were positive. For stringency purposes, we requested positivity for two MV antigens, for a case to be considered positive. A murine neuroblastoma cell line permanently infected by MV was used as a positive control; the same cell line, but non-infected was used as a negative control. These cell lines were also the controls used for the RT-PCR and ISH assays. Twenty-five cases of non-Hodgkin lymphoma were also

**2.2 Reverse transcriptase-polymerase chain reaction**

**140**

tested by IHC.

Total RNA was also retrieved from several FFPE tissues from CHL patients, using the Paraffin Block RNA Isolation Kit (Ambion Inc., The RNA Company, Austin, TX. USA) and RT-PCR followed by nested PCR carried out with 10 μl RNA, as described above. As controls, we used seven cases of non-Hodgkin lymphomas, one CHL which was negative for MV by IHC. As housekeeping genes, we used β-actin and protein phosphatase 2C β (pp2Cβ) considered appropriate for experiments related with low amounts of RNA.

To confirm our RT-PCR findings, the cDNA products of the nested PCR were blotted to GeneScreen (NEN Research Products, Boston, MA, USA) and hybridized to either an NP-MV probe (*CAATCCCTGGAGATTCCTCA)* or to a HA-MV probe (*GCTGGAAGCTGACACCTTTC)*, labeled [γ-32 P] ATP (Amersham).

#### **2.3** *In situ* **hybridization on paraffin sections**

The digoxigenin (DIG)-labeled antisense and sense probes to MV-specific mRNAs were prepared by linearizing the plasmids containing NP-MV including positions 105–586, and those including HA-MV encompassing positions 8147– 8482 in PGEM-T Easy Vectors (Promega), with *Sal*I or *Sac*II. *In vitro* transcription was performed with the DIG-RNA Labeling kit, with SP6 for NP antisense and HA sense probes and T7 for NP sense and HA antisense DIG-labeled probes. The quality of the probes was determined by dot blotting, using a DIG-DNA detection kit (Roche Molecular Biochemicals), according to the manufacturer instructions, with minor modifications. *In situ* hybridization was performed, using Ogata's technique with certain modifications [32]. Paraffin sections were deparaffinized, rehydrated, and submitted to microwave in the presence of 10 mM MgCl2 buffer (pH 6) for 5 min at 750 W. Sections were allowed to cool down for 20 min and then digested with 20 μg/ml proteinase K for 10 min at 37**°**C. They were then fixed in paraformaldehyde in 0.1 M phosphate buffer containing 50% formamide, 10 mM Tris-HCl (pH 7.6), 200 μg/ml tRNA, 1 x Denhardt's solution, 10% dextran sulphate, 600 mM NaCl, 0.25% SDS, and 1 mM EDTA, pH 8.0. Hybridized DIG-labeled probes were detected by the DIG-Nucleic Acid Detection kit (Roche Molecular Biochemicals), followed by blocking with 1.5% of the blocking reagent included in the kit. The color reaction was stopped with 10 mM Tris-HCl (pH 7.6) and 1 mM EDTA. Sections were then fixed in 4% paraformaldehyde in phosphate-buffered saline and stained with hematoxylin. In addition to the neuroblastoma cell lines used as positive and negative controls, CHL cases negative for MV by IHC and NHL served as controls.

#### **2.4 Comparison between IHC, RT-PCR, and ISH**

This was performed on a sampling of cases. For this purpose, IHC positivity to a single NP-MV or HA-MV antibody was considered sufficient.

#### **2.5 Clinicopathological correlations**

Emphasis was given to MV IHC and to EBV-LMP1 expression, as RT-PCR and ISH disclosed much less results. For contingency table analysis, the χ2 or Fisher's exact test was used as appropriate.

### **3. Preliminary results**

The lymph node tissues of 82 of 154 CHL patients were positive for at least two MV antigens (**Figure 3**). Neutralization of immunostaining was made possible by

incubation of the primary anti-NP-MV antibody with the corresponding NP-MV peptide (**Figure 3b**). This assay confirms the specificity of the anti-NP-MV antibody in this context. Twenty-five cases of non-Hodgkin lymphoma (NHL) were totally negative for MV antigens.

A Southern blotting of the nested PCR shows several bands, some stronger, others faint, disclosing more hemagglutinin (HA, H) MV positivity, as compared with NP-MV positivity (**Figure 4**). Samples from 7 NHL were negative. Thus, in our laboratory, HA-MV RNA was positive in 4 of 15 cases, while only 2 of 16 NP-MV RNAs were expressed by the Southern blot in CHL.

Upon sequencing and alignment of three of the experiment products, we displayed an almost complete homology with the control (see [3] for details on the homology).

