Alterations in the mitochondrial genome have been chronicled in most solid tumors, including breast cancer. The intent of this paper is to compare and document somatic mitochondrial D-loop mutations in paired samples of ductal carcinoma
In 2010, close to 207,090 new cases of IBC were diagnosed in the United States, and DCIS was identified in an additional 54,010 women who did not yet have IBC [
Although DCIS masses are often small by comparison to IBC masses, DCIS is typically detected through mammography or self-examination. However, there are significant shortcomings to these methods. Mammography is generally used to identify breast masses within a resolution limit of 1 cm, and the Van Nuys prognostic index (VNPI) for DCIS does not score tumors less than 1.5 cm. This means that a subset of smaller lesions which may have significant future clinical impact remain undetected and/or evaluated. Breast self-examination has also been extensively shown to be an ineffective detection tool for Asian women [
Currently, there is no clinical means to distinguish between the heterogeneous types of DCIS and recognize the carcinomas that will progress into invasive, metastatic breast cancer. The mechanism that drives this transformation from DCIS to IBC is also not well understood. Hence, when mammography detects DCIS, a full diagnostic workup and treatment is required [
The progression of DCIS is poorly understood because the technology used to detect it relies on tissue mass. Indeed, the identification of DCIS via mammography is low compared to larger tumors. If a significant proportion of IBC cases originate as DCIS, then successful detection and stratification of these lesions will assist the clinician and the patient with determining potential monitoring and treatment strategies. A recent review articulated the need for a combined research effort directed towards this clinical need [
This study proposes using somatic mitochondrial D-loop mutations in paired samples of DCIS and IBC to identify a potential breast ductal epithelial “cancerization” field effect. Alterations in the mitochondrial genome have been chronicled in most solid tumors, including breast cancer [
Mitochondrial D-loop mutations can be evaluated using tissue samples from solid tumors. Using biofluids with low cellularity such as nipple aspirate fluid (NAF) or ductal lavage (DL) represents a much less invasive route for developing early detection tests. The mtGenome is ideal for these investigations because it has a high copy number per cell, when compared to the nuclear archive of DNA.
There are other characteristics suggesting that the mtGenome may be an ideal “biosensor” as follows: (1) each copy of the mtGenome is clonal; (2) the mtGenome has a maternal inheritance pattern which precludes generational recombination; (3) somatic mutations appearing in a subset of mtGenomes, known as heteroplasmy, afford early disease detection; (4) the modest size of the mtGenome (16,568 bp) allows inexpensive, targeted, and concentrated genetic analyses; (5) the mtGenome has a 10–100-fold copy advantage over the nuclear genome; (6) the mitochondrial organelle is the center of ATP synthesis and is the mediator of cell apoptosis, and for successful tumorigenesis to occur, energy production must be replaced by an alternative process and apoptosis must be by-passed; (7) mitochondrial DNA (mtDNA) has an accelerated somatic mutation rate in which mutations occur within years, and perhaps months, from when molecular pathways are altered by early molecular changes associated with malignant transformation; (8) mutations in the mtGenome have been attested in a wide variety of solid tumors.
Women who were referred to a surgical oncologist for a clinical breast examination and had a biopsy with positive results were recruited to this study. Patients having a biopsy, lumpectomy, or mastectomy were selected based on a pathology report which identified both DCIS and IBC. Two patients had both a biopsy and a secondary procedure (lumpectomy and mastectomy). All patients were procured in accordance with the ethical guidelines of the Thunder Bay Regional Health Sciences Research Ethics Board in adherence to the Tri-Council Policy Statement on Ethical Conduct for Research Involving Humans. Written consent was obtained from the patients for publication of the study. Patients were selected based on review of biopsy and/or surgical pathology reports. A total of 34 patients were identified, however, upon sectioning of requested samples, only 15 had sufficient quantities of both IBC and DCIS to warrant LCM. After complete sample processing (extraction through sequencing), 5 patients were further eliminated due to sample drop-out. A total of 34 samples, including blood, were contributed by a suite of 10 patients (Table
Clinical, pathological information with parallel mutation sites in common between both DCIS and IBC for each patient, inclusive of all patient tissue samples. Heteroplasmic sites are scored as mutations.
