1034 other words, our policy has been affected by the development of reliable cytology and we hope to expedite admission, simplify the management, and avoid frozen section in those patients with a negative cytological report. This procedure seems to us reasonable and safe. It should be noted that some centres are having considerable success in obtaining histological confirmation of the nature of breast lumps using punchtype (’Trucut’) needles to obtain a good core of tissue. There is no doubt that in experienced hands this is a good method of approach, but it has some disadvantages and, in particular, it has not been successful for the smaller breast mass. Dr Lever and Mr Webb suggest that our use of aspiration cytology is a glorified form of occupational therapy. This is not only untrue but takes no account of the fact that cytologists and surgeons in this country have not yet arrived at a working format which has led to wide acceptance. Our work is a serious effort to establish a procedure which may be practical enough to attract a wider following than has been achieved by the few enthusiasts who have to date claimed good results. Department of Surgery, Royal Infirmary, Glasgow G4 OSF.
L. H. BLUMGART HELENA E. HUGHES
Label is mainly label in fibroblasts,
METHOD FOR STUDYING FUNCTIONAL ACTIVITY OF BREAST-CANCER CELLS
SiR,—This letter describes an alternative to the procedure described by Chayen et al. for studying the functional activity of malignant breast epithelium. It satisfies the following necessary criteria: (1) sufficient simplicity for use as a standard laboratory procedure; (2) applicability to cells of an individual’s own tumour and to small amounts of tumour; (3) the malignant cell population studied should be representative of that in the tumour (cells obtained by mechanical or enzymatic dissociation do not always satisfy this criterion); (4) the activity studied (i.e., protein synthesis) is specialised and yet basic enough for one to assume with some validity that most cells are undertaking it in varying degrees at any one time; (5) it permits verification by microscopic visualisation that the results obtained pertain to the malignant cell population and not to admixed non-malignant cells-breast tumours often contain much non-malignant epithelium in addition to lymphoid cells,
macrophages, fibroblasts, &c. Fresh tumour is minced and washed three times with Hank’s balanced salt solution (H.B.s.s.; contains no aminoacids). Either then or after three days’ incubation (37°C in 5% CO2) in the individual’s own serum (see later) and three subsequent washes in H.B.S.S., the fragments of an experimental group (4-5 fragments; wet weight approximately 10 mg) are exposed for two hours (37°C in 5% CO2) to 0.5 ml volumes of H.B.S.S. containing 20% fetal calf serum (F.C.S.; previously dialysed to remove aminoacids and unbound hormones), 5 i1.C of mixture of 15 tritium-labelled L-aminoacids in 0 IN HCI (New England Nuclear; NET 250) and bicarbonate to restore the pH to 7.3. After two washes in medium containing 10% dialyzed the fragments are fixed in formalin and sectioned at 6:1. intervals. Two consecutive sections near the surface are placed on a slide and stained with haematoxylin and eosin. One is mounted while the other is dipped in Kodak NTB2 nuclear track emulsion (50% solution in distilled water), developed after three days (twenty-four hours when greater sensitivity is needed), restained with haematoxylin (two minutes) and eosin (two minutes) and mounted. An area of obvious malignancy is located on the first section and the intensity of labelling (0 to +++) of this area is noted on the adjacent section. This is an extremely simple procedure and can give much interesting information about the biological activity of malignant breast epithelium. Labelling of lymphoid cells and fibroblasts is often not much greater than background levels (see accompanying
F.C.S
figure). 2. Preece, P.
E., Williams, G., Teddy, P. G., Bolton, P. M. Lancet, 1975,
of fragment of scirrhous breast carcinoma labelled with tritiated aminoacids.
Autoradiograph
incorporated by malignant cells. Note paucity of lymphoid cells, and vascular structures.
This convenient procedure is currently being used to identify at the time of putatively curative surgery those individuals who are at risk with respect to the development of metastatic lesions at a later date or indeed, may already have subclinical metastatic deposits. Certain obvious questions merit study. For example: (1) Is there a positive correlation between the functional activity of malignant cells and their capacity to form metastatic foci? More important, (2) Can the existence of intravascular defence mechanisms inimical to the survival of malignant cells which might enter the circulation4 be detected by comparing the functional activity of an individual’s tumour cells in her own undiluted serum on the one hand and, on the other, in a dialysate of her serum containing 5% of her undiluted serum (the latter solution closely resembles the composition of the extravascular fluid in which malignant cells of a solid tumour are located) and (3) Does the existence of such mechanisms decrease the risk of metastatic lesions developing? (4) Will a comparison of the functional activity of tumour cells in areas where they are admixed with lymphoid cells and where they are not give some idea about the capacity of an individual’s cellular immunity to retard the expansion of established subclinical metastatic foci? In the circulation the shearing forces and turbulence would probably militate against the close and prolonged contact between target and effector cell needed for the manifestations of cellular immunity to be demonstrable. This work
supported by U.S.P.H.S. grants CA-08856, an appropriation from the Commonwealth of Pennsylvania. was
CA-06927, RR-05539, and CA-05255 and Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pa 19111, U.S.A.
