408 N. E. GARDIN

by histological or cytological tests, with a deficit of cellular or humoral immunity demonstrated by laboratory tests, and with adequate renal and hepatic function values (respectively: serum creatinine 1.5 mg/dL or lower, serum bilirubin 2 mg/dL or lower). Other inclusion criteria were the absence of chemotherapy, radiotherapy, immuno- therapy with corticosteroids or other immunosuppres- sive drugs, or any other experimental treatment in the 30 days before study entry, and the absence of granulocyte or granulocyte-macrophage colony-stimulating factors given in the previous 10 days. Patients were ineligible if they had had a recent positive pregnancy test, were breast-feeding, there was a possibility of a future preg- nancy, or if they had a psychiatric or neurological disor- der including dementia that could affect the compliance with the protocol.

Laboratory evaluation. Patients underwent laboratory analysis after clinical evaluation. Tests included white blood cell count, T lymphocyte count, CD4+ and CD8+ T cell subsets, plasma concentration of complement com- ponent C3 and C4, measurement of serum immunoglobulin (Ig) A, IgG and IgM, and then intradermal tests of delayed hypersensitivity with antigens inoculation (candidin, trichophytin and purified protein derivative – PPD). Skin test indurations were measured 48 to 72 h after inoculation, and after this VA treatment began. All the tests were performed before the VA treatment and 2 or 3 days after. Then 4 weeks after the last dose of VA, blood tests were performed again. Skin tests were made only twice because of sensitization risks.

VA treatment. Ampoules of 20 mg of VA Qu were sup- plied by Weleda do Brasil Laboratório & Farmácia Ltda (São Paulo, Brazil). The mistletoe extract Viscum album Qu (Quercus) 20 mg is an aqueous sterile preparation derived from Viscum album L. grown on oak and fermented with Lactobacillus plantarum. VA was subcutaneously injected in the abdominal or gluteal region, twice a week, with an interval of 3 or 4 days; a total of seven injections. All patients were monitored weekly by the responsible investigator and adverse events were checked closely at each visit. Common terminology criteria for adverse events were used to classify reactions associated with the use of VA (National Cancer Institute, 2003).

RESULTS

Four patients were enrolled in this study and their char- acteristics are shown in Table 1. The first patient was also diagnosed with squamous cell carcinoma of the epiglottis, stage II, treated with surgery and radiotherapy

24 months before the VA tests; and basal cell carci- noma of the nose and upper limb, treated with surgery 2 months before the VA tests.

The second patient was the only smoker (8 cigarettes/ day for 20 years), and the fourth patient was the only one taking other drugs during the VA tests (atenolol 100 mg and nifedipine 40 mg daily for hypertension). This patient had to be admitted to the hospital 2 weeks after the second test because of a new chemotherapy cycle and was not submitted to the third test.

Immunological response

Tables 2–5 show all laboratory parameters examined before the VA use (‘before’), 2 or 3 days after the VA treatment (‘second tests’) and 4 weeks after the last dose of VA (‘third tests’). Values out of the normal range are in bold type. The fourth patient was not submitted to PPD for a second test because he had a strong posi- tive reaction to the first. The first patient showed the best response, resulting in the enhancement of all analysed parameters apart from the intradermal tests. Similarly, the fourth patient showed an increase in all indices except trichophytin inoculation. All parameters, which were initially below the normal range, had increased by the final tests, except for some intradermal reactions (three skin tests of the first and second patients and one of the third and fourth patients).

Adverse events

The VA treatment was well tolerated and were adminis- tered in the outpatient setting. Systemic symptoms did not occur. All patients reported mild induration on the injection site (Table 6). Only one patient had moderate pain and erythema. All toxicities reversed spontaneously without sequela, generally after one day. There was no need to use symptomatic medication, interrupting VA, or changing VA dose or frequency.

DISCUSSION

The present study investigated immune stimulation by VA in four cancer patients who had immune impairment. These patients, who received seven subcutaneous doses of VA 20 mg, showed improvement in several laboratory parameters, confirming that VA can improve the immune response and restore suppressed cellular and humoral immunity to some extent. There is evidence, supported by clinical studies, that VA has positive benefits for

Table 1. Patient characteristics

Age Patient Gender (years)

1. 1 Male 57

2. 2 Male 18

3. 3 Female 44

4. 4 Male 46

Cancer diagnosis

Hodgkin disease (nodular sclerosis) Hodgkin disease (mixed cellularity) Breast (infiltrative ductal)
Multiple myeloma (IgG)

Stage

III-B II-A II III-A

Months from diagnosisa

43 50 30 16

Months from Treatment end of treatmenta

CT+RT 31 CT+RT 33 SR+RT+CT 20 CT4

Phytother. Res. 23, 407–411 (2009) DOI: 10.1002/ptr

a To VA tests.
CT, chemotherapy; RT, radiotherapy; SR, surgery.

