Background: Granulomatous lobular mastitis (GLM) is a rare, benign, and complex breast disease that can be easily misdiagnosed as breast cancer. The etiology of GLM is unclear, and optimal treatment has not been established. Methods: Medical records for 333 patients with GLM in recent 5 years at Longhua Hospital, Shanghai, China, were analyzed. Potential pathogens in 33 fresh tissue specimens were also analyzed using 16S rDNA sequencing technology, matrix-assisted laser desorption ionization time of flight mass spectrometry, and bacterial cultures. Results: The median age of patients was 32 years (range 22–47 years). Among 333 patients, 38.7% displayed elevated prolactin, while 23.7% displayed high interleukin-2. In the granulomatous lesion, CD3-positive T lymphocytes were significantly more than CD20-positive B lymphocytes around the vacuoles or microabscesses. Gram-positive organisms were observed in 82 cases, including in 22 cases from fresh tissue specimens. Thirty-three cases yielded associated pathogens and all displayed multiple pathogenic infections, as identified using 16S rDNA sequencing technology. Pathogenic infections were further identified as belonging to 16 main genera and 8 main pathogenic species. Conclusions: GLM displays distinct histological and clinical features similar to those that have been previously reported in the literature. Using 16S rDNA sequencing technology, all of our cases demonstrated multiple pathogenic infections, which provided more useful information for clinical treatment.

Granulomatous lobular mastitis (GLM), first described by Kessler and Wolloch in 1972 [1], is a rare, benign, chronic inflammatory disease of the breast. GLM predominantly affects young women of childbearing age with a history of parity and lactation [2, 3]. Common histopathological characteristics of GLM are noncaseating granulomas centered on a lobule, often with suppurative granulomas centered on vacuoles. Due to the absence of specific clinical and radiological findings, GLM can mimic breast cancer. Researchers have also reported cases where GLM and breast cancer coexist [4‒7].

At present, the etiology of GLM has not been completely defined. GLM has been linked to several possible etiologies and pathogeneses such as autoimmunity, microbiological infection, hyperprolactinemia, alpha-1 antitrypsin deficiency, oral contraceptive use, gestation and breastfeeding, smoking, diabetes mellitus, local trauma, and exposure to irritants [8, 9]. Recently, studies have revealed increased evidence for an association with corynebacterial infection, especially in the distinct pattern known as cystic neutrophilic granulomatous mastitis (CNGM) [10‒14]. One study indicated that MTHFR gene polymorphism is associated with GLM [15]. However, many potential pathogens remain unevaluated. The management of GLM remains controversial, with some opting for traditional Chinese medicine (TCM), steroid therapy, antibiotic therapy, or surgical treatment [16‒20]. To date, there is no consensus regarding an optimal treatment for GLM. Therefore, revealing the etiology and treatment for the disease is important.

In this study, we report a series of medical records for 333 patients diagnosed with GLM and the analysis of 33 fresh tissue specimens. Potential pathogens in the 33 fresh tissue samples were additionally analyzed using 16S rDNA sequencing technology, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and bacterial cultures. The goals of this study were to further improve our understanding of GLM and explore pathogens that potentially contribute to GLM. CNGM is a rare subtype of GLM with a highly distinct histological pattern often associated with Gram-positive organisms. Most granulomas are composed of central lipid vacuoles rimmed by neutrophils and an outer cuff of epithelioid histiocytes. Some of the lipid vacuoles may contain Gram-positive organisms. All of our cases had multiple pathogenic infections demonstrated by using 16S rDNA sequencing technology, which provided useful information for clinical treatment.

Patient Selection

We performed a search of medical records for patients diagnosed with GLM in recent 5 years at Longhua Hospital in Shanghai, China. A total of 333 patients were recruited into our study. Thirty-three fresh tissue specimens were also obtained. All specimens were surgically removed. Collected data included clinical presentations, radiological findings, laboratory investigations, histopathological findings, the duration of follow-up visits, and microbiological workups.

