542

Plasma NPY concentrations were very high in the

phaeochromocytoma patients (460±68 pmo1/1 ; range120-1080) while healthy controls had levels below 100

pmol/1 (55±12 pmol/1 ; range 10-90). Plasma noradrenalinelevels were, in some cases, only just above the upper limit ofnormal and there was no significant correlation in thesepatients between NPY and noradrenaline concentrations(r=0’45; p = 0 . 1 ; n= 13).

Discussion

The high concentrations of NPY in the nervous systemtogether with its potent biological actions, suggest that NPYis an important neuropeptide.1,4,6

NPY is present in the sympathetic chain and its productionby tumours arising from this system was not unexpected. Theconcentrations of the peptide in most of these tumours was fargreater than concentrations found in normal tissues.Abnormal multiple molecular forms of peptideimmunoreactivities are frequently found in neuroendocrinetumours and this probably reflects defects in post-translational enzymic processing of the hormoneprecursors.1O The detection of variable amounts ofimmunoreactive material eluting earlier than the NPY peakon the HPLC column probably represents such biosyntheticprecursors of the NPY molecule or some alteration in post-translational modification.

NPY has recently been shown to be an importantcomponent of the nervous system of the human heart.11 Thepeptide has visceral vasoconstrictor activity in the cat, andpreliminary experiments in an isolated heart preparationsuggest that NPY may be involved in the regulation ofmyocardial perfusion.12 The presence of high circulatingconcentrations of NPY may, therefore; contribute in part tothe cardiovascular features of phaeochromocytoma.

Phaeochromocytomas are responsible for up to 0’ 5% of allcases of hypertension but because these tumours are usuallybenign and curable they represent an important group of thehypertensive population.13,14 Ganglioneuroblastomas are

uncommon in adults, though they are one of the mostfrequent neoplasms in early childhood,15 and resection maybe curative. Ganglioneuromas are not normally associatedwith hypertension, though this may be due to simultaneoussecretion of other hypotensive agents such as dopamine orvasoactive intestinal peptide. Whether NPY is responsiblefor any of the many symptoms of phaeochromocytomas organglioneuromas will have to await further study of theactions of NPY in man.

Phaeochromocytomas are diagnosed by measuring plasmaor urinary catecholamines or the urinary metabolites such asvanillylmandelic acid. 16 NPY, on the other hand, appears tobe substantially raised even in patients with noradrenalinelevels just above the upper limit of normal or just above,therefore NPY assays may be of value in the diagnosis of thesetumours.

J. M. A. is in receipt of a Wellcome Trust training fellowship. The work wassupported by the Medical Research Council.

Correspondence to S. R. B., Department of Medicine, Royal PostgraduateMedical School, Du Cane Road, London W12 OHS.

References at foot of next column

1. Tatemoto K, Carlquist M, Mutt V. Neuropeptide Y: a novel brain peptide withstructural similarities to peptide YY and pancreatic polypeptide. Nature 1982; 296:659-60.

2. Tatemoto K. Neuropeptide Y: Complete ammo acid sequence of the brain peptideProc Natl Acad Sci USA 1982; 79: 5485-89.

3. Adrian TE, Allen JM, Bloom SR, Ghatei MA, Rosser MN, Roberts GW, Crow TJ,Tatemoto K, Polak JM. Massive concentrations of neuropeptide Y in componentsof human basal ganglia. Nature (in press).

4. Lundberg JM, Terenius L, Hökfelt T, et al. Neuropeptide Y (NPY)-likeimmunoreactivity in peripheral noradrenergic neurons and effects of NPY onsympathetic function. Acta Physiol Scand 1982; 116: 477-80.

5. Hökfelt T, Lundberg JM, Tatemoto K, et al. Neuropeptide Y (NPY)- and FMRF amideneuropeptide-like immunoreactivities in catecholamine neuron of the rat medullaoblongata. Acta Physiol Scand 1983; 117: 315-18.

6. Allen JM, Adrian TE, Tatemoto K, Polak JM, Hughes J, Bloom SR. Two novel relatedpeptides, neuropeptide Y (NPY). and peptide YY (PYY) inhibit the contraction ofthe electrically-stimulated mouse vas deferens. Neuropeptides 1982; 3: 71-77

7. Pearse AGE, Polak JM. Bifunctional reagents as vapour and liquid-phase fixative forimmunohistochemistry. Histochem J 1975; 7: 179-86.

