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Extramedullary haematopoiesis in the human infant breast
The Breast (1992) 1,182-186
Extramedullary haematopoiesis in the human infant breast R. Anbazhagan,
J. Bartkova*,
B. Nathan and B. A. Gusterson
Institute of Cancer Research, Haddow Laboratories, Experimental Pathology, Bmo, Czechoslovakia
Sutton, Surrey UK. *Research
Institute of Clinical and
S UMMA R Y. During a study of breast development in the infant, we noticed that extramedullary haematopoiesis was a frequent occurrence.’ As haematopoiesis occurring in the infant breast has received little attention, we carried out a detailed histological study. Prominent foci of haematopoiesis were histologically demonstrated in 18 out of 58 samples obtained from infants less than 3.5 months of age. These foci were composed of both erythroid and myeloid cells. A positive correlation between the age of the infant and sequential maturation of red cell precursors was demonstrated. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) showing strong positive staining of the nuclei of the haematopoietic cells in some cases. Occasional megakaryocytes were identified by immunohistochemical staining for von Willebrand factor. A strong correlation between the development of lobules and the occurrence of haematopoiesis was demonstrated. The influence of intra-lobular connective tissue on the development of lobules and haematopoiesis is discussed.
INTRODUCTION
MATERIAL AND METHODS
Our current understanding of haematopoiesis in the fetus is based mainly on two studies published in the early 1940s. Bloom and Bartelmez (1940), studied a series of young human embryos starting with the 13-day stage, and described erythropoiesis in the yolk sac, liver and bone marrow .2 Gilmour (1942) reviewed the literature regarding haematopoiesis in intrauterine and neonatal life and also included his own material in his study. He described other areas of haematopoiesis including lymph node, spleen, tbymus, kidney and connective tissue including meninges mesentery, lymph plexuses and retroperitoneal tissue.3 During a study of infant breast development we noticed that haematopoiesis was a frequent occurrence’ and felt this deserved more attention. A detailed histological study has therefore being undertaken and has included histochemical staining for myeloid and megakaryocyte markers. A search of the older literature revealed that haematopoiesis in the infant breast was described as early as 1921 by Gruber.“ The aim of this article is to emphasize that in infants, the breast is one of the important sites of extramedullary haematopoiesis and to discuss the significance of some of our findings related to breast development and the occurrence of haematopoiesis in this organ.
Collection of specimens The material used was the same as that reported in the previous study.’ Necropsy specimens of infant breasts from 72 individuals (aged from newborn to 2 years) were collected. One breast from each case was fixed in 2% glutaraldehyde plus 2% formaldehyde.’ and the other was fixed in modified methacarn (60% methanol, 30% inhibisol, 10% acetic acid v/v).~
Processing for light microscopy Slices of tissue 2-4 mm thick were processed for paraffin wax embedding. Three pm thick sections were cut and stained with haematoxylin and eosin for microscopic examination. Sections from methacarn fixed material were used for immunohistochemistry and for chloroacetate esterase staining.
Antibodies used 1. Anti-human von Willebrand factor antibody. This is a rabbit polyclonal antibody from Dakopatts (Denmark), which recognises endothelial cells and megakaryocytes. It was supplied as purified immunoglobulin fraction and used at a dilution of 1 in 200.
Address for correspondence: Professor B. A. Gusterson, Section of Cell Biology and Experimental Pathology, Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, SM2 5NG, UK 182
Extramedullary
haematopoiesis
in the human infant breast
183
2. Anti-PCNA antibody (PC 10). This mouse monoclonal antibody which recognises proliferating cell nuclear antigen,’ was a generous gift from Dr. David P. Lane. It was used at a dilution of 1 in 200.