Histopathological Assessment

All specimens were fixed in 10% neutral formaldehyde. Paraffin-embedded sections, 4 μm in thickness, were mounted onto 3-aminopropyltriethoxysilane-coated glass slides. To evaluate histopathological features, hematoxylin and eosin staining was performed. Prepared samples were examined by two experienced pathologists. Gram staining was performed for 149 cases. One to two wax blocks containing lipid vacuoles were analyzed in each case.

Immunohistochemistry

Immunohistochemistry was performed on the Dako Autostainer Link 48 (DAKO, Agilent Technologies Inc., Santa Clara, USA) to reveal the expression of CD3 (monoclonal antibody, MX036, Fuzhou Maixin Biotechnology Co.), CD20 (monoclonal antibody, L26, DAKO, Agilent Technologies Inc.), CD68 (monoclonal antibody, PG-M1, Beijing Zhongshan Jinqiao Biotechnology Co.), IgG (polyclonal antibody, Fuzhou Maixin Biotechnology Co.), IgG4 (monoclonal antibody, EP138, Fuzhou Maixin Biotechnology Co.), CD38 (monoclonal antibody, MX044, Fuzhou Maixin Biotechnology Co.), and CD138 (monoclonal antibody, MI15, Fuzhou Maixin Biotechnology Co.). All specimens were fixed in 10% neutral formaldehyde. Paraffin-embedded sections, 3 μm in thickness, were mounted onto 3-aminopropyltriethoxysilane-coated glass slides. Prepared samples were examined by two experienced pathologists. Tonsil tissue was used as an external positive control. The staining steps were as follows: (1) all tissue sections were baked at 65°C for 2 h; (2) dewaxing: xylene 6 times, 5–10 min each time; anhydrous ethanol 3 times; 95% ethanol, running water flushing; (3) antigen retrieval: preheat tissue sections at 65°C and 97°C for 18 min, then cool to 65°C; (4) antibody applications: add primary antibody and incubate for 40 min; after block agent for 10 min, add second antibody for 20 min; and DAB color development for 5–10 min; (5) redyeing: hematoxylin staining for 30 s.

Bacterial Culture

Samples from 32 cases of GLM were cultured on blood agar plates, and samples from 24 cases were cultured on blood agar plates that contained 1% polysorbate (Tween) 80. All incubations occurred at 35°C for 72 h.

MALDI-TOF Mass Spectrometry

Species cultured from 32 cases were identified via MALDI-TOF-MS. A small amount of a colony was transferred to a metallic MALDI-TOF MSP 96 plate (Bruker Daltonik GmbH, Germany). Sample locations and numbers were recorded on the record sheet. Standard solution (1 μL, 50% acetonitrile, 47.5% water, and 2.5% trifluoroacetic acid) was added to each sample hole and dried naturally. One microlitre 70% formic acid was added to each sample, and 1 μL matrix solution (a-cyano-4-hydroxycinnamic acid, 2.5 mg/tube, adding 250 μL standard solution to dissolve) was then added and dried naturally. Measurements were performed using a Microflex mass spectrometer (Bruker Daltonic, Wissembourg, France) via the Flex Control software. The spectrum was imported into the BioTyper software (revision 2.0; Bruker, Karlsruhe, Germany). Identification score criteria used as recommended by the manufacturer: a score of 2.300–3.000 indicated species identification with high confidence level, a score of 2.000–2.299 indicated species identified at genus level and possible species level, a score of 1.700–1.999 indicated possible genus identification, and a score of 0.000–1.699 indicated unreliable species identification.

16S rDNA Sequencing Technology

To detect bacterial organisms, fresh tissue samples from 33 GLMs were analyzed using 16S rDNA sequencing technology. The microbial community DNA was extracted using NucleoSpin Soil Kit (Macherey-Nagel, Germany) following the manufacturer’s instructions. DNA was quantified with a Qubit Fluorometer by using Qubit® dsDNA BR Assay Kit (Invitrogen, USA) and the quality was checked by running aliquot on 1% agarose gel.