Patient | Age | Sample | Est | Pro | HER2 | Grade | NG | TF | MS | BR | EIC | Parallel mutations | Haplotype |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
33 | 65 | Mast | − | − | + | 3 | 3 | 2 | 3 | 8 | + | 192, 224, 304, 311 | U5a |
| |||||||||||||
36 | 67 | Lump | + | − | − | 1 | 1 | 1 | 1 | 3 | − | 188, 189, 223, 224, 270, 291, 311, 319, 362, 390 | H |
| |||||||||||||
43 | 51 | Biopsy | 2 | 93, 189, 298 | V | ||||||||
52 | Lump | + | + | 2 | 2 | 2 | 2 | 6 | − | ||||
| |||||||||||||
46 | 66 | Biopsy | − | + | − | 2 | 2 | 3 | 1 | 6 | − | 126, 189, 223, 291, 357, 362, 390 | J |
| |||||||||||||
57 | 41 | Lump | + | + | − | 3 | 2 | 3 | 3 | 8 | + | 189, 291, 390 | H |
| |||||||||||||
64 | 51 | Biopsy | 182, 183, 249 | H | |||||||||
51 | Mast | − | − | + | 3 | 3 | 3 | 3 | 9 | − | |||
| |||||||||||||
74 | 39 | Biopsy | − | − | + | 203, 304 | K | ||||||
40 | Mast | 2 | 3 | 2 | 1 | 6 | + | ||||||
| |||||||||||||
83 | 49 | Lump | + | − | − | 2 | 2 | 3 | 1 | 6 | − | 189 | H |
| |||||||||||||
89 | 41 | Lump | − | − | − | 3 | 3 | 3 | 3 | 9 | − | 189, 223, 224, 291, 298, 311, 362, 390 | K |
| |||||||||||||
94 | 50 | Lump | + | + | 1 | 2 | 2 | 1 | 5 | − | 93, 189, 224, 291, 311 | H |
Unavailable information is left blank. NG: nuclear grade; MS: mitotic score; BR: modified Bloom-Richardson grade; EIC: extensive intraductal component.
Requested tissues (biopsy and mastectomy samples) were sectioned from formalin-fixed paraffin-embedded (FFPE) blocks and processed for LCM. LCM was performed by two qualified, gowned, gloved, and masked technicians who captured both DCIS and IBC from each patient. By direct observation of the process, about 3-4 cells were harvested per laser pulse, or capture event, and approximately 2,000 captures were recovered from each tissue type. DNA was liberated from LCM samples by an overnight digestion at 65°C in 50
A portion of the D-loop was amplified with primer sets MT1, 2, 3 forward and reverse (MitoScreen Assay Kit, Transgenomic, Omaha, NE) using the following reagent concentrations per reaction: 1X FastStart High Fidelity Reaction Buffer, 1.8 mM MgCl2, and 0.25 U FastStart High Fidelity Enzyme Blend (Roche, Burgess Hill, UK); 0.2 mM of each dNTP; 0.3
Following amplification, DHPLC was used to identify areas of mtGenome alterations. Sample analysis temperatures were predicted using Navigator software (Transgenomic, Omaha, NE). The gradient mobile phase consisted of Buffer A (0.1 M Triethylammonium Acetate pH 7.0) and Buffer B (0.1 M Triethylammonium Acetate pH 7.0) and 25% Acetonitrile. These buffers were mixed to provide a linear gradient varying Buffer B, for MT1 59–67, MT2 57–65, and MT3 59–67 over a 4-minute period. After each analysis, the column was cleaned with 100% Buffer B and then equilibrated for 2 minutes with Buffers A : B 54 : 56 (MT1 and MT3) 52 : 58 (MT2). Analysis temperatures were 58°C for MT1, 60°C for MT2, and 57°C and 59°C for MT3. Prior to injection, the samples were heteroduplexed by heating to 95°C for 5 minutes and then cooling at 1.5°C per minute down to 25°C.
Due to the low amount of template recovered from the LCM procedure, sequencing efforts were limited to a target sequence through and around hypervariable region 1 (HV1; 16,024–16,383). HV1 has a 2-fold higher mutation rate than HV2 [
This provided a low yield product which was amplified for a smaller sequence (627 bp) with nested primer D1 [
Reaction conditions were again the same as mentioned previously with the following changes: primer concentrations were increased to 0.6
Primer set MT2/MT19 (15424-102) was used to generate template for nested amplification with D1 primers (15898–16525). Both sets of primers were tested for null amplification against Rho 0 derived template [
Amplified template was sequenced at Genevision (Newcastle Upon Tyne, UK). Both Geneious bioinformatics software (Biomatters) and Sequencher 4.5 (Gene Codes) were used for sequence analyses.
Analyses were performed on HV1 mutation patterns and all applicable parameters listed in the pathology report: age, receptor status, tumor grade, nuclear grade, tubule formation, mitotic score, modified Bloom-Richardson grade, and presence or absence of extensive intraductal component. Attempts to correlate the diagnostic rankings and per-site mutation results were made using point-biserial and rank-biserial statistics. Pearson rank correlation was used to identify the strength of the relationship between HVR1 relative substitution rates and the prevalence of each mutation site in the patient data. IBC and DCIS sample populations were considered separately in order to determine if any patterns existed in the mutation load of the individual sample types as well as to discover the presence of any interactions between the two tissue types. Again, Pearson correlations were used as statistics for this analysis.