Immunoperoxidase staining Tissue sections from methacarn fixed specimens were treated with 0.3% hydrogen peroxide in phosphatebuffered saline (PBS) for 30 min to inhibit endogenous peroxidase and then washed with PBS. The sections were incubated for 1 h at room temperature with 200 /LI of the primary antibody, diluted in 0.5% bovine serum albumin (BSA) in PBS. Sections were then washed in two changes of PBS and incubated for 30 min at room temperature with peroxidase-conjugated rabbit antimouse or anti-rabbit 1gG (Amersham), diluted l/l00 in 0.5% BSA in PBS. The slides were washed in several changes of PBS and rinsed in distilled water before being treated with substrate consisting of 100 mg of 3,3_diaminobenzidine, 100 ml Tris buffer (pH 7.2), 100 ml distilled water and 66 ~1 of hydrogen peroxide. Control sections were stained without the first antibody.
Staining for chloroacetate esterase A solution of hexazonium pararosanilin was prepared by mixing equal volumes of 4% sodium nitrite in distilled water and 4% basic fuchsin in N/2 hydrochloric acid. This was left in a refrigerator at 4°C for 2-10 min. A buffer solution was prepared by adding 2 drops of hexazonium pararosanilin to 10 ml of Verona1 acetate buffer (pH 9.2) and the final pH was adjusted to 6.3. Substrate solution was prepared by adding the above buffer to 5 mg of naphthol AS-D chloroacetate dissolved in one drop of dimethyl formamide. Paraffin sections were dewaxed and taken to distilled water and incubated in freshly prepared substrate solution at room temperature for 30 min. They were then washed in water. air dried, cleared in inhibisol and mounted in DPX.
RESULTS Of the 72 infant breasts, 18 showed prominent foci of haematopoiesis in haematoxylin and eosin stained sections. All these were in individuals younger than 3.5 months. There was a total of 58 individuals younger than 3.5 months and approximately 31% in this group showed haematopoietic foci. In our previous study of morphology of the infant breast, marked heterogeneity in the morphological development of the infant breast was noted. Some had a less developed ductal system without lobular development (defined as morphological type I & II); others had a well-developed ductal system with numerous branching ducts and terminal lobules
Fig. l-Infant haematopoietic 550).
breast, 15 days old. Adipose tissue with elements. (Haematoxylin
and eosin stain, x-
(defined as morphological type III). It was noted that 16 out of the 18 breasts which showed haematopoietic foci belonged to morphological type III, i.e. those with lobule formation indicating a correlation between lobule development and the presence of haematopoietic foci. These haematopoietic foci were composed of erythroid and myeloid cells and varied from small collections of 3-5 cells arranged in a chain-like pattern to larger clusters containing 20-30 cells in groups distributed predominantly at the junction between the intralobular and interlobular connective tissue. This region is rich in blood vessels and the haematopoietic foci were situated in close vicinity to these. There were no distinguishing features between the erythroid and myeloid elements with regard to distribution in relation to stromal or parenchymal tissue. In a few instances small areas of adipose tissue in the interlobular connective tissue were seen which contained strands of haematopoietic elements (2-4 cells wide) running between the adipocytes (Fig. I). Haematopoietic cells occurring in newborn breasts were mainly composed of immature erythroid cells with a few myeloid elements. These cells showed large nuclei and scanty cytoplasm. Not infrequently a few histiocytes with large irregular nuclei and moderate amounts of cytoplasm could be seen in the immediate vicinity. A gradual differentiation of erythroid cells could be seen in relation to age. Thus erythroid cells occurring in infants 1 or 2 months old showed large nuclei and varying amounts of basophilic cytoplasm (Fig. 2). In infants older than 2 months these cells showed small pyknotic nuclei and large amounts of haemoglobinised eosinophilic cytoplasm. In some instances the nuclei showed karyorrhexis. Myeloid elements were more difficult to identify although eosinophilic granules were clearly seen in some of the cells. Staining for chloroacetate esterase showed strong positive staining in the cytoplasm of
Fig. 2-Infant breast, 1.5 months old. A group of erythroid and myeloid cells. (Haematoxylin and eosin stain, x 1000).