Variable regions V4 of bacterial 16S rRNA gene were amplified using degenerate PCR primers, 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Both forward and reverse primers were tagged with Illumina adapter, pad, and linker sequences. PCR enrichment was performed in a 50 μL reaction containing 30 ng template, fusion PCR primer, and PCR master mix. PCR cycling conditions were as follows: 95°C for 3 min, 30 cycles of 95°C for 45 s, 56°C for 45 s, 72°C for 45 s, and final extension for 10 min at 72°C for 10 min. The PCR products were purified using Agencourt AMPure XP beads and eluted in Elution buffer. Libraries were quality controlled using the Agilent Technologies 2100 bioanalyzer. The validated libraries were used for sequencing on Illumina HiSeq 2500 platform (BGI, Shenzhen, China) following the standard pipelines of Illumina and generating 2 × 250 bp paired-end reads. Specific microbiological information is provided in Table 1.

Table 1.

Microbial data of GLM

 Microbial data of GLM
 Microbial data of GLM

Statistical Analysis

SPSS (NY, USA) 24.0 software was used for the statistical analysis. Frequency (%) was determined for quantifiable data. The data were reviewed and approved by all authors.

Clinical Characteristics

Clinical and demographic characteristics of investigated patients are provided in Table 2. All of the patients were female. Patients ranged in age from 22 to 47 years (mean age: 32 years, median age: 32 years). The majority of patients were in the 30–39 age group (63.7%). Three hundred twenty-seven (327, 98.2%) patients were parous, and 318 (95.5%) patients had a breastfeeding experience with a lactation duration ranging from 1 month to 2 years. Patients were postpartum diagnosed with GLM over an interval of 1 month to 10 years. One hundred sixty-five (165, 49.5%) of determined lesions were located within the left breast and 147 (44.1%) were located within the right breast. In 21 patients (6.4%), bilateral lesions were determined. Laboratory investigations indicated that 129 (38.7%) patients had elevated prolactin, 79 (23.7%) patients had high interleukin-2, and 131 (39.3%) patients had a high erythrocyte sedimentation rate.

Table 2.

Clinical and demographic data of GLM

 Clinical and demographic data of GLM
 Clinical and demographic data of GLM

One hundred ninety-nine (199, 59.8%) patients underwent magnetic resonance imaging. Magnetic resonance imaging results indicated nontumor enhancement foci, and, secondarily, a cluster of ring-shaped, non-uniform enhancement, and a high signal in T2WI. One hundred sixty-two cases were assigned a grade of BI-RADS, 51 (31.5%) cases were grade 2, 80 (49.4%) cases were grade 3, 26 (16.0%) cases were grade 4a, and 5 (3.1%) cases were grade 4b.

Two (0.6%) patients were additionally complicated by a pituitary adenoma. Two (0.6%) patients had a history of oral antipsychotics, of which one was nulliparous. Twenty-three (23, 6.9%) patients were complicated by a catheter dilatation. Forty-two (42, 12.6%) patients were complicated by fistula formation. Forty-eight (48, 14.4%) patients were complicated by erythema nodosum. Fifty-nine (59, 17.7%) patients were complicated by nipple retraction.

Pathological Characteristics and Immunohistochemistry

In all cases, noncaseating granulomatous inflammation centered on a lobule was observed, often with suppurative granulomas centered on vacuoles (Fig. 1). Most granulomas were centered on vacuoles and surrounded by neutrophils, with a background of acute, chronic inflammation that included the presence of lymphocytes, plasma cells, epithelioid histiocytes, multinucleated giant cells, and eosinophils (Fig. 2), which form is thought to be CNGM. The size and number of vacuoles varied. Most common microcysts displayed a combination of multiple adipocytes of different sizes. Some granulomatous lesions were fused, resulting in obliteration of the normal architecture.

Fig. 1.

Noncaseating granulomas centered on a lobule and vacuoles (×40 magnification).

Fig. 1.

Noncaseating granulomas centered on a lobule and vacuoles (×40 magnification).

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Fig. 2.

Suppurative granulomas centered on vacuolated spaces rimmed by neutrophils and surrounded by lymphocytes, plasma cells, epithelioid, histiocytes, multinucleated giant cells, and eosinophils (×200 magnification).

Fig. 2.

Suppurative granulomas centered on vacuolated spaces rimmed by neutrophils and surrounded by lymphocytes, plasma cells, epithelioid, histiocytes, multinucleated giant cells, and eosinophils (×200 magnification).