*In situ* hybridization on paraffin sections shows a strong cytoplasmic staining in the positive control (persistently MV infected murine neuroblastoma cell line) (upper left). The same cell line uninfected by MV was negative. An H/RS cell shows strong cytoplasmic positivity with NP-MV (lower left). Positive granular staining of several H/RS cells is disclosed in the right lower quadrant (**Figure 5**). Altogether, 2 of 7 CHL sections submitted for ISH were positive for HA-MV RNAs, while 8 of 21 were positive for NP-MV RNAs.

#### **Figure 3.**

*Classic Hodgkin lymphoma-nucleoprotein-MV expression. a. Positive cytoplasmic immunostain of Hodgkin/ Reed-Sternberg cells. b. Immunostaining following neutralization of antibody with a relevant MV peptide (Immunoperoxidase with DAB × 320).*

**143**

**Figure 5.**

**Figure 4.**

*of expression of MV in non-Hodgkin lymphomas.*

*Southern blotting based on cDNA from RT-PCR: A. A few cases of CHL show bands positive for* 

*hemagglutinin-MV (PC—positive control). B. Isolated bands, positive for nucleoprotein-MV. C and D. Absence* 

*In situ hybridization with MV-RNA. a. Positive control, using a murine, MV-transfected cell line. b. Negative control, with a non-transfected cell line. c. A Hodgkin/Reed-Sternberg cell disclosing positive cytoplasm for* 

*MV-NP-RNA. d. H/RS cells showing a granular cytoplasmic positivity for MV-HA-RNA.*

*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 4.**

*Viruses and Viral Infections in Developing Countries*

RNAs were expressed by the Southern blot in CHL.

totally negative for MV antigens.

were positive for NP-MV RNAs.

homology).

incubation of the primary anti-NP-MV antibody with the corresponding NP-MV peptide (**Figure 3b**). This assay confirms the specificity of the anti-NP-MV antibody in this context. Twenty-five cases of non-Hodgkin lymphoma (NHL) were

A Southern blotting of the nested PCR shows several bands, some stronger, others faint, disclosing more hemagglutinin (HA, H) MV positivity, as compared with NP-MV positivity (**Figure 4**). Samples from 7 NHL were negative. Thus, in our laboratory, HA-MV RNA was positive in 4 of 15 cases, while only 2 of 16 NP-MV

Upon sequencing and alignment of three of the experiment products, we displayed an almost complete homology with the control (see [3] for details on the

*In situ* hybridization on paraffin sections shows a strong cytoplasmic staining in the positive control (persistently MV infected murine neuroblastoma cell line) (upper left). The same cell line uninfected by MV was negative. An H/RS cell shows strong cytoplasmic positivity with NP-MV (lower left). Positive granular staining of several H/RS cells is disclosed in the right lower quadrant (**Figure 5**). Altogether, 2 of 7 CHL sections submitted for ISH were positive for HA-MV RNAs, while 8 of 21

*Classic Hodgkin lymphoma-nucleoprotein-MV expression. a. Positive cytoplasmic immunostain of Hodgkin/ Reed-Sternberg cells. b. Immunostaining following neutralization of antibody with a relevant MV peptide* 

**142**

**Figure 3.**

*(Immunoperoxidase with DAB × 320).*

*Southern blotting based on cDNA from RT-PCR: A. A few cases of CHL show bands positive for hemagglutinin-MV (PC—positive control). B. Isolated bands, positive for nucleoprotein-MV. C and D. Absence of expression of MV in non-Hodgkin lymphomas.*

#### **Figure 5.**

*In situ hybridization with MV-RNA. a. Positive control, using a murine, MV-transfected cell line. b. Negative control, with a non-transfected cell line. c. A Hodgkin/Reed-Sternberg cell disclosing positive cytoplasm for MV-NP-RNA. d. H/RS cells showing a granular cytoplasmic positivity for MV-HA-RNA.*

A clinicopathological correlation was based on the immunophenotypic findings, as the specificity of the antibodies had been sustained by Western blotting and since, in each case, the immunostain had been confirmed with more than two antibodies. Last, the immunohistochemical (IHC) study had released the most manageable results.

**Table 1** shows a statistically significant expression of MV antigens in female patients, in nodular sclerosis (NS) CHL, as compared with mixed cellularity (MC) CHL. A significant correlation was exhibited between negative CD15 and p53, and the negative expression of MV. **Table 2** displays a statistically significant expression of MV antigens compounded by the expression of EBV/LMP1, in NS-CHL, significantly more than that in MC-CHL, in early clinical stages, with apoptotic index lower than median and with the positive expression of MDM2.

A multivariable logistic regression model of the association of prognostic factors with dying from CHL, in 89 patients, is shown. Stage IV, as compared with the remainder, carried a significantly poorer prognosis, thus confirming that our cohort was representative. The compound expression of MV and EBV, specifically of MV+; EBV−, as well as MV−; EBV− was displayed as poor risk factors in our patients (**Table 3**).