Fig. %-Infant breast, 1 month old, stained with anti-human von Willebrand factor antibody. A megakaryocyte with multilobed nucleus and large amount of granular cytolplasm. x 1000.
these cells. Megakaryocytes were rare and seen in only two breasts. Three megakaryocytes were seen in one and two in the other. These were related to blood vessels but no relationship with any other structure was evident. These megakaryocytes were large with multiple nuclei and large amounts of cytoplasm and were hard to recognise in haematoxylin and eosin stained sections, although they were easily identified in sections stained with anti-human von Willebrand factor antibody because of their strong cytoplasmic staining (Fig. 3.). Staining with anti-PCNA antibody showed positive nuclear staining of haematopoietic foci in 7 breasts (Fig. 4). The staining was heterogenous within individual foci and varied between foci in the same section.
1979 that Valdes-Dapena reported further observations on the occurrence of haematopoiesis in two infant breasts in his book ‘Histology of the fetus and newborn’.’ although Zuntova ( 1983)9 and McKiernan et al (1988)” also recorded the presence the haematopoietic foci in the neonates and infants we know of no other references reporting the occurrence of haematopoiesis in the newborn or infant breast. Haematopoiesis is known to occur in the infant breast” and in the adult breastiz-17 in association with abnormalities of haematopoiesis in the marrow. The occurrence of haematopoiesis in the normal adult breast has been reported recently. Malberger et al (1988) found haematopoietic elements in 9 women out of 10 000 fine need aspirations of breast lesions.” There was no evidence at the time of fine needle aspirations or on subsequent follow up of myelofibrosis with myeloid metaplasia in these patients. They concluded that
DISCUSSION Haematopoiesis begins in the 19-day-old embryo in the yolk sac and occurs later in the liver and bone marrow. Extramedullary haematopoiesis has been reported to occur in a variety of other sites including spleen, lymph nodes, thymus, kidney, breast and connective tissue plexuses and (meninges, mesentery, lymph retroperitoneal tissue). Extramedullary haematopoiesis occurring in the neonatal breast was reported as early as 1921. Gruber studied breasts from 50 newborns histologically4 and in his paper entitled ‘About the mammary gland of the newborn with special reference to blood formation’ Gruber gave a detailed description of foci of haematopoiesis in the newborn breast. He described the nuclear and cytoplasmic features of erythroid, myeloid and megakaryocytic cells. He also described the disappearance of these cells in infants after a few weeks of life and concluded that these cells were not due to an inflammatory cell infiltrate but due to local proliferation. After the initial observation by Gruber it was not until
Fig. 44nfant breast 1.5 months old, stained with anti-PCNA antibody. Strong positive staining of nuclei in the haematopoietic cells. x 1000.
Extramedullary
microfoci of extramedullary haematopoiesis in the normal breasts of healthy women do occur. To our knowledge there is no published literature on the incidence of extramedullary haematopoiesis in any other exocrine gland, under physiological conditions. Our results clearly indicate that in infants, the breast is one of the important sites where haematopoiesis occurs frequently (approximately 3 1%) and continues until 3.5 months of age. The erythroid elements which occur in the early post-natal period showed a large proportion of immature cells whereas in later periods the cells showed better differentiation. Thus any new foci of haematopoietic foci occurring later is unlikely, as they would be expected to show immature elements. Occurrence of erythroid elements nuclei with undergoing karyorrhexis indicate that these cells undergo degeneration after maturation explaining the disappearance of these after 3.5 months. For the first time we have confirmed the presence of myeloid and megakaryocytic elements in the infant breast by enzyme histochemial and immunohistochemical staining for the respective cell lineages. Positive staining for PCNA correlates well with cell proliferation and thymidine labelling.” The heterogeneous reactivity of the haematopoietic elements with anti-PCNA antibody indicate that these foci are not quiescent but are active and undergo cell cycle changes. A further interesting observation is the relationship between occurrence of haematopoiesis and development of lobules in the infant breast, with 16 out of 18 breasts which showed haematopoiesis having lobules. Haematopoietic foci also showed a close relationship either with intra-lobular connective tissue or interlobular adipose tissue. Intralobular connective tissue is known to be capable of supporting haematopoiesis and this has been referred to by Bassler as ‘mantle tissue’. He suggested that it was capable of producing blood cells during the whole of life.