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Seventy-seven (77, 23.1%) patients’ tissue samples were stained using immunohistochemistry. CD3, CD20, and CD38 were stained on cytomembrane. CD68, IgG, and IgG4 were stained on cytoplasm. CD138 was stained on the cytomembrane and cytoplasm. In the granulomatous lesion, CD20-positive B lymphocytes were significantly more than CD3-positive T lymphocytes in the peripheral area (CD3 [31.49 ± 11.50]%, CD20 [68.51 ± 11.50]%, p < 0.05), CD3-positive T lymphocytes (Fig. 3) were significantly more than CD20-positive B lymphocytes (Fig. 4) around the vacuoles or microabscesses (CD3 [78.51 ± 10.00]%, CD20 [21.49 ± 10.00]%, p < 0.05). There was a zonal distribution of lymphocytes around vacuoles or microabscesses.

Fig. 3.

CD3-positive T lymphocytes within a granulomatous lesion (En Vision, ×100 magnification).

Fig. 3.

CD3-positive T lymphocytes within a granulomatous lesion (En Vision, ×100 magnification).

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Fig. 4.

CD20-positive B lymphocytes within a granulomatous lesion (En Vision, ×100 magnification).

Fig. 4.

CD20-positive B lymphocytes within a granulomatous lesion (En Vision, ×100 magnification).

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Gram Stain

Among the 333 cases of GLM, 149 cases are CNGM with typical lipid vacuoles. Lipid vacuoles may contain sparse, rod-shaped, Gram-positive bacilli. To increase the positive rate, Gram staining was performed on the 149 cases of CNGM. Samples from 82 (55%) cases were observed to contain Gram-positive organisms, of which 22 cases had fresh tissue specimens available for analysis. Gram-positive organisms were only identified within the optically empty lipid solubility central vacuoles of granulomas as rod-shaped pleomorphic coccobacilli. Gram-positive organisms were commonly arranged in groups conforming to small clusters and were sometimes found to be very scarce in vacuoles. Bacteria within vacuoles demonstrated various formations, for example, in palisades, “V” shapes, or “Chinese characters,” and partial internally present bacteria were “rosary bead-like” or “bamboo-like” in appearance (Fig. 5). The number of organisms in vacuoles greatly varied, and, occasionally, Gram-negative organisms were also identified.

Fig. 5.

Gram-positive coryneform bacteria arranged within lipid solubility vacuoles of variable shapes and sizes (Gram stain, ×400 magnification).

Fig. 5.

Gram-positive coryneform bacteria arranged within lipid solubility vacuoles of variable shapes and sizes (Gram stain, ×400 magnification).

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Detection and Characterization of Pathogens

The analysis of microbial data is provided in Table 1. To further detect pathogens in patients with GLM, 16S rDNA sequencing technology was utilized for analyzing fresh tissue specimens obtained from 33 GLM patients. A bioinformatics analysis revealed that 16 main genera and 8 main species of pathogens were present in the 33 GLM patients (Fig. 6). Species with abundances of less than 0.5% in all samples were combined into a group designated as “others.” All of the pathogens were bacteria. Thirty-three patients had multiple pathogens.

Fig. 6.

Taxonomic profiles for granulomatous mastitis microbiota at the genera and species level. A bioinformatics analysis compared the relative abundance of bacteria in 33 patients with granulomatous mastitis at the genera (a) and species (b) levels. Each bar represents a subject. Taxa names are provided on the right.

Fig. 6.

Taxonomic profiles for granulomatous mastitis microbiota at the genera and species level. A bioinformatics analysis compared the relative abundance of bacteria in 33 patients with granulomatous mastitis at the genera (a) and species (b) levels. Each bar represents a subject. Taxa names are provided on the right.

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The top five genera of pathogens detected were Serratia (33/33), Citrobacter (33/33), Acinetobacter (33/33), Pseudomonas (27/33), and Sphingomonas (22/33). Using species classification, the top five species of pathogens were Serratia marcescens (33/33), Pseudomonas fragi (24/33), Pseudomonas veronii (22/33), Sphingomonas azotifigens (20/33), and Pelomonas puraquae (16/33) (Fig. 7).