8. Sternberger LA. Immunocytochemistry, 2nd ed. New York: John Wiley and Sons,1979

9. Bloom SR, Long RG. Radioimmunoassay of gut regulatory peptides. LondonSaunders, 1982.

10. Irvine GB, Murphy RF. Multiple forms of gastroenteropancreatic hormones. Gut1981, 22: 1048-69.

11. Gu J, Polak JM, Adrian TE, Allen JM, Tatemoto K, Bloom SR. Neuropeptide tyrosine(NPY): a major new cardiac neuropeptide. Lancet 1983; i: 1008-10.

12. Allen JM, Birchman PMM, Edwards AV, Tatemoto K, Bloom SR. Neuropeptide Y(NPY) reduces myocardial perfusion and inhibits the force of contraction of theisolated perfused rabbit heart. Regul Pept 1983; 6: 247-54.

13. Goldenberg M, Aranow H, Smith AA, Faber M. Pheochromocytoma and essentialhypertensive vascular disease. Arch Intern Med 1950; 86: 823-36.

14. Hermann H, Mornex R. Human tumours secreting catecholamines. Oxford

Pergamon Press, 1964.15. Hall R, Anderson J, Smart GA, Besser M. Fundamentals of clinical endocrinology

London: Pitman, 1980: 208-78.16. Causon RC, Brown MJ. Catecholamine measurements in phaeochromocytoma: a

reivew. Ann Clin Biochem 1982, 19: 396-404.

Preliminary Communications

TOXIC EFFECTS OF EARLY ADJUVANTCHEMOTHERAPY FOR BREAST CANCER

Ludwig Breast Cancer Study Group*

Summary Combination cytotoxic chemotherapy(intravenous cyclophosphamide, metho-

trexate, and fluorouracil) was administered within 36 h ofmastectomy to 368 women with operable breast cancer in arandomised, controlled clinical trial. The control group of187 patients received either no chemotherapy or

conventionally timed chemotherapy. Unpredictable andsevere toxic effects were significantly more common in

patients aged ≥50 who had received at least 80% of the fullchemotherapy dose and in patients who had received chemo-therapy within 6 h of mastectomy than in other patients.Methotrexate was believed to be the principal cause of thesetoxic effects, because of potentiation by nitrous oxide anaes-thesia. Leucovorin rescue was therefore added to the

regimen.

*Participating centres-Auckland, We-llington (New Zealand), Cape Town(South Africa), Essen, Dusseldorf (West Germany), Goteborg (Sweden),Ljubljana (Yugoslavia), Madrid (Spain), Melbourne, Perth, Sydney(Australia), and the Swiss Group for Clinical Cancer Research (Switzerland).Coordinating centre-Ludwig Institute for Cancer Research (Bern Branch),Bern, Switzerland. Statistical Centre-Dana-Farber Cancer Center, Boston,USA. Writing committee-A. Goldhirsch, R. Gelber, B. Davis, C.-M.

Rudenstam, M. H. N. Tattersall.

543

INTRODUCTION

POSTOPERATIVE adjuvant endocrine or cytotoxic chemo-therapy in operable breast cancer prolongs disease-freesurvival and produces a survival advantage in certain sub-groups of patientsl,2 (and Ludwig Breast Cancer StudyGroup, unpublished). Studies of adjuvant chemotherapy inanimal tumours and mathematical models have shown thatthe timing of chemotherapy in relation to surgery is a criticaldeterminant of outcome.3-S Two clinical trials have used

chemotherapy in the immediate postoperative period andthere were survival advantages in at least some of the patientstreated.6,7 We are conducting a controlled, randomisedclinical trial to find out whether early commencement ofadjuvant chemotherapy in operable breast cancer is morebeneficial than conventionally timed combination chemo-therapy, which starts 3-5 weeks after mastectomy. We reportthe development of unexpected toxic effects in patientsreceiving chemotherapy immediately after surgery.

PATIENTS AND METHODS

From August to October, 1981, 65 patients were treated in a pilotstudy with chemotherapy given immediately after surgery(immediate chemotherapy). From November, 1981, to October,1982, 555 premenopausal and postmenopausal patients withoperable breast cancer (stages i and 11) were randomly allocated toreceive immediate chemotherapy (186), conventionally timedcombination chemotherapy or observation only if pathological stageI (187), or immediate chemotherapy followed by conventionallytimed chemotherapy (182). As of February, 1983, data were missingfor 46 patients, 6 entries were cancelled, 1 patient had receivedimmediate chemotherapy by mistake, and 9 patients had refusedimmediate chemotherapy. Of the remaining 493 patients 327 hadreceived immediate chemotherapy and 166 had not. 480 patientsreceived nitrous oxide during anaesthesia. Other anaestheticinformation was not available.