‘j He also referred to it as the ‘cytogenic stroma of the mammary gland’ to indicate its ability to support the development of cells. These observations might suggest that the microenvironment which influences differentiation of terminal duct lobular units of the fetal breast might also play a role in the development of haematopoietic foci. The importance of functional differences in the inter and intralobular stroma is indicated by a recent report that the fibroblasts of these two compartments express different ectopeptidases.‘” In this context we have also demonstrated that in infant breasts with developing lobules also have a defined subpopulation of fibroblasts (Anbazhagan and Atherton. unpublished observation). This is consistent with evidence from bone-marrow where marrow stromal cells appear to actively support haematopoiesis. Zontova” has reported a relationship between birth weight and extent of haematopoiesis in the breast. No haematopoiesis was observed in his study in babies weighing around 1OOOgm at birth, whereas in babies weighing over 1500gm haematopoietic foci were
haematopoiesis
in the human infant breast
185
consistently found in the breast. In pregnancy oestrogen concentrations are known to vary widely between individuals”. Oestrogen levels in pregnancy are considered as important determinants of birth weight “and high oestrogen levels might be expected to be associated with greater birth weight. We have suggested that the morphological variation observed in the newborn breast might result from differences in hormonal environment in utero.’ The association of haematopoietic foci with lobular development in the breast supports the view that higher oestrogen levels might contribute to the combination of high birth weight, lobular development in the breast and the occurrence of haematopoietic foci. Local hypoxia is an important factor in inducing haematopoiesis. Furthermore actively proliferating cells utilise oxygen and create a local hypoxic condition. Taking these two into consideration we postulate that increased proliferative activity (induced by oestrogen) leading to the development of lobules could lead to a relative local hypoxic environment which could be an important factor in the induction of haematopoiesis in the neonatal breast.
References I. Anbazhagan R, Bartek J, Monaghan P. Gusterson B A. Growth and development of the human infant breast. Am J Anat I9Y I : 192:407417. Bloom W. Bartelmez G W. Haematopoiesis in young human embryos. Am J Anat 1940; 67:21-53. Gilmour J R. Normal haematopoiesis in intrauterine and neonatal life. J Pathol Bact 1942; 52:25-55. Gruber G B. iiber die Milchdrilsenschwellung bei Neugeborenen. Z Kinderheilk 1921; 30:336-362. Monaghan P and Roberts J D B. Immunocytochemical evidence for neuroendocrine differentiation in human breast carcinomas. J Pathol 1985: 147:281-2. 6. Mitchell D. Ibrahim S and Gusterson B A. Improved immunohistochemical localization of tissue antigens using modified methacarn fixation. J Histochem Cytochem 1985: 33:491-%95. 7. Hall P A. Levison D A. Woods A L et al. Proliferating cell nuclear antigen (PCNA) immunolocalisation in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. J Pathol 1990; 162:285-2Y4. 8. Valdes-Dapena M D. Histology of the fetus and the new horn. Philadelphia, Saunders. 1979, pp 504-509. 9. Zuntova A. [Extra medullary haematopoiesis in the mammary gland of neonates and young infants]. Cesk Patol 1983: 19(3X 138-148. 10. McKiernan J. Coyne J, Cahalane S. Histology of breast development in early life. Arch Dis Childhood 1988; 63: 13gl3Y. II. Brannan D. Extramedullary haematopoiesis in anaemias. Bull Johns Hopkins Hosp 1927; 41:104-136. 12. Dieterick H. Studien iiber extramedullare Blutbildung bei chirungischen Erkrankurgen. Arch Klin Chir 1925; 134: 166-l 78. 13. Seifert G. iiber Gewebsreaktionen der menschlichen Brustdtise bei Leuklmien. Virchows Arch (Path Anat) 1952: 322:336-357. 14. Bassler R. The morphology of hormone induced structural changes in the female breast. Curr Top Pathol 1970; 53: I-8Y. 15. Glew H. Haese W H, McIntyre P A. Myeloid metaplasia with myelofihrosis: The clinical spectrum of extramedullary haematopoiesis and tumour formation. Johns Hopkins Med .I 1973: 132:253-265. 16. Brooks J J. Krugman D T, Damjanov I. Myeloid metaplasia presenting as a breast mass. Am J Surg Path01 1980; 4:28l-285. 17. Martinelli G, Santini D, Bazzocchi F, Pileri S. Casanova S. Myeloid metaplasia of the breast. Virchows Archiv (Pathoi Anat) 1983: 401:20?-207.