Fig. 7.

Number of cases for each pathogen detected in 33 patients with granulomatous mastitis at the genus (a) and species (b) levels. Taxa names are provided on the left.

Fig. 7.

Number of cases for each pathogen detected in 33 patients with granulomatous mastitis at the genus (a) and species (b) levels. Taxa names are provided on the left.

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Bacterial culture was performed for 32 patients with available fresh tissue specimens. Identified species, using a blood agar plate, were as follows: Staphylococcus epidermidis (18/32, 56.3%), Staphylococcus aureus (2/32, 6.3%), and Dermabacter hominis (1/32, 3.1%). We also observed the presence of Staphylococcus epidermidis (15/24, 62.5%) and multiple bacteria (2/24, 8.3%) on blood agar plates containing 1% polysorbate (Tween) 80 (Fig. 8). We further identified Staphylococcus epidermidis (18/32, 56.3%), Staphylococcus aureus (2/32, 6.3%), Dermabacter hominis (1/32, 3.1%), and multiple bacteria (3/32, 9.4%) using MALDI-TOF-MS. The multiple bacteria observed in case 2 were largely: Staphylococcus epidermidis, Bacillus endophyticus, Acinetobacter pittii, Rhodococcus fascians, Candida boidinii, Sphingobacterium mizutaii, Bacillus flexus, and Bacteroides fragilis. The multiple bacteria observed in case 15 were largely: Listeria monocytogenes, Plesiomonas shigelloides, Penicillium chrysogenum, Streptococcus mutans, Bordetella parapertussis, Lactobacillus brevis, Dermabacter hominis, and urinate rod bacillus. Multiple bacteria identified in case 16 were largely: Staphylococcus epidermidis, Staphylococcus lugdunensis, Paenibacillus polymyxa, and Bacillus cereus.

Fig. 8.

Bacterial culture of fresh specimens. a Staphylococcus aureuson a blood agar plate (case 9). b Dermabacter hominison a blood agar plate (case 23). c Staphylococcus epidermidison a blood agar plate containing 1% polysorbate (Tween) 80 (case 20). d Multiple bacteria on a blood agar plate containing 1% polysorbate (Tween) 80 (case 2).

Fig. 8.

Bacterial culture of fresh specimens. a Staphylococcus aureuson a blood agar plate (case 9). b Dermabacter hominison a blood agar plate (case 23). c Staphylococcus epidermidison a blood agar plate containing 1% polysorbate (Tween) 80 (case 20). d Multiple bacteria on a blood agar plate containing 1% polysorbate (Tween) 80 (case 2).

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Follow-Up and Outcomes

Follow-up information was obtained by telephone and during outpatient visits. We received follow-up data from 314 patients. For 19 patients, no follow-up data were available. The follow-up duration was 6–32 months. Two hundred seventy-eight (278, 88.5%) patients made a full recovery, 28 (8.9%) patients had an improved condition, and 8 (2.5%) patients made no recovery or had a recurrence.

GLM, also known as idiopathic granulomatous mastitis (IGM), is a rare, chronic inflammatory breast disease of unknown etiology. Although GLM is a benign disease, mastectomy may be performed, which may result in loss of the breast and cause serious impacts on patients’ physical and mental health. GLM generally affects young women of childbearing age with a history of parity and lactation and can occasionally occur in the nulliparous and in men [21‒24]. In our study, lesions were consistent with previous studies [25]. The most common presentation for GLM is breast mass and pain, generally accompanied by skin ulceration, sinus formation, abscess, erythema nodosum, nipple retraction, and axillary lymphadenopathy [26‒29].

GLM has the distinct histological characteristics of noncaseating granulomas centered on a lobule. In our study, most granulomas were centered on vacuoles surrounded by neutrophils, with a background of acute, chronic inflammation, as determined by the presence of lymphocytes, plasma cells, epithelioid histiocytes, multinucleated giant cells, and eosinophils, which are considered to be CNGM. Intralobular ducts were reduced or absent. Some granulomatous lesions were fused and resulted in the obliteration of normal architecture. Cases of GLM can also be accompanied by catheter dilatation, fibroadenoma, erythema nodosum, etc.