Immediate chemotherapy consisted of cyclophosphamide (400mg/m2), methotrexate (40 mg/m2), and 5-fluorouracil (600 mg/m2)given intravenously within 36 h of mastectomy (day 1) and again 1week later (day 8). Day 8 dosage was modified on the basis ofhaematological parameters and mucositis. Conventionally timedchemotherapy consisted of the same drugs (at the same doses) asimmediate chemotherapy, in 6-month cycles starting 25-36 daysafter mastectomy, with the addition of prednisone (7’ 5 mg/day)and, in postmenopausal patients, tamoxifen (20 mg/day).For the purpose of this analysis toxic effects considered to be

dangerous or potentially dangerous were denoted as toxicity X.These were wound-healing difficulties (wound-healing delaybeyond 4 weeks, dehiscence, necrosis, extensive haematoma orseroma, or wound infection requiring drainage or antibiotics);systemic infection requiring antibiotics; severe myelosuppression(white blood cell count <109/1 and/or platelet count <5x 10/1;stomatitis of any grade (included after a preliminary analysisdemonstrated that stomatitis was associated to a great extent withother severe and lethal toxic effects).Associations between toxic effects and various patient and

treatment characteristics were assessed by chi-square analysis. Therelation between toxicity X and time of drug administration aftermastectomy and age of the patient were evaluated by logisticregression models. 8

RESULTS

1 of the 65 pilot study patients suffered severe toxic effects,consisting of wound infection, sepsis, and severe stomatitisafter receiving the full immediate chemotherapy doses. Sherecovered within 10 days.

In the randomised trial, 4 deaths occurred after immediatechemotherapy. The first patient, aged 70 years, died ofpneumonia with severe myelosuppression on day 15. She hadreceived only the first day’s chemotherapy (700 mgcyclophosphamide, 70 mg methotrexate, and 1075 mg

fluorouracil). Renal failure was detected on day 2 (creatinine300 mol/1). Presumably, low preoperative creatinine valueswere incorrect. The second patient, aged 54 years, died ofrespiratory, hepatic, and renal failure on day 40. She hadreceived 680 mg cyclophosphamide, 68 mg methotrexate,and 1020 mg fluorouracil on day 1, and half doses on day 8because her white blood count was 3.2 X 109/1. A woundinfection was drained of purulent fluid in the 3rd week. Thethird patient, 65 years old, died of septic shock with

myelosuppression on day 15. Her day 8 dosage was 45% ofthe day 1 dosage (650 mg cyclophosphamide, 65 mgmethotrexate, and 950 mg fluorouracil) because her whiteblood count was 3 - 8 x 109/1. The 4th patient who died (aged41 years) entered the trial after Oct 1, 1982, but was treatedbefore protocol modification and is not included in the

analyses. She had iron-deficiency anaemia (haemoglobin 60g/1) and thrombocytosis (7-6x10" platelets/I). She received675 mg cyclophosphamide, 70 mg methotrexate, and 1025mg fluorouracil on day 1. Deep vein thrombosis was observedon day 5 and she died of a massive lung embolism on day 7.Local and systemic infection as well as dehiscence and

delay in wound healing were more frequent in the groupreceiving immediate chemotherapy than in the other patients(table I). Stomatitis was present in 30% of the 47 patients whoexperienced any of the other disorders denoted as toxicity Xor severe toxic effects, other than vomiting. In contrast only

TABLE I-POSTOPERATIVE COMPLICATIONS AND

CHEMOTHERAPY-RELATED TOXIC EFFECTS

*Severe: leucopema 10"/I; thrombocytopenia 5xJO"*/I; intractable nauseaand vomiting; stomatitis ulcers, cannot eat; anaemia with symptoms requiringtransfusions; infection requiring antibiotics, surgery; renal impairment,>3x’normal creatinine and blood urea nitrogen; liver impairment, >5xnormal function tests; wound-healing problems, necrosis, local infection,haematoma requiring surgery for healing.tNot considered a severe effect.