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The Breast
18. Malberger E, Hazzani A, Lemberg S. Extramedullary haematopoiesis in breast aspirates. Acta Cytol 1988; 32835-837. 19. Monagahan P, Perusinghe P, Nicholson RI, McClelland RA, O’Hare M J, Lane D P et al. Growth factor stimulation of proliferating cell nuclear antigen (PCNA) in human breast epithelium in organ culture. Cell Biol Int Reports 1991; 15:561-570. 20. Atherton A J, Monaghan P, Warburton M J, Robertson D, Kenny A J, Gusterson B A. Dipeptidyl peptidase IV expression identities a functionalsub-population of breast tibroblasts. Int J Cancer 1992; 50:15-19.
21. Genuth S M. The reproductive glands. In: Berne R M, Levy M N, eds. Physiology. St. Louis: C V Mosby Co, 1983: 1069-l 115. 22. Petridou E, Panagiotopoulou K, Katsouyanni K, Spanos E. Trichopoulos D. Tobacco smoking, pregnancy estrogens and birth weight. Epidemiology 1990; 1:247-250.
______~___ Date received 28 April 1992 Date accepted 26 June 1992
Extramedullary haematopoiesis in the human infant breast R. Anbazhagan,
J. Bartkova*,
B. Nathan and B. A. Gusterson
Institute of Cancer Research, Haddow Laboratories, Experimental Pathology, Bmo, Czechoslovakia
Sutton, Surrey UK. *Research
Institute of Clinical and
S UMMA R Y. During a study of breast development in the infant, we noticed that extramedullary haematopoiesis was a frequent occurrence.’ As haematopoiesis occurring in the infant breast has received little attention, we carried out a detailed histological study. Prominent foci of haematopoiesis were histologically demonstrated in 18 out of 58 samples obtained from infants less than 3.5 months of age. These foci were composed of both erythroid and myeloid cells. A positive correlation between the age of the infant and sequential maturation of red cell precursors was demonstrated. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) showing strong positive staining of the nuclei of the haematopoietic cells in some cases. Occasional megakaryocytes were identified by immunohistochemical staining for von Willebrand factor. A strong correlation between the development of lobules and the occurrence of haematopoiesis was demonstrated. The influence of intra-lobular connective tissue on the development of lobules and haematopoiesis is discussed.
INTRODUCTION
MATERIAL AND METHODS
Our current understanding of haematopoiesis in the fetus is based mainly on two studies published in the early 1940s. Bloom and Bartelmez (1940), studied a series of young human embryos starting with the 13-day stage, and described erythropoiesis in the yolk sac, liver and bone marrow .2 Gilmour (1942) reviewed the literature regarding haematopoiesis in intrauterine and neonatal life and also included his own material in his study. He described other areas of haematopoiesis including lymph node, spleen, tbymus, kidney and connective tissue including meninges mesentery, lymph plexuses and retroperitoneal tissue.3 During a study of infant breast development we noticed that haematopoiesis was a frequent occurrence’ and felt this deserved more attention. A detailed histological study has therefore being undertaken and has included histochemical staining for myeloid and megakaryocyte markers. A search of the older literature revealed that haematopoiesis in the infant breast was described as early as 1921 by Gruber.“ The aim of this article is to emphasize that in infants, the breast is one of the important sites of extramedullary haematopoiesis and to discuss the significance of some of our findings related to breast development and the occurrence of haematopoiesis in this organ.