H. F. JEEJEFBHOY
HORMONE SENSITIVITY IN BREAST CANCER
SIR,-Miss Wilson and Dr Carr (Sept. 6, p. 465), using visual
assessment
of
histochemistry of
vitro, detected in 44%
a
response
to a
Bitensky, L., Daly, J.
i,
R. Lancet, 1970, i, 868.
hormone,
in but using arise from
cancers
assessment claimed that great errors variation in section thickness. Dr Daly and others (Oct. 4, p. 662) pointed out that the above anomaly could not be accounted for by inconsistent section thickness. In quantifying the histochemical dehydrogenase, no method
’Quantimet’
1184. 3. Chayen, J., Altman, F. P.,
80 breast
4.
Jeejeebhoy,
H. F. Int.
J. Cancer, 1975, 15, 867.
1035 is without its
problems. Homogenisation
averages
over
a
heterogeneous section of tumour and the advantage of histochemical localisation is lost; yet, despite this, the results of Beeby et al.’ do suggest that 5 of their tumours (nos. 4, 7, 13, and
of 12 may have responded to a hormone. The measurement made by mechanical densitometers is essentially that of the area covered by formazan ; the relationship to mass and hence amount of enzyme is unknown. A variety of numerical results can be obtained by different settings, but in this work it is especially difficult to find a method of validation of the number obtained. We agree completely with Daly et al. that the use of inappropriate instrumentation or incorrect adjustment can lead to meaningless numerical results. We ourselves have twice used the quantimet under expert supervision-either Metals Research Ltd. or Dr S. Bradbury, (Oxford)—in "blind" comparisons to visually made readings. For each test condition 10-15 separate non-overlapping fields of three consecutive sections were scanned. The instrument’s controls were adjusted to ensure measurement primarily of the formazan granules, and were kept constant during measurement. Throughout 145 readings, the quantimet results confirmed the visual assessment that had been made in that tumours scored as independent showed no significant statistical difference between sections from cultures with or without hormones, and that sections scored as dependent showed sigin mean density. Coefficients of nificant increases (p
L. H. BLUMGART HELENA E. HUGHES
Label is mainly label in fibroblasts,
METHOD FOR STUDYING FUNCTIONAL ACTIVITY OF BREAST-CANCER CELLS
SiR,—This letter describes an alternative to the procedure described by Chayen et al. for studying the functional activity of malignant breast epithelium. It satisfies the following necessary criteria: (1) sufficient simplicity for use as a standard laboratory procedure; (2) applicability to cells of an individual’s own tumour and to small amounts of tumour; (3) the malignant cell population studied should be representative of that in the tumour (cells obtained by mechanical or enzymatic dissociation do not always satisfy this criterion); (4) the activity studied (i.e., protein synthesis) is specialised and yet basic enough for one to assume with some validity that most cells are undertaking it in varying degrees at any one time; (5) it permits verification by microscopic visualisation that the results obtained pertain to the malignant cell population and not to admixed non-malignant cells-breast tumours often contain much non-malignant epithelium in addition to lymphoid cells,
macrophages, fibroblasts, &c. Fresh tumour is minced and washed three times with Hank’s balanced salt solution (H.B.s.s.; contains no aminoacids). Either then or after three days’ incubation (37°C in 5% CO2) in the individual’s own serum (see later) and three subsequent washes in H.B.S.S., the fragments of an experimental group (4-5 fragments; wet weight approximately 10 mg) are exposed for two hours (37°C in 5% CO2) to 0.5 ml volumes of H.B.S.S. containing 20% fetal calf serum (F.C.S.; previously dialysed to remove aminoacids and unbound hormones), 5 i1.C of mixture of 15 tritium-labelled L-aminoacids in 0 IN HCI (New England Nuclear; NET 250) and bicarbonate to restore the pH to 7.3. After two washes in medium containing 10% dialyzed the fragments are fixed in formalin and sectioned at 6:1. intervals. Two consecutive sections near the surface are placed on a slide and stained with haematoxylin and eosin. One is mounted while the other is dipped in Kodak NTB2 nuclear track emulsion (50% solution in distilled water), developed after three days (twenty-four hours when greater sensitivity is needed), restained with haematoxylin (two minutes) and eosin (two minutes) and mounted. An area of obvious malignancy is located on the first section and the intensity of labelling (0 to +++) of this area is noted on the adjacent section. This is an extremely simple procedure and can give much interesting information about the biological activity of malignant breast epithelium. Labelling of lymphoid cells and fibroblasts is often not much greater than background levels (see accompanying
F.C.S
figure). 2. Preece, P.
