TW201623963A

TW201623963A - Methods of treating hepatocellular carcinoma

Info

Publication number: TW201623963A
Application number: TW104105779A
Authority: TW
Inventors: 薛峰; 布蘭登西葛斯; 一鴻姚; 佳齊黃; 克里斯摩爾豪斯; 維朱
Original assignee: 麥迪紐有限責任公司; 中國上海交通大學醫學院
Priority date: 2014-02-20
Filing date: 2015-02-17
Publication date: 2016-07-01
Also published as: WO2015127103A1

Abstract

This application provides methods of treating hepatocellular carcinoma in a patient, methods of evaluating the recurrence risk profile of a hepatocellular carcinoma, and diagnostic agents for the diagnosis of hepatocellular carcinoma.

Description

治療肝細胞癌之方法 Method for treating hepatocellular carcinoma

肝細胞癌(hepatocellular carcinoma，HCC)係最常見之原發性肝惡性腫瘤及世界範圍內第三大癌症死亡原因，且B型肝炎病毒係主要致病因素。El-Serag,H.B.及Rudolph,K.L.,Gastroenterology 132：2557-2576(2007)；Parkin,D.M.,Int J Cancer 118,3030-3044(2010)。除僅在少數HCC患者人口中最低限度地有效之索拉非尼(sorafenib)以外，尚未批準用於HCC之治療。患者之治療選擇受到限制。外科手術切除、燒灼或正位肝移植(orthotopic liver transplantation，OLT)對於某些患者係可行的，但在該等程式之後復發率可高達40%。Chang,M.H.等人，J.Natl.Cancer Inst.101：1348-1355(2009)；Marsh,J.W.等人，Hepatology 26：444-450(1997)。在符合諸如Milan、Toronto或UCSF準則等臨床準則之患者中，OLT後之HCC復發率為20-40%。Chang等人，J Natl Cancer Inst 101：1348-1355(2009)；Marsh等人，Hepatology 26：444-450(1997)。供給者在屍體器官移植中傳播惡性腫瘤之情形極其罕見且其係在移植器官中重新出現或早已存於供給者器官中。在兩種情形中，腫瘤來源獨立於接受者原發性癌。OLT後之復發HCC病例之最普遍事件係源於接受者之腫瘤，其中復發腫瘤之遺傳組成與原發性腫瘤相匹配。因此，業內需要基於原發性腫瘤之性質確定復發之風險。 Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver and the third leading cause of cancer death worldwide, and the main cause of hepatitis B virus. El-Serag, HB and Rudolph, KL , Gastroenterology 132: 2557-2576 (2007); Parkin, DM , Int J Cancer 118, 3030-3044 (2010). Treatment for HCC has not been approved except for sorafenib, which is minimally effective in only a small number of HCC patient populations. The patient's treatment options are limited. Surgical resection, cauterization, or orthotopic liver transplantation (OLT) is feasible for some patients, but the recurrence rate can be as high as 40% after these procedures. Chang, MH et al, J. Natl. Cancer Inst. 101:1348-1355 (2009); Marsh, JW et al, Hepatology 26: 444-450 (1997). In patients who meet clinical criteria such as the Milan, Toronto, or UCSF guidelines, the HCC recurrence rate after OLT is 20-40%. Chang et al, J Natl Cancer Inst 101:1348-1355 (2009); Marsh et al, Hepatology 26: 444-450 (1997). It is extremely rare for a supplier to transmit a malignant tumor in a cadaveric organ transplant and its re-emergence in the transplanted organ or already in the donor's organ. In both cases, the tumor source is independent of the recipient's primary cancer. The most common event of recurrent HCC cases after OLT is derived from the recipient's tumor, in which the genetic composition of the recurrent tumor matches the primary tumor. Therefore, the industry needs to determine the risk of recurrence based on the nature of the primary tumor.

研究已顯示，使用臨床病理學變數(尤其例如腫瘤大小、血管侵犯、腫瘤狀態、腫瘤等級及甲型胎兒蛋白含量)對具有高HCC復發風險之患者進行分類之一致性之水準有變化。Clavien等人，Lancet Oncol.13(1)：e11-e22(2012)。在未確立輔佐治療之致命疾病中，實質缺少供給者器官且經濟負擔較高，在分子水準上隨附當前臨床準則鑑別OLT之復發風險較低之合格患者，極有可能顯著改良患者之臨床結果。 Studies have shown that the use of clinical pathology variables (especially, for example, tumor size, vascular invasion, tumor status, tumor grade, and alpha-fetal protein content) has a consistent level of consistency in classifying patients with a high risk of HCC recurrence. Clavien et al., Lancet Oncol. 13(1): e11-e22 (2012). In the case of a fatal disease in which no adjuvant treatment is established, the donor organ is substantially absent and the financial burden is high. It is highly likely that the current clinical criteria will be accompanied by the current clinical criteria to identify qualified patients with a lower risk of recurrence of OLT, and it is highly likely that the clinical outcome of the patient will be significantly improved. .

根據闡述，治療患者肝細胞癌之方法包含：(a)自患者獲得肝細胞癌試樣；(b)確定試樣是否具有正常的HERC5 mRNA表現程度；及(c)若試樣具有正常的HERC5 mRNA表現程度，則使患者有資格進行正位肝移植。在一個實施例中，將試樣中HERC5 mRNA之含量與歷史對照組中HERC5 mRNA之含量進行比較。在一些情況中，歷史對照包含來自至少10名、25名或50名個體之數據。 According to the description, a method for treating a patient with hepatocellular carcinoma comprises: (a) obtaining a hepatocellular carcinoma sample from a patient; (b) determining whether the sample has a normal degree of HERC5 mRNA expression; and (c) if the sample has a normal HERC5 The degree of mRNA expression allows the patient to be eligible for orthotopic liver transplantation. In one embodiment, the amount of HERC5 mRNA in the sample is compared to the amount of HERC5 mRNA in the historical control. In some cases, historical controls contain data from at least 10, 25, or 50 individuals.

在一個實施例中，歷史對照組具有正常個體。在另一實施例中，歷史對照組具有所患肝細胞癌在兩年內未復發之患者。在一個實施例中，若患者試樣中之HERC5 mRNA含量較歷史對照組之平均值低等於或高於兩個標準偏差，則該HERC5 mRNA含量指定為正常的。在另一模式中，若患者試樣中之HERC5 mRNA含量較歷史對照組之平均值低等於或高於一個標準偏差，則該HERC5 mRNA含量指定為正常的。 In one embodiment, the historical control group has a normal individual. In another embodiment, the historical control group has a patient whose hepatocellular carcinoma has not relapsed within two years. In one embodiment, the HERC5 mRNA content is designated as normal if the HERC5 mRNA content in the patient sample is less than or equal to two standard deviations from the mean of the historical control. In another mode, the HERC5 mRNA content is designated as normal if the HERC5 mRNA level in the patient sample is less than or equal to one standard deviation from the mean of the historical control group.

在另一實施例中，歷史對照組具有所患肝細胞癌在兩年內復發之患者。在一個態樣中，若患者試樣中之HERC5 mRNA含量較歷史對照組之平均值高等於或高於一個標準偏差，則該HERC5 mRNA含量指定為正常的。 In another embodiment, the historical control group has a patient with hepatocellular carcinoma recurring within two years. In one aspect, the HERC5 mRNA content is designated as normal if the HERC5 mRNA level in the patient sample is greater than or equal to one standard deviation from the mean of the historical control group.

在另一態樣中，歷史對照組具有已患肝細胞癌之患者。在一個實施例中，若患者試樣中之HERC5 mRNA含量高於歷史組之中值，則該HERC5 mRNA含量指定為正常的。 In another aspect, the historical control group has patients who have developed hepatocellular carcinoma. In one embodiment, if the HERC5 mRNA content in the patient sample is higher than the historical group, The HERC5 mRNA content is then designated as normal.

根據一種方法，將試樣中之HERC5 mRNA含量與自患者獲得之正常組織之試樣中之HERC5 mRNA含量進行比較。在一個實施例中，正常組織係肝組織。在一種模式中，肝細胞癌試樣中HERC5 mRNA之含量高於正常組織試樣中之含量之80%且該患者有資格進行正位肝移植。在另一模式中，肝細胞癌試樣中HERC5 mRNA之含量低於正常組織試樣中之含量之50%且該患者沒有資格進行正位肝移植。 According to one method, the HERC5 mRNA content in the sample is compared to the HERC5 mRNA content in a sample of normal tissue obtained from the patient. In one embodiment, the normal tissue is liver tissue. In one mode, the amount of HERC5 mRNA in a hepatocellular carcinoma sample is higher than 80% of the amount in a normal tissue sample and the patient is eligible for orthotopic liver transplantation. In another mode, the amount of HERC5 mRNA in the hepatocellular carcinoma sample is less than 50% of the amount in the normal tissue sample and the patient is not eligible for orthotopic liver transplantation.

在一個實施例中，患者沒有資格使用索拉非尼。在另一實施例中，利用索拉非尼治療患者且該治療不成功。 In one embodiment, the patient is not eligible to use sorafenib. In another embodiment, the patient is treated with sorafenib and the treatment is unsuccessful.

在一個實施例中，肝細胞癌試樣具有HERC5 DNA缺失且該患者沒有資格進行正位肝移植。在另一實施例中，肝細胞癌試樣具有HERC5 mRNA過低表現且該患者沒有資格進行正位肝移植。 In one embodiment, the hepatocellular carcinoma sample has a deletion of HERC5 DNA and the patient is not eligible for orthotopic liver transplantation. In another embodiment, the hepatocellular carcinoma sample has a low expression of HERC5 mRNA and the patient is not eligible for orthotopic liver transplantation.

在另一實施例中，患者有資格進行正位肝移植之決策亦包括評估臨床準則。在一個態樣中，臨床準則包含以下各項中之至少一者或多者：病灶大小，病灶數量，是否具有肝外表現，是否具有血管侵犯，基於生檢HCC是否分化不良，腫瘤狀態，腫瘤等級及甲型胎兒蛋白含量。在另一態樣中，臨床準則包含(a)單一病灶不超過5cm，或不多於3個病灶，該等病灶皆不大於3cm；及(b)無大血管侵犯。在另一態樣中，臨床準則包含UCSF準則且包括：(a)單一病灶不大於6.5cm；或(b)不多於3個病灶，該等病灶皆不超過4.5cm且其總腫瘤直徑不超過8cm。 In another embodiment, the decision of the patient to qualify for orthotopic liver transplantation also includes evaluating clinical criteria. In one aspect, the clinical criteria include at least one or more of the following: lesion size, number of lesions, presence or absence of extrahepatic manifestations, vascular invasion, biopsy based on biopsy, tumor status, tumor Grade and type A fetal protein content. In another aspect, the clinical criteria comprise (a) no more than 5 cm, or no more than 3 lesions in a single lesion, none of the lesions being greater than 3 cm; and (b) no macrovascular invasion. In another aspect, the clinical guidelines include UCSF criteria and include: (a) a single lesion no greater than 6.5 cm; or (b) no more than 3 lesions, none of the lesions exceeding 4.5 cm and the total tumor diameter not More than 8cm.

在另一實施例中，臨床準則包含Toronto準則且包括任一腫瘤大小或數量，及無因HCC所致之全身症狀，及基於組織學排除分化不良之HCC(僅超過Milan腫瘤)。在另一模式中，臨床準則包含Hangzhou準則且包括：a)總腫瘤直徑小於或等於8cm；(b)總腫瘤直徑大於8 cm，且組織病理學等級為I級或II級，且手術前AFP含量小於或等於400ng/ml。 In another embodiment, the clinical criteria include the Toronto criteria and includes any tumor size or number, and no systemic symptoms due to HCC, and histological exclusion of poorly differentiated HCC (only more than Milan tumors). In another mode, the clinical criteria include the Hangzhou criteria and include: a) the total tumor diameter is less than or equal to 8 cm; (b) the total tumor diameter is greater than 8 Cm, and the histopathology grade is grade I or grade II, and the preoperative AFP content is less than or equal to 400 ng / ml.

在一個實施例中，甲型胎兒蛋白濃度小於400ng/mL(其中較低含量指示OLT後之潛在正面結果)。 In one embodiment, the alpha-type fetal protein concentration is less than 400 ng/mL (where lower levels indicate potential positive results after OLT).

在一種模式中，具有正常HERC5 mRNA含量之患者在移植等待列表上具有優先於任何沒有正常HERC5 mRNA含量之肝細胞癌患者之優先權。在一個實施例中，患者接受肝移植。 In one mode, patients with normal HERC5 mRNA levels have priority over any of the hepatocellular carcinoma patients who do not have normal HERC5 mRNA content on the transplant waiting list. In one embodiment, the patient receives a liver transplant.

根據闡述，評估肝細胞癌之復發風險概況之方法包含：(a)提供肝細胞癌試樣；(b)在活體外確定試樣是否具有正常HERC5 mRNA表現量；(c)若試樣具有正常的HERC5 mRNA表現量，且該確定係結合至少一個臨床準則作出的，則將肝細胞癌定性為具有低復發風險。在一個實施例中，在正位肝移植後肝細胞癌具有低復發風險。在一些情況中，歷史對照包含來自至少10名、25名或50名個體之數據。 According to the description, the method for assessing the risk of recurrence of hepatocellular carcinoma comprises: (a) providing a sample of hepatocellular carcinoma; (b) determining whether the sample has normal HERC5 mRNA expression in vitro; (c) if the sample has normal The amount of HERC5 mRNA expressed, and the determination is made in conjunction with at least one clinical criterion, identifies hepatocellular carcinoma as having a low risk of recurrence. In one embodiment, hepatocellular carcinoma has a low risk of recurrence after orthotopic liver transplantation. In some cases, historical controls contain data from at least 10, 25, or 50 individuals.

根據一種方法，將試樣中之HERC5 mRNA含量與自患者獲得之正常組織之試樣中之HERC5 mRNA含量進行比較。在一個實施例中，正常組織為肝組織。在一種模式中，肝細胞癌試樣中HERC5 mRNA之含量高於正常組織試樣中之含量之80%且該患者有資格進行正位肝移植。在另一模式中，肝細胞癌試樣中HERC5 mRNA之含量低於正常組織試樣中之含量之50%且該患者沒有資格進行正位肝移植。 According to one method, the HERC5 mRNA content in the sample is compared to the HERC5 mRNA content in a sample of normal tissue obtained from the patient. In one embodiment, the normal tissue is liver tissue. In one mode, the amount of HERC5 mRNA in a hepatocellular carcinoma sample is higher than 80% of the amount in a normal tissue sample and the patient is eligible for orthotopic liver transplantation. In another mode, the amount of HERC5 mRNA in the hepatocellular carcinoma sample is less than 50% of the amount in the normal tissue sample and the patient is not eligible for orthotopic liver transplantation.

在一個實施例中，肝細胞癌試樣具有HERC5 DNA缺失且該患者沒有資格進行正位肝移植。在另一實施例中，肝細胞癌試樣具有mRNA HERC5過低表現且該患者沒有資格進行正位肝移植。 In one embodiment, the hepatocellular carcinoma sample has a deletion of HERC5 DNA and the patient is not eligible for orthotopic liver transplantation. In another embodiment, the hepatocellular carcinoma sample has a low expression of mRNA HERC5 and the patient is not eligible for orthotopic liver transplantation.