Collection of specimens The material used was the same as that reported in the previous study.’ Necropsy specimens of infant breasts from 72 individuals (aged from newborn to 2 years) were collected. One breast from each case was fixed in 2% glutaraldehyde plus 2% formaldehyde.’ and the other was fixed in modified methacarn (60% methanol, 30% inhibisol, 10% acetic acid v/v).~
Processing for light microscopy Slices of tissue 2-4 mm thick were processed for paraffin wax embedding. Three pm thick sections were cut and stained with haematoxylin and eosin for microscopic examination. Sections from methacarn fixed material were used for immunohistochemistry and for chloroacetate esterase staining.
Antibodies used 1. Anti-human von Willebrand factor antibody. This is a rabbit polyclonal antibody from Dakopatts (Denmark), which recognises endothelial cells and megakaryocytes. It was supplied as purified immunoglobulin fraction and used at a dilution of 1 in 200.
Address for correspondence: Professor B. A. Gusterson, Section of Cell Biology and Experimental Pathology, Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, SM2 5NG, UK 182
Extramedullary
haematopoiesis
in the human infant breast
183
2. Anti-PCNA antibody (PC 10). This mouse monoclonal antibody which recognises proliferating cell nuclear antigen,’ was a generous gift from Dr. David P. Lane. It was used at a dilution of 1 in 200.
Immunoperoxidase staining Tissue sections from methacarn fixed specimens were treated with 0.3% hydrogen peroxide in phosphatebuffered saline (PBS) for 30 min to inhibit endogenous peroxidase and then washed with PBS. The sections were incubated for 1 h at room temperature with 200 /LI of the primary antibody, diluted in 0.5% bovine serum albumin (BSA) in PBS. Sections were then washed in two changes of PBS and incubated for 30 min at room temperature with peroxidase-conjugated rabbit antimouse or anti-rabbit 1gG (Amersham), diluted l/l00 in 0.5% BSA in PBS. The slides were washed in several changes of PBS and rinsed in distilled water before being treated with substrate consisting of 100 mg of 3,3_diaminobenzidine, 100 ml Tris buffer (pH 7.2), 100 ml distilled water and 66 ~1 of hydrogen peroxide. Control sections were stained without the first antibody.
Staining for chloroacetate esterase A solution of hexazonium pararosanilin was prepared by mixing equal volumes of 4% sodium nitrite in distilled water and 4% basic fuchsin in N/2 hydrochloric acid. This was left in a refrigerator at 4°C for 2-10 min. A buffer solution was prepared by adding 2 drops of hexazonium pararosanilin to 10 ml of Verona1 acetate buffer (pH 9.2) and the final pH was adjusted to 6.3. Substrate solution was prepared by adding the above buffer to 5 mg of naphthol AS-D chloroacetate dissolved in one drop of dimethyl formamide. Paraffin sections were dewaxed and taken to distilled water and incubated in freshly prepared substrate solution at room temperature for 30 min. They were then washed in water. air dried, cleared in inhibisol and mounted in DPX.
RESULTS Of the 72 infant breasts, 18 showed prominent foci of haematopoiesis in haematoxylin and eosin stained sections. All these were in individuals younger than 3.5 months. There was a total of 58 individuals younger than 3.5 months and approximately 31% in this group showed haematopoietic foci. In our previous study of morphology of the infant breast, marked heterogeneity in the morphological development of the infant breast was noted. Some had a less developed ductal system without lobular development (defined as morphological type I & II); others had a well-developed ductal system with numerous branching ducts and terminal lobules
Fig. l-Infant haematopoietic 550).