544

12% of the 280 patients without such disorders had stomatitis(p=0’003). Stomatitis was therefore included as a

component of toxicity X.The frequency of toxicity X was higher in patients >50

years old than in younger patients (p=0’06; table II),

TABLE II-FREQUENCY OF TOXICITY X BY AGE CORRELATED WITHTHE TIME FROM END OF MASTECTOMY TO START OF CHEMOTHERAPY

&mdash;

*Numbers in parentheses number with toxicity X/total.t 5% of patients received chemotherapy while still on the operating table afterwound was closed.

particularly in those who had received at least 80% of the fulldose of drugs on day 1 (19% in those aged <50 years vs 30% inthose aged 50 years; p=0’03). 28% of patients over age 65(10/36) received reduced doses on day 1, primarily owing tophysician concern about giving the protocol-specified day 1dose to elderly patients; none had toxicity X. In contrast, 35%(9/26) of the patients receiving the full day 1 dose in this agegroup had toxicity X.The initial white blood count, initial leucocytosis (>10"/1)

or a 50% fall in white blood count from day 1 to day 8 (butremaining j4 x 109/1 on day 8) did not help to predict theeventual toxic effects.97% of the patients assigned to immediate chemotherapy

received the drugs within 36 h of mastectomy. A logisticregression analysis of the downward trend in the frequency oftoxicity X with increasing time from mastectomy wassignificant at p = 0 - 02, even when controlling for the effectsof age.Analysis of the frequency of toxic effects by randomisation

centre (one for Switzerland, one for the rest of Europe andSouth Africa, and one for Australasia) showed a difference inthe frequency of toxicity X between the various centres(p = 0 - 0002; table III). The difference in the frequency

TABLE III-FREQUENCY OF TOXIC EFFECTS IN EACH RANDOMISATIONCENTRE

* Randomisation centres randomly coded A, B, C.

reported between the institutions might be due to a higherfrequency of wound-healing problems, infections, andstomatitis. Rates of reported wound-healing problems inpatients who did not receive immediate chemotherapy also

varied (from 2% to 9%) between randomisation centres, butthis variation was not statistically significant.

DISCUSSION

Several published reports have drawn attention to the-effects of nitrous oxide anaesthesia on bone-marrow

function,9-" and one has suggested that nitrous oxideanaesthesia might potentiate the toxicity of methotrexategiven a few hours later by interfering with folate metabolismthrough inhibition of methionine synthetase." Moreover,nitrous oxide has been reported to reduce the motility ofhuman neutrophils in vitro,12 so qualitative as well as

quantitative granulocyte defects may result.

Our findings that toxic effects on mucosa and the timing ofimmediate chemotherapy were correlated with serious toxiceffects led us to believe that methotrexate was the principalcause of the toxic effects. The enhancement of methotrexate

toxicity may result either from potentiation by nitrous oxideanaesthesia or from a transient and undetected renal

impairment. We decided therefore to modify our immediatechemotherapy to include 15 mg leucovorin (5-formyltetrahydrofolate) 24 h after the day 1 drug administration.Leucovorin was administered intravenously to avoidconversion of formyl tetrahydrofolate to methyltetrahydrofolate, whose utilisation requires methionine

synthetase action.13 In addition, the upper age limit forpatients receiving immediate chemotherapy has been set at 65years, and intravenous hydration for 36 h after mastectomyhas been added as a requirement for patients receivingimmediate chemotherapy. We are currently investigatingother possible causes of these unexpected toxic effects relatedto the observed geographical differences. The revised

programme has been in effect for the past 8 months. Anevaluation of the toxic effects observed with the revised

regimen will be conducted in a year, when a comparablenumber of patients will have been treated.

Correspondence ’should be addressed to Dr Aron Goldhirsch, LudwigInstitute for Cancer Research, Inselspital, CH-3010 Bern, Switzerland.

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haemopoietic toxicity of nitrous oxide in man. Br J Haematol 1983; 53: 189-20010. Kano Y, Sakomoto S, Sakuraya K, et al. Effect of nitrous oxide on human bone marrow

cells and its synergistic effect with methionine and methotrexate on functional folatedeficiency. Cancer Res 1981; 41: 4698-701.

11. Kano Y, Sakamoto S, Sakuraya K. Effects of nitrous oxide on human cell lines. CancerRes 1983; 43: 1493-96.

12. Nunn JF, O’Morain C. Nitrous oxide decreases motility of human neutrophils in vitroAnesthesiology 1982; 56: 45-48.

13. Dudman NPB, Slowiaczek P, Tattersall MHN. Methotrexate rescue by 5-methyl THFor 5-formyl THF in lymphoblastoid cell lines. Cancer Res 1982; 42: 502-07