JP7840705B2 - How to diagnose the prognosis of triple-negative breast cancer - Google Patents

Description

This invention broadly relates to methods for diagnosing the prognosis of triple-negative breast cancer, etc.

When diagnosed with breast cancer, patients are typically classified into subtypes based on their characteristics, and treatment methods are determined accordingly. Breast cancer that does not express estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HER2) is called triple-negative breast cancer (TNBC).

Triple-negative breast cancer (HMCC) is the most aggressive subtype of breast cancer, accounting for approximately 10-15% of all breast cancers, and is characterized by early recurrence. Unlike hormone receptor-positive and HER2-positive breast cancers, hormone therapy and drugs targeting the HER2 protein receptor are ineffective in treating triple-negative breast cancer. Furthermore, triple-negative breast cancer has a poorer prognosis and a higher recurrence rate compared to other breast cancer subtypes, making prognosis prediction extremely important.

Zhang et al., "Association Analysis of Somatic Copy Number Alteration Burden With Breast Cancer Survival", Front. Genet., 01 October 2018

Non-Patent Literature 1 describes that the amount of gene copy number change (CNA burden; hereinafter also referred to as "CNA amount" to distinguish it from "CNA") in breast cancer patients, specifically the ratio of the total size of regions with CNA calls of 1 kb or more on autosomes to the total size of autosomes, can serve as a prognostic biomarker for breast cancer. Non-Patent Literature 1 classifies breast cancer patients into a high-CNA group and a low-CNA group, suggesting that the high-CNA group has a higher mortality risk.

However, Non-Patent Document 1 only examined a small number of cases regarding CNA levels, and merely stated that there was a difference in prognosis between the group with high and low CNA levels. Therefore, the proposed cutoff value may not be appropriate. Consequently, those skilled in the art cannot predict the prognosis of triple-negative breast cancer solely based on the amount of CNA.

The inventors of this invention discovered that the sum of copy number variations (CNAs) of cancer-related genes can serve as an indicator of prognosis in patients with triple-negative breast cancer, leading to the completion of this invention.

In other words, this application encompasses the following inventions.
(1)
A method to assist in the prognosis diagnosis of breast cancer patients in whom the expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor, and progesterone receptor is negative,
The process involves measuring the copy number variation (CNA) of cancer-related genes contained in the sample derived from the breast cancer patient,
This includes a step of calculating the sum of the number of genes whose copy number is increasing and the number of genes whose copy number is decreasing.
The prognosis is poor if the sum of the copy number changes is greater than or equal to a predetermined cutoff value, and good if the sum of the copy number changes is less than the predetermined cutoff value.
A method in which the cutoff value is determined by ROC curve (Receiver Operating Characteristic curve) analysis.
(2)
The method according to (1), wherein a gene is determined to have an increasing copy number if the copy number change is 2.5 or more, and a gene is determined to have a decreasing copy number if the copy number change is 0.75 or less.
(3)
The method according to (1) or (2), wherein the cancer-related gene is selected from the group consisting of the following 435 genes.
(4)
The method according to any of (1) to (3), wherein the prognosis is a prediction of the survival rate 1000 days or more after it is determined that the expression of HER2, estrogen receptor, and progesterone receptor is negative.
(5)
The method according to any one of (1) to (4), wherein the sample is derived from a patient with first-time breast cancer.
(6)
The method according to any of (1) to (5), wherein the cutoff value is 20.5.

While there have been reports suggesting that specific genes, such as TP53, have the greatest impact on breast cancer prognosis, no data has been reported indicating that the sum of CNAs has a stronger influence on prognosis than such specific genes.

At the time of sample collection, there was no clear difference in the stage or progression of triple-negative breast cancer between the high and low CNA groups. However, subsequent follow-up studies revealed that the group with higher CNA was more prone to metastasis and recurrence, and chemotherapy was less effective. Therefore, the sum of CNAs of cancer-related genes may be a valuable indicator of prognosis for patients with triple-negative breast cancer.