breast, 15 days old. Adipose tissue with elements. (Haematoxylin
and eosin stain, x-
(defined as morphological type III). It was noted that 16 out of the 18 breasts which showed haematopoietic foci belonged to morphological type III, i.e. those with lobule formation indicating a correlation between lobule development and the presence of haematopoietic foci. These haematopoietic foci were composed of erythroid and myeloid cells and varied from small collections of 3-5 cells arranged in a chain-like pattern to larger clusters containing 20-30 cells in groups distributed predominantly at the junction between the intralobular and interlobular connective tissue. This region is rich in blood vessels and the haematopoietic foci were situated in close vicinity to these. There were no distinguishing features between the erythroid and myeloid elements with regard to distribution in relation to stromal or parenchymal tissue. In a few instances small areas of adipose tissue in the interlobular connective tissue were seen which contained strands of haematopoietic elements (2-4 cells wide) running between the adipocytes (Fig. I). Haematopoietic cells occurring in newborn breasts were mainly composed of immature erythroid cells with a few myeloid elements. These cells showed large nuclei and scanty cytoplasm. Not infrequently a few histiocytes with large irregular nuclei and moderate amounts of cytoplasm could be seen in the immediate vicinity. A gradual differentiation of erythroid cells could be seen in relation to age. Thus erythroid cells occurring in infants 1 or 2 months old showed large nuclei and varying amounts of basophilic cytoplasm (Fig. 2). In infants older than 2 months these cells showed small pyknotic nuclei and large amounts of haemoglobinised eosinophilic cytoplasm. In some instances the nuclei showed karyorrhexis. Myeloid elements were more difficult to identify although eosinophilic granules were clearly seen in some of the cells. Staining for chloroacetate esterase showed strong positive staining in the cytoplasm of
Fig. 2-Infant breast, 1.5 months old. A group of erythroid and myeloid cells. (Haematoxylin and eosin stain, x 1000).
Fig. %-Infant breast, 1 month old, stained with anti-human von Willebrand factor antibody. A megakaryocyte with multilobed nucleus and large amount of granular cytolplasm. x 1000.
these cells. Megakaryocytes were rare and seen in only two breasts. Three megakaryocytes were seen in one and two in the other. These were related to blood vessels but no relationship with any other structure was evident. These megakaryocytes were large with multiple nuclei and large amounts of cytoplasm and were hard to recognise in haematoxylin and eosin stained sections, although they were easily identified in sections stained with anti-human von Willebrand factor antibody because of their strong cytoplasmic staining (Fig. 3.). Staining with anti-PCNA antibody showed positive nuclear staining of haematopoietic foci in 7 breasts (Fig. 4). The staining was heterogenous within individual foci and varied between foci in the same section.
1979 that Valdes-Dapena reported further observations on the occurrence of haematopoiesis in two infant breasts in his book ‘Histology of the fetus and newborn’.’ although Zuntova ( 1983)9 and McKiernan et al (1988)” also recorded the presence the haematopoietic foci in the neonates and infants we know of no other references reporting the occurrence of haematopoiesis in the newborn or infant breast. Haematopoiesis is known to occur in the infant breast” and in the adult breastiz-17 in association with abnormalities of haematopoiesis in the marrow. The occurrence of haematopoiesis in the normal adult breast has been reported recently. Malberger et al (1988) found haematopoietic elements in 9 women out of 10 000 fine need aspirations of breast lesions.” There was no evidence at the time of fine needle aspirations or on subsequent follow up of myelofibrosis with myeloid metaplasia in these patients. They concluded that
DISCUSSION Haematopoiesis begins in the 19-day-old embryo in the yolk sac and occurs later in the liver and bone marrow. Extramedullary haematopoiesis has been reported to occur in a variety of other sites including spleen, lymph nodes, thymus, kidney, breast and connective tissue plexuses and (meninges, mesentery, lymph retroperitoneal tissue). Extramedullary haematopoiesis occurring in the neonatal breast was reported as early as 1921. Gruber studied breasts from 50 newborns histologically4 and in his paper entitled ‘About the mammary gland of the newborn with special reference to blood formation’ Gruber gave a detailed description of foci of haematopoiesis in the newborn breast. He described the nuclear and cytoplasmic features of erythroid, myeloid and megakaryocytic cells. He also described the disappearance of these cells in infants after a few weeks of life and concluded that these cells were not due to an inflammatory cell infiltrate but due to local proliferation. After the initial observation by Gruber it was not until
Fig. 44nfant breast 1.5 months old, stained with anti-PCNA antibody. Strong positive staining of nuclei in the haematopoietic cells. x 1000.