在另一實施例中，使患者有資格進行正位肝移植之決策亦包括評估臨床準則。在一個態樣中，臨床準則包含以下各項中之至少一者：病灶大小，病灶數量，是否具有肝外表現，是否具有血管侵犯，基於生檢HCC是否分化不良，腫瘤狀態，腫瘤等級及甲型胎兒蛋白含量。在另一態樣中，臨床準則包含(a)單一病灶不超過5cm，或不多於3個病灶，該等病灶皆不超過3cm；及(b)無大血管侵犯。在另一態樣中，臨床準則包含UCSF準則且包括：(a)單一病灶不大於6.5cm；或(b)不多於3個病灶，該等病灶皆不超過4.5cm且其總腫瘤直徑不超過8cm。 In another embodiment, the decision to qualify the patient for orthotopic liver transplantation also includes evaluating clinical criteria. In one aspect, the clinical criteria include at least one of the following: lesion size, number of lesions, presence of extrahepatic manifestations, vascular invasion, poor differentiation based on biopsy HCC, tumor status, tumor grade, and Type fetal protein content. In another aspect, the clinical criteria comprises (a) no more than 5 cm, or no more than 3 lesions in a single lesion, none of the lesions exceeding 3 cm; and (b) no macrovascular invasion. In another aspect, the clinical guidelines include UCSF criteria and include: (a) a single lesion no greater than 6.5 cm; or (b) no more than 3 lesions, none of the lesions exceeding 4.5 cm and the total tumor diameter not More than 8cm.

在另一實施例中，臨床準則包含Toronto準則且包括任一腫瘤大小或數量，及無因HCC所致之全身症狀，及基於組織學排除分化不良之HCC(僅超過Milan腫瘤)。在另一模式中，臨床準則包含Hangzhou Zhou準則且包括a)總腫瘤直徑小於或等於8cm；(b)總腫瘤直徑超過8cm，且組織病理學等級為I級或II級，且手術前AFP含量小於或等於400ng/mL。 In another embodiment, the clinical criteria include the Toronto criteria and includes any tumor size or number, and no systemic symptoms due to HCC, and histological exclusion of poorly differentiated HCC (only more than Milan tumors). In another mode, the clinical criteria include the Hangzhou Zhou criteria and include a) the total tumor diameter is less than or equal to 8 cm; (b) the total tumor diameter is over Over 8 cm, and histopathological grade is grade I or grade II, and the preoperative AFP content is less than or equal to 400 ng / mL.

在一種模式中，具有正常HERC5 mRNA含量之患者在移植等待列表上具有優於任何沒有正常HERC5 mRNA含量之肝細胞癌患者之優先權。在一個實施例中，患者接受肝移植。 In one mode, patients with normal HERC5 mRNA levels have priority over any of the hepatocellular carcinoma patients who do not have normal HERC5 mRNA content on the transplant waiting list. In one embodiment, the patient receives a liver transplant.

另一實施例包括使用HERC5正向引子及反向引子(其每一者係自HERC5之mRNA生成)產生用於活體外診斷可利用正位肝移植進行治療之肝細胞癌之診斷液。 Another embodiment includes the use of a HERC5 forward primer and a reverse primer, each of which is generated from mRNA of HERC5, to produce a diagnostic fluid for in vitro diagnosis of hepatocellular carcinoma that can be treated with orthotopic liver transplantation.

另一實施例包括使用HERC5特異性探針產生用於活體外診斷可利用正位肝移植進行治療之肝細胞癌之診斷液。 Another embodiment includes the use of a HERC5-specific probe to generate a diagnostic fluid for in vitro diagnosis of hepatocellular carcinoma that can be treated with orthotopic liver transplantation.

另一實施例包括使用HERC5核酸序列作為定量對照以產生用於活體外診斷可利用正位肝移植進行治療之肝細胞癌之診斷液。 Another embodiment includes the use of a HERC5 nucleic acid sequence as a quantitative control to generate a diagnostic fluid for in vitro diagnosis of hepatocellular carcinoma that can be treated with orthotopic liver transplantation.

其他目標及優點將在下文說明中部分地加以陳述，且根據本說明將部分地顯而易見，或者可藉由實踐而知曉。該等目標及優點將藉助隨附申請專利範圍中特定指出之要素及組合來實現及達成。 Other objects and advantages will be set forth in part in the description which follows. These objectives and advantages will be realized and attained by the <RTIgt;

應瞭解，前述一般說明及以下詳細說明兩者僅具有例示性及解釋性且並非限制申請專利範圍。 It is to be understood that both the foregoing general description and

併入本說明書中並構成本說明書之一部分的附圖圖解說明一個(若干個)實施例，且與本說明一起用於解釋本文所闡述之原理。 BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in FIG

序列之闡述Sequence description

圖1A-D顯示在A)HCC1、B)HCC4、C)HCC5及D)HCC11之原發性(內部跡線)及復發(外部跡線)腫瘤中鑑別之體細胞CN擴增(紅色)或缺失(綠色)；E)3/4患者中基因組內CN擴增或缺失之共用及特有區域。色彩編碼係如下：原發性腫瘤特有(紅色=擴增；藍色=缺失)；復發腫瘤特有(褐色=擴增)；及原發性腫瘤與復發腫瘤共用(綠色=擴增；紫色=缺失)。 Figure 1A-D shows somatic CN amplification (red) identified in A ) HCC1, B ) HCC4, C ) HCC5 and D ) HCC11 primary (internal trace) and recurrent (external trace) tumors or Deletion (green); E ) Common and endemic areas of CN amplification or deletion in the genome of 3/4 patients. The color coding system is as follows: primary tumor specific (red = amplification; blue = deletion); recurrent tumor specific (brown = amplification); and primary tumor shared with recurrent tumor (green = amplification; purple = missing) ).

圖2顯示受4名HCC患者之原發性及/或復發腫瘤中之基因驅動及/或基因表現活化或阻抑影響之Wnt/β-連環蛋白信號傳導及直接相關路徑。 Figure 2 shows Wnt/β-catenin signaling and direct correlation pathways affected by gene-driven and/or gene expression activation or repression in primary and/or recurrent tumors in 4 HCC patients.

圖3A-F顯示，使用兩個獨立研究，HERC5 mRNA表現程度預測HCC中之腫瘤復發、總體存活及無進展存活。A)來自正常健康對照之肝組織(紅色；n=239)與來自HCC患者之腫瘤生檢(綠色；n=247)間之基因表現log₂倍數改變分佈，其中藍色線指示正常對照之平均值-2SD；B)比較低HERC5 mRNA表現組(n=62)與高HERC5 mRNA表現組(n=180)之Kaplan-Meier(KM)曲線，其中HCC復發作為反應(對數秩，p=0.0198)；C)比較低HERC5mRNA表現組(n=62)與高HERC5 mRNA表現組(n=180)之KM曲線，其中總體存活作為反應(對數秩，p=0.0063)。除相等性之KM測試以外，亦使用多變數Cox PH回歸模型用於調整年齡、肝硬化狀態、性別、HBV/HCV狀態及TNM分期之兩種模型。數據係自Roessler等人之Cancer Res 70(24)：10202-12(2010)獲取。D)來自HCC患者之腫瘤生檢之基因表現log₂強度分佈(綠色)，其中藍色線指示65名患者之中值log₂強度；E)比較低HERC5 mRNA表現組(n=20)與高HERC5 mRNA表現組(n=24)之KM曲線，其中無進展存活作為反應(對數秩，p=0.067)；F)比較低HERC5 mRNA表現組(n=20)與高HERC5 mRNA表現組(n=24)之KM曲線，其中總體存活作為反應(對數秩，p=0.023)。除相等性之KM測試以外，亦使用多變數Cox PH回歸模型用於調整年齡、性別及HBV狀態之兩種模型。數據係自Boyault等人之Hepatology 45(1)：42-52(2007)獲取。 Figures 3A-F show that the degree of HERC5 mRNA expression predicts tumor recurrence, overall survival, and progression free survival in HCC using two independent studies. A ) Gene expression log ₂ fold change distribution between normal healthy controls (red; n = 239) and tumor biopsies from HCC patients (green; n = 247), with blue lines indicating the average of normal controls Value -2SD ; B ) Kaplan-Meier (KM) curve comparing the low HERC5 mRNA expression group (n=62) with the high HERC5 mRNA expression group (n=180), in which HCC recurrence was used as a response (logarithmic rank, p=0.0198) C ) Comparison of the KM curves of the low HERC5 mRNA expression group (n=62) and the high HERC5 mRNA expression group (n=180), with overall survival as a response (log rank, p=0.0063). In addition to the KM test of equality, a multivariate Cox PH regression model was also used to adjust the two models of age, cirrhosis status, gender, HBV/HCV status, and TNM stage. The data was obtained from Roessler et al., Cancer Res 70(24): 10202-12 (2010). D ) Gene expression from the HCC patients showed a log ₂ intensity distribution (green), with a blue line indicating the median log ₂ intensity in 65 patients; E ) a lower HERC5 mRNA performance group (n=20) and higher The KM curve of the HERC5 mRNA expression group (n=24), with progression-free survival as a response (log rank, p=0.067); F ) comparison of low HERC5 mRNA expression group (n=20) with high HERC5 mRNA expression group (n= 24) KM curve in which overall survival was performed as a response (log rank, p=0.023). In addition to the KM test of equality, a multivariate Cox PH regression model was also used to adjust the two models of age, gender, and HBV status. The data was obtained from Boyault et al., Hepatology 45(1): 42-52 (2007).

圖4顯示4名HCC患者之原發性及復發腫瘤中之WES體細胞SNV型式。 Figure 4 shows the WES somatic SNV pattern in primary and recurrent tumors of 4 HCC patients.

圖5顯示原發性腫瘤與復發腫瘤間共用之SNV顯示優於4名HCC患者中之原發性腫瘤特有之彼等SNV之純系優點。誤差槓代表95%信賴區間。 Figure 5 shows that the SNV shared between the primary tumor and the recurrent tumor shows superior advantages over their SNV specific to the primary tumor in the four HCC patients. The error bars represent 95% confidence intervals.

圖6A-D顯示HCC1、HCC4、HCC5及HCC11之復發腫瘤(y軸)與原發性腫瘤(x軸)間之非沉默體細胞SNV Vf，及顯示原發性腫瘤特有(橙紅色)、原發性腫瘤與復發腫瘤間共用(淺藍色)或復發腫瘤特有(綠色)之計數比例之柱狀圖。 Figure 6A-D shows non-silent somatic cell SNV Vf between recurrent tumor (y-axis) of HCC1, HCC4, HCC5 and HCC11 and primary tumor (x-axis), and shows primary tumor-specific (orange-red), original A histogram of the ratio of the number of common (light blue) or recurrent tumor specific (green) between the tumor and the recurrent tumor.

圖7提供來自使用Roessler等人之研究針對染色體4q缺失內之基因預測HCC復發之Cox PH回歸模型之HERC5高/低組之轉化之p值之分佈(x軸係物理座標)。 Figure 7 provides a distribution of p-values (x-axis physical coordinates) from the transformation of the HERC5 high/low group of the Cox PH regression model predicting HCC recurrence for genes within the chromosome 4q deletion using Roessler et al.

圖8A-B圖解說明用於預測肝樣本中具有HCV-陽性肝硬化之患者之預後(良好=紅色；綠色=中等；淺藍色=較差)之與186-基因印記相關之HERC5之表現譜。A)自Hoshida等人之研究確定之3個預後水準之分佈(良好：n=60；中等：n=101；較差：n=55)。B)自Hoshida等人之Gastroenterology 144：1024-30(2013)確定之2個預後水準之分佈(良好：n=109；較差：n=107)。表指示使用186-基因印記自事件發生時間分析計算之風險。 8A-B illustrate the performance profile of HERC5 associated with the 186-gene imprint for predicting prognosis (good = red; green = medium; light blue = poor) in patients with HCV-positive cirrhosis in a liver sample. A) Distribution of 3 prognostic levels determined by Hoshida et al. (good: n=60; medium: n=101; poor: n=55). B) Distribution of 2 prognostic levels as determined by Hoshida et al. Gastroenterology 144: 1024-30 (2013) (good: n = 109; poor: n = 107). The table indicates the risk of using the 186-gene imprint to calculate the time from the occurrence of the event.

圖9A-B提供A)使用自1000個基因組專案資料庫鑑別之300個異型接合SNP，每名患者5個樣本(原發性腫瘤、接受者正常鄰近組織、復發腫瘤、供給者正常鄰近組織及接受者血液)之WES DNA生殖系SNP相關熱圖；及B)相同患者(沒有接受者血液樣本)之mRNA相關熱圖。褐色/黃色/白色方塊及每一軸上之聚類圖指示每一樣本一式五份用於DNA及一式四份用於mRNA之分組之類似性。 Figure 9A-B provides A) 300 heterozygous SNPs identified from 1000 genome project databases, 5 samples per patient (primary tumor, recipient normal adjacent tissue, recurrent tumor, normal adjacent tissue of the donor and Receiver blood) WES DNA germline SNP-related heat map; and B) mRNA-related heat map of the same patient (no recipient blood sample). The brown/yellow/white squares and clustering plots on each axis indicate that each sample is used in triplicate for DNA and quadruplicate similarity for the grouping of mRNAs.

圖10圖解說明每一患者5個樣本(原發性腫瘤、接受者正常鄰近組織、復發腫瘤、供給者正常鄰近組織及接受者血液)之WES DNA生殖系SNV熱圖。 Figure 10 illustrates the WES DNA germline SNV heat map for 5 samples per patient (primary tumor, recipient normal adjacent tissue, recurrent tumor, normal adjacent tissue of the donor, and recipient blood).

圖11展示使用純系關係值計算之導算及具有中等-至-高polyphen預測之所有非沉默SNV對腫瘤來源之確定。 Figure 11 shows the determination of tumor source using all of the non-silent SNVs calculated using the pure line relationship values and all medium-to-high polyphen predictions.

圖12A-D提供使用HCC1、HCC4、HCC5及HCC11之復發腫瘤(y軸)與原發性腫瘤(x軸)間之所有體細胞SNV Vf之斜率(m)之供給者污染計算。已自該等圖移除原發性腫瘤或復發腫瘤特有之所有體細胞變體，如同已移除原發性腫瘤或復發腫瘤試樣中任何Vf<5%者一般。紅色虛線指示x=y且藍色實線指示最小平方擬合。 Figures 12A-D provide supplier contamination calculations for the slope (m) of all somatic SNV Vf between recurrent tumors (y-axis) and primary tumors (x-axis) using HCC1, HCC4, HCC5, and HCC11. All somatic variants characteristic of primary or recurrent tumors have been removed from these maps as if any Vf < 5% in the primary tumor or recurrent tumor specimen had been removed. The red dashed line indicates x=y and the solid blue line indicates the least squares fit.