Copyright © 2008 John Wiley & Sons, Ltd.

Table 2. Results of patient 1

Parameter

Before VA

1670 868 701 109 244 0.45 66 21 188 882 63 0 0 0 Normal/Evaluatedb 4/14

a Due to a technical problem.
b Number of normal parameters/number of parameters evaluated.

2–3 days after VA

Conclusion

Improvement Improvement Improvement Improvement Kept normal Improvement Normal

Kept normal Kept normal Improvement Improvement Unaltered Unaltered Unaltered Improvement

Conclusion

Improvement Kept normal Kept normal Kept normal Kept normal Improvement Kept normal Kept normal Kept normal Kept normal Kept normal Unaltered Unaltered Unaltered Improvement

Conclusion

Kept normal Kept normal Kept normal Kept normal Kept normal Kept high Kept normal Kept normal Kept high Kept normal Kept normal Unaltered Improvement Improvement Improvement

Leucocytes × 109/L Neutrophils × 109/L Lymphocytes × 109/L CD4 (cells/μL)

4000–11000 1800–7700 800–4950 240–1800 120–1110 0.9–2.2

2280 1254 889 Not donea Not donea Not donea 84 26 227 963 63
0
0
0 5/11

2–3 days after

3600

2160 1116 234 333 0.7 98 21 216 1430 119 0 0 0 8/14

2–3 days after

      9360
      5616
      2433
      1166

521

2.24 128 32 478 1490

3440

2476

791 126 249 0.51

94

26 210 968 78

–
–
– 7/11

4 weeks after VA

     4730
     2606
     1466

408

522

0.78

76

       20
      211
     1250
      128

–
–
– 10/11

4 weeks after VA

     8230
     4608
     2798
     1556

585

2.66

89

26

449

CD8 (cells/μL) CD4/CD8 ratio
C3 (mg/dL)
C4 (mg/dL)
IgA (mg/dL)
IgG (mg/dL)
IgM (mg/dL) Candidin (mm) PPD (mm) Trichophytin (mm)

50–120 10–40 60–400

Normal range

4 weeks after VA

900–1500 70–320 5–10 5–10 5–10

IMMUNE RESPONSE TO MISTLETOE

409

Table 3. Results of patient 2

Parameter

Leucocytes × 109/L Neutrophils × 109/L Lymphocytes × 109/L CD4 (cells/μL)

CD8 (cells/μL)
CD4/CD8 ratio
C3 (mg/dL)
C4 (mg/dL)
IgA (mg/dL)
IgG (mg/dL)
IgM (mg/dL)
Candidin (mm)
PPD (mm)
Trichophytin (mm)
Normal/Evaluateda 9/14

a Number of normal parameters/number of parameters evaluated.

Normal range

Before VA

3840

VA

4000–11000 1800–7700 800–4950 240–1800 120–1110 0.9–2.2

1958 1612 324 555 0.58 65 24 213 1460 109 0 0 0

50–120 10–40 60–400

900–1500 70–320 5–10 5–10 5–10

Table 4. Results of patient 3

Parameter

Leucocytes × 109/L Neutrophils × 109/L Lymphocytes × 109/L CD4 (cells/μL)

CD8 (cells/μL)
CD4/CD8 ratio
C3 (mg/dL)
C4 (mg/dL)
IgA (mg/dL)
IgG (mg/dL)
IgM (mg/dL)
Candidin (mm)
PPD (mm)
Trichophytin (mm)
Normal/Evaluateda 9/14

a Number of normal parameters/number of parameters evaluated.

Copyright © 2008 John Wiley & Sons, Ltd.

VA

Normal range

Before VA

  9670
  6285
  2417
  1184

4000–11000 1800–7700 800–4950 240–1800 120–1110 0.9–2.2

491

2.41

89

30

455

50–120 10–40 60–400

900–1500 70–320 5–10 5–10 5–10

1103 131 0 2 2

1310 135 125

0– 9– 9–

10/14 9/11

Phytother. Res. 23, 407–411 (2009) DOI: 10.1002/ptr

410

N. E. GARDIN

Table 5. Results of patient 4

Parameter

Leucocytes × 109/L Neutrophils × 109/L Lymphocytes × 109/L CD4 (cells/μL)

CD8 (cells/μL)
CD4/CD8 ratio
C3 (mg/dL)
C4 (mg/dL)
IgA (mg/dL)
IgG (mg/dL)
IgM (mg/dL)
Candidin (mm)
PPD (mm)
Trichophytin (mm)
Normal/Evaluateda 8/14

a Number of normal parameters/number of parameters evaluated.