Figure 1 shows a prognosis prediction workflow in one embodiment. Figure 2 shows a prognosis prediction method in one embodiment. Figure 3 shows the survival curves for 53 cases of confirmed triple-negative breast cancer, categorized as follows: the CNA sum value of 20.5 or higher is classified as the CNA sum value High group, and the CNA sum value of less than 20.5 is classified as the CNA sum value Low group. The horizontal axis represents the number of days after confirmed diagnosis.

The following describes embodiments of the present invention (hereinafter referred to as "these embodiments"), but the scope of the present invention is not limited to these embodiments.

(Methods to assist in prognosis diagnosis)
In a first embodiment, a method to assist in the prognosis diagnosis of breast cancer patients in whom the expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor, and progesterone receptor is negative,
The process involves measuring the copy number variation (CNA) of cancer-related genes contained in the sample derived from the breast cancer patient,
This includes a step of calculating the sum of the number of genes whose copy number is increasing and the number of genes whose copy number is decreasing.
The prognosis is poor if the sum of the copy number changes is greater than or equal to a predetermined cutoff value, and good if the sum of the copy number changes is less than the predetermined cutoff value.
A method is provided in which the cutoff value is determined by ROC curve (Receiver Operating Characteristic curve) analysis.

Triple-negative breast cancer is a type of breast cancer in which none of the human epidermal growth factor receptor 2 (HER2), estrogen receptor, or progesterone receptor is expressed. Samples containing cancer-related genes are collected from patients diagnosed with negative (non-expression) results for all three receptors through pathological examination.

Examples of samples containing cancer-related genes include biopsy specimens and surgical specimens derived from lesions in breast cancer patients. Preferably, the specimen is from the initial diagnosis, and preferably, it is a specimen that has been pathologically confirmed to be breast cancer. Breast cancer is diagnosed through cytological examinations such as fine-needle aspiration cytology, secretion cytology, and imprint cytology, as well as histological examinations such as needle biopsy and vacuum-assisted breast tissue biopsy.

The specimens are further subtyped based on indicators such as hormone receptors, HER2, and Ki67. For example, specimens that are negative for HER2, estrogen receptor, and progesterone receptor expression are judged to be triple-negative breast cancer. Known methods such as IHC (immunohistochemistry) and FISH (fluorescence in situ hybridization) can be used to diagnose triple-negative breast cancer.

The appropriate treatment for breast cancer varies depending on its subtype, both pre- and post-operatively. Treatment options for triple-negative breast cancer include surgery, radiation therapy, and chemotherapy, but chemotherapy is often administered pre-operatively. Examples of such chemotherapy drugs include anthracycline-based chemotherapy (e.g., Adriamycin, epirubicin), taxane-based chemotherapy (e.g., paclitaxel, docetaxel), the PARP inhibitor Lynparza (olaparib), and the PD-L1 inhibitor Tecentriq® (atezolizumab). These drugs may also be used in combination.

Triple-negative breast cancer is highly invasive and carries a high risk of early recurrence. Patients often experience recurrence within a few years of surgery, and their prognosis is poor. Therefore, it is crucial to perform prognostic assessments on samples determined to originate from patients diagnosed with and treated for triple-negative breast cancer.

In one embodiment, a sample derived from a patient diagnosed with triple-negative breast cancer is subjected to a process to measure the copy number variation (CNA) of cancer-related genes.

The sample from which copy number changes are measured may be a biopsy specimen or a surgical specimen. The sample only needs to be in a state where copy number changes can be measured; it may have undergone prior preparation such as formalin fixation and paraffin embedding, or staining with antibodies or fluorescent labeling substances. The sample may be from either a primary tumor or a metastatic tumor.

As used herein, "CNA" is an abbreviation for Copy Number Alterations, which refers to acquired changes in the copy number of DNA in somatic cells.
Copy number (CNA) is one of the items commonly reported in gene panel testing, whole-genome testing, and cancer genome testing, and refers to acquired changes in the copy number of DNA in somatic cells. CNAs are divided into copy number increases and decreases; a high copy number increase is called amplification, a relatively low level increase is called gain, and a decrease is called deletion or loss. A deletion of one allele is called a hemi-deletion, and a deletion of both alleles is called a homo-deletion.