圖13A-C圖解說明使用HCC1、HCC4及HCC5之接受者特有之同型接合單一核苷酸多型性(SNP)得到之供給者污染B-等位元基因頻率(BAF)長條圖。在移除由CNV影響之所有等位基因後，將供給者細胞之污染計算為中值(1-復發腫瘤中接受者特有的同型接合等位基因之Vf)。對供給者組織污染之估計如下：HCC1=72%，HCC4=3%，且HCC5=48%。HCC11原發性及復發腫瘤源於接受者，故此處不包括該試樣。 Figures 13A-C illustrate a supplier-contaminated B-allelic gene frequency (BAF) bar graph obtained using homozygous single nucleotide polymorphism (SNP) unique to the recipient of HCC1, HCC4, and HCC5. After removal of all alleles affected by CNV, the contamination of the donor cells was calculated as the median (1 - Vf of the homozygous allele of the recipient in the recurrent tumor). Estimates of supplier tissue contamination are as follows: HCC1 = 72%, HCC4 = 3%, and HCC5 = 48%. HCC11 primary and recurrent tumors originate from the recipient, so the sample is not included here.

I. 治療肝細胞癌之方法I. Method for treating hepatocellular carcinoma A. 評估HERC5 mRNA表現 A. Evaluation of HERC5 mRNA performance

可藉助正位肝移植(OLT)治療肝細胞癌(HCC)。由於即使在使用使患者有資格進行OLT之臨床準則後原發性腫瘤之復發可負面地影響OLT之成功率，故需要輔助醫師選擇適當的OLT候選者(具體而言彼等具有較低復發風險者)之其他資訊。 Hepatocellular carcinoma (HCC) can be treated by orthotopic liver transplantation (OLT). Since the recurrence of primary tumors can negatively impact the success of the OLT even after using the patient's eligibility for clinical criteria for OLT, the physician is required to select the appropriate OLT candidates (specifically, they have a lower risk of recurrence) Other information.

在一個實施例中，治療患者肝細胞癌之方法包含自患者獲得肝細胞癌試樣；確定試樣是否具有正常HERC5 mRNA表現量；及若試樣具有正常HERC5 mRNA表現量，則使患者有資格進行正位肝移植。在一個實施例中，肝細胞癌試樣具有HERC5 DNA缺失且患者沒有資格進行正位肝移植。在另一實施例中，肝細胞癌試樣具有HERC5 mRNA過低表現且患者沒有資格進行正位肝移植。在另一實施例中，具有正常HERC5 mRNA含量之患者在移植等代列表上具有優先於至少一個沒有正常HERC5 mRNA含量之肝細胞癌患者之優先權。在另一實施例中，具有正常HERC5 mRNA含量之患者在移植等待列表上具有優先於任何沒有正常HERC5 mRNA含量之任一肝細胞癌患者之優先權。在另一實施例中，具有正常HERC5 mRNA含量之患者接受肝移植。 In one embodiment, a method of treating a patient with hepatocellular carcinoma comprises obtaining a hepatocellular carcinoma sample from a patient; determining whether the sample has a normal HERC5 mRNA expression level; and qualifying the patient if the sample has a normal HERC5 mRNA expression level Orthotopic liver shift plant. In one embodiment, the hepatocellular carcinoma sample has a deletion of HERC5 DNA and the patient is not eligible for orthotopic liver transplantation. In another embodiment, the hepatocellular carcinoma sample has a low expression of HERC5 mRNA and the patient is not eligible for orthotopic liver transplantation. In another embodiment, a patient with a normal HERC5 mRNA content has priority over a hepatocellular carcinoma patient who has at least one normal HERC5 mRNA content on a transplant et al. list. In another embodiment, a patient with a normal HERC5 mRNA content has priority over any hepatocellular carcinoma patient with no normal HERC5 mRNA content on the transplant waiting list. In another embodiment, a patient having a normal HERC5 mRNA level receives a liver transplant.

B. 量測HERC5 mRNA含量B. Measuring HERC5 mRNA content 1. 獲得活體外試樣1. Obtaining an in vitro sample

在一些實施例中，為量測HERC5 mRNA之含量，自患者移除腫瘤試樣。在一個實施例中，對出於其他目的(例如為診斷HCC或對腫瘤進行分期)移除之生檢試樣實施此步驟。可以外科手術移除生檢或可藉助針刺生檢移除其。在一個實施例中，可使用生檢後減少腫瘤細胞播種之技術，例如使用同軸切割針技術採用具有較寬直徑之引導器(例如，17規引導器)及具有較小直徑之生檢針(例如，18規生檢針)從而使得針引導器在至少一次或多次切割針通過期間保持在適當位置且沿生檢之跡線保護正常組織。在一個實施例中，可採用針核心生檢，且在另一實施例中，可使用細針抽吸細胞學。在一個實施例中，可使用腹腔鏡外科手術技術來獲得生檢。在其他實施例中，可使用超音波或其他成像技術輔助獲得試樣。 In some embodiments, to measure the amount of HERC5 mRNA, a tumor sample is removed from the patient. In one embodiment, this step is performed on a biopsy sample that is removed for other purposes, such as for diagnosing HCC or staging the tumor. The biopsy can be surgically removed or removed by a needle biopsy. In one embodiment, techniques for reducing tumor cell seeding after a biopsy can be used, such as using a coaxial cutting needle technique using a guide having a wider diameter (eg, a 17 gauge guide) and a biopsy needle having a smaller diameter ( For example, the 18 gauge needle is such that the needle guide remains in place during at least one or more passes of the cutting needle and protects normal tissue along the trace of the biopsy. In one embodiment, a needle core biopsy can be employed, and in another embodiment, fine needle aspiration cytology can be used. In one embodiment, a laparoscopic surgical technique can be used to obtain a biopsy. In other embodiments, the sample may be assisted using ultrasound or other imaging techniques.

2. 確定活體外試樣中之HERC5 mRNA含量2. Determination of HERC5 mRNA content in in vitro samples

可使用各種標準分子生物技術來確定活體外試樣中之HERC5含量且本發明實施例並非限於檢測試樣中HERC5含量之任一方式或方法。在一種模式中，可檢測mRNA含量。在另一模式中，可檢測DNA含量。在一個實施例中，患者具有單一肝細胞癌病灶。在另一實施例中，患者具有多個肝細胞癌病灶。在患者具有多個病灶之實施例中，可在一個、一些或所有病灶中確定HERC5含量。 Various standard molecular biotechnologies can be used to determine HERC5 content in ex vivo samples And the embodiments of the invention are not limited to any manner or method of detecting the HERC5 content in a sample. In one mode, the mRNA content can be detected. In another mode, the DNA content can be detected. In one embodiment, the patient has a single hepatocellular carcinoma lesion. In another embodiment, the patient has multiple hepatocellular carcinoma lesions. In embodiments where the patient has multiple lesions, the HERC5 content can be determined in one, some, or all of the lesions.

在一種模式中，可在試樣中使用微陣列(例如DNA或mRNA微陣列)檢測HERC5 mRNA之含量。在DNA或mRNA微陣列中，結合至目標HERC5序列之探針可藉助表面工程藉由共價鍵結合至微陣列之固體表面。固體表面可為玻璃或矽晶片或其可包括微珠。可藉由使用螢光團標記、銀標記或化學發光標記之靶標檢測及定量探針靶標雜交，以確定靶標中核酸序列之相對豐度。 In one mode, the amount of HERC5 mRNA can be detected in a sample using a microarray, such as a DNA or mRNA microarray. In a DNA or mRNA microarray, a probe that binds to a target HERC5 sequence can be covalently bonded to the solid surface of the microarray by surface engineering. The solid surface can be a glass or tantalum wafer or it can comprise microbeads. The relative abundance of the nucleic acid sequences in the target can be determined by detecting and quantifying probe target hybridization using a target of fluorophore labeling, silver labeling or chemiluminescent labeling.

此實施例中可採用各種市售DNA或mRNA微陣列，例如(但不限於)Affymetrix^®晶片或Illumina^® BeadArray微陣列技術。 It may employ various commercially available DNA or mRNA microarray embodiment of this embodiment, for example (but not limited to) Affymetrix ^® wafer or Illumina ^® BeadArray microarray technology.

在另一模式中，可在試樣中使用PCR(例如定量PCR(qPCR)，亦稱為即時PCR)檢測HERC5 mRNA之含量。qPCR使得能夠同時進行檢測及定量，其可係序列之絕對拷貝數或正規化為DNA輸入時之相對量。亦可藉由首先利用反轉錄酶將mRNA序列反轉錄成cDNA(反轉錄酶定量PCR或RT-qPCR)使用基於mRNA之PCR。 In another mode, the amount of HERC5 mRNA can be detected in a sample using PCR (eg, quantitative PCR (qPCR), also known as real-time PCR). qPCR enables simultaneous detection and quantification, which can be the absolute copy number of a sequence or normalized to the relative amount of DNA input. mRNA-based PCR can also be used by first reverse transcribed the mRNA sequence into cDNA using reverse transcriptase (reverse transcriptase quantitative PCR or RT-qPCR).

隨著反應即時進展，可藉助一或多種方法(包括(但不限於)使用***標記有在探針與其互補序列雜交後允許檢測之螢光報告基因之任何雙鏈DNA及/或序列特異性DNA探針(例如寡核苷酸)之非特異性螢光染料)檢測擴增之DNA(來自DNA試樣或自mRNA反轉錄之cDNA)。在非特異性模式中，DNA結合染料(例如SYZBR Green)將結合至所有dsDNA PCR產品，且在此模式中，僅所需要之序列特異性試劑係用於擴增HERC5之引子。在序列特異性DNA探針模式中，螢光報告基因僅檢測含有探針序列之DNA，因而即便在非特異性DNA擴增存在下亦可增加特異性且使得能夠達成定量。在一些模式中，序列特異性DNA探針在一個末端具有螢光報告基因且在另一末端具有淬滅體以阻止螢光，除非其結合至靶標DNA鏈。 As the reaction progresses immediately, any double-stranded DNA and/or sequence-specific DNA that allows for detection of the fluorescent reporter gene after hybridization of the probe to its complementary sequence can be performed by one or more methods including, but not limited to, the use of an insertion marker. Amplified DNA (from DNA samples or cDNA reverse transcribed from mRNA) is detected by a non-specific fluorescent dye of a probe (eg, an oligonucleotide). In a non-specific mode, a DNA binding dye (eg, SYZBR Green) will bind to all dsDNA PCR products, and in this mode, only the sequence-specific reagents required are used to amplify the primer for HERC5. In the sequence-specific DNA probe mode, the fluorescent reporter gene detects only the DNA containing the probe sequence, and thus even in the presence of non-specific DNA amplification It is also possible to increase the specificity and enable the quantification to be achieved. In some modes, the sequence-specific DNA probe has a fluorescent reporter gene at one end and a quencher at the other end to prevent fluorescence unless it binds to the target DNA strand.

在此實施例中可採用各種市售qPCR套組，例如(但不限於)基於GoTaq^®染料之qPCR及RT-qPCR、基於GoTaq^®探針之qPCR及RT-qPCR，及Plexor^® qPCR及RT-qPCR系統，所有套組皆來自Promega^®。 Examples of various commercially available qPCR kit may be employed in this embodiment, for example (but not limited to) the dye-based qPCR GoTaq ^® and RT-qPCR, the probe-based qPCR GoTaq ^® and RT-qPCR, and Plexor ^® qPCR and RT- qPCR system, all kits are from Promega ^® .

C. 比較HERC5 mRNA表現C. Comparison of HERC5 mRNA expression 1. 用於比較之對照人群及技術1. Control population and technology for comparison

在已對HERC5含量進行定量後，可對比截止含量或對照評估該HERC5含量。可藉由在對照組中實施足夠數量之實驗獲得截止含量。因此，對照可為並行對照實驗或其可為歷史對照。在一個實施例中，將試樣中之HERC5 mRNA含量與歷史對照組中之HERC5 mRNA之含量進行比較。在一個實施例中，對照係健康肝組織試樣或來自評估健康肝組織試樣之歷史資料。 After the HERC5 content has been quantified, the HERC5 content can be assessed against the cut-off content or control. The cut-off content can be obtained by performing a sufficient number of experiments in the control group. Thus, the control can be a parallel control experiment or it can be a historical control. In one embodiment, the HERC5 mRNA content in the sample is compared to the amount of HERC5 mRNA in the historical control. In one embodiment, the control is a healthy liver tissue sample or a historical data from a sample of a healthy liver tissue.

在一個實施例中，歷史對照組具有正常個體。在另一實施例中，歷史對照組具有所患肝細胞癌在至少5年、6年、7年、8年、9年或10年內未復發之患者。該等對照組可稱作展示「正常」含量之陰性對照組，由於該等對照組無肝細胞癌或由於其患有在指定時間段內未復發之肝細胞癌。在一個實施例中，若HERC5患者試樣含量較歷史陰性對照組之平均值低等於或高於兩個標準偏差，則該含量指定為正常的。在另一實施例中，若HERC5患者試樣比歷史陰性對照組之平均值低等於或高於一個標準偏差，則該試樣指定為正常的。 In one embodiment, the historical control group has a normal individual. In another embodiment, the historical control group has patients whose hepatocellular carcinoma has not relapsed for at least 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years. Such controls may be referred to as negative controls displaying "normal" levels, as these controls have no hepatocellular carcinoma or because they have hepatocellular carcinoma that has not relapsed within a specified period of time. In one embodiment, the content is designated as normal if the HERC5 patient sample content is less than or equal to two standard deviations from the mean of the historical negative control group. In another embodiment, the sample is designated as normal if the HERC5 patient sample is less than or equal to one standard deviation from the mean of the historical negative control group.

在另一實施例中，歷史對照組具有所患肝細胞癌在1年、2年、3年、4年或5年內復發之患者。此對照組可稱作展示「低」含量之陽性對照組，由於該對照組患有在指定時間段內復發之肝細胞癌。在一種模式中，若HERC5患者試樣含量較歷史陽性對照組之平均值高等於或高於一個標準偏差，則該含量指定為正常的。 In another embodiment, the historical control group has patients with hepatocellular carcinoma that relapse within 1 year, 2 years, 3 years, 4 years, or 5 years. This control group can be referred to as a positive control group displaying a "low" content, since the control group has hepatocellular carcinoma that relapses within a specified period of time. In one mode, if the HERC5 patient sample is higher than the historical positive control group, the average is equal to or Above a standard deviation, this content is specified as normal.

在再一實施例中，歷史對照組包含已患肝細胞癌之患者，其中在已具有復發者或沒有復發者之間無區別。此歷史對照組可稱作all-HCC對照組。在一個實施例中，若HERC5患者試樣含量較all-HCC對照組之平均值高等於或高於一個標準偏差，則該含量指定為正常的。在另一實施例中，若HERC5患者試樣高於all-HCC對照組之中值，則該試樣指定為正常的。 In still another embodiment, the historical control group comprises patients who have developed hepatocellular carcinoma, wherein there is no difference between those who have or who have relapsed. This historical control group can be referred to as the all-HCC control group. In one embodiment, the amount is specified as normal if the HERC5 patient sample content is greater than or equal to one standard deviation from the average of the all-HCC control group. In another embodiment, the sample is designated as normal if the HERC5 patient sample is above the middle of the all-HCC control.

在該等模式中之任一模式中，歷史對照組可包含來自至少10名、25名、50名、100名、250名、500名或1000名個體之數據。在一個實施例中，可採用一種以上技術或對照。 In either of these modes, the historical control group can contain data from at least 10, 25, 50, 100, 250, 500, or 1000 individuals. In one embodiment, more than one technique or control may be employed.