Table 6. Adverse events associated with the use of VA

Normal range

Before VA

  4210
  2273
  1599

427

2–3 days after VA

Conclusion

4000–11000 1800–7700 800–4950 240–1800 120–1110 0.9–2.2

677

0.63

  4570
  2467
  1965

662

792

0.84 131 16 72 7590 36 5

Not done

0

8/13

Kept normal Kept normal Kept normal Kept normal Kept normal Improvement Become high Kept normal Kept normal Kept high Improvement Improvement –

Unaltered Improvement

50–120 10–40 60–400

117 15 63 7380 31 0 30 0

900–1500 70–320 5–10 5–10 5–10

Patient Adverse event

1. 1 Induration and erythema

2. 2 Pain and erythema

   Pain Induration Itching

3. 3 Pain and erythema Induration

4. 4 Pain Induration

some cancer patients although efficacy is still not con- sidered to have been conclusively demonstrated (Ernst et al., 2003). In 1989, Kiene published the first meta- analysis about VA clinical studies (Kiene, 1989), which included 46 studies and trials of VA therapy for carci- nomatous diseases. There were 35 controlled studies, of which 12 were considered conclusive, and all of these showed an advantage of the mistletoe group in survival time and survival rate. Nine of those 12 studies were statistically significant. Kleijnen and Kipschild (1994) also analysed VA clinical studies, but were more criti- cal about methodological aspects. They uncovered 11 controlled trials, four of which showed significance with a positive result for mistletoe as a treatment for cancer, six trials showed a positive trend and one with no benefit. Finally, in 2007 Kienle and Kiene evaluated only prospective clinical trials on the effectiveness of anthroposophic mistletoe therapy for cancer (Kienle and Kiene, 2007). Thirty seven studies were identified: 16 randomized, nine non-randomized and 12 single-arm cohort studies. Among 25 controlled trials evaluated for clinically relevant outcome measures, a statistically sig- nificant benefit for survival was reported in eight of 17 trials, for remission of tumor and malignant effusion in two of four controlled trials, for quality of life in three of five studies, and for quality of life and reduction of side effects of cytoreductive therapies (chemotherapy, radiation or surgery) in five of seven trials. Among 12 single-arm cohort studies, five of seven studies found substantial tumor remission, one study reported remis- sion of carcinoma in intra-epithelial neoplasm, and four

Copyright © 2008 John Wiley & Sons, Ltd.

Grade

I II I I I I I I I

Period

Duration

1 day
2 days
1 day
1 day
1 day
1 day 8–10 days 1 day
1 day

After dose 4 After dose 1 After dose 2 After dose 3 After dose 5 After all 7 doses After all 7 doses After all 7 doses After dose 1

studies reported remission of malignant pleural effusion or ascites.

In the present study, almost all immune indices improved after VA therapy. This supports the work of Chernyshov et al. who showed previously that VA therapy reduced recurrent respiratory infections and improved immune parameters in more than 70% of 92 children living in areas exposed to the radioactive fallout from Chernobyl (Chernyshov et al., 2000). The immunomodu- lating and anticancer activities of VA are attributed to its three main classes of biologically active components: lectins, viscotoxins and polysaccharides (Romagnoli et al., 2003; Stein et al., 1999b; Coulon et al., 2003; Frantz et al., 2000). The lectins especially have well recognized antitumor and immunomodulating activities.

The incidence of adverse effects was small, most of them transient and mild, and none systemic. Previous clinical studies showed the same results (Gorter et al., 1999, Stein and Berg, 2000), consequently, complemen- tary treatment with VA has been considered safe.

In conclusion, although this study has had only four cases, the VA therapy showed immune benefits in labo- ratory tests and suggests that VA can enhance humoral and cellular immune responses. However, new studies attesting to the clinical impact of these immunological effects in cancer patients are needed.

Acknowledgement

The author is grateful to Dr José Roberto Lazzarini Neves and Dr Bernardo Kaliks Litvak and Dr Ricardo Caponero for their scientific support.

Phytother. Res. 23, 407–411 (2009) DOI: 10.1002/ptr