Extramedullary
microfoci of extramedullary haematopoiesis in the normal breasts of healthy women do occur. To our knowledge there is no published literature on the incidence of extramedullary haematopoiesis in any other exocrine gland, under physiological conditions. Our results clearly indicate that in infants, the breast is one of the important sites where haematopoiesis occurs frequently (approximately 3 1%) and continues until 3.5 months of age. The erythroid elements which occur in the early post-natal period showed a large proportion of immature cells whereas in later periods the cells showed better differentiation. Thus any new foci of haematopoietic foci occurring later is unlikely, as they would be expected to show immature elements. Occurrence of erythroid elements nuclei with undergoing karyorrhexis indicate that these cells undergo degeneration after maturation explaining the disappearance of these after 3.5 months. For the first time we have confirmed the presence of myeloid and megakaryocytic elements in the infant breast by enzyme histochemial and immunohistochemical staining for the respective cell lineages. Positive staining for PCNA correlates well with cell proliferation and thymidine labelling.” The heterogeneous reactivity of the haematopoietic elements with anti-PCNA antibody indicate that these foci are not quiescent but are active and undergo cell cycle changes. A further interesting observation is the relationship between occurrence of haematopoiesis and development of lobules in the infant breast, with 16 out of 18 breasts which showed haematopoiesis having lobules. Haematopoietic foci also showed a close relationship either with intra-lobular connective tissue or interlobular adipose tissue. Intralobular connective tissue is known to be capable of supporting haematopoiesis and this has been referred to by Bassler as ‘mantle tissue’. He suggested that it was capable of producing blood cells during the whole of life.‘j He also referred to it as the ‘cytogenic stroma of the mammary gland’ to indicate its ability to support the development of cells. These observations might suggest that the microenvironment which influences differentiation of terminal duct lobular units of the fetal breast might also play a role in the development of haematopoietic foci. The importance of functional differences in the inter and intralobular stroma is indicated by a recent report that the fibroblasts of these two compartments express different ectopeptidases.‘” In this context we have also demonstrated that in infant breasts with developing lobules also have a defined subpopulation of fibroblasts (Anbazhagan and Atherton. unpublished observation). This is consistent with evidence from bone-marrow where marrow stromal cells appear to actively support haematopoiesis. Zontova” has reported a relationship between birth weight and extent of haematopoiesis in the breast. No haematopoiesis was observed in his study in babies weighing around 1OOOgm at birth, whereas in babies weighing over 1500gm haematopoietic foci were
haematopoiesis
in the human infant breast
185
consistently found in the breast. In pregnancy oestrogen concentrations are known to vary widely between individuals”. Oestrogen levels in pregnancy are considered as important determinants of birth weight “and high oestrogen levels might be expected to be associated with greater birth weight. We have suggested that the morphological variation observed in the newborn breast might result from differences in hormonal environment in utero.’ The association of haematopoietic foci with lobular development in the breast supports the view that higher oestrogen levels might contribute to the combination of high birth weight, lobular development in the breast and the occurrence of haematopoietic foci. Local hypoxia is an important factor in inducing haematopoiesis. Furthermore actively proliferating cells utilise oxygen and create a local hypoxic condition. Taking these two into consideration we postulate that increased proliferative activity (induced by oestrogen) leading to the development of lobules could lead to a relative local hypoxic environment which could be an important factor in the induction of haematopoiesis in the neonatal breast.
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18. Malberger E, Hazzani A, Lemberg S. Extramedullary haematopoiesis in breast aspirates. Acta Cytol 1988; 32835-837. 19. Monagahan P, Perusinghe P, Nicholson RI, McClelland RA, O’Hare M J, Lane D P et al. Growth factor stimulation of proliferating cell nuclear antigen (PCNA) in human breast epithelium in organ culture. Cell Biol Int Reports 1991; 15:561-570. 20. Atherton A J, Monaghan P, Warburton M J, Robertson D, Kenny A J, Gusterson B A. Dipeptidyl peptidase IV expression identities a functionalsub-population of breast tibroblasts. Int J Cancer 1992; 50:15-19.
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______~___ Date received 28 April 1992 Date accepted 26 June 1992