2. 用於比較之健康組織對照及技術2. Healthy tissue comparison and technology for comparison

在另一模式中，可自患有肝細胞癌之患者獲得健康組織且可將健康組織中HERC5之mRNA含量與肝細胞癌試樣中HERC5之mRNA含量進行比較。在一個實施例中，正常組織係肝組織。 In another mode, healthy tissue can be obtained from a patient with hepatocellular carcinoma and the mRNA content of HERC5 in healthy tissue can be compared to the mRNA content of HERC5 in a hepatocellular carcinoma sample. In one embodiment, the normal tissue is liver tissue.

在一個態樣中，若肝細胞癌試樣中HERC5之mRNA含量高於正常組織試樣中含量之70%、75%、80%、85%、90%，則HERC5含量經提供為指示患者有資格進行正位肝移植之積極因素。在一些情況中，亦使用其他臨床因素。 In one aspect, if the mRNA content of HERC5 in the hepatocellular carcinoma sample is higher than 70%, 75%, 80%, 85%, 90% of the content in the normal tissue sample, the HERC5 content is provided to indicate that the patient has Qualified for positive factors in orthotopic liver transplantation. In some cases, other clinical factors are also used.

在另一態樣中，當肝細胞癌試樣中HERC5之mRNA含量低於正常組織試樣中含量之40%、45%、50%、55%、60%或65%時，HERC5含量經提供為抵抗患者有資格進行正位肝移植之消極因素。 In another aspect, when the mRNA level of HERC5 in the hepatocellular carcinoma sample is lower than 40%, 45%, 50%, 55%, 60% or 65% of the content in the normal tissue sample, the HERC5 content is provided. To counter the negative factors that patients are eligible for orthotopic liver transplantation.

D. 納入臨床準則D. Inclusion of clinical guidelines

在本文所闡述之任一模式中，另一實施例可包括在製作決策過程中考慮患者是否有資格或沒資格進行正位肝移植之臨床準則。在一個實施例中，臨床準則包含以下各項中之至少一者：病灶大小，病灶數量，是否具有肝外表現，是否具有血管侵犯，基於生檢HCC是否分化不良，腫瘤狀態，腫瘤等級及甲型胎兒蛋白含量。 In any of the modes set forth herein, another embodiment may include clinical criteria for considering whether a patient is eligible or not eligible for orthotopic liver transplantation during a decision making process. In one embodiment, the clinical criteria comprises at least one of the following: lesion size, number of lesions, presence of extrahepatic manifestations, vascular invasion, based on biopsy HCC scores Poor dysfunction, tumor status, tumor grade and alpha fetal protein content.

例如，在一個態樣中，臨床準則包含Milan準則：(a)單一病灶不超過5cm，或不多於3個病灶，該3個病灶皆不超過3cm；及(b)無大血管侵犯。在另一態樣中，臨床準則包含UCSF準則：(a)單一病灶不大於6.5cm；或(b)不多於3個病灶，該3個病灶皆不超過4.5cm且其總腫瘤直徑不超過8cm。 For example, in one aspect, the clinical criteria include the Milan criteria: (a) a single lesion no more than 5 cm, or no more than 3 lesions, none of the 3 lesions exceed 3 cm; and (b) no macrovascular invasion. In another aspect, the clinical criteria include UCSF criteria: (a) a single lesion no greater than 6.5 cm; or (b) no more than 3 lesions, none of the 3 lesions exceed 4.5 cm and the total tumor diameter does not exceed 8cm.

在另一態樣中，臨床準則包含Toronto準則且包括任一腫瘤大小或數量，及無因HCC所致之全身症狀，及基於組織學排除分化不良HCC(僅超過Milan腫瘤)。 In another aspect, the clinical criteria include the Toronto criteria and includes any tumor size or number, and no systemic symptoms due to HCC, and histological exclusion of poorly differentiated HCC (only more than Milan tumors).

在另一態樣中，臨床準則包含Hangzhou準則且包括a)總腫瘤直徑小於或等於8cm；(b)總腫瘤直徑超過8cm，且組織病理學等級為I級或II級，且手術前AFP含量小於或等於400ng/mL。 In another aspect, the clinical criteria include the Hangzhou criteria and include a) the total tumor diameter is less than or equal to 8 cm; (b) the total tumor diameter is greater than 8 cm, and the histopathology grade is grade I or grade II, and the preoperative AFP content Less than or equal to 400 ng/mL.

在一種模式中，準則亦包括甲型胎兒蛋白濃度是否小於400ng/mL(其中較低含量指示OLT後之潛在陽性結果)。 In one mode, the criteria also include whether the A-type fetal protein concentration is less than 400 ng/mL (where lower levels indicate potentially positive results after OLT).

II. 評估肝細胞癌復發風險之方法II. Methods for assessing the risk of recurrence of hepatocellular carcinoma

在另一態樣中，實施例包括評估肝細胞癌復發風險概況之方法，該方法包含：a. 提供肝細胞癌試樣；b. 在活體外確定試樣是否具有正常的HERC5 mRNA表現量；c. 若試樣具有正常的HERC5 mRNA表現量且該確定係結合至少一個臨床準則作出的，則將肝細胞癌定性為具有低復發風險。 In another aspect, the embodiments comprise a method of assessing a risk profile for hepatocellular carcinoma recurrence, the method comprising: a. providing a hepatocellular carcinoma sample; b. determining whether the sample has normal HERC5 mRNA expression in vitro; c. Hepatocellular carcinoma is characterized as having a low risk of recurrence if the sample has a normal amount of HERC5 mRNA expression and the determination is made in conjunction with at least one clinical criterion.

一個實施例涵蓋使用HERC5正向及反向引子(其每一者係自HERC5之mRAN序列生成)產生用於活體外診斷可利用正位肝移植進行治療之肝細胞癌之診斷液。另一實施例涵蓋使用HERC5特異性探針產生用於活體外診斷可利用正位肝移植進行治療之肝細胞癌之診斷液。另一態樣涵蓋使用HERC5核酸序列作為定量對照以產生用於活體外診斷可利用正位肝移植進行治療之肝細胞癌之診斷液。在實施例中，診斷藥劑可與其他臨床準則結合。在另一實施例中，在已作出初始診斷後使用診斷液來評估肝細胞癌之復發風險並輔助確定治療計畫。 One embodiment encompasses the use of HERC5 forward and reverse primers, each of which is generated from the mRAN sequence of HERC5, to generate a diagnostic fluid for in vitro diagnosis of hepatocellular carcinoma that can be treated with orthotopic liver transplantation. Another embodiment encompasses the use of HERC5 specific probes A diagnostic solution for hepatocellular carcinoma that can be used for in vitro diagnosis and can be treated with orthotopic liver transplantation. Another aspect encompasses the use of the HERC5 nucleic acid sequence as a quantitative control to generate a diagnostic fluid for in vitro diagnosis of hepatocellular carcinoma that can be treated with orthotopic liver transplantation. In embodiments, the diagnostic agent can be combined with other clinical criteria. In another embodiment, the diagnostic fluid is used to assess the risk of recurrence of hepatocellular carcinoma after an initial diagnosis has been made and to aid in determining a treatment plan.

可使用與在上述章節I.B至I.D中相同之用於評估HERC5含量、比較HERC5含量及納人臨床準則之技術。 Techniques for assessing HERC5 content, comparing HERC5 levels, and human clinical criteria can be used as in Sections I.B through I.D above.

現在將詳細參考本發明例示性實施例，在附圖中圖解說明該等實施例的實例。在可能的情況下，將遍及各附圖使用相同參考號來指代相同或相似部件。藉由考量本說明書及本文中所揭示之實踐，熟習此項技術者將明瞭其他實施例。在以下實例中進一步解釋該等實施例。該等實例並不限制申請專利範圍之範圍，只是僅僅用於闡明某些實施例。本說明書及各實例意欲僅視為例示性，且真實範圍及精神係藉由以下申請專利範圍來指示。 Reference will now be made in detail to the exemplary embodiments embodiments Wherever possible, the same reference numerals are used to the Other embodiments will be apparent to those skilled in the art from a <RTIgt; These embodiments are further explained in the examples below. The examples are not intended to limit the scope of the claims, but are merely illustrative of certain embodiments. The description and the examples are intended to be illustrative only, and the true scope and spirit are indicated by the following claims.

實例Instance 實例1. DNA序列讀長(read)映射及變體識別(calling)Example 1. DNA sequence read length mapping and variant recognition (calling) A)全外顯子組及RNA測序A) Whole exome and RNA sequencing

利用來自四名中國HCC OLT患者之原發性及復發腫瘤(P_T及R_T)、正常鄰近組織(P_NAT及R_NAT)及接受者血液(P_B)實施全外顯子組及RNA測序(WES及RNASeq)，且對主要樣本(表2A(復發患者)及表2B(非復發患者))實施全基因組測序。所有四名患者皆在UCSF準則內，兩名在OLT測試時為B型肝炎病毒陽性；三名患者患有復發肝腫瘤且一名患者在OLT後的24個月內患有肺轉移。DNA全基因組序列(WGS)及全外顯子組序列(WES)資料係使用Illumina標準文庫製備及測序方案生成。提供兩種序列資料類型之90聚體序列讀長之成對末端FASTQ檔案。 Full exome and RNA sequencing using primary and recurrent tumors (P _T and R _T ), normal adjacent tissues (P _NAT and R _NAT ), and recipient blood (P _B ) from four Chinese HCC OLT patients (WES and RNASeq), and genome-wide sequencing was performed on the primary samples (Table 2A (relapsed patients) and Table 2B (non-relapsing patients)). All four patients were within the UCSF guidelines, two were positive for hepatitis B virus at the OLT test; three patients had recurrent liver tumors and one patient had lung metastases within 24 months after OLT. DNA Whole Genome Sequence (WGS) and Whole Exome Sequence (WES) data were generated using the Illumina standard library preparation and sequencing protocol. A paired-end FASTQ file of the 90-mer sequence of the two sequence data types is provided.

對於讀長計數、品質值、kmer用法、GC含量及FastQC(v0.10.1)內之所有其他相關參數而言，所有序列資料皆為QCd。使用Bowtie2(v2.0.0-beta7；Langmead等人，Nat Methods 9(4)：2357-9(2012))將DNA序列與人類基因組(UCSC hg19；Feb 2009 release；Genome Reference Consortium GRCh37)進行比對，且分別使用GATK(v2.3.4；McKenna等人，Genome Res 20：1297-303(2010))及Picard(v1.85)實施***缺失重新排列(indel realignment)及PCR複製移除。每一樣本來自WES之所有映射總結皆提供於表3中。對於RNASeq及WES資料，使用SAMtools(v0.1.18；Li等人，Bioinformatics 25：2078-9(2009))mpileup(Qphred>30及映射品質>30)利用VarScan2(v2.3.2；Koboldt等人，Genome Res 22(3)：568-76(2012))相對於人類參考利用以下參數識別單一核苷酸變體(SNV)及***/缺失(indel)：變體最小頻率>5%，用於變體識別之VarScan2 p值(基於費希爾精確測試(Fisher’s exact test))<0.01，最小覆蓋=20，最小讀長=3，最小平均品質=30，純合子之最小頻率=0.8。對於WGS資料，倘若深度遠低於WES資料，則使用以下論證：最小覆蓋=15，最小讀長=2，最小平均品質=13，Qphred>13且映射品質>1。20個樣本之平均WES深度在66X至109X之範圍內(平均值=92.1X)，且在最小10X深度下，平均覆蓋率為73.4%(Qphred>30)(表3)。 For sequence length counts, quality values, kmer usage, GC content, and all other relevant parameters within FastQC (v0.10.1), all sequence data is QCd. The DNA sequence was aligned with the human genome (UCSC hg19; Feb 2009 release; Genome Reference Consortium GRCh37) using Bowtie 2 (v2.0.0-beta7; Langmead et al, Nat Methods 9(4): 2357-9 (2012)). Insertion deletion realignment and PCR replication removal were performed using GATK (v2.3.4; McKenna et al., Genome Res 20: 1297-303 (2010)) and Picard (v1.85), respectively. A summary of all mappings from each sample from WES is provided in Table 3 . For RNASeq and WES data, use VarScan2 (v2.3.2; Koboldt et al., Genome) using SAMtools (v0.1.18; Li et al., Bioinformatics 25:2078-9 (2009)) mpileup (Qphred>30 and mapping quality >30). Res 22(3): 568-76 (2012)) Identification of single nucleotide variants (SNV) and insertions/deletions (indel) using the following parameters relative to human reference: variant minimum frequency > 5%, for variants The identified VarScan2 p value (based on Fisher's exact test) <0.01, minimum coverage = 20, minimum read length = 3, minimum average quality = 30, and minimum frequency of homozygote = 0.8. For WGS data, if the depth is much lower than the WES data, the following arguments are used: minimum coverage = 15, minimum read length = 2, minimum average quality = 13, Qphred > 13 and mapping quality > 1. Average WES depth of 20 samples In the range of 66X to 109X (average = 92.1X), and at a minimum depth of 10X, the average coverage was 73.4% (Qphred > 30) (Table 3).

在P_Ts及R_Ts中鑑別出1,145種體細胞變體，包括影響567個基因之616種體細胞非沉默(非同義取代、終止密碼子獲得(stop-gain)取代、終止密碼子缺失(stop-loss)取代或框移取代)單一核苷酸變體(SNV)或***/缺失(indel)(表4且未顯示資料)。體細胞SNV分佈具有最高的C>T/G>A出現率及最低的T>G/A>C出現率(圖4)，此與先前的HCC報導(Totoki等人，Nat Genet 43：464-71(2011))一致，且匹配的P_T及R_T對具有類似的轉換/顛換取代分佈，其中HCC11具有最高的T>A/A>T發生率。非沉默-至-沉默SNV比率在P_Ts中平均為2.9且在R_Ts中平均為2.3，R_Ts中之比率較低係供給者-組織污染之結果。在P_Ts及R_Ts中鑑別出1,145種體細胞變體，包括影響567個基因之616種體細胞非沉默(非同義取代、終止密碼子獲得取代、終止密碼子缺失取代或框移取代)單一核苷酸變體(SNV)或***/缺失(indel)(表4且未顯示資料)。體細胞SNV分佈具有最高的C>T/G>A出現率及最低的T>G/A>C出現率(圖4)，此與先前的HCC報導(Totoki等人，Nat Genet 43：464-71(2011))一致，且匹配之P_T及R_T對具有類似的轉換/顛換取代分佈，其中HCC11具有最高的T>A/A>T發生率。非沉默-至-沉默SNV比率在P_Ts中平均為2.9且在R_Ts中平均為2.3，RTs中之比率較低係供給者-組織污染之結果。 1,145 somatic variants were identified in P _T s and R _T s, including 616 somatic cells that affect 567 genes are non-silencing (non-synonymous substitution, stop-gain substitution, stop codon deletion) (stop-loss) substitution or frame shift substitution) single nucleotide variant (SNV) or insertion/deletion (indel) (Table 4 and no data shown). The somatic SNV distribution has the highest C>T/G>A occurrence rate and the lowest T>G/A>C occurrence rate (Fig. 4), which is consistent with previous HCC reports ( Totoki et al., Nat Genet 43:464- 71 (2011)) Consistent, and matched P _T and R _T pairs have similar conversion/transversion substitution profiles, where HCC 11 has the highest T>A/A>T incidence. Non-silent - to - the silence SNV ratio P _T s and an average of 2.9 in the average R _T s of 2.3, the ratio R _T s in the lower line provider - result of tissue contamination. 1,145 somatic variants were identified in P _T s and R _T s, including 616 somatic cells that affect 567 genes are non-silencing (non-synonymous substitution, stop codon substitution, stop codon deletion substitution or frame shift substitution) ) Single nucleotide variant (SNV) or insertion/deletion (indel) (Table 4 and no data shown). The somatic SNV distribution has the highest C>T/G>A occurrence rate and the lowest T>G/A>C occurrence rate (Fig. 4), which is consistent with previous HCC reports ( Totoki et al., Nat Genet 43:464- 71 (2011)) Consistent, and matching P _T and R _T pairs have similar conversion/transversion substitution profiles, where HCC 11 has the highest T>A/A>T incidence. The non-silent-to-silent SNV ratio averaged 2.9 in P _T s and 2.3 in R _T s, and the lower ratio in RTs was the result of donor-tissue contamination.