Because of the absence of specific clinical and radiological findings, confusing GLM with breast cancer is common [30, 31]. Furthermore, differential diagnoses of GLM include plasma cell mastitis, duct ectasia, sclerosing lymphocytic lobular mastitis, IgG4-related sclerosing mastitis, and other known causes of granulomatous diseases such as tuberculosis, sarcoidosis, fat necrosis, foreign body granuloma, etc. [32, 33]. However, in our study, we found that CD3-positive T lymphocytes were present at higher levels than CD20-positive B lymphocytes around vacuoles or microabscesses. This result is different than expected for sclerosing lymphocytic lobular mastitis in which CD20-positive B lymphocytes are mainly expressed. We also observed a few scattered plasma cells expressing IgG4 and IgG4/IgG <40%, suggesting that our cases did not suffer from IgG4-related sclerosing mastitis.

The etiology and pathogenesis of GLM have, to date, not been fully defined. GLM has possible etiologies and mechanisms of pathogenesis such as autoimmunity, microbiological infection, hyperprolactinemia, alpha-1 antitrypsin deficiency, oral contraceptive use, gestation and breastfeeding, smoking, diabetes mellitus, local trauma, and exposure to irritants. Nikolaev et al. [34] reported that intracranial lesion-induced hyperprolactinemia may be directly causal for granulomatous mastitis. Non-lactation causes have also been proposed. For example, hyperprolactinemia caused by antipsychotic medication for psychiatric illness is a risk factor for granulomatous mastitis [21, 22, 35]. In our study, we also had patients with elevated prolactin levels; we postulate that there is an association between elevated prolactin and GLM, whereby hypersensitivity induced by extravasated lactation secretions may be responsible for GLM. The specific mechanism should be further investigated.

Recently, increasing evidence for an association between GLM and corynebacterial infection, especially in regards to a distinct pattern termed CNGM, has been discussed [36, 37]. Naik et al. [38] reported that Gram-positive bacilli were identified in 20/24 cases and that MALDI-TOF-MS identified Corynebacterium kroppenstedtii in 2 cases and Corynebacterium tuberculostearicum in 2 cases, with no identified bacteria for 1 case. Corynebacterium were also identified using 16s rRNA sequencing in DNA that extracted from formalin-fixed paraffin-embedded tissue in 12/23 cases. Oddo et al. [39] reported that 28 of 57 cases diagnosed with GLM had coryneform bacteria, identified using Gram staining, within central vacuoles. However, no specific pathogens have been detected in some studies regarding GLM [16, 25, 40]. The number of organisms identified within vacuoles varied, and, sometimes, Gram-negative organisms were concurrently observed. Due to this variability, careful analysis was required so no specimens were missed. Organism-specific locations within the granuloma were sufficient to eliminate the possibility of sample contamination. No significant difference between Gram stain positive and negative groups in terms of skin ulcer, puncture, fever, or axillary lymph node enlargement was determined.

To further detect pathogens, we initially used formalin-fixed paraffin-embedded tissue; however, this was unsuccessful. We, therefore, collected fresh tissue specimens from 33 patients with GLM. 16S rDNA sequencing technology, bacterial cultures, and MALDI-TOF-MS were used for microbiological studies. We found that 33 cases were associated with pathogens. All of the cases were multiple pathogenic bacterial infections, a finding consistent with previous studies [41]; the pathogens detected should have a certain guiding effect on clinical treatment. We found Corynebacterium in 11/33 cases in relatively low abundance. In contrast to previous studies, Corynebacterium was not the main pathogen. S. marcescens was in relatively high abundance in this study. Serratia species are gram-negative bacteria, which can cause a heterogeneous spectrum of diseases in humans and animals. Giulia Fazio et al. [42] described a challenging case of a S. marcescens’ pulmonary infection, which occurred in a patient with breast cancer. Gram-negative organisms were also observed in the vacuoles of GLM samples.