CNA can be measured using gene mutation detection methods such as next-generation sequencers. The abnormal copy number is determined by the following procedure, which converts the value to an integer. This is then compared to the normal copy number, and the resulting multiple is the CNA. For example, if the normal copy number is set to "1," then a multiple of "3" is considered the "changed multiple."
1) Normalization of coverage calculated by the sequencer;
2) Correction of bias (GC content and repeating sequences);
3) Integerization by segmenting the region captured by the capture reagent (on-target region and off-target region).

As used herein, "cancer-related genes" refers to a group of genes involved in the development of cancer, including oncogenes and tumor suppressor genes. Oncogenes are genes that primarily perform functions necessary for cell proliferation and differentiation, while tumor suppressor genes are genes that exert functions such as inhibiting proliferation in cells in which mutations have occurred in such oncogenes. Cancer-related genes may also be genes targeted in cancer gene panel testing.

Examples of cancer-related genes include those listed in the table below.

Next, for the measured CNA, the total number of genes with increased copy numbers and the total number of genes with decreased copy numbers are calculated. The calculation process may include steps for counting the number of cancer-related genes with decreased copy numbers and counting the number of cancer-related genes with increased copy numbers.

While not intended to be limiting, CNA calculation can be performed using gene panel testing with next-generation sequencers, etc. In panel testing, the copy number changes in each gene are calculated, and the genes with copy number changes for each patient are listed.

The CNA in a sample is calculated as an absolute value for each gene. For example, in sample X, the CNA change of gene A is 3.8 times (meaning the copy number is 3.8 times higher than the normal portion), and the CNA change of gene B is 5.5 times. In contrast, conventional techniques express CNA changes as a percentage rather than an absolute value.

In one embodiment, a gene is determined to have an increasing copy number if the copy number change is 2.5 or greater, and a gene is determined to have a decreasing copy number if the copy number change is 0.75 or less.

While there is no clear difference in stage or progression between samples based on CNA changes at the time of sample collection, using the sum of CNA changes as an indicator can assist in prognostic diagnosis of triple-negative breast cancer. For example, a poor prognosis is judged when the sum of copy number changes is above a predetermined cutoff value, and a good prognosis is judged when the sum of copy number changes is below the predetermined cutoff value.

The cutoff value is determined by ROC curve (Receiver Operating Characteristic Curve) analysis, where sensitivity is plotted on the vertical axis and (1 - specificity) on the horizontal axis. For example, based on the value calculated by plotting an ROC curve for a continuous variable (number of genes undergoing copy number variation), the data is divided into a high (High) and low (Low) group for analysis, and the optimal point of sensitivity - (1 - specificity) is derived as the cutoff value. A preferred cutoff value is 20.5.

As used herein, "prognostic diagnosis" refers to the prediction of the prognosis, or the survival rate after a specified period following treatment or diagnosis, for example, 1000 days after definitive diagnosis. For example, without intent to limit, if the sum of the CNAs of the target patients is High relative to 20.5, it is possible to predict a survival rate of approximately 60% at 3 years and 40% at 5 years. Conversely, if it is Low, it is possible to predict a survival rate of 80% or higher at 3, 4, and 5 years.

(Treatment method)
In a second embodiment, a method for treating breast cancer patients in whom the expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor, and progesterone receptor is negative,
The process involves measuring the copy number variation (CNA) of cancer-related genes contained in the sample derived from the breast cancer patient,
A process to calculate the total number of genes whose copy number is increasing and the total number of genes whose copy number is decreasing,
A process to determine that the prognosis is poor if the sum of the copy number changes is greater than or equal to a predetermined cutoff value, and that the prognosis is good if the sum of the copy number changes is less than a predetermined cutoff value,
The process involves treating patients with a poor prognosis using a high-intensity regimen, and patients with a good prognosis using a low-intensity regimen.
Includes,
A method is provided in which the cutoff value is determined by ROC curve (Receiver Operating Characteristic curve) analysis.

If a poor prognosis can be predicted in advance, it becomes possible to implement more intensive regimens, such as multi-drug chemotherapy, during the perioperative period before recurrence occurs, thereby reducing the risk of recurrence. Furthermore, if a good prognosis is predicted, standard chemotherapy may be administered, or chemotherapy may be omitted, and the patient's condition monitored. For example, it may be possible to reduce the burden on the patient by using drug therapy with reduced intensity, or even by omitting chemotherapy in some cases, thereby ensuring a low recurrence rate while minimizing side effects and improving the patient's quality of life.