B)體細胞SNV之鑑別B) Identification of somatic SNV 1. 方法Method

對批次識別中之所有試樣進行變體識別且將其輸出為變體識別格式(VCF)(v4.1)檔案。然後，根據以下規則針對P_T對P_B、P_NAT對P_B、R_T對P_B及R_NAT對P_B之對之間之體細胞SNV及indel識別實施變體識別細化，其中P=原發性，R=復發，T=腫瘤，NAT=正常鄰近組織，且B=血液，如下所述：1)在正常試樣及腫瘤中，對於WES，參考或變體(或二者)之平均Qphred>30(對於WGS，13)，2)來自正常/腫瘤與參考/變體間之讀長計數之費希爾精確測試之P值<0.01，3)正常試樣中之變體頻率(Vf)<3%，且4)腫瘤Vf比正常試樣之Vf大至少5%。 Variant recognition was performed on all samples in the batch identification and output as a variant identification format (VCF) (v4.1) file. Then, the variant identification refinement is performed for the somatic SNV and indel identification between the P _T pair P _B , the P _NAT pair P _B , the R _T pair P _B and the R _NAT pair P _B pair according to the following rules, wherein P= Primary, R = recurrence, T = tumor, NAT = normal adjacent tissue, and B = blood, as follows: 1) in normal samples and tumors, for WES, reference or variant (or both) Average Qphred > 30 (for WGS, 13), 2) Fisher's exact test from normal/tumor versus reference/variant counts P value <0.01, 3) Variant frequency in normal samples ( Vf) < 3%, and 4) The tumor Vf is at least 5% larger than the Vf of the normal sample.

為鑑別每一患者之P_T及R_T試樣特有之體細胞SNV，移除與P_T對P_B SNV之列表共用的在P_NAT對P_B之間鑑別的彼等SNV，如同移除與R_T對P_B SNV之列表共用的在R_NAT對P_B之間鑑別的彼等SNV一般。然後，使用ANNOVAR(v2013-07-28；Wang等人，Nucleic Acids Res 38(16)：e164(2010))實施註解及對基因之變體效應，且基於以下各項選擇所有非沉默(即非同義，終止密碼子獲得或終止密碼子缺失)SNV或非沉默indel(即框移取代)：1)在利用所有種族之1000個基因組中，未知或最小等位元基因頻率(MAF)<0.05，且2)不位於UCSC基因組資料庫(http：//genome.ucsc.edu)之縱排重複或破壞重複區域內。 To identify the somatic SNV specific to the P _T and R _T samples of each patient, remove the SNVs identified between the P _{NAT and} the P _B that are shared with the P _T pair of P _B SNVs, as if removed and R _{T is} common to the SNVs identified by the list of P _B SNVs between R _{NAT and} P _B . Then, using ANNOVAR (v2013-07-28; Wang et al., Nucleic Acids Res 38(16):e164 (2010)) to perform annotations and variant effects on genes, and select all non-silencing based on the following (ie, non- Synonymous, stop codon gain or stop codon deletion) SNV or non-silent indel (ie, frame shift substitution): 1) In the 1000 genomes of all races, the unknown or minimum allele frequency (MAF) <0.05, And 2) not located in the tandem repeat or disrupted repeat region of the UCSC Genome Database ( http://genome.ucsc.edu ).

所有沉默SNV及indel皆係基於預測之外顯子功能(同義)及上述第2條準則來選擇。每一患者之體細胞SNV及indel計數提供於表4中。 All silent SNVs and indels were selected based on the predicted exon function (synonymous) and the above criteria. The somatic SNV and indel counts for each patient are provided in Table 4.

亦根據以下規則針對上文所闡述之對細化生殖系SNV識別：1)在正常試樣及腫瘤中，對於WES，參考或變體(或二者)之平均Qphred>30(對於WGS，13)，2)在正常試樣及腫瘤中，來自VarScan2費希爾精確測試之P值(相對於參考基因組)<0.01，且3)正常試樣及腫瘤中之最小Vf>5%。 The identification of the genital germline SNV as described above is also based on the following rules: 1) In normal samples and tumors, for the WES, the reference or variant (or both) has an average Qphred > 30 (for WGS, 13 2) In normal samples and tumors, the P value from the VarScan2 Fisher exact test (relative to the reference genome) <0.01, and 3) the minimum Vf > 5% in normal samples and tumors.

所有非沉默(即非同義，終止密碼子獲得或終止密碼子缺失)SNV或非沉默indel(即框移取代)皆係基於以下各項來選擇：1)所有種族之1000個基因組中，未知或最小等位元基因頻率(MAF)<0.05，且2)不位於UCSC基因組資料庫(http：//genome.ucsc.edu)之縱排重複或破壞重複區域內。 All non-silencing (ie non-synonymous, stop codon gain or stop codon deletion) SNV or non-silent indel (ie, frame shift substitution) are selected based on the following: 1) 1000 genomes of all races, unknown or The minimum allele frequency (MAF) < 0.05, and 2) is not located in the tandem repeat or disrupted repeat region of the UCSC Genome Database ( http://genome.ucsc.edu ).

此提供生殖系SNV列表。 This provides a list of germline SNVs.

2. 結果2. Results

具有驅動突變之純系作為腫瘤中之主要純系具有選擇優點。對每一患者在P_Ts特有之基因座與P_Ts與R_Ts間共用之彼等基因座之間評估所有非沉默SNV及indel中之變體頻率(Vf)分佈(圖5)。P_Ts與R_Ts間共用之變體之Vf(平均值=39.4)顯著高於彼等為P_Ts特有者(平均值=23.4；所有四名患者p<0.001；表4)。亦在患者中比較P_T、R_T特有或共用之非沉默變體，從而指示與每一獨特集合相比，共用之純系之比例大幅增加(圖6)。此表明共用之體細胞突變很可能為驅動突變。 A pure line with a drive mutation has the advantage of being the major pure line in a tumor. The variant frequency (Vf) distribution in all non-silent SNVs and indels was assessed for each patient between the P _T s specific loci and their loci shared between P _T s and R _T s (Figure 5). The Vf of the variant shared between P _T s and R _T s (mean = 39.4) was significantly higher than those of P _T s (mean = 23.4; all four patients p <0.001; Table 4). Non-silent variants of P _T , R _T specific or shared were also compared in patients, indicating that the proportion of shared pure lines was significantly increased compared to each unique set (Figure 6). This suggests that shared somatic mutations are likely to be driving mutations.

亦在P_Ts及R_Ts內鑑別體細胞CNV(未顯示資料)。針對P_T或R_T之獨特性或該兩者間所共用選擇顯著擴增或缺失之區域，所有皆在4名患者之至少3名內(圖1)。鑑別P_Ts與R_Ts間共用之DNA擴增，其中彼等標記有星號(*)者係在先前報導(Guichard等人，Nat Genetics 44：694-699(2012))在HCC患者之P_Ts中觀察到的：1q*、6p*、8q*、17q*及20p*，而共用之DNA缺失包括：4q*及17p*。在17q及20q內鑑別R_T特有之擴增區域。 Somatic cell CNV was also identified in P _T s and R _T s (data not shown). The regions that are significantly amplifying or deleting for the uniqueness of P _T or R _T or the combination of the two are all within at least 3 of 4 patients (Fig. 1). Identification of DNA amplification shared between P _T s and R _T s, where those labeled with an asterisk (*) were previously reported ( Guidard et al., Nat Genetics 44:694-699 (2012)) in patients with HCC Observed in _T s: 1q*, 6p*, 8q*, 17q*, and 20p*, while shared DNA deletions include: 4q* and 17p*. The amplified regions specific to R _T were identified in 17q and 20q.

實例2. RNA序列讀長映射及差異表現分析Example 2. RNA sequence read length mapping and differential performance analysis

使用Illumina標準文庫製備及測序方案藉由BGI生成RNA全轉錄組序列資料。將90聚體序列讀長之成對末端FASTQ檔案提供給MedImmune。對於讀長計數、品質值、kmer用法、GC含量及FastQC(v0.10.1)內之所有其他相關參數而言，序列資料為QCd。每個配對物之平均讀長計數為5000萬。使用TopHat2(v2.0.9；Kim等人，Genome Biol 14(4)：R36(2013))及人類參考gtf註解檔案(GRCh37.68)將RNA讀長映射至人類基因組(UCSC hg19；Feb 2009 release；Genome Reference Consortium GRCh37)。使用htseq-count及DESeq(v1.12.1；Anders等人，Genom Biol 11：R106(2010))計算並正規化轉錄本計數。使用DESeq負二項分佈，使用p<0.01及｜倍數改變｜>2作為臨限值，計算P_T與P_NAT以及R_T與R_NAT之間之p值及倍數改變。 RNA full transcriptome sequence data was generated by BGI using the Illumina standard library preparation and sequencing protocol. The paired end FASTQ file of the 90-mer sequence was read and provided to MedImmune. For read length counts, quality values, kmer usage, GC content, and all other relevant parameters within FastQC (v0.10.1), the sequence data is QCd. The average read length of each pair is 50 million. RNA read length was mapped to the human genome using TopHat2 (v2.0.9; Kim et al., Genome Biol 14(4): R36 (2013)) and human reference gtf annotation file (GRCh37.68) (UCSC hg19; Feb 2009 release; Genome Reference Consortium GRCh37). Transcript counts were calculated and normalized using htseq-count and DESeq (v1.12.1; Anders et al, Genom Biol 11: R106 (2010)). Using the DESeq negative binomial distribution, using p < 0.01 and | multiple change | > 2 as the threshold, calculate the p-value and fold change between P _T and P _NAT and R _T and R _NAT .

實例3. 體細胞拷貝數變異(CNV)分析Example 3. Somatic cell copy number variation (CNV) analysis

基於源於WES比對之讀長深度使用R包DNAcopy及ExomeCNV鑑別CNV。基於以下三個原因選擇1Mb視窗大小來鑑別CN片段：1)鑑別大型片段而非局部變異之區域，2)編碼區域基因結構在外顯子組捕獲中可為成百上千個千鹼基長，需要大視窗來涵蓋全部基因而非基因之部分，及3)所有患者之總結顯示在1Mb臨限值下不包括<3%之片段。在基因組內計算CNV之Log₂ R比率(LRR)作為所有腫瘤與正常比較之間之差異(參見上文針對相關配對所闡述)。使用ABSOLUTE軟體(Carter等人，Nature Biotechnol 30(5)：413-21(2012))評估腫瘤純度且針對每一腫瘤與病理學評價進行比較。所有CNV結果皆提供於圖1中(未顯示原始資料)。 The CNV was identified using R-package DNAcopy and ExomeCNV based on the read length from the WES alignment. The 1Mb window size is selected to identify CN fragments based on three reasons: 1) identifying large fragments rather than regions of local variation, and 2) coding region gene structures can be hundreds of kilobases long in exome capture, A large window is required to cover all genes, not part of the gene, and 3) a summary of all patients shows that the fragment of <3% is not included at the 1Mb threshold. The Log ₂ R ratio (LRR) of CNV was calculated within the genome as the difference between all tumors compared to normal (see above for related pairings). Tumor purity was assessed using ABSOLUTE software ( Carter et al, Nature Biotechnol 30(5): 413-21 (2012)) and compared for pathology evaluation for each tumor. All CNV results are provided in Figure 1 (original data not shown).

實例4. 路徑分析Example 4. Path Analysis A)方法A) Method

為進一步鑑別關鍵體細胞遺傳改變，採用整合路徑分析。使用Ingenuity Pathway Analysis(IPA；Ingenuity,Redwood City,CA)實施路徑分析，同時在R(v3.0.1；R：A Language and Environment for Statistical Computing，R Development Core Team,R Foundation for Statistical Computing,Vienna,Austria,2013)中實施圖解析腳本及其他分析。實施兩種類型之路徑分析輸出。計算第一種路徑分析輸出以關聯對路徑調節之上游遺傳效應。對於此方法，將具有個體患者之原發性腫瘤與復發腫瘤之間共用之具有中等-至-高PolyPhen功能效應之非沉默體細胞SNV及indel之所有基因與在顯著擴增或缺失之CNV區域內且基因表現｜倍數改變｜>2(P_T/P_NAT及R_T/NAT_T)；來自DESeq之p值<0.05之基因組合。然後，將該等基因用於使用IPA之規範路徑富集分析中。使用IPA中之比較工具來組合四名患者中之每一者之所有路徑資訊，且按路徑將合成分數計算為一路徑在患者中之-log₁₀(pvs)之總和(未顯示資料)。僅對於所有四名患者中復發腫瘤特有之彼等情況，亦針對彼等基因使用此相同準則實施此同一路徑富集分析(未顯示資料)。第二種路徑分析輸出包括來自IPA之上游分析輸出及差異表現之轉錄本(在P_T對P_NAT及R_T對R_NAT之間共用)，從而鑑別最富集轉錄因子(TF)調節之基因之彼等TF(如藉由文獻所確定)。此稱作上游基因印記(表5)。 To further identify key somatic genetic changes, integrated pathway analysis was used. Path analysis was performed using Ingenuity Pathway Analysis (IPA; Ingenuity, Redwood City, CA), while at R (v3.0.1; R: A Language and Environment for Statistical Computing, R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria) , 2013) implementation of the graph analysis script and other analysis. Implement two types of path analysis outputs. The first path analysis output is calculated to correlate the upstream genetic effects of the path adjustment. For this method, all genes of non-silencing somatic cells SNV and indel with medium-to-high PolyPhen functional effects shared between primary and recurrent tumors of individual patients and CNV regions in significant amplification or deletion Internal gene expression | fold change |> 2 (P _T /P _NAT and R _T /NAT _T ); gene combination of p value <0.05 from DESeq. These genes were then used in the canonical path enrichment analysis using IPA. The comparison tool in IPA was used to combine all path information for each of the four patients, and the composite score was calculated by path as the sum of -log ₁₀ (pvs) in the patient (data not shown). This same path enrichment analysis was performed using this same criterion for each of the four patients with recurrence-only tumors (data not shown). The second path analysis output includes a transcript from the upstream analysis output and differential performance of the IPA (shared between P _{T and} P _NAT and R _T to R _NAT ) to identify the most abundant transcription factor (TF) regulated gene. Their TFs (as determined by the literature). This is called the upstream gene imprint (Table 5).