Mutations were identified in HV1 which was reamplified with Rho 0 null primers and sequenced. All patients in this study demonstrate heteroplasmy in all of the associated histologies in comparison to germ plasma, or blood. It is important to note that 18 sites had homoplasmic and/or heteroplasmic mutation sites in common between DCIS and IBC lesions recovered from the same patient. All patients had at least 1 corresponding homoplasmic and/or heteroplasmic site in both DCIS and IBC. These results parallel similar observations noting that other biomarkers are held in common between DCIS and IBC [
HV1 somatic mutations are bolded, while mutations persisting in all patient samples are also italicized. Patient histologies are compared to the corresponding sequence of their germplasm or blood (B) to detect mutations. Only those sites appearing in all histologies for a given patient are identified.
93 | 126 | 188 | 189 | 192 | 203 | 223 | 224 | 249 | 270 | 291 | 298 | 304 | 311 | 319 | 357 | 362 | 390 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
RCRS | T | T | C | T | C | A | C | T | T | C | C | T | T | T | G | T | T | G |
| ||||||||||||||||||
33 B | T | T | C | T | T | A | C | T | T | T | C | T | C | T | G | T | T | G |
33 MIBC | N | N | C | T |
|
A | C |
|
T | T | C | T |
|
|
G | T | T | G |
33 MDCIS | T |
|
C | T |
|
A | C |
|
T |
|
C | T |
|
|
G | T | T | G |
| ||||||||||||||||||
36 B | T | T | T | T | C | A | C | T | T | C | C | T | T | T | A | T | T | G |
36 LIBC | T | T |
|
|
C | A |
|
|
T |
|
|
T | T |
|
|
T |
|
|
36 LDCIS | T | T |
|
|
C | A |
|
|
T |
|
|
T | T |
|
|
T |
|
|
| ||||||||||||||||||
43 B | C | T | C | T | C | A | C | T | T | C | C | C | T | T | G | T | T | G |
43 BIBC |
|
T | C |
|
C | A | C |
|
T |
|
C |
|
T | T | G | T | T | G |
43 BDCIS |
|
T | C |
|
C | A | C |
|
T | C | C |
|
T | T | G | T | T | G |
43 LDCIS |
|
|
C |
|
C | A |
|
T | T | C |
|
|
|
T | G | T |
|
G |
| ||||||||||||||||||
46 B | T | C | C | T | C | A | C | T | T | C | C | T | T | T | G | C | T | G |
46 BIBC | T |
|
C |
|
C | A |
|
T | T | C |
|
T | T | T | G |
|
|
|
46 BDCIS | T |
|
C |
|
C | A |
|
T | T | C |
|
T | T | T | G |
|
|
|
| ||||||||||||||||||
57 B | T | T | C |
|
C | A | C | T | T | C | C | T | T | T | G | T | T | G |
57 LIBC | N | N | C |
|
C | A |
|
T | T | C |
|
T | T | T | G | T |
|
|
57 LDCIS | T | T | C |
|
C | A | C |
|
T |
|
|
T | T | Y | G | T | T |
|
| ||||||||||||||||||
64 B | T | T | C | C | C | A | C | T | C | C | C | T | T | T | G | T | T | G |
64 MIBC1 | N | N | C | C | C | A |
|
|
|
|
|
T | T |
|
G | T |
|
|
64 MIBC2 | T | T | C |
|
C | A |
|
T |
|
C |
|
T | T | T | G | T |
|
|
64 MDCIS | T | T | C |
|
C | A | C |
|
|
|
C | T | T |
|
G | T | T | G |
| ||||||||||||||||||
74 B | T | T | C | T | C | G | C | T | T | C | C | T | C | T | G | T | T | G |
74 BIBC | T | T | C |
|
C |
|
|
T | T | C |
|
T |
|
T | G | T |
|
|
74 BDCIS | T | T | C |
|
C |
|
|
T | T | C |
|
T |
|
T | G | T |
|
|
74 MIBC | T | T | C | T | C |
|
C |
|
T |
|
C | T |
|
T | G | T | T | G |
74 MDCIS | T | T | C |
|
C |
|
|
T | T | C |
|
T |
|
T | G | T |
|
|
| ||||||||||||||||||
83 B | T | T | C | C | C | A | C | T | T | C | C | T | T | T | G | T | T | G |
83 BIBC | T | T | C |
|
C | A |
|
T | T | C | C | T | T | T | G | T | T | G |
83 BDCIS | T | T | C |
|
C | A | C |
|
T | C | C | T | T |
|
G | T | T | G |
| ||||||||||||||||||
89 B | T | T | C | T | C | A | C | C | T | C | C | C | T | C | G | T | T | G |
89 BIBC | N | N | C |
|
C | A |
|
|
T | C |
|
|
T |
|
G | T |
|
|
89 BDCIS | N | N | C |
|
C | A |
|
|
T |
|
|
|
|
|
G | T |
|
|
| ||||||||||||||||||
94 B | C | T | C | C | C | A | C | T | T | T | T | T | T | T | G | T | T | G |
94 BIBC |
|
T | C |
|
C | A | C |
|
T |
|
|
T | T |
|
G | T | T | G |
94 BDCIS |
|
T | C |
|
C | A | C |
|
T | T |
|
T | T |
|
G | T | T | G |
IBC: invasive breast carcinoma, DCIS: ductal carcinoma