E., Williams, G., Teddy, P. G., Bolton, P. M. Lancet, 1975,
of fragment of scirrhous breast carcinoma labelled with tritiated aminoacids.
Autoradiograph
incorporated by malignant cells. Note paucity of lymphoid cells, and vascular structures.
This convenient procedure is currently being used to identify at the time of putatively curative surgery those individuals who are at risk with respect to the development of metastatic lesions at a later date or indeed, may already have subclinical metastatic deposits. Certain obvious questions merit study. For example: (1) Is there a positive correlation between the functional activity of malignant cells and their capacity to form metastatic foci? More important, (2) Can the existence of intravascular defence mechanisms inimical to the survival of malignant cells which might enter the circulation4 be detected by comparing the functional activity of an individual’s tumour cells in her own undiluted serum on the one hand and, on the other, in a dialysate of her serum containing 5% of her undiluted serum (the latter solution closely resembles the composition of the extravascular fluid in which malignant cells of a solid tumour are located) and (3) Does the existence of such mechanisms decrease the risk of metastatic lesions developing? (4) Will a comparison of the functional activity of tumour cells in areas where they are admixed with lymphoid cells and where they are not give some idea about the capacity of an individual’s cellular immunity to retard the expansion of established subclinical metastatic foci? In the circulation the shearing forces and turbulence would probably militate against the close and prolonged contact between target and effector cell needed for the manifestations of cellular immunity to be demonstrable. This work
supported by U.S.P.H.S. grants CA-08856, an appropriation from the Commonwealth of Pennsylvania. was
CA-06927, RR-05539, and CA-05255 and Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pa 19111, U.S.A.
H. F. JEEJEFBHOY
HORMONE SENSITIVITY IN BREAST CANCER
SIR,-Miss Wilson and Dr Carr (Sept. 6, p. 465), using visual
assessment
of
histochemistry of
vitro, detected in 44%
a
response
to a
Bitensky, L., Daly, J.
i,
R. Lancet, 1970, i, 868.
hormone,
in but using arise from
cancers
assessment claimed that great errors variation in section thickness. Dr Daly and others (Oct. 4, p. 662) pointed out that the above anomaly could not be accounted for by inconsistent section thickness. In quantifying the histochemical dehydrogenase, no method
’Quantimet’
1184. 3. Chayen, J., Altman, F. P.,
80 breast
4.
Jeejeebhoy,
H. F. Int.
J. Cancer, 1975, 15, 867.
1035 is without its
problems. Homogenisation
averages
over
a
heterogeneous section of tumour and the advantage of histochemical localisation is lost; yet, despite this, the results of Beeby et al.’ do suggest that 5 of their tumours (nos. 4, 7, 13, and
of 12 may have responded to a hormone. The measurement made by mechanical densitometers is essentially that of the area covered by formazan ; the relationship to mass and hence amount of enzyme is unknown. A variety of numerical results can be obtained by different settings, but in this work it is especially difficult to find a method of validation of the number obtained. We agree completely with Daly et al. that the use of inappropriate instrumentation or incorrect adjustment can lead to meaningless numerical results. We ourselves have twice used the quantimet under expert supervision-either Metals Research Ltd. or Dr S. Bradbury, (Oxford)—in "blind" comparisons to visually made readings. For each test condition 10-15 separate non-overlapping fields of three consecutive sections were scanned. The instrument’s controls were adjusted to ensure measurement primarily of the formazan granules, and were kept constant during measurement. Throughout 145 readings, the quantimet results confirmed the visual assessment that had been made in that tumours scored as independent showed no significant statistical difference between sections from cultures with or without hormones, and that sections scored as dependent showed sigin mean density. Coefficients of nificant increases (p