B)結果B) Results

鑑別每一患者具有中等-至-高PolyPhen分值之非沉默SNV及indel及在顯著擴增或缺失CN區域內亦過表現或過低表現(｜倍數｜>2；p<0.05)且在P_Ts與R_Ts(P_T/P_NAT ∩ R_T/R_NAT)之間共用之基因。個別合併每一患者之該等基因並分析富集該等因數之路徑(未顯示資料)。亦鑑別每一患者之藉由差異性表現之針對共用腫瘤調節基因(P_T/P_NAT ∩ R_T/R_NAT)之轉錄靶標(表示為上游基因印記)富集之轉錄因子(p<0.01及｜倍數｜>2；表4)。 Identification of non-silent SNV and indel with moderate-to-high PolyPhen scores for each patient and over or under performance in significant amplified or absent CN regions (|multiples |>2;p<0.05) and at P A gene shared between _T s and R _T s (P _T /P _NAT ∩ R _T /R _NAT ). Individually combine the genes for each patient and analyze the path to enrich the factors (data not shown). Transcription factors (p<0.01) enriched by transcriptional targets (represented as upstream gene imprints) for the shared tumor regulatory gene (P _T /P _NAT ∩ R _T /R _NAT ) by differential expression were also identified for each patient. |Multiples>>2; Table 4).

儘管在患者之間共用之具有體細胞突變之基因極少，但Wnt/β-連環蛋白信號傳導係所有患者中在P_Ts及R_Ts二者中受影響之最上游路徑(圖2(原始資料未顯示)。例如，在P_Ts與R_Ts之間共用之有害體細胞遺傳或基因組改變包括：HCC1：TP53及AKT2中之非沉默突變，CTNB1、E2F1及TCF4中上游基因印記之活化，及CDKN2A上游基因印記之阻抑；HCC4：CDH11及TGFBR3終止密碼子突變，POU5F1及UBD中之DNA擴增，及GNAO1中之DNA缺失，伴隨E2F1及TXB2上游基因印記之活化及上游基因印記CDKN2A及RB1之阻抑；HCC5：CDH1中之DNA缺失及TP53中之非沉默突變，伴隨E2F1及MYC上游基因印記之活化及上游基因印記CDKN2A及TP53之阻抑；HCC11：CTNNB1中之活化突變S45Y，LRP1中之終止密碼子突變及CDKN2A之ANK2結構域中之非沉默突變，伴隨SOX9中之DNA擴增。所有4名患者關於腫瘤阻抑劑CDKN2A(p14ARF)之P_Ts及R_Ts中之共用不活化而會聚。使用與鑑別所有4名患者中R_Ts特有之最富集路徑相同之策略來鑑別細胞週期信號傳導(未顯示資料)。 Although there are very few genes with somatic mutations shared among patients, the most upstream path affected by both P _T s and R _T s in all patients with Wnt/β-catenin signaling (Figure 2 (original The data are not shown. For example, the harmful somatic or genomic alterations shared between P _T s and R _T s include: HCC1: non-silent mutations in TP53 and AKT2 , activation of upstream gene imprints in CTNB1, E2F1 and TCF4 and upstream of the repressor gene signature CDKN2A; HCC4: CDH11 and TGFBR3 stop codon mutation, and POU5Fl UBD in the DNA amplification, and DNA in the absence of GNAO1 accompanied E2F1 gene signature of TXB2 and upstream activating genes upstream and imprinting CDKN2A And inhibition of RB1 ; HCC5: DNA deletion in CDH1 and non-silent mutation in TP53 , accompanied by activation of E2F1 and MYC upstream gene imprinting and suppression of upstream gene imprinting CDKN2A and TP53 ; HCC11: activating mutation S45Y in CTNNB1 , The stop codon mutation in LRP1 and the non-silent mutation in the ANK2 domain of CDKN2A , accompanied by DNA amplification in SOX9 . All 4 patients were involved in the P _T s and R _T s of the tumor suppressor CDKN2A ( p14ARF ) Shared not Activated to converge. Cell cycle signaling was identified using the same strategy as identifying the most enriched pathway unique to R _T s in all 4 patients (data not shown).

實例5. 患者一致性QCExample 5. Patient consistency QC

為驗證P_T、P_B及P_NAT以及R_T及R_NAT之一致性，自1000個基因組資料庫選擇300種MAF>0.3且<0.7之異型接合單一核苷酸多型性(SNP)。然後聚集每名患者之所有5個DNA試樣(4個試樣用於RNA)以觀察個體或樣本標記中之任何顯著偏差(圖4)。另外，在熱圖中聚集針對原發性及復發腫瘤試樣二者識別之生殖系變體以驗證在4名患者中之每一者之一式五份之DNA樣本內變體識別之適當阻斷(圖9及10)。 To verify the consistency of P _T , P _B and P _NAT and R _T and R _NAT , 300 MAF>0.3 and <0.7 heterotypic single nucleotide polymorphisms (SNPs) were selected from 1000 genomic databases. All 5 DNA samples (4 samples for RNA) from each patient were then pooled to observe any significant deviations in the individual or sample markers (Figure 4). In addition, germline variants identified for both primary and recurrent tumor samples were pooled in a heat map to verify proper blockade of variant recognition within five copies of each of the four patients. (Figures 9 and 10).

實例6. 純系關係(CR)值Example 6. Pure relationship (CR) values

藉由獨立列舉每一腫瘤中具有中等或高PolyPhen標識之所有非沉默SNV來評價每一原發性及復發腫瘤對之純系關係。測定在兩個匹配個體腫瘤之間共有之匹配基因座變體數，並計算針對每一腫瘤類型鑑別之共用變體相對於變體總數之比率。認為CR值大於0.5之個體源於接受者(圖11)。 The relationship between each primary and recurrent tumor pair was evaluated by independently listing all non-silent SNVs with a medium or high PolyPhen signature in each tumor. Determine the number of matched locus variants shared between two matched individual tumors and calculate for each tumor type The ratio of other variant variants to the total number of variants. Individuals with a CR value greater than 0.5 were considered to be from the recipient (Figure 11).

實例7. 復發腫瘤內之供給者存在Example 7. The presence of a supplier within a recurrent tumor

在HCC中，研發預測腫瘤復發及/或存活之一致性生物標記具有顯著臨床重要性。使用來自4名患者之WES及RNASeq資料以及來自顯示腫瘤復發(n=9，包括4；7/9 HBV⁺)或不顯示腫瘤復發(n=12；12/12 HBV⁺)之更大HCC患者群之P_Ts。鑑別在匹配P_Ts及R_Ts中之DNA缺失區域內且在復發HCC患者之P_Ts中顯著過低表現之基因(p<0.001；倍數<-2；未顯示資料)。此獲得4個基因，其中一者用於事件發生時間分析中：HERC5。 In HCC, the development of consistent biomarkers predicting tumor recurrence and/or survival has significant clinical importance. Use WES and RNASeq data from 4 patients and larger HCC patients from tumor recurrence (n=9, including 4;7/9 HBV ⁺ ) or no tumor recurrence (n=12; 12/12 HBV ⁺ ) Group P _T s. Genes that were significantly under-represented in the DNA deletion regions matching P _T s and R _T s and in P _T s of relapsed HCC patients were identified (p <0.001; fold <-2; no data shown). This resulted in 4 genes, one of which was used in the time of event analysis: HERC5 .

假定供給者傳播之惡性腫瘤之發病率極低(<0.05%)，則供給者腫瘤生檢之癌症純系組合物應主要由原發性腫瘤組成(Kauffman等人，Transplantation 73：358-362(2002))。然而，很難使用射頻燒灼(HCC1、HCC4及HCC5)或視訊輔助胸腔鏡外科手術(HCC11)技術自供給者組織獲得純接受者腫瘤生檢，如在該等患者中所實施。為鑑別供給者肝生檢內原發性腫瘤(基於接受者)之比例，實施兩次獨立計算。第一計算涉及回歸在復發腫瘤中識別之所有體細胞(非沉默及沉默)變體對原發性腫瘤中之彼等之Vf。移除原發性腫瘤或復發腫瘤特有之體細胞變體以不偏移最佳擬合線。然後用最小平方計算此曲線之斜率並自1減去以指示原發性腫瘤中之「供給者組織污染」比例(圖12)。 Assuming that the incidence of malignant tumors transmitted by the donor is extremely low (<0.05%), the cancer-only composition of the donor's tumor biopsy should consist primarily of primary tumors ( Kauffman et al., Transplantation 73:358-362 (2002). )). However, it is difficult to obtain pure recipient tumor biopsy from donor tissue using radiofrequency ablation (HCC1, HCC4, and HCC5) or video-assisted thoracoscopic surgery (HCC11) techniques, as implemented in such patients. To determine the proportion of primary tumors (based on recipients) within the donor liver bioassay, two independent calculations were performed. The first calculation involves regression of all of the somatic (non-silent and silent) variants identified in the recurrent tumor to their Vf in the primary tumor. The somatic cell variants characteristic of the primary or recurrent tumor are removed to not shift the best fit line. The slope of this curve is then calculated using the least squares and subtracted from 1 to indicate the "supplier tissue contamination" ratio in the primary tumor (Figure 12).

第二種方法利用一組接受者特有之同型接合SNP且將供給者細胞之污染計算為(1-復發腫瘤中接受者特有同型接合等位基因之Vf)之平均值，其中排除受CNV影響或位於chr X/Y中之等位基因。接受者特有之同型接合等位基因係彼等在接受者原發性血液中具有>90% Vf及在供給者中具有<10% Vf之等位基因，其對每一患者具有特異性(圖13)。 The second method utilizes a set of recipient-specific homozygous SNPs and calculates the contamination of the donor cells as an average of (1 - Vf of the recipient-specific homozygous allele in the recurrent tumor), excluding CNV effects or The allele located in chr X/Y. Receiver-specific homozygous alleles have an allele of >90% Vf in the recipient's primary blood and <10% Vf in the donor, which is specific for each patient (figure 13).

實例8. 預後標記之鑑別Example 8. Identification of prognostic markers

使用RNASeq資料，藉由每一患者內之匹配正常鄰近組織來換算原發性腫瘤中之DESeq正規化log₂基因計數。然後使用每一患者之該等倍數改變值來對比經歷腫瘤復發之患者(n=9)及未經歷腫瘤復發之患者(n=12)。鑑別總計273個基因之｜倍數改變｜>2且p<0.01。然後使在染色體4q(542個基因)中，3/4患者中之原發性及復發腫瘤樣本內彼等具有共用CNV缺失之基因與273個差異性表現之基因相交。根據此分析，鑑別以下基因：NAA11、HERC5、DDX60及HERC6。 Using the RNASeq data, the DESeq normalized log ₂ gene count in the primary tumor was converted by matching normal adjacent tissues within each patient. These fold change values for each patient were then used to compare patients who experienced tumor recurrence (n=9) and those who did not experience tumor recurrence (n=12). A total of 273 genes were identified | fold change |> 2 and p < 0.01. Then, among chromosome 4q (542 genes), the genes with shared CNV deletions in the primary and recurrent tumor samples in 3/4 patients intersected with 273 differentially expressed genes. Based on this analysis, the following genes were identified: NAA11, HERC5, DDX60, and HERC6 .

實例9. 事件發生時間分析Example 9. Analysis of the time of occurrence of an event A)方法A) Method

使用事件發生時間分析來關聯兩個可公開獲得之微陣列資料集中HERC5之表現與總體存活及HCC復發(Roessler等人，Cancer Res 70(24)：10202-12(2010)[GSE14520]；Boyault等人，Hepatology 45(1)：42-52(2007)[E-TABM-36])。每個研究實施三種不同分析：使用相等性之卡方(chi-squared)測試之兩個存活曲線之間之差異的Kaplan-Meier(KM)分析、多變數Cox比例風險(PH)回歸模型及針對每一變數之單變數Cox PH回歸模型。對於Roessler研究，使用健康常態分佈(n=239)之平均值-2SD作為切點將HERC5(在HGU133A陣列上之219863_at)分為高或低患者組。在低組中有62名HCC患者，且在高組中有180名患者。對於多變數Cox PH回歸模型，在針對年齡、肝硬化狀態(二元)、性別(二元)、HBV/HCV狀態(活性病毒複製慢性載體=2；慢性載體=1；無=0)及TNM分期(I=1，II=2，IIIA/IIIB/IIIC=3)調整後，使用分組係數、風險比以及似然比測試之p值來評價每一基因在高及低表現組之間之差異。亦在單變數Cox PH回歸模型中個別評價相同變數。在兩個單獨分析中評價總體存活及腫瘤復發時間(圖3及表6A-C)。對於Boyault研究，無正常健康個體試樣，故使用HCC患者表現值之中值將HERC5(在HGU133A陣列上之219863_at)分為高及低組。在低組中有20名HCC患者且在高組中有24名患者。然後計算針對性別(二元)、年齡及HBV(負效價=0；正效價=1)狀態調整之高及低患者組之間之總體存活及無進展存活二者(圖3及表7A-C)。 Event time analysis was used to correlate the performance and overall survival of HERC5 in two publicly available microarray datasets with HCC recurrence ( Roessler et al, Cancer Res 70(24): 10202-12 (2010) [GSE14520]; Boyault et al. Human, Hepatology 45(1): 42-52 (2007) [E-TABM-36]). Three different analyses were performed for each study: Kaplan-Meier (KM) analysis using the difference between the two survival curves of the chi-squared test of equality, the multivariate Cox proportional hazard (PH) regression model, and Single variable Cox PH regression model for each variable. For the Roessler study, HERC5 (219863_at on the HGU133A array) was divided into high or low patient groups using the mean -2 SD of the healthy normal distribution (n=239) as the cut point. There were 62 HCC patients in the low group and 180 patients in the high group. For multivariate Cox PH regression models, for age, cirrhosis status (binary), gender (binary), HBV/HCV status (active viral replication chronic vector = 2; chronic vector = 1; no = 0) and TNM After adjustment (I=1, II=2, IIIA/IIIB/IIIC=3), the grouping coefficient, risk ratio, and p-value of the likelihood ratio test were used to evaluate the difference between each gene in the high and low performance groups. . The same variables were also individually evaluated in a single variable Cox PH regression model. Overall survival and tumor recurrence time were evaluated in two separate analyses (Figure 3 and Tables 6A-C). For the Boyault study, there were no normal healthy individual samples, so HERC5 (219863_at on the HGU133A array) was divided into high and low groups using the median value of HCC patient performance. There were 20 HCC patients in the low group and 24 patients in the high group. Then calculate both the overall survival and the progression-free survival between the high and low patient groups for gender (binary), age, and HBV (negative titer = 0; positive titer = 1) status adjustment (Figure 3 and Table 7A). -C).