For triple-negative breast cancer, combination therapy using anthracycline and taxane drugs is well-known. Examples of anthracycline drugs include doxorubicin (Adriamycin) and epirubicin (Farmorubicin). Examples of taxane drugs include paclitaxel (Taxol) and docetaxel (Taxotere). However, these are merely examples, and known drugs can be appropriately combined for the treatment of triple-negative breast cancer.

Examples of low-intensity treatment regimens include combination therapy with anthracycline drugs and cyclophosphamide before or after surgery, followed by Taxol, and treatment that omits all chemotherapy. When chemotherapy is omitted, after surgical resection of the tumor, further treatment may be omitted, and observation may suffice.

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Following the flow chart shown in Figure 1, we conducted breast cancer screening and prognosis prediction evaluations for patients suspected of having breast cancer. First, for cases where breast cancer was definitively diagnosed pathologically via needle biopsy, hormone receptor and HER2 levels were evaluated in the tissue samples obtained from the needle biopsies. As a result, CNA measurements were performed on 53 cases definitively diagnosed with triple-negative breast cancer using the CANCERPLEX® (registered trademark) genome gene testing system (manufactured by Denka Co., Ltd.).

Prognosis prediction was performed according to the flow chart shown in Figure 2. First, for each of the 53 samples, the total number of genes with increased copy numbers and those with decreased copy numbers was calculated, and the total CNA value for each sample was determined.

A ROC curve was created using the calculated sum of CNAs as the test variable and the presence or absence of death during the observation period as the status variable. The sum of CNAs that maximized the Youden index (sensitivity + specificity - 1) was calculated and used as the cutoff value. Since the obtained cutoff value was 20.5, the sum of CNAs of 20.5 or higher were classified as the High group, and the sum of CNAs less than 20.5 as the Low group. Prognosis was predicted by determining whether the group was High or Low.

The results are shown in Figure 3.

Since CNA accurately reflects the biological malignancy of cancer, cases with a high CNA at diagnosis are expected to have a high recurrence rate and potentially lead to death, due to the nature of the cancer. For such cases with a high CNA, it is possible to reduce the risk of recurrence by administering a high-intensity perioperative regimen of drug therapy before recurrence occurs. On the other hand, in the group with a low CAN (cancer recurrence rate), the risk of cancer recurrence is low. Therefore, by using standard perioperative drug therapy for triple-negative breast cancer, or by reducing the intensity of treatment to minimize side effects, it is possible to reduce side effects and improve the patient's quality of life while ensuring a low recurrence rate.

Claims (5)

A method to assist in the prognosis diagnosis of breast cancer patients in whom the expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor, and progesterone receptor is negative,
The following 435 cancer-related genes are included in the sample derived from the aforementioned breast cancer patient:
A process for measuring the copy number change (CNA),
This includes a step of calculating the sum of the number of genes whose copy number is increasing and the number of genes whose copy number is decreasing among the 435 genes .
A poor prognosis is indicated when the sum of the number of genes with increasing copy numbers and the number of genes with decreasing copy numbers exceeds a predetermined cutoff value, and a good prognosis is indicated when the sum of the number of genes with increasing copy numbers and the number of genes with decreasing copy numbers is less than a predetermined cutoff value.
A method in which the cutoff value is determined by ROC curve (Receiver Operating Characteristic curve ) analysis. The method according to claim 1, wherein a gene is determined to have an increasing copy number if the copy number change is 2.5 or more, and a gene is determined to have a decreasing copy number if the copy number change is 0.75 or less. The method according to claim 1 or 2 , wherein the prognosis is a prediction of the survival rate 1000 days or more after it is determined that the expression of HER2, estrogen receptor, and progesterone receptor is negative. The method according to any one of claims 1 to 3 , wherein the sample is derived from a patient with first-diagnosis breast cancer. The method according to any one of claims 1 to 4 , wherein the cutoff value is 20.5.