#### **Table 1.**

*Associations of classic Hodgkin lymphoma with the measles virus.*


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**4. Interim discussion**

**Table 3.**

the malignancy.

**5. The refutation episode**

The results of the first part of our study (2004) are summarized [3]. The association between MV and CHL, in a descriptive investigation, does not contain, so far, causal elements. This linkage is sustained by older epidemiologic studies describing brain and spinal cord tumors, as well as CHL, which followed exposure to the MV around the time of birth [33, 34]. They also suggest sporadic cases of CHL regression, occurring after acute measles infection or an MV vaccine [35–38]. Moreover, viral childhood infections,

*Multivariable logistic regression model of association of prognostic factors with dying of HL in 89 patients.*

**Factor Odds ratio 95% CI** *p* **Value** Gender 1.32 0.34–5.18 0.69 Stage **13.67** 2.3–80.5 0.004 Bcl-2 0.34 0.08–1.44 0.34 MV+; EBV− **10.05** 0.98–103.47 0.05 MV−; EBV+ 1.53 0.07–35.44 0.79 MV−; EBV− **15.2** 1.05–220.3 0.04

Early clinical correlations exhibit more females, more NS-CHL, and more early stage cases, among patients with positive MV expression in their lymph node tissues. Nevertheless, positive MV expression in CHL seems to be related with a worse prognosis. One cannot refute the loss of the advantage conferred to the above variables by an MV infection, as expressed in the H/RS cells of CHL. Any mechanism involving the MV in CHL, if uncovered, should correlate with a worse outcome of

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

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

including measles, have been reported to protect from CHL occurrence [39].

available to him, mainly those which showed an MV-antigen positivity.

RNA quality and the classic H/RS cell features [40].

#### **Table 2.**

*Hodgkin lymphoma—association with measles and Epstein-Barr viruses.*

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


**Table 3.**

*Viruses and Viral Infections in Developing Countries*

manageable results.

A clinicopathological correlation was based on the immunophenotypic findings, as the specificity of the antibodies had been sustained by Western blotting and since, in each case, the immunostain had been confirmed with more than two antibodies. Last, the immunohistochemical (IHC) study had released the most

**Table 1** shows a statistically significant expression of MV antigens in female patients, in nodular sclerosis (NS) CHL, as compared with mixed cellularity (MC) CHL. A significant correlation was exhibited between negative CD15 and p53, and the negative expression of MV. **Table 2** displays a statistically significant expression of MV antigens compounded by the expression of EBV/LMP1, in NS-CHL, significantly more than that in MC-CHL, in early clinical stages, with apoptotic index

A multivariable logistic regression model of the association of prognostic factors with dying from CHL, in 89 patients, is shown. Stage IV, as compared with the remainder, carried a significantly poorer prognosis, thus confirming that our cohort was representative. The compound expression of MV and EBV, specifically of MV+; EBV−, as well as MV−; EBV− was displayed as poor risk factors in our patients (**Table 3**).

Gender F 40 (**64.5**) 28 (35.5) 0.036 M 42 (47.2) 47 (52.8) Subtype NS 58 (**68.2**) 27 (31.8) 0.0013 MC 21 (40.4) 31 (59.6) CD15 pos 77 (59.7) 52 (40.3) 0.015 neg 4 (26.7) 11 (**73.3**) p53 pos 72 (61.5) 45 (38.5) 0.017 neg 5 (31.3) 11 (**68.8**)

Subtype NS 45 (**86.5**) 7 (13.5) 0.00008 MC 11 (44) 14 (56) Stage I–IIA 22 (**84.6**) 4 (15.4) 0.023 IIB–IVB 22 (57.9) 16 (42.1) BCL-2 Pos. 31 (**93.9**) 2 (6.1) 0.0001 Neg. 27 (54) 23 (46) MDM-2 Pos. 36 (**87.8**) 5 (12.2) 0.0013 Neg. 18 (54.5) 15 (45.5)

Apop index <Median 31 (**81.6**) 7 (18.4) 0.024 >Median 22 (42.1) 16 (57.9)

*Hodgkin lymphoma—association with measles and Epstein-Barr viruses.*

*Associations of classic Hodgkin lymphoma with the measles virus.*

**MV+; LMP− MV−; LMP+** *p* **Value**

**MV+ MV−** *p* **Value**

n (%) n (%)

lower than median and with the positive expression of MDM2.

Total 82 (54.3) 69 (45.7)

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**Table 2.**

**Table 1.**

*Multivariable logistic regression model of association of prognostic factors with dying of HL in 89 patients.*