為展示HERC5與染色體4q中之相同DNA缺失區域內其他基因相比之預後特異性，對在來自獨立研究中之一者之此缺失區域內發現之每一基因實施類似事件發生時間分析(Roessler等人，Cancer Res 70(24)：10202-12(2010))。在4q上鑑別及在陣列上表示之262個基因中，與正常對照之組織相比，在腫瘤生檢中118個基因過低表現。在獨立事件發生時間模型中使用該118個基因，HAND2與HCC復發之關聯最為顯著，但其高/低患者之分佈高度不平衡(3.6%之患者於低組中)。HERC5與HCC復發之關聯為第二顯著(圖7)，其展示此基因獨立於染色體4q中其他具有CN缺失之基因之生物特異性。 To demonstrate the prognostic specificity of HERC5 compared to other genes in the same DNA deletion region in chromosome 4q, a similar event occurrence time analysis was performed for each gene found in this deleted region from one of the independent studies ( Roessler et al. Person, Cancer Res 70 (24): 10202-12 (2010)). Of the 262 genes identified on the 4q and represented on the array, 118 genes were understated in tumor biopsy compared to normal control tissues. Using the 118 genes in the independent event time model, HAND2 was most significantly associated with HCC recurrence, but the distribution of high/low patients was highly unbalanced (3.6% of patients were in the low group). The association of HERC5 with HCC recurrence was the second significant (Figure 7), which shows that this gene is independent of the biospecificity of other genes with CN deletions in chromosome 4q.

B)結果B) Results

所有經阻抑基因之間之最高級上游基因印記包括IFNα2及IFN-λ。在Roessler等人及Boyault等人之事件發生時間分析中對HERC5 mRNA表現加以分類，其中使用健康對照肝組織(n=239)(Roessler)或HCC患者分佈之中值(低n=20；高n=24)(Boyault)將HERC5 mRNA表現分類為高(n=180)或低(n=62)HCC患者組。在Roessler研究中，在針對年齡、肝硬化狀態、性別、HBV/HCV狀態及TNM分期調整後，用於預測總體存活及HCC復發之高/低分組變數分別為p=0.004；HR=2.02[1.26,3.25]及p=0.004；HR=1.80[1.20,2.69](圖3及表6A-C)；且在Boyault研究中，在針對年齡、性別及HBV狀態調整後，用於預測總體存活及無進展存活之高/低分組變數分別為p=0.02；HR=3.31[1.22,8.96]及p=0.01；HR=3.80[1.38,10.43](圖3及表7A-C)。評價HERC5預後關聯與染色體4q之缺失區域中之其他基因相比之特異性(圖7)。在患有HCV-陽性肝硬化之患者中，與藉由Hoshida等人，Gastroenterology 144：1024-30(2013)鑑別之較差預後組(n=107)相比，在以分子方式測定之良好預後(n=109)中，HERC5亦顯著過表現(p<0.0001)(圖8)。 The highest upstream gene signature between all repressed genes includes IFNα2 and IFN-λ. HERC5 mRNA expression was classified in the time-of-day analysis of events by Roessler et al. and Boyault et al., using healthy controls liver tissue (n=239) (Roessler) or HCC patients with median distribution (low n=20; high n =24) (Boyault) classifies HERC5 mRNA performance as a high (n=180) or low (n=62) HCC patient group. In the Roessler study, the high/low group variables used to predict overall survival and HCC recurrence after adjustment for age, cirrhosis status, gender, HBV/HCV status, and TNM stage were p=0.004; HR=2.02 [1.26] , 3.25] and p=0.004; HR=1.80 [1.20, 2.69] (Figure 3 and Table 6A-C); and in the Boyault study, adjusted for age, gender, and HBV status, used to predict overall survival and none The high/low grouping variables for progression survival were p = 0.02; HR = 3.31 [1.22, 8.96] and p = 0.01; HR = 3.80 [1.38, 10.43] (Figure 3 and Table 7A-C). The specificity of the HERC5 prognosis association compared to other genes in the deleted region of chromosome 4q was evaluated (Fig. 7). In patients with HCV-positive cirrhosis, a good prognosis measured in a molecular manner compared to the poor prognosis group identified by Hoshida et al., Gastroenterology 144: 1024-30 (2013) (n=107) Among n=109), HERC5 also showed significant overexpression (p<0.0001) (Fig. 8).

結果指示與HCC中之P_Ts相比，在OLT後R_Ts中之純系優點，且在兩種腫瘤中Wnt/β-連環蛋白信號傳導活化程度最高。HERC5係作為HCC復發風險及存活之預測子來鑑別且在Liverome資料庫編目之14個基因印記中之任一者中不存在(Lee等人，BMC Genomics 12增刊3：S3(2011))。此基因編碼之蛋白質催化ISG15之偶聯且驅使IRF3持續活化。因此，HERC5之存在活化宿主抗病毒反應，且其不存在降低該等反應，從而表明其與調節先天性免疫反應(HCC中導致腫瘤復發之潛在關鍵功能)之關聯；此假說將在今後研究中嚴格地評估。 The results indicate a pure advantage in R _T s after OLT compared to P _T s in HCC, and Wnt/β-catenin signaling activation is highest in both tumors. The HERC5 line was identified as a predictor of HCC recurrence risk and survival and did not exist in any of the 14 gene imprints cataloged in the Liverome database ( Lee et al, BMC Genomics 12 Supplement 3: S3 (2011)). The protein encoded by this gene catalyzes the coupling of ISG15 and drives the continuous activation of IRF3. Thus, the presence of HERC5 activates the host antiviral response, and its absence reduces these responses, suggesting its association with the regulation of the innate immune response (a potentially key function leading to tumor recurrence in HCC); this hypothesis will be in future studies. Strictly evaluated.

實例10. DNA及RNA B型肝炎病毒整合Example 10. DNA and RNA Hepatitis B virus integration

對於B型肝炎病毒(HBV)整合至人類基因組中之DNA位點，將90聚體WES成對末端序列讀長映射至參考索引檔案，包括人類基因組(UCSC hg19；Feb 2009版；Genome Reference Consortium GRCh37)、A型肝炎病毒(NC_001489)、B型肝炎病毒(NC_003977)、C型肝炎病毒(NC_004102)、δ型肝炎病毒(NC_001653)、E型肝炎病毒(NC_001434)及G型肝炎病毒(NC_001710)，使用Bowtie2(v2.0.0-beta7；Langmead等人，Nat Methods 9(4)：357-9(2012))。保留含有一個與人類基因組之配對比對及另一個與HBV基因組之配對比對之讀長對，如同兩個讀長對與HBV基因組比對之情形一般。每種方法必須觀察到至少2個事件才可視為顯著，與先前報導一致(Sung等人，Nat Genet 27(44)：765-9(2012))。對於RNA讀長，使用90聚體成對末端讀長及TopHat2作為讀長比對器實施類似方法(v2.0.9；Kim等人，Genome Biol 14(4)：R36(2013))。結果顯示於表8A-B中。 For the integration of hepatitis B virus (HBV) into the DNA locus in the human genome, the 90-mer WES paired end sequences are read and mapped to the reference index file, including the human genome (UCSC hg19; Feb 2009; Genome Reference Consortium GRCh37) ), hepatitis A virus (NC_001489), hepatitis B virus (NC_003977), hepatitis C virus (NC_004102), δ hepatitis virus (NC_001653), hepatitis E virus (NC_001434), and hepatitis G virus (NC_001710), Bowtie 2 (v2.0.0-beta7; Langmead et al, Nat Methods 9(4):357-9 (2012)) was used. The retention pair has a pair of pairs with the human genome and another pair with the HBV genome, as if the two pairs were aligned with the HBV genome. At least 2 events must be observed for each method to be considered significant, consistent with previous reports ( Sung et al., Nat Genet 27(44): 765-9 (2012)). A similar approach was performed for RNA read length using a 90-mer paired end read length and TopHat2 as a read length comparator (v2.0.9; Kim et al., Genome Biol 14(4): R36 (2013)). The results are shown in Tables 8A-B.

使用先前已報導之類似方法，使用WES及RNASeq資料來鑑別人類基因組整合之兩個位點及具有映射至病毒之兩個配對之讀長(Sung等人，Nat Genet 27(44)：765-9(2012)；Jiang等人，Genome Res 22：593-601(2012))。對於HBV存在，HCC4原發性及復發腫瘤具有最多DNA及RNA HBV計數(對於原發性及復發腫瘤DNA/RNA，分別為11/14,450及12/16,000)，此與臨床評價一致。HCC1僅在P_T中而非R_T中具有DNA HBV計數，與陰性病毒效價一致，但在此R_T中觀察到RNA HBV計數(14個計數；表8A)。HCC5及HCC11二者在P_T或R_T中皆無DNA HBV計數，與臨床評價一致。在HCC1 P_T DNA之3個特有區域中觀察到HBV整合位點，且用RNA之一個區域中觀察到該等位點。對於HCC4，在P_T RNA中觀察到3個特有位點，且在HBV聚合酶/X基因與染色體8q23.2之間之整合計數最多。在HCC4 R_T RNA中亦觀察到此相同高整合位點。(表8B)。 Using similar methods previously reported, WES and RNASeq data were used to identify two sites of human genome integration and two pairs of readings mapped to the virus ( Sung et al, Nat Genet 27(44): 765-9 (2012); Jiang et al., Genome Res 22: 593-601 (2012)). For the presence of HBV, HCC4 primary and recurrent tumors have the most DNA and RNA HBV counts (11/14, 450 and 12/16,000 for primary and recurrent tumor DNA/RNA, respectively), which is consistent with clinical evaluation. HCC1 has a DNA HBV count only in P _T but not R _T , consistent with the negative virus titer, but RNA HBV counts were observed in this R _T (14 counts; Table 8A). Both HCC5 and HCC11 had no DNA HBV count in either P _T or R _T , which was consistent with clinical evaluation. The HBV integration site was observed in three unique regions of HCC1 P _T DNA, and was observed in one region of RNA. For HCC4, three unique sites were observed in P _T RNA, and the integration count between HBV polymerase/X gene and chromosome 8q23.2 was the highest. This same high integration site was also observed in HCC4 R _T RNA. (Table 8B).

實例11. HCC試樣中HERC5含量之測定Example 11. Determination of HERC5 content in HCC samples

第一個患者經診斷患有肝細胞癌且作出關於此患者之治療選擇之決策。評估該患者之傳統臨床準則，包括腫瘤大小、腫瘤數、大血管侵犯及是否存在任何肝外病灶。發現該患者無大血管侵犯，無肝外病灶且有兩個直徑各自為2cm之肝病灶。 The first patient was diagnosed with hepatocellular carcinoma and made a decision regarding the treatment options for this patient. The patient's traditional clinical criteria were assessed, including tumor size, number of tumors, macrovascular invasion, and the presence of any extrahepatic lesions. The patient was found to have no macrovascular invasion, no extrahepatic lesions and two liver lesions each having a diameter of 2 cm.

製備一個病灶之針刺生檢，所選病灶係在針刺生檢期間最易到達者。使用qPCR及對HERC5具有特異性之經標記探針測定HERC5 mRNA表現程度。將肝細胞癌HERC5 mRNA表現程度與標準化截止值相比較。 A needle acupuncture biopsy was prepared, and the selected lesions were the most easily reachable during the acupuncture biopsy. The degree of expression of HERC5 mRNA was determined using qPCR and labeled probes specific for HERC5. The degree of expression of HERC5 mRNA in hepatocellular carcinoma was compared to a standardized cut-off value.

截止值係藉由使用相同qPCR技術測定一組正常患者之HERC5 mRNA表現程度來測定。截止值經測定較正常患者對照組值之平均值低兩個標準偏差。 The cut-off value was determined by measuring the extent of HERC5 mRNA expression in a group of normal patients using the same qPCR technique. The cut-off value was determined to be two standard deviations below the mean of the normal patient control values.

第一個患者之HERC5含量低於截止值及降低其在移植列表上之優先順序。 The first patient's HERC5 content is below the cutoff and reduces it on the transplant list. Priority order.

第二個患者經診斷患有肝細胞癌且評估進展類似於第一個患者。發現該患者無大血管侵犯，無肝外病灶且有3個直徑分別為1cm、1.5cm及2cm之肝病灶。第二個患者之HERC5含量高於截止值且提高其在移植列表上之優先順序。 The second patient was diagnosed with hepatocellular carcinoma and assessed for progression similar to the first patient. The patient was found to have no major vascular invasion, no extrahepatic lesions and three liver lesions with diameters of 1 cm, 1.5 cm and 2 cm, respectively. The second patient's HERC5 content was above the cut-off value and increased its priority on the transplant list.

等效形式Equivalent form

認為上述書面說明書足以使熟習此項技術者能實踐該等實施例。上述說明及實例詳述某些實施例且闡述本發明者預期之最佳模式。然而，應理解，不管上文文本可呈現得多麼詳細，可以多種方式實踐該等實施例且申請專利範圍包括其任何等效形式。 It is believed that the above written description is sufficient to enable those skilled in the art to practice the embodiments. The above description and examples are illustrative of certain embodiments and set forth the best mode contemplated by the present invention. However, it should be understood that the embodiments may be practiced in a variety of ways, and the scope of the claims includes any equivalents thereof.

Claims

一種治療患者之肝細胞癌之方法，其包含：a. 自該患者獲得肝細胞癌試樣；b. 確定該試樣是否具有正常HERC5 mRNA表現量；c. 若該試樣具有正常HERC5 mRNA表現量，則使該患者具有正位肝移植資格。 A method for treating hepatocellular carcinoma in a patient, comprising: a. obtaining a hepatocellular carcinoma sample from the patient; b. determining whether the sample has normal HERC5 mRNA expression; c. if the sample has normal HERC5 mRNA expression The amount makes the patient eligible for orthotopic liver transplantation.

如請求項1之方法，其中將該試樣中之HERC5 mRNA含量與歷史對照組中之HERC5 mRNA含量相比較。 The method of claim 1, wherein the HERC5 mRNA content in the sample is compared to the HERC5 mRNA content in a historical control group.

如請求項2之方法，其中該歷史對照組具有正常個體。 The method of claim 2, wherein the historical control group has a normal individual.

如請求項2之方法，其中該歷史對照組具有所患肝細胞癌在兩年內未復發之患者。 The method of claim 2, wherein the historical control group has a patient whose hepatocellular carcinoma has not relapsed within two years.

如請求項3至4中任一項之方法，其中若該HERC5患者試樣含量較該歷史對照組之平均值低等於或高於兩個標準偏差，則將該含量指定為正常。 The method of any one of claims 3 to 4, wherein the content of the HERC5 patient sample is designated as normal if the sample content is lower than the average of the historical control group by two standard deviations or more.

如請求項3至4中任一項之方法，其中若該HERC5患者試樣較該歷史對照組之平均值低等於或高於一個標準偏差，則將該試樣指定為正常。 The method of any one of claims 3 to 4, wherein the sample is designated as normal if the HERC5 patient sample is lower than the average of the historical control group by one standard deviation or more.

如請求項2之方法，其中該歷史對照組具有所患肝細胞癌在兩年內復發之患者。 The method of claim 2, wherein the historical control group has a patient whose hepatocellular carcinoma relapses within two years.

如請求項7之方法，其中若該HERC5患者試樣含量較該歷史對照組之平均值高等於或高於一個標準偏差，則將該含量指定為正常。 The method of claim 7, wherein the content of the HERC5 patient sample is designated as normal if the content of the HERC5 patient sample is higher than or equal to a standard deviation of the historical control group.

如請求項2之方法，其中該歷史對照組具有已患肝細胞癌之患者。 The method of claim 2, wherein the historical control group has a patient who has developed hepatocellular carcinoma.

如請求項9之方法，其中若該HERC5患者試樣高於該歷史組之中值，則將該試樣指定為正常。 The method of claim 9, wherein the HERC5 patient sample is higher than the historical group Value, the sample is designated as normal.