Of the patients in this study, 9/10 had a mutation at positions 16189 and 16224 in at least one of their samples. Site 16189 may be of particular interest, as it is found in both DCIS and IBC in every patient. Mutations at position 16270 occur in 80% of the patients studied, but only about one third of these patients had this mutation in both histopathologies. 37.5% of the patients had this mutation in DCIS only, and 37.5% had this mutation in IBC exclusively. Three other sites, 16223, 16270, and 16291, occur in 8 patients, 2 sites (16311, 16362) in 7 patients, 1 site (16390) in 6 patients, 1 site (16304) in 4 patients, 1 site (16126) in 3 patients, and 2 sites (16093 and 16298) in 2 patients. Finally, 6 sites (16188, 16192, 16203, 16249, 16319, and 16357) are exclusive to 6 individual patients.
There appears to be no statistically significant correlation between single individual mutation sites and specific gradings; namely, the modified Bloom-Richardson grade, nuclear grade, tubule formation, and mitotic score.
The mutation loads of the IBC and DCIS samples were similar, even though up to a third of the mutations for a given patient differed. The average mutation load per patient was the same.
Considering IBC and DCIS mutation load from a per-site perspective, the two populations strongly correlate (
The observed frequency of mutations in the study population indicates a medium correlation with the relative mutation rates in HV1. All of the identified sites have estimated relative rates greater than zero, and 65% of the sites are classified as “fast” by multiple studies since they have a greater tendency to mutate than other neighboring sites. Using the same metric (substitution rate >2), 88% of the identified sites could be classified as “fast” [
The mutation sites identified by this study appear predisposed towards mutation. Since sites such as 16189 and 16224 are present in almost every patient, they demonstrate near confluence in this small cohort. This is perhaps due to a biological propensity to rapid mutation. As such, this attribute could be used as a breast cancer marker if this behavior is consistent in transforming breast tissue.
These results are consistent with a field effect demonstrated in epithelial tissues in general, including those cells lining the mammary ducts [
Unfortunately, only patients 43 and 74 had follow-up procedures allowing this level of comparative analyses. The IBC and DCIS from the remaining 8 study participants were associated with 1 procedure, a biopsy, lumpectomy, or mastectomy. Absence of a 1 : 1 correlation between the mutation patterns of IBC and DCIS for a given patient and between separate procedures is likely a result of capturing ducts from tissue cross-sections and the convoluted anatomy of ductal tissue (i.e., patient 43). The extent and effect of the field may vary among associated, parallel ducts. Also, heteroplasmic signal detection up to 20% may not have been reached in all comparative patient samples.
Both telomere content (TC) and allelic imbalance (AI) have been documented in histologically normal breast tissue at 1 cm from a tumor focus. At 5 cm from a focus, TC and AI reflect normal parameters. This field could be much wider than 1 cm, since data was collected only at 1 and 5 cm intervals [
It has been reported that D-loop mutations are associated with tumors which are both estrogen and progesterone receptor negative in women 50 years of age or older [
In other work, NAF was successfully retrieved from 82% of the participants with 96% yielding fluid from both breasts [
This study was able to identify mtGenome alterations that occur in both DCIS and IBC within individual patients that are suggestive of a cancerization field effect, and DCIS that may be aggressive in nature. Other work demonstrates that large amounts of genetic information can be recovered from the high-copy-number mtGenome in low volume biofluids [
A. Maggrah, K. Robinson, J. Creed, R. Wittock, H. Brown, A. Harbottle, D. Klein, B. Reguly, and R. Parr work for Mitomics Inc. and A. Harbottle, D. Klein, and R. Parr own stocks in Mitomics Inc.
All funding sources are Canadian and contributed equally to this study: Industrial Research Assistance Program, FedNor, Northern Ontario Heritage Foundation, Mitomics Inc.