Although it remains unclear which if any of these pathogens causes GLM, the discovery of pathogens could aid the guidance of clinical treatment. The species detected in our study differ from those described in previous reports. One possible explanation for our findings is that the organisms were only present in vacuoles with abundances different from other locations. Close attention to detail was employed in order to identify bacteria within vacuoles. Another possible explanation is that Corynebacterium are rare fastidious lipophilic bacterium that can survive in harsh conditions for long periods. A limitation to our study was the fact that our sample capacity was relatively small. Therefore, whether or not the identified pathogens, or secondary infections, cause GLM requires further investigation. Larger patient numbers in a future study would be useful for furthering our understanding of GLM.

To date, no established consensus on optimal treatment for GLM exists. Treatments can be designated according to a timescale and patient preference [17, 28]. Treatments for GLM include TCM, steroid therapy, antibiotic therapy, and surgical treatment. Multiple treatments are recommended in order to improve therapeutic effects [16, 40]. Some studies have shown that lipophilic antibiotics, such as trimethoprim, clarithromycin, and rifampicin, are effective [43, 44]. Topical steroids may be the first-line treatment for many patients [45], and other immunosuppressive drugs, such as methotrexate, may be used in patients with steroid resistance or steroid side effects [46]. Wang et al. [47] found that surgery following steroid therapy was an effective and more satisfactory treatment than only using steroid therapy. Liao et al. [48] found that ultrasound classification-guided minimally invasive rotary cutting, a safe and effective method, helped determine suitable patients, especially those with localized abscess and localized hypoechoic mass types with low recurrence rates. Wang et al. [49] found minimally invasive comprehensive treatment was a new method for the treatment of GLM, ensuring a therapeutic effect while maintaining breast completeness.

Most of the patients enrolled in our study had undergone repeated treatment in other hospitals and had a long course of disease and severe lesions. All patients were treated with surgery, using procedures such as drainage or mastectomy combined with TCM applied orally or locally. Such treatments mostly led to positive results. Wang et al. [47] found that the rate of recurrence of GLM was lower for the surgically treated group (5.1%) as compared to those treated with steroids (22.7%). However, only 2.5% of our patients had no recovery or recurrence.

Our hospital has strong, distinctive characteristics and the prominent advantage of TCM. Using Chinese medicine, Professor Liu concluded that the pathogenesis of GLM is liver stagnation transforming into fire and turbidity due to spleen deficiency. Disease treatment using Chinese medicine is largely based on a resolving method that is focused on the liver and spleen, performed in stages, and focused on local tissue. Internal treatment is the priority, while external treatment is used in a complementary manner. In this treatment scheme, achieving recovery is of utmost importance.

In summary, in this study, we reported clinical data regarding 333 patients diagnosed with GLM. We employed 16S rDNA sequencing technology, MALDI-TOF-MS, and bacterial cultures to detect potential pathogens in 33 fresh tissue specimens. Based on our analyses, we concluded that GLM has distinct histological and clinical features. Most granulomas were composed of central lipid vacuoles rimmed by neutrophils and an outer cuff of epithelioid histiocytes. Some lipid vacuoles contained Gram-positive organisms, which were considered as CNGM. Thirty-three of the analyzed cases yielded multiple pathogenic infections by using 16S rDNA sequencing technology, which provided useful information for clinical treatment. To explore potential pathogens involved in GLM and to provide an accurate diagnosis and subsequent therapeutic methodologies, further study is required.

This study is in accordance with the Declaration of Helsinki. All of the patients signed an informed consent form and the Ethical Committee approved this research. The study protocol was reviewed and approved by the Ethical Committee of Longhua Hospital Shanghai University of Traditional Chinese Medicine, approval number 2022LCSY023.

The authors have no conflicts of interest to declare.

The study was supported by National Natural Science Foundation (81403429), “seedling plan” and “science and technology innovation projects” of Longhua Hospital Shanghai University of Traditional Chinese Medicine.

Xiaoyun Zhang: conception and design; Yunyuan Li: material preparation, data collection and analysis, and drafting of the manuscript; Ling Chen, Chunyan Zhang, Yanwen Wang, Jun Hu, and Mengyun Zhou: material preparation, data collection; and Xiaoyun Zhang: critical revision of the manuscript. All authors read and approved the submitted version.

All data are accessible upon reasonable request to the corresponding author.

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