如請求項2至10中任一項之方法，其中該歷史對照包含來自至少10個個體之資料。 The method of any one of clauses 2 to 10, wherein the historical control comprises data from at least 10 individuals.

如請求項2至10中任一項之方法，其中該歷史對照包含來自至少25個個體之資料。 The method of any one of clauses 2 to 10, wherein the historical control comprises data from at least 25 individuals.

如請求項2至10中任一項之方法，其中該歷史對照包含來自至少50個個體之資料。 The method of any one of clauses 2 to 10, wherein the historical control comprises data from at least 50 individuals.

如請求項1之方法，其中將該試樣中之HERC5mRNA含量與自該患者獲得之正常組織之試樣中之HERC5 mRNA含量相比較。 The method of claim 1, wherein the HERC5 mRNA content in the sample is compared to the HERC5 mRNA content in a sample of normal tissue obtained from the patient.

如請求項14之方法，其中該正常組織係肝組織。 The method of claim 14, wherein the normal tissue is liver tissue.

如請求項14至15中任一項之方法，其中該肝細胞癌試樣中之HERC5 mRNA含量高於該正常組織試樣中之含量之80%，且該患者有資格進行正位肝移植。 The method of any one of claims 14 to 15, wherein the HERC5 mRNA content in the hepatocellular carcinoma sample is higher than 80% of the content in the normal tissue sample, and the patient is eligible for orthotopic liver transplantation.

如請求項14至15中任一項之方法，其中該肝細胞癌試樣中之HERC5 mRNA含量低於該正常組織試樣中之含量之50%，且該患者無資格進行正位肝移植。 The method of any one of claims 14 to 15, wherein the HERC5 mRNA content in the hepatocellular carcinoma sample is less than 50% of the content in the normal tissue sample, and the patient is not eligible for orthotopic liver transplantation.

如請求項1至17中任一項之方法，其中該患者無資格使用索拉非尼(sorafenib)。 The method of any one of claims 1 to 17, wherein the patient is ineligible to use sorafenib.

如請求項1至17中任一項之方法，其中利用索拉非尼治療該患者且該治療不成功。 The method of any one of claims 1 to 17, wherein the patient is treated with sorafenib and the treatment is unsuccessful.

如請求項1至19中任一項之方法，其中該肝細胞癌試樣具有HERC缺失且該患者無資格進行正位肝移植。 The method of any one of claims 1 to 19, wherein the hepatocellular carcinoma sample has a HERC deficiency and the patient is not eligible for orthotopic liver transplantation.

如請求項1至19中任一項之方法，其中該肝細胞癌試樣具有HERC5過低表現且該患者無資格進行正位肝移植。 The method of any one of claims 1 to 19, wherein the hepatocellular carcinoma sample has a low expression of HERC5 and the patient is not eligible for orthotopic liver transplantation.

如請求項1至21中任一項之方法，其中使患者有資格進行正位肝移植之該決策亦包括評估臨床準則。 The method of any one of claims 1 to 21, wherein the decision to qualify the patient for orthotopic liver transplantation also includes assessing clinical criteria.

如請求項22之方法，其中該等臨床準則包含以下中之至少一者：病灶大小、病灶數、是否具有肝外表現、是否具有血管侵犯、基於生檢HCC是否分化不良、腫瘤狀態、腫瘤等級及甲型胎兒蛋白含量。 The method of claim 22, wherein the clinical criteria comprise at least one of the following: lesion size, number of lesions, presence or absence of extrahepatic manifestations, vascular invasion, poor differentiation based on biopsy HCC, tumor status, tumor grade And the type A fetal protein content.

如請求項22至23中任一項之方法，其中該等臨床準則包含：a. 單一病灶不超過5cm或不多於3個病灶，皆不大於3cm；及b. 無大血管侵犯。 The method of any one of claims 22 to 23, wherein the clinical criteria comprise: a. a single lesion no more than 5 cm or no more than 3 lesions, no greater than 3 cm; and b. no large vessel invasion.

如請求項22至23中任一項之方法，其中該等臨床準則包含：a. 單一病灶不大於6.5cm；或b. 不多於3個病灶，其皆不超過4.5cm且其總腫瘤直徑不超過8cm。 The method of any one of claims 22 to 23, wherein the clinical criteria comprise: a. a single lesion no greater than 6.5 cm; or b. no more than 3 lesions, no more than 4.5 cm and a total tumor diameter Not more than 8cm.

如請求項22至23中任一項之方法，其中該等臨床準則包含Toronto準則。 The method of any one of claims 22 to 23, wherein the clinical criteria comprise a Toronto criterion.

如請求項22至23中任一項之方法，其中該等臨床準則包含Hangzhou準則。 The method of any one of clauses 22 to 23, wherein the clinical criteria comprise a Hangzhou criterion.

如請求項22至27中任一項之方法，其中該甲型胎兒蛋白濃度小於400ng/mL。 The method of any one of clauses 22 to 27, wherein the alpha-type fetal protein concentration is less than 400 ng/mL.

如請求項1至28中任一項之方法，其中具有正常HERC5 mRNA含量之患者在移植等待列表上具有優先於任何沒有正常HERC5 mRNA含量之肝細胞癌患者之優先權。 The method of any one of claims 1 to 28, wherein the patient having a normal HERC5 mRNA content has priority over a transplant waiting list on any hepatocellular carcinoma patient who does not have a normal HERC5 mRNA content.

如請求項1至16、18至19、22至29中任一項之方法，其中該患者接受肝移植。 The method of any one of claims 1 to 16, 18 to 19, 22 to 29, wherein the patient receives a liver transplant.

一種評估肝細胞癌之復發風險概況之方法，其包含：a. 提供肝細胞癌試樣；b. 在活體外測定該試樣是否具有正常HERC5 mRNA表現量；c. 若該試樣具有正常HERC5 mRNA表現量，則將該肝細胞癌定性為具有低復發風險，且該測定係結合至少一個臨床準則作出。 A method for assessing a risk of recurrence of hepatocellular carcinoma, comprising: a. providing a hepatocellular carcinoma sample; b. determining whether the sample has normal HERC5 mRNA expression in vitro; c. if the sample has normal HERC5 Hepatocellular carcinoma It is characterized as having a low risk of recurrence and the assay is made in conjunction with at least one clinical criterion.

如請求項31之方法，其中該肝細胞癌在正位肝移植後具有低復發風險。 The method of claim 31, wherein the hepatocellular carcinoma has a low risk of recurrence after orthotopic liver transplantation.

如請求項31至32之方法，其中將該試樣中之HERC5 mRNA含量與歷史對照組中之HERC5 mRNA含量相比較。 The method of claims 31 to 32, wherein the HERC5 mRNA content in the sample is compared to the HERC5 mRNA content in a historical control group.

如請求項33之方法，其中該歷史對照組具有正常個體。 The method of claim 33, wherein the historical control group has a normal individual.

如請求項33之方法，其中該歷史對照組具有所患肝細胞癌在兩年內未復發之患者。 The method of claim 33, wherein the historical control group has a patient whose hepatocellular carcinoma has not relapsed within two years.

如請求項34至35中任一項之方法，其中若該HERC5患者試樣含量較該歷史對照組之平均值低等於或高於兩個標準偏差，則將該含量指定為正常。 The method of any one of claims 34 to 35, wherein the content of the HERC5 patient sample is designated as normal if the sample content is lower than the average of the historical control group by two standard deviations or more.

如請求項35至35中任一項之方法，其中若該HERC5患者試樣較該歷史對照組之平均值低等於或高於一個標準偏差，則將該試樣指定為正常。 The method of any one of claims 35 to 35, wherein the sample is designated as normal if the HERC5 patient sample is less than or equal to one standard deviation from the mean of the historical control.

如請求項33之方法，其中該歷史對照組具有所患肝細胞癌在兩年內復發之患者。 The method of claim 33, wherein the historical control group has a patient whose hepatocellular carcinoma has recurred within two years.

如請求項38之方法，其中若該HERC5患者試樣含量較該歷史對照組之平均值高等於或高於一個標準偏差，則將該含量指定為正常。 The method of claim 38, wherein the content of the HERC5 patient sample is designated as normal if the sample content is higher than or equal to a standard deviation of the historical control group.

如請求項33之方法，其中該歷史對照組具有已患肝細胞癌之患者。 The method of claim 33, wherein the historical control group has a patient who has developed hepatocellular carcinoma.

如請求項40之方法，其中若該HERC5患者試樣高於該歷史組之中值，則將該試樣指定為正常。 The method of claim 40, wherein the sample is designated as normal if the HERC5 patient sample is above the value in the historical group.

如請求項33至41中任一項之方法，其中該歷史對照包含來自至少10個個體之資料。 The method of any one of clauses 33 to 41, wherein the historical control comprises data from at least 10 individuals.

如請求項33至41中任一項之方法，其中該歷史對照包含來自至少25個個體之資料。 The method of any one of clauses 33 to 41, wherein the historical control comprises data from at least 25 individuals.

如請求項33至41中任一項之方法，其中該歷史對照包含來自至少50個個體之資料。 The method of any one of clauses 33 to 41, wherein the historical control comprises data from at least 50 individuals.

如請求項31之方法，其中將該試樣中之HERC5 mRNA含量與自該患者獲得之正常組織之試樣中之HERC5 mRNA含量相比較。 The method of claim 31, wherein the HERC5 mRNA content in the sample is compared to the HERC5 mRNA content in a sample of normal tissue obtained from the patient.

如請求項45之方法，其中該正常組織係肝組織。 The method of claim 45, wherein the normal tissue is liver tissue.

如請求項45至46中任一項之方法，其中該肝細胞癌試樣中之HERC5 mRNA含量高於該正常組織試樣中之含量之80%，且該患者有資格進行正位肝移植。 The method of any one of claims 45 to 46, wherein the HERC5 mRNA content in the hepatocellular carcinoma sample is higher than 80% of the content in the normal tissue sample, and the patient is eligible for orthotopic liver transplantation.

如請求項45至46中任一項之方法，其中該肝細胞癌試樣中之HERC5 mRNA含量低於該正常組織試樣中之含量之50%，且該患者無資格進行正位肝移植。 The method of any one of claims 45 to 46, wherein the HERC5 mRNA content in the hepatocellular carcinoma sample is less than 50% of the content in the normal tissue sample, and the patient is not eligible for orthotopic liver transplantation.

如請求項31至48中任一項之方法，其中該患者無資格使用索拉非尼。 The method of any one of claims 31 to 48, wherein the patient is ineligible to use sorafenib.

如請求項31至48中任一項之方法，其中利用索拉非尼治療該患者且該治療不成功。 The method of any one of claims 31 to 48, wherein the patient is treated with sorafenib and the treatment is unsuccessful.

如請求項31至48中任一項之方法，其中該肝細胞癌試樣具有HERC5缺失且在正位肝移植後具有高復發風險。 The method of any one of claims 31 to 48, wherein the hepatocellular carcinoma sample has a HERC5 deletion and has a high risk of recurrence after orthotopic liver transplantation.

如請求項31至48中任一項之方法，其中該肝細胞癌試樣具有HERC5過低表現且在正位肝移植後具有高復發風險。 The method of any one of claims 31 to 48, wherein the hepatocellular carcinoma sample has a low expression of HERC5 and a high risk of recurrence after orthotopic liver transplantation.

如請求項31至52中任一項之方法，其中評估該復發風險亦包括評估臨床準則。 The method of any one of claims 31 to 52, wherein assessing the risk of recurrence also includes evaluating clinical criteria.

如請求項22之方法，其中該等臨床準則包含以下中之至少一者：病灶大小、病灶數量、是否具有肝外表現、是否具有血管侵犯、基於生檢HCC是否分化不良、腫瘤狀態、腫瘤等級及甲型胎兒蛋白含量。 The method of claim 22, wherein the clinical criteria comprise at least one of the following: lesion size, number of lesions, presence or absence of extrahepatic manifestations, vascular invasion, poor differentiation based on biopsy HCC, tumor status, tumor grade And type A Fetal protein content.

如請求項22至23中任一項之方法，其中該等臨床準則包含a. 單一病灶不超過5cm或不多於3個病灶，皆不大於3cm；及b. 無大血管侵犯。 The method of any one of claims 22 to 23, wherein the clinical criteria comprise a. a single lesion no more than 5 cm or no more than 3 lesions, no greater than 3 cm; and b. no macrovascular invasion.

如請求項22至23中任一項之方法，其中該等臨床準則包含該Toronto準則，且包括任何腫瘤大小或數目，及無因HCC所致之全身症狀，及基於組織學排除分化不良HCC(僅超過Milan腫瘤)。 The method of any one of claims 22 to 23, wherein the clinical criteria include the Toronto criteria and include any tumor size or number, and no systemic symptoms due to HCC, and exclusion of poorly differentiated HCC based on histology ( Only more than Milan tumors).

如請求項22至23中任一項之方法，其中該等臨床準則包含該Hangzhou準則。 The method of any one of clauses 22 to 23, wherein the clinical criteria comprise the Hangzhou criteria.

一種HERC5正向及反向引子之用途，其用於產生用於活體外診斷可利用正位肝移植治療之肝細胞癌之診斷液。 A use of HERC5 forward and reverse primers for the production of diagnostic fluids for the diagnosis of hepatocellular carcinoma which can be treated in vitro by orthotopic liver transplantation.

一種HERC5特異性探針之用途，其用於產生用於活體外診斷可利用正位肝移植治療之肝細胞癌之診斷液。 A use of a HERC5-specific probe for the production of a diagnostic solution for in vitro diagnosis of hepatocellular carcinoma which can be treated by orthotopic liver transplantation.

一種HERC5核酸序列作為定量對照之用途，其用於產生用於活體外診斷可利用正位肝移植治療之肝細胞癌之診斷液。 A use of a HERC5 nucleic acid sequence as a quantitative control for the production of a diagnostic fluid for the diagnosis of hepatocellular carcinoma which can be treated in vitro by orthotopic liver transplantation.

一種鑑別作為正位肝移植候選者之患者之方法，其包含：a. 自該患者獲得肝細胞癌試樣；b. 測定該試樣是否具有正常HERC5 mRNA表現量。 A method of identifying a patient as a candidate for orthotopic liver transplantation, comprising: a. obtaining a hepatocellular carcinoma sample from the patient; b. determining whether the sample has a normal HERC5 mRNA expression level.

如請求項63之方法，其中將該試樣中之HERC5 mRNA含量與歷史對照組中之HERC5 mRNA含量相比較。 The method of claim 63, wherein the content of HERC5 mRNA in the sample is The HERC5 mRNA levels in the historical control group were compared.

如請求項64之方法，其中該歷史對照組具有正常個體。 The method of claim 64, wherein the historical control group has a normal individual.

如請求項64之方法，其中該歷史對照組具有所患肝細胞癌在兩年內未復發之患者。 The method of claim 64, wherein the historical control group has a patient whose hepatocellular carcinoma has not relapsed within two years.

如請求項65至66中任一項之方法，其中若該HERC5患者試樣含量較該歷史對照組之平均值低等於或高於兩個標準偏差，則將該含量指定為正常。 The method of any one of clauses 65 to 66, wherein the content of the HERC5 patient sample is designated as normal if the sample content is lower than the average of the historical control group by two standard